hypothesis in photosynthesis

Microbe Notes

Photosynthesis: Equation, Steps, Process, Diagram

Photosynthesis is defined as the process, utilized by green plants and photosynthetic bacteria, where electromagnetic radiation is converted into chemical energy and uses light energy to convert carbon dioxide and water into carbohydrates and oxygen.

• The carbohydrates formed from photosynthesis provide not only the necessary energy form the energy transfer within ecosystems, but also the carbon molecules to make a wide array of biomolecules.
• Photosynthesis is a light-driven oxidation-reduction reaction where the energy from the light is used to oxidize water, releasing oxygen gas and hydrogen ions, followed by the transfer of electrons to carbon dioxide, reducing it to organic molecules.
• Photosynthetic organisms are called autotrophs because they can synthesize chemical fuels such as glucose from carbon dioxide and water by utilizing sunlight as an energy source.
• Other organisms that obtain energy from other organisms also ultimately depend on autotrophs for energy.
• One of the essential requirements for photosynthesis is the green pigment ‘chlorophyll’ which is present in the chloroplasts of green plants and some bacteria.
• The pigment is essential for ‘capturing’ sunlight which then drives the overall process of photosynthesis.

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Interesting Science Videos

Photosynthesis equations/reactions/formula

• The process of photosynthesis differs in green plants and sulfur bacteria.
• In plants, water is utilized along with carbon dioxide to release glucose and oxygen molecules.
• In the case of sulfur bacteria, hydrogen sulfide is utilized along with carbon dioxide to release carbohydrates, sulfur, and water molecules.

Oxygenic Photosynthesis

The overall reaction of photosynthesis in plants is as follows:

Carbon dioxide + Water  + solar energy → Glucose + Oxygen

6CO 2 + 6H 2 O  +  solar energy   →   C 6 H 12 O 6 + 6O 2

Carbon dioxide + Water  + solar energy → Glucose + Oxygen + Water

6CO 2 + 12H 2 O+ solar energy    →    C 6 H 12 O 6 + 6O 2 + 6H 2 O

Anoxygenic Photosynthesis

The overall reaction of photosynthesis in sulfur bacteria is as follows:

CO 2 + 2H 2 S + light energy   →    (CH 2 O)  + H 2 O  + 2S

Video Animation: Photosynthesis (Crash Course)

Photosynthetic pigments

• Photosynthetic pigments are the molecules involved in absorbing electromagnetic radiation, transferring the energy of the absorbed photons to the reaction center, resulting in photochemical reactions in the organisms capable of photosynthesis.
• The molecules of photosynthetic pigments are quite ubiquitous and are always composed of chlorophylls and carotenoids.
• In addition to chlorophyll, photosynthetic systems also contain another pigment, pheophytin (bacteriopheophytin in bacteria), which plays a crucial role in the transfer of electrons in photosynthetic systems.
• Moreover, other pigments can be found in particular photosynthetic systems, such as xanthophylls in plants.

Image Source: Simply Science .

Chlorophyll

• Chlorophyll is the pigment molecule, which is the principal photoreceptor in the chloroplasts of most green plants.
• Chlorophylls consist of a porphyrin ring, which is bounded to an ion Mg 2+ , attached to a phytol chain.
• Chlorophylls are very effective photoreceptors because they contain networks of alternating single and double bonds.
• In chlorophyll, the electrons are not localized to a particular atomic nucleus and consequently can more readily absorb light energy.
• In addition, chlorophylls also have solid absorption bands in the visible region of the spectrum.
• Chlorophylls are found either in the cytoplasmic membranes of photosynthetic bacteria, or thylakoid membranes inside plant chloroplasts.

Bacteriorhodopsin

• Bacteriorhodopsin is another class of photosynthetic pigment that exists only in halobacteria.
• It is composed of a protein attached to a retinal prosthetic group.
• This pigment is responsible for the absorption of light photons, leading to a conformational change in the protein, which results in the expulsion of the protons from the cell.

Phycobilins

• Cyanobacteria and red algae employ phycobilins such as phycoerythrobilin and phycocyanobilin as their light-harvesting pigments.
• These open-chain tetrapyrroles have the extended polyene system found in chlorophylls, but not their cyclic structure or central Mg 2+ .
• Phycobilins are covalently linked to specific binding proteins, forming phycobiliproteins, which associate in highly ordered complexes called phycobilisomes that constitute the primary light-harvesting structures in these microorganisms.

Carotenoids

• In addition to chlorophylls, thylakoid membranes contain secondary light-absorbing pigments, or accessory pigments, called carotenoids.
• Carotenoids may be yellow, red, or purple. The most important are β -carotene, which is a red-orange isoprenoid, and the yellow carotenoid lutein.
• The carotenoid pigments absorb light at wavelengths not absorbed by the chlorophylls and thus are supplementary light receptors.

Factors affecting photosynthesis

Blackman formulated the Law of limiting factors while studying the factors affecting the rate of photosynthesis. This Law states that the rate of a physiological process will be limited by the factor which is in the shortest supply. In the same way, the rate of photosynthesis is also affected by a number of factors, which are namely;

• As the intensity of light increases, the rate of light-dependent reactions of photosynthesis and in turn, the rate of photosynthesis increases.
• With increased light intensity, the number of photons falling on a leaf also increases. As a result, more chlorophyll molecules are ionized, and more ATPs and NADH are generated.
• After a point, however, the rate of photosynthesis remains constant as the light intensity increases. At this point, photosynthesis is limited by some other factors.
• Besides, the wavelength of light also affects the rate of photosynthesis.
• Different photosynthetic systems absorb light energy more effectively at different wavelengths.

Carbon dioxide

• An increase in the concentration of carbon dioxide increases the rate at which carbon is incorporated into carbohydrates in the light-independent reactions of photosynthesis.
• Thus, increasing the concentration of carbon dioxide in the atmosphere rapidly increases the rate of photosynthesis up to a point after which it is limited by some other factors.

Temperature

• The light-independent reactions of photosynthesis are affected by changes in temperature as they are catalyzed by enzymes, whereas the light-dependent reactions are not.
• The rate of the reactions increases as the enzymes reach their optimum temperature, after which the rate begins to decrease as the enzymes tend to denature.

Process/ Steps of Photosynthesis

The overall process of photosynthesis can be objectively divided into four steps/ process:

1. Absorption of light

• The first step in photosynthesis is the absorption of light by chlorophylls that are attached to the proteins in the thylakoids of chloroplasts.
• The light energy absorbed is then used to remove electrons from an electron donor like water, forming oxygen.
• The electrons are further transferred to a primary electron acceptor, quinine (Q) which is similar to CoQ in the electron transfer chain.

2. Electron Transfer

• The electrons are now further transferred from the primary electron acceptor through a chain of electron transfer molecules present in the thylakoid membrane to the final electron acceptor, which is usually NADP + .
• As the electrons are transferred through the membrane, protons are pumped out of the membrane, resulting in the proton gradient across the membrane.

3. Generation of ATP

• The movement of protons from the thylakoid lumen to the stroma through the F 0 F 1 complex results in the generation of ATP from ADP and Pi.
• This step is identical to the step of the generation of ATP in the electron transport chain .

4. Carbon Fixation

• The NADP and ATP generated in steps 2 and 3 provide energy, and the electrons drive the process of reducing carbon into six-carbon sugar molecules.
• The first three steps of photosynthesis are directly dependent on light energy and are thus, called light reactions, whereas the reactions in this step are independent of light and thus are termed dark reactions.

Types/ Stages/ Parts of photosynthesis

Figure: Photosynthesis takes place in two stages: light-dependent reactions and the Calvin cycle. Light-dependent reactions, which take place in the thylakoid membrane, use light energy to make ATP and NADPH. The Calvin cycle, which takes place in the stroma, uses energy derived from these compounds to make GA3P from CO 2 . Image Source: OpenStax (Rice University) .

Photosynthesis is divided into two stages based on the utilization of light energy:

1. Light-dependent reactions

• The light-dependent reactions of photosynthesis only take place when the plants/ bacteria are illuminated.
• In the light-dependent reactions, chlorophyll and other pigments of photosynthetic cells absorb light energy and conserve it as ATP and NADPH while simultaneously, evolving O 2 gas.
• In the light-dependent reactions of photosynthesis, the chlorophyll absorbs high energy, short-wavelength light, which excites the electrons present inside the thylakoid membrane.
• The excitation of electrons now initiates the transformation of light energy into chemical energy.
• The light reactions take in two photosystems that are present in the thylakoid of chloroplasts.

Figure: Light-dependent reactions of photosynthesis in the thylakoid membrane of plant cells. Image Source: Wikipedia (Somepics) .

Photosystem II

• Photosystem II is a group of proteins and pigments that work together to absorb light energy and transfer electrons through a chain of molecules until it finally reaches an electron acceptor.
• Photosystem II has a pair of chlorophyll molecules, also known as P680 as the molecules best absorb light of the wavelength 680 nm.
• The P680 donates a pair of electrons after absorbing light energy, resulting in an oxidized form of P680.
• Finally, an enzyme catalyzes the splitting of a water molecule into two electrons, two hydrogen ion, and oxygen molecules.
• This pair of electrons then are transferred to P680, causing it to return to its initial stage.

Photosystem I

• Photosystem I is a similar complex like photosystem II except for that photosystem I have a pair of chlorophyll molecules known as P700 as they best absorb the wavelength of 700 nm.
• As photosystem I absorb light energy, it also becomes excited and transfers electrons.
• The now oxidized form of P700 then accepts an electron from photosystem II, restring back to its initial stage.
• The electrons from photosystem I are then passed in a series of redox reactions through the protein ferredoxin.
• The electrons finally reach NADP + , reducing them to NADPH.

2 H 2 O + 2 NADP +  + 3 ADP + 3 P i  + light → 2 NADPH + 2 H +  + 3 ATP + O 2

Video Animation: The Light Reactions of Photosynthesis (Ricochet Science)

2. Light independent reactions (Calvin cycle)

Light independent reactions of photosynthesis are anabolic reactions that lead to the formation of a sex-carbon compound, glucose in plants. The reactions in this stage are also termed dark reactions as they are not directly dependent on the light energy but do require the products formed from the light reactions.

Figure: Overview of the Calvin cycle pathway. Image Source: Wikipedia (Mike Jones) .

This stage consists of 3 further steps that lead to carbon fixation/ assimilation.

Step 1: Fixation of CO 2 into 3-phosphoglycerate

• In this step, one CO 2 molecule is covalently attached to the five-carbon compound ribulose 1,5-biphosphate catalyzed by the enzyme ribulose 1,5-biphosphate carboxylase, also called rubisco.
• The attachment results in the formation of an unstable six-carbon compound that is then cleaved to form two molecules of 3-phosphoglycerate.

Step 2: Conversion of 3-phosphoglycerate to glyceraldehydes 3-phosphate

• The 3-phosphoglycerate formed in step 1 is converted to glyceraldehyde 3-phosphate by two separate reactions.
• At first, enzyme 3-phosphoglycerate kinase present in the stroma catalyzes the transfer of a phosphoryl group from ATP to 3-phosphoglycerate, yielding 1,3-bisphosphoglycerate.
• Next, NADPH donates electrons in a reaction catalyzed by the chloroplast-specific isozyme of glyceraldehyde 3-phosphate dehydrogenase, producing glyceraldehyde 3-phosphate and phosphate (Pi).
• Most of the glyceraldehyde 3-phosphate thus produced is used to regenerate ribulose 1,5-bisphosphate.
• The rest of the glyceraldehyde is either converted to starch in the chloroplast and stored for later use or is exported to the cytosol and converted to sucrose for transport to growing regions of the plant.

Step 3: Regeneration of ribulose 1,5-biphosphate from triose phosphates

• The three-carbon compounds formed in the previous steps are then converted into the five-carbon compound, ribulose 1,5-biphosphate through a series of transformations with intermediates of three-, four,-, five-, six-, and seven-carbon sugar.
• As the first molecules in the process, if regenerated, this stage of photosynthesis results in a cycle (Calvin cycle).

3 CO 2 + 9 ATP + 6 NADPH + 6 H +     →     glyceraldehyde-3-phosphate (G3P) + 9 ADP + 8 P i  + 6 NADP +  + 3 H 2 O

A G3P molecule contains three fixed carbon atoms, so it takes two G3Ps to build a six-carbon glucose molecule. It would take six turns of the cycle to produce one molecule of glucose.

Video Animation: The Calvin Cycle (Ricochet Science)

Products of Photosynthesis

The outcomes of light-dependent reactions of photosynthesis are:

The products of light-independent reactions (Calvin cycle) of photosynthesis are:

• glyceraldehyde-3-phosphate (G3P) / Glucose (carbohydrates)

The overall products of photosynthesis are:

• Glucose (carbohydrates)
• Sulfur (in photosynthetic sulfur bacteria)

Photosynthesis Examples

Photosynthesis in green plants or oxygenic bacteria.

• In plants and oxygenic bacteria like cyanobacteria, photosynthesis takes place in the presence of green pigment, chlorophyll.
• It takes place in the thylakoids of the chloroplasts, resulting in products like oxygen gas, glucose, and water molecules.
• Most of the glucose units in plants are linked to form starch or fructose or even sucrose.

Photosynthesis in sulfur bacteria

• In purple sulfur bacteria, photosynthesis takes place in the presence of hydrogen sulfur rather than water.
• Some of these bacteria like green sulfur bacteria have chlorophyll whereas other purple sulfur bacteria have carotenoids as photosynthetic pigments.
• The result of photosynthesis in these bacteria are carbohydrates (not necessarily glucose), sulfur gas, and water molecules.

Importance of photosynthesis

• Photosynthesis is the primary source of energy in autotrophs where they make their food by utilizing carbon dioxide, sunlight, and photosynthetic pigments.
• Photosynthesis is equally essential for heterotrophs, as they derive their energy from the autotrophs.
• Photosynthesis in plants is necessary to maintain the oxygen levels in the atmosphere.
• Besides, the products of photosynthesis contribute to the carbon cycle occurring in the oceans, land, plants, and animals.
• Similarly, it also helps maintain a symbiotic relationship between plants, animals, and humans.
• Sunlight or solar energy is the primary source of all other forms of energy on earth, which is utilized through the process of photosynthesis.

Artificial photosynthesis

Artificial photosynthesis is a chemical process that mimics the biological process of utilization of sunlight, water and carbon dioxide to produce oxygen and carbohydrates.

Image Source: Phys .

• In artificial photosynthesis, photocatalysts are utilized that are capable of replicating the oxidation-reduction reactions taking place during natural photosynthesis.
• The main function of artificial photosynthesis is to produce solar fuel from sunlight that can be stored and used under conditions, where sunlight is not available.
• As solar fuels are prepared, artificial photosynthesis can be used to produce just oxygen from water and sunlight, resulting in clean energy production.
• The most important part of artificial photosynthesis is the photocatalytic splitting of a water molecule, resulting in oxygen and large quantities of hydrogen gas.
• Further, light-driven carbon reduction can also be performed to replicate the process of natural carbon fixation, resulting in carbohydrates molecules.
• Thus, artificial photosynthesis has applications in the production of solar fuels, photoelectrochemistry, engineering of enzymes, and photoautotrophic microorganisms for the production of microbial biofuel and biohydrogen from sunlight.

Video Animation: Learning from leaves: Going green with artificial photosynthesis

Photosynthesis vs. Cellular respiration

Image Source: Khan Academy .

Video Animation: Photosynthesis vs. Cellular Respiration Comparison (BOGObiology)

FAQs (Revision Questions)

Where does photosynthesis occur? Photosynthesis occurs in the thylakoid membrane of the chloroplasts.

What are the products of photosynthesis? The products of photosynthesis are carbohydrates (glucose), oxygen, and water molecules.

What are the reactants of photosynthesis? The reactants of photosynthesis are carbon dioxide, water, photosynthetic pigments, and sunlight.

How are photosynthesis and cellular respiration related? Photosynthesis and cellular respiration are essentially the reverses of one another where photosynthesis is an anabolic process resulting in the formation of organic molecules. In contrast, cellular respiration is a catabolic process resulting in the breaking down of organic molecules to release energy.

• Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 17.2, Entry to the Citric Acid Cycle and Metabolism Through It Are Controlled.Available from: https://www.ncbi.nlm.nih.gov/books/NBK22347/
• Nelson DL and Cox MM. Lehninger Principles of Biochemistry. Fourth Edition.
• Montero F. (2011) Photosynthetic Pigments. In: Gargaud M. et al. (eds) Encyclopedia of Astrobiology. Springer, Berlin, Heidelberg
• Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 16.3, Photosynthetic Stages and Light-Absorbing Pigments.Available from: https://www.ncbi.nlm.nih.gov/books/NBK21598/

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Anupama Sapkota

1 thought on “Photosynthesis: Equation, Steps, Process, Diagram”

How can we say that 6 calvin cycles are needed to produce 1 glucose molecule why not 2?

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Article Contents

Origins of photosynthesis, photosynthetic pigments, reaction centers, electron transport chains, antenna systems, carbon fixation pathways, transition to oxygenic photosynthesis, literature cited.

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Early Evolution of Photosynthesis

This work was supported by the Exobiology Program from the U.S. National Aeronautics and Space Administration (grant no. NNX08AP62G).

E-mail [email protected] .

www.plantphysiol.org/cgi/doi/10.1104/pp.110.161687

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Robert E. Blankenship, Early Evolution of Photosynthesis, Plant Physiology , Volume 154, Issue 2, October 2010, Pages 434–438, https://doi.org/10.1104/pp.110.161687

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Photosynthesis is the only significant solar energy storage process on Earth and is the source of all of our food and most of our energy resources. An understanding of the origin and evolution of photosynthesis is therefore of substantial interest, as it may help to explain inefficiencies in the process and point the way to attempts to improve various aspects for agricultural and energy applications.

A wealth of evidence indicates that photosynthesis is an ancient process that originated not long after the origin of life and has evolved via a complex path to produce the distribution of types of photosynthetic organisms and metabolisms that are found today ( Blankenship, 2002 ; Björn and Govindjee, 2009 ). Figure 1 shows an evolutionary tree of life based on small-subunit rRNA analysis. Of the three domains of life, Bacteria, Archaea, and Eukarya, chlorophyll-based photosynthesis has only been found in the bacterial and eukaryotic domains. The ability to do photosynthesis is widely distributed throughout the bacterial domain in six different phyla, with no apparent pattern of evolution. Photosynthetic phyla include the cyanobacteria, proteobacteria (purple bacteria), green sulfur bacteria (GSB), firmicutes (heliobacteria), filamentous anoxygenic phototrophs (FAPs, also often called the green nonsulfur bacteria), and acidobacteria ( Raymond, 2008 ). In some cases (cyanobacteria and GSB), essentially all members of the phylum are phototrop2hic, while in the others, in particular the proteobacteria, the vast majority of species are not phototrophic.

Small subunit rRNA evolutionary tree of life. Taxa that contain photosynthetic representatives are highlighted in color, with green highlighting indicating a type I RC, while purple highlighting indicates a type II RC. The red arrow indicates the endosymbiotic event that formed eukaryotic chloroplasts. Tree adapted from Pace (1997) .

Overwhelming evidence indicates that eukaryotic photosynthesis originated from endosymbiosis of cyanobacterial-like organisms, which ultimately became chloroplasts ( Margulis, 1992 ). So the evolutionary origin of photosynthesis is to be found in the bacterial domain. Significant evidence indicates that the current distribution of photosynthesis in bacteria is the result of substantial amounts of horizontal gene transfer, which has shuffled the genetic information that codes for various parts of the photosynthetic apparatus, so that no one simple branching diagram can accurately represent the evolution of photosynthesis ( Raymond et al., 2002 ). However, there are some patterns that can be discerned from detailed analysis of the various parts of the photosynthetic apparatus, so some conclusions can be drawn. In addition, the recent explosive growth of available genomic data on all types of photosynthetic organisms promises to permit substantially more progress in unraveling this complex evolutionary process.

While we often talk about the evolution of photosynthesis as if it were a concerted process, it is more useful to consider the evolution of various photosynthetic subsystems, which have clearly had distinct evolutionary trajectories. In this brief review we will discuss the evolution of photosynthetic pigments, reaction centers (RCs), light-harvesting (LH) antenna systems, electron transport pathways, and carbon fixation pathways. These subsystems clearly interact with each other, for example both the RCs and antenna systems utilize pigments, and the electron transport chains interact with both the RCs and the carbon fixation pathways. However, to a significant degree they can be considered as modules that can be analyzed individually.

We know very little about the earliest origins of photosynthesis. There have been numerous suggestions as to where and how the process originated, but there is no direct evidence to support any of the possible origins ( Olson and Blankenship, 2004 ). There is suggestive evidence that photosynthetic organisms were present approximately 3.2 to 3.5 billion years ago, in the form of stromatolites, layered structures similar to forms that are produced by some modern cyanobacteria, as well as numerous microfossils that have been interpreted as arising from phototrophs ( Des Marais, 2000 ). In all these cases, phototrophs are not certain to have been the source of the fossils, but are inferred from the morphology or geological context. There is also isotopic evidence for autotrophic carbon fixation at 3.7 to 3.8 billion years ago, although there is nothing that indicates that these organisms were photosynthetic. All of these claims for early photosynthesis are highly controversial and have engendered a great deal of spirited discussion in the literature ( Buick, 2008 ). Evidence for the timing of the origin of oxygenic photosynthesis and the rise of oxygen in the atmosphere is discussed below. The accumulated evidence suggests that photosynthesis began early in Earth’s history, but was probably not one of the earliest metabolisms and that the earliest forms of photosynthesis were anoxygenic, with oxygenic forms arising significantly later.

Chlorophylls are essential pigments for all phototrophic organisms. Chlorophylls are themselves the product of a long evolutionary development, and can possibly be used to help understand the evolution of other aspects of photosynthesis. Chlorophyll biosynthesis is a complex pathway with 17 or more steps ( Beale, 1999 ). The early part of the pathway is identical to heme biosynthesis in almost all steps and has clearly been recruited from that older pathway. The later steps include the insertion of magnesium and the elaboration of the ring system and its substituents. The earliest version of the pathway (and that used by most modern anoxygenic photosynthetic organisms) almost certainly was anaerobic, both not requiring and not tolerating the presence of O 2 . However, all modern oxygenic photosynthetic organisms now require O 2 as an oxidant at several steps in the pathway. This has been explained in terms of gene replacement of the genes coding for the enzymes at these steps, with the result that the overall pathway is unchanged but the enzymes at key steps are completely different in different groups of phototrophs ( Raymond and Blankenship, 2004 ).

A key concept in using chlorophyll biosynthesis pathways to infer the evolution of photosynthesis is the Granick hypothesis, which states that the biosynthetic pathway of chlorophyll recapitulates the evolutionary sequence ( Granick, 1965 ). This is an appealing idea and probably at least partly true. However, in some cases, in particular the situation of chlorophyll and bacteriochlorophyll, it has been argued that the strict version of the Granick hypothesis is misleading and other interpretations are more likely ( Blankenship, 2002 ; Blankenship et al., 2007 ).

All photosynthetic organisms contain carotenoids, which are essential for photoprotection, usually also function as accessory pigments, and in many cases serve as key regulatory molecules. Carotenoids, unlike chlorophylls, are also found in many other types of organisms, so their evolutionary history may reflect many other functions in addition to photosynthesis ( Sandman, 2009 ).

The RC complex is at the heart of photosynthesis; so much attention has been paid to understand the evolution of RCs. A wealth of evidence, including structural, spectroscopic, thermodynamic, and molecular sequence analysis, clearly segregates all known RCs into two types of complexes, called type I and type II ( Blankenship, 2002 ). Anoxygenic phototrophs have just one type, either type I or II, while all oxygenic phototrophs have one of each type. The primary distinguishing feature of the two types of RCs are the early electron acceptor cofactors, which are FeS centers in type I RCs and pheophytin/quinone complexes in type II RCs. The distribution of RC types on the tree of life is shown in Figure 1 and a comparative electron transport diagram that compares the different RCs in different types of organisms is shown in Figure 2 , with type I RCs color coded green and type II RCs color coded purple.

Electron transport diagram indicating the types or RCs and electron transport pathways found in different groups of photosynthetic organisms. The color coding is the same as for Figure 1 and highlights the electron acceptor portion of the RC. Figure courtesy of Martin Hohmann-Marriott.

Further analysis strongly suggests that all RCs have evolved from a single common ancestor and have a similar protein and cofactor structure. This is clearly seen when structural overlays of both type I and II RCs are made, showing a remarkably conserved three-dimensional protein and cofactor structure, despite only minimal residual sequence identity ( Sadekar et al., 2006 ). These comparisons have been used to derive structure-based evolutionary trees that do not rely on sequence alignments. Figure 3 shows a schematic evolutionary tree of RCs that is derived from this sort of analysis. It proposes that the earliest RC was intermediate between type I and II (type 1.5) and that multiple gene duplications have given rise to the heterodimeric (two related yet distinct proteins that form the core of the RC) complexes that are found in most modern RCs.

Schematic evolutionary tree showing the development of the different types of RC complexes in different types of photosynthetic organisms. This tree is based on structural comparisons of RCs by Sadekar et al. (2006) . Blue color coding indicates protein homodimer, while red indicates protein heterodimer complexes. Red stars indicate gene duplication events that led to heterodimeric RCs. Helio, Heliobacteria; GSB, green sulfur bacteria; FAP, filamentous anoxygenic phototroph.

A second important issue that relates to RC evolution is the question of how both type I and II RCs came to be in cyanobacteria, while all other photosynthetic prokaryotes have only a single RC. The various proposals that have been made to explain this fact can all be divided into either fusion or selective loss scenarios or variants thereof ( Blankenship et al., 2007 ). In the fusion hypothesis, the two types of RCs develop separately in anoxygenic photosynthetic bacteria and are then brought together by a fusion of two organisms, which subsequently developed the ability to oxidize water. In the selective loss hypothesis, the two types of RCs both evolved in an ancestral organism and then loss of one or the other RC gave rise to the organisms with just one RC, while the ability to oxidize water was added later. Both scenarios have proponents, and it is not yet possible to choose between them.

The primary photochemistry and several of the early secondary electron transfer reactions take place within the RC complex. However, additional electron transfer processes are necessary before the process of energy storage is complete. These include the cytochrome bc   1 and b   6 f complexes. These complexes oxidize quinols produced by photochemistry in type II RCs or via cyclic processes in type I RCs and pumps protons across the membrane that in turn contribute to the proton motive force that is used to make ATP. All phototrophic organisms have a cytochrome bc   1 or b   6 f complex of generally similar architecture, with the exception of the FAP phylum of anoxygenic phototrophs ( Yanyushin et al., 2005 ). This group contains instead a completely different type of complex that is called alternative complex III. The evolutionary origin of this complex is not yet clear. While the cytochrome bc   1 and b   6 f complexes are similar in many ways, the cytochrome c   1 and f subunits are very different and are almost certainly of distinct evolutionary origin ( Baniulis et al., 2008 ).

All photosynthetic organisms contain a light-gathering antenna system, which functions to collect excitations and transfer them to the RC where the excited state energy is used to drive photochemistry ( Green and Parson, 2003 ). While the presence of an antenna is universal, the structure of the antenna complexes and even the types of pigments used in them is remarkably varied in different types of photosynthetic organisms. This very strongly suggests that the antenna complexes have been invented multiple times during the course of evolution to adapt organisms to particular photic environments. So while evolutionary relationships are clear among some categories of antennas, such as the LH1 and LH2 complexes of purple bacteria and the LHCI and LHCII complexes of eukaryotic chloroplasts, it is not possible to relate these broad categories of antennas to each other in any meaningful way. This is in contrast to the RCs, where all available evidence clearly points to a single origin that has subsequently undergone a complex evolutionary development.

Most phototrophic organisms are capable of photoautotrophic metabolism, in which inorganic substrates such as water, H 2 S, CO 2 , or HCO 3   − are utilized along with light energy to produce organic carbon compounds and oxidized donor species. However, there are some groups of phototrophs that cannot carry out photoautotrophic metabolism and there are at least three entirely separate autotrophic carbon fixation pathways that are found in different types of organisms ( Thauer, 2007 ). By far the dominant carbon fixation pathway is the Calvin-Benson cycle, which is found in all oxygenic photosynthetic organisms, and also in most purple bacteria. The GSB use the reverse tricarboxylic acid cycle, and many of the FAPs use the 3-hydroxypropionate cycle ( Zarzycki et al., 2009 ). The Gram-positive heliobacteria lack any known autotrophic carbon fixation pathway and usually grow photoheterotrophically ( Asao and Madigan, 2010 ). Similarly, the aerobic anoxygenic phototrophs, which are closely related to the purple bacteria, lack any apparent ability to fix inorganic carbon. In the latter case, it seems most likely that the ancestor of this group contained the Calvin-Benson cycle but lost the genes because of their obligate aerobic lifestyle ( Swingley et al., 2007 ).

The carbon fixation machinery is thus similar to the antennas, in that several entirely separate solutions have been adopted by different classes of phototrophic organisms. This would be consistent with the idea that the earliest phototrophs were photoheterotrophic, using light to assimilate organic carbon, instead of being photoautotrophic. The ability to fix inorganic carbon was then added to the metabolism somewhat later during the course of evolution, possibly borrowing carbon fixation pathways that had developed earlier in autotrophic nonphotosynthetic organisms.

Perhaps the most widely discussed yet poorly understood event in the evolution of photosynthesis is the invention of the ability to use water as an electron donor, producing O 2 as a waste product and giving rise to what is now called oxygenic photosynthesis. The production of O 2 and its subsequent accumulation in the atmosphere forever changed the Earth and permitted the development of advanced life that utilized the O 2 during aerobic respiration. Several lines of geochemical evidence indicate that free O 2 began to accumulate in the atmosphere by 2.4 billion years ago, although the ability to do oxygenic photosynthesis probably began somewhat earlier ( Buick, 2008 ). In order for O 2 to accumulate, it is necessary that both the biological machinery needed to produce it has evolved, but also the reduced carbon produced must be buried by geological processes, which are controlled by geological processes such as plate tectonics and the buildup of continents. So the buildup of O 2 in the atmosphere represents a coming together of the biology that gives rise to O 2 production and the geology that permits O 2 to accumulate.

Oxygen is produced by PSII in the oxygen evolving center, which contains a tetranuclear manganese complex. The evolutionary origin of the oxygen evolving center has long been a mystery. Several sources have been suggested, but so far no convincing evidence has been found to resolve this issue ( Raymond and Blankenship, 2008 ). The possibility that functional intermediate stages existed that connect the anoxygenic type II RCs to PSII seems likely ( Blankenship and Hartman, 1998 ).

The process of photosynthesis originated early in Earth’s history, and has evolved to its current mechanistic diversity and phylogenetic distribution by a complex, nonlinear process. Current evidence suggests that the earliest photosynthetic organisms were anoxygenic, that all photosynthetic RCs have been derived from a single source, and that antenna systems and carbon fixation pathways have been invented multiple times.

Asao M   Madigan MT ( 2010 ) Taxonomy, phylogeny, and ecology of the heliobacteria . Photosynth Res   104 : 103 – 111

Google Scholar

Baniulis D   Yamashita E   Zhang H   Hasan SS   Cramer WA ( 2008 ) Structure-function of the cytochrome b   6   f complex . Photochem Photobiol   84 : 1349 – 1358

Beale S ( 1999 ) Enzymes of chlorophyll biosynthesis . Photosynth Res   60 : 43 – 73

Björn LO   Govindjee ( 2009 ) The evolution of photosynthesis and chloroplasts . Curr Sci   96 : 1466 – 1474

Blankenship RE ( 2002 ) Molecular Mechanisms of Photosynthesis . Blackwell Science, Oxford

Blankenship RE   Hartman H ( 1998 ) The origin and evolution of oxygenic photosynthesis . Trends Biochem Sci   23 : 94 – 97

Blankenship RE   Sadekar S   Raymond J ( 2007 ) The evolutionary transition from anoxygenic to oxygenic photosynthesis . In   Falkowski P   Knoll AN , eds , Evolution of Aquatic Photoautotrophs . Academic Press , New York , pp 21–35

Buick R ( 2008 ) When did oxygenic photosynthesis evolve?   Philos Trans R Soc Lond B Biol Sci   363 : 2731 – 2743

Des Marais DJ ( 2000 ) Evolution: When did photosynthesis emerge on Earth?   Science   289 : 1703 – 1705

Granick S ( 1965 ) Evolution of heme and chlorophyll . In   Bryson G   Vogel HJ , eds , Evolving Genes and Proteins . Academic Press , New York , pp 67–88

Green BR   Parson WW   eds ( 2003 ) Light-Harvesting Antennas . Kluwer, Dordrecht , The Netherlands

Margulis L ( 1992 ) Symbiosis in Cell Evolution . WH Freeman , San Francisco

Olson JM   Blankenship RE ( 2004 ) Thinking about the evolution of photosynthesis . Photosynth Res   80 : 373 – 386

Pace NR ( 1997 ) A molecular view of microbial diversity and the biosphere . Science   276 : 734 – 740

Raymond J ( 2008 ) Coloring in the tree of life . Trends Microbiol   16 : 41 – 43

Raymond J   Blankenship RE ( 2004 ) Biosynthetic pathways, gene replacement and the antiquity of life . Geobiology   2 : 199 – 203

Raymond J   Blankenship RE ( 2008 ) The origin of the oxygen-evolving complex . Coord Chem Rev   252 : 377 – 383

Raymond J   Zhaxybayeva O   Gerdes S   Gogarten JP   Blankenship RE ( 2002 ) Whole genome analysis of photosynthetic prokaryotes . Science   298 : 1616 – 1620

Sadekar S   Raymond J   Blankenship RE ( 2006 ) Conservation of distantly related membrane proteins: photosynthetic reaction centers share a common structural core . Mol Biol Evol   23 : 2001 – 2007

Sandman G ( 2009 ) Evolution of carotenoid desaturation: the complication of a simple pathway . Arch Biochem Biophys   483 : 169 – 174

Swingley WD   Gholba S   Mastrian SD   Matthies HJ   Hao J   Ramos H   Acharya CR   Conrad AL   Taylor HL   Dejesa LC  et al.  ( 2007 ) The complete genome sequence of Roseobacter denitrificans reveals a mixotrophic rather than photosynthetic metabolism . J Bacteriol   189 : 683 – 690

Thauer RK ( 2007 ) A fifth pathway of carbon metabolism . Science   318 : 1732 – 1733

Yanyushin MF   del Rosario M   Brune DC   Blankenship RE ( 2005 ) A new class of bacterial membrane oxidoreductases . Biochemistry   44 : 10037 – 10045

Zarzycki J   Brecht V   Muller M   Fuchs G ( 2009 ) Identifying the missing steps of the autotrophic 3-hydroxypropionate CO 2 fixation cycle in Chloroflexus aurantiacus . Proc Natl Acad Sci USA   106 : 21317 – 21322

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ENCYCLOPEDIC ENTRY

Photosynthesis.

Photosynthesis is the process by which plants use sunlight, water, and carbon dioxide to create oxygen and energy in the form of sugar.

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Learning materials, instructional links.

• Photosynthesis (Google doc)

Most life on Earth depends on photosynthesis .The process is carried out by plants, algae, and some types of bacteria, which capture energy from sunlight to produce oxygen (O 2 ) and chemical energy stored in glucose (a sugar). Herbivores then obtain this energy by eating plants, and carnivores obtain it by eating herbivores.

The process

During photosynthesis, plants take in carbon dioxide (CO 2 ) and water (H 2 O) from the air and soil. Within the plant cell, the water is oxidized, meaning it loses electrons, while the carbon dioxide is reduced, meaning it gains electrons. This transforms the water into oxygen and the carbon dioxide into glucose. The plant then releases the oxygen back into the air, and stores energy within the glucose molecules.

Chlorophyll

Inside the plant cell are small organelles called chloroplasts , which store the energy of sunlight. Within the thylakoid membranes of the chloroplast is a light-absorbing pigment called chlorophyll , which is responsible for giving the plant its green color. During photosynthesis , chlorophyll absorbs energy from blue- and red-light waves, and reflects green-light waves, making the plant appear green.

Light-dependent Reactions vs. Light-independent Reactions

While there are many steps behind the process of photosynthesis, it can be broken down into two major stages: light-dependent reactions and light-independent reactions. The light-dependent reaction takes place within the thylakoid membrane and requires a steady stream of sunlight, hence the name light- dependent reaction. The chlorophyll absorbs energy from the light waves, which is converted into chemical energy in the form of the molecules ATP and NADPH . The light-independent stage, also known as the Calvin cycle , takes place in the stroma , the space between the thylakoid membranes and the chloroplast membranes, and does not require light, hence the name light- independent reaction. During this stage, energy from the ATP and NADPH molecules is used to assemble carbohydrate molecules, like glucose, from carbon dioxide.

C3 and C4 Photosynthesis

Not all forms of photosynthesis are created equal, however. There are different types of photosynthesis, including C3 photosynthesis and C4 photosynthesis. C3 photosynthesis is used by the majority of plants. It involves producing a three-carbon compound called 3-phosphoglyceric acid during the Calvin Cycle, which goes on to become glucose. C4 photosynthesis, on the other hand, produces a four-carbon intermediate compound, which splits into carbon dioxide and a three-carbon compound during the Calvin Cycle. A benefit of C4 photosynthesis is that by producing higher levels of carbon, it allows plants to thrive in environments without much light or water. The National Geographic Society is making this content available under a Creative Commons CC-BY-NC-SA license . The License excludes the National Geographic Logo (meaning the words National Geographic + the Yellow Border Logo) and any images that are included as part of each content piece. For clarity the Logo and images may not be removed, altered, or changed in any way.

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Photosynthetic Physiology of Blue, Green, and Red Light: Light Intensity Effects and Underlying Mechanisms

Associated data.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Red and blue light are traditionally believed to have a higher quantum yield of CO 2 assimilation ( QY , moles of CO 2 assimilated per mole of photons) than green light, because green light is absorbed less efficiently. However, because of its lower absorptance, green light can penetrate deeper and excite chlorophyll deeper in leaves. We hypothesized that, at high photosynthetic photon flux density ( PPFD ), green light may achieve higher QY and net CO 2 assimilation rate ( A n ) than red or blue light, because of its more uniform absorption throughtout leaves. To test the interactive effects of PPFD and light spectrum on photosynthesis, we measured leaf A n of “Green Tower” lettuce ( Lactuca sativa ) under red, blue, and green light, and combinations of those at PPFD s from 30 to 1,300 μmol⋅m –2 ⋅s –1 . The electron transport rates ( J ) and the maximum Rubisco carboxylation rate ( V c,max ) at low (200 μmol⋅m –2 ⋅s –1 ) and high PPFD (1,000 μmol⋅m –2 ⋅s –1 ) were estimated from photosynthetic CO 2 response curves. Both QY m,inc (maximum QY on incident PPFD basis) and J at low PPFD were higher under red light than under blue and green light. Factoring in light absorption, QY m,abs (the maximum QY on absorbed PPFD basis) under green and red light were both higher than under blue light, indicating that the low QY m,inc under green light was due to lower absorptance, while absorbed blue photons were used inherently least efficiently. At high PPFD , the QY inc [gross CO 2 assimilation ( A g )/incident PPFD ] and J under red and green light were similar, and higher than under blue light, confirming our hypothesis. V c,max may not limit photosynthesis at a PPFD of 200 μmol m –2 s –1 and was largely unaffected by light spectrum at 1,000 μmol⋅m –2 ⋅s –1 . A g and J under different spectra were positively correlated, suggesting that the interactive effect between light spectrum and PPFD on photosynthesis was due to effects on J . No interaction between the three colors of light was detected. In summary, at low PPFD , green light had the lowest photosynthetic efficiency because of its low absorptance. Contrary, at high PPFD , QY inc under green light was among the highest, likely resulting from more uniform distribution of green light in leaves.

Introduction

The photosynthetic activity of light is wavelength dependent. Based on McCree’s work ( McCree, 1971 , 1972 ), photosynthetically active radiation is typically defined as light with a wavelength range from 400 to 700 nm. Light with a wavelength shorter than 400 nm or longer than 700 nm was considered as unimportant for photosynthesis, due to its low quantum yield of CO 2 assimilation, when applied as a single waveband ( Figure 1 ). Within the 400–700 nm range, McCree (1971) showed that light in the red region (600–700 nm) resulted in the highest quantum yield of CO 2 assimilation of plants. Light in the green region (500–600 nm) generally resulted in a slightly higher quantum yield than light in the blue region (400–500 nm) ( Figure 1 ; McCree, 1971 ). The low absorptance of green light is partly responsible for its low quantum yield of CO 2 assimilation. Within the visible spectrum, green leaves have the highest absorptance in the blue region, followed by red. Green light is least absorbed by green leaves, which gives leaves their green appearance ( McCree, 1971 ; Zhen et al., 2019 ).

The normalized action spectrum of the maximum quantum yield of CO 2 assimilation for narrow wavebands of light from ultra-violet to far-red wavelengths ( McCree, 1971 ). Redrawn using data from Sager et al. (1988).

Since red and blue light are absorbed more strongly by photosynthetic pigments than green light, they are predominantly absorbed by the top few cell layers, while green light can penetrate deeper into leaf tissues ( Nishio, 2000 ; Vogelmann and Evans, 2002 ; Terashima et al., 2009 ; Brodersen and Vogelmann, 2010 ), thus giving it the potential to excite photosystems in deeper cell layers. Leaf photosynthesis may benefit from the more uniform light distribution throughout a leaf under green light. Absorption of photons by chloroplasts near the adaxial surface may induce heat dissipation of excess excitation energy in those chloroplasts, while chloroplasts deeper into the leaf receive little excitation energy ( Sun et al., 1998 ; Nishio, 2000 ). Blue and red photons, therefore, may be used less efficiently and are more likely to be dissipated as heat than green photons.

The misconception that red and blue light are used more efficiently by plants than green light still occasionally appears ( Singh et al., 2015 ), often citing McCree’s action spectrum or the poor absorption of green light by chlorophyll extracts. The limitations of McCree’s action spectrum were explained in his original paper: the quantum yield was measured under low photosynthetic photon flux density ( PPFD ), using narrow waveband light, and expressed on an incident light basis ( McCree, 1971 ), but these limitations are sometimes ignored. The importance of green light for photosynthesis has been well established in more recent studies ( Sun et al., 1998 ; Nishio, 2000 ; Terashima et al., 2009 ; Hogewoning et al., 2012 ; Smith et al., 2017 ).

From those studies, one trend has emerged that has not received much attention: there is an interactive effect of light quality and intensity on photosynthesis ( Sun et al., 1998 ; Evans and Vogelmann, 2003 ; Terashima et al., 2009 ). At low PPFD , green light has the lowest QY inc (quantum yield of CO 2 assimilation on incident light basis) because of its low absorptance; at high PPFD , on the other hand, red and blue light have a lower QY inc than green light, because of their high absorptance by photosynthetic pigments, which shifts much of the light absorption closer to the upper leaf surface. This reduces both the quantum yield of CO 2 assimilation in cells in the upper part of a leaf and light availability in the bottom part of a leaf.

The interactive effect between light quality and intensity was illustrated in an elegant study that quantified the differential quantum yield, or the increase in leaf CO 2 assimilation per unit of additional light ( Terashima et al., 2009 ). The differential quantum yield was measured by adding red or green light to a background illumination of white light of different intensities. At low background white light levels, the differential quantum yield of red light was higher than that of green light, due to the low absorptance of green light. But as the background light level increased, the differential quantum yield of green light decreased more slowly than that of red light, and was eventually higher than that of red light ( Terashima et al., 2009 ). The red light was absorbed efficiently by the chloroplasts in the upper part of leaves. With a high background level of white light, those chloroplasts already received a large amount of excitation energy from white light and up-regulated non-photochemical quenching (NPQ) to dissipate excess excitation energy as heat, causing the additional red light to be used inefficiently. Green light, on the other hand, was able to reach the chloroplasts deeper in the mesophyll and excited those chloroplasts that received relatively little excitation energy from white light. Therefore, with high background white light intensity, additional green light increased leaf photosynthesis more efficiently than red light ( Terashima et al., 2009 ).

In this paper, we present a comprehensive study to explore potential interactive effect of light intensity and light quality on C 3 photosynthesis and underlying processes. We quantified the photosynthetic response of plants to blue, green, and red light over a wide PPFD range to better describe how light intensity and waveband interact. In addition, we examined potential interactions among blue, green, and red light, using light with different ratios and intensities of the three narrow waveband lights. To get a better understanding of the biochemical reasons for the effects of light spectrum and intensity on CO 2 assimilation, we constructed assimilation – internal leaf CO 2 ( C i ) response curves ( A/C i curves) under blue, green, and red light, as well as combinations of the three narrow waveband lights at both high and low PPFD . We hypothesized that effects of different light spectra would be reflected in the electron transport rate ( J ) required to regenerate consumed ribulose 1,5-bisphosphate (RuBP), rather than the maximum carboxylation rate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) ( V c,max ).

Materials and Methods

Plant material.

Lettuce “Green Towers” plants were grown from seed in 1.7 L round pots filled with soilless substrate (Fafard 4P Mix, Sun Gro Horticulture, Agawam, MA, United States). The plants were grown in a growth chamber (E15, Conviron, Winnipeg, Manitoba, Canada) at 23.2 ± 0.8°C (mean ± SD), under white fluorescent light with a 14-hr photoperiod, vapor pressure deficit (VPD) of 1.20 ± 0.43 kPa and a PPFD of 200–230 μmol⋅m –2 ⋅s –1 at the floor level, and ambient CO 2 concentration. Plants were sub-irrigated when necessary with a nutrient solution containing 100 mg⋅L –1 N, made with a complete, water-soluble fertilizer (Peter’s Excel 15-5-15 Cal-Mag fertilizer, Everris, Marysville, OH, United States).

Leaf Absorptance, Transmittance, and Reflectance

Leaf absorptance was determined using a method similar to that of Zhen et al. (2019) . Three plants were randomly selected. A newly expanded leaf from each plant was illuminated with a broad-spectrum halogen bulb (70W; Sylvania, Wilmington, MA, United States) for leaf transmittance measurement. Transmittance was measured with a spectroradiometer (SS-110, Apogee, Logan, UT, United States). The halogen light spectrum was taken as reference measurement with the spectroradiometer placed directly under the halogen bulb in a dark room. Then, a lettuce leaf was placed between the halogen bulb and spectroradiometer, with its adaxial side facing the halogen bulb and transmitted light was measured. Leaf transmittance was then calculated on 1 nm resolution. Light reflectance of the leaves was measured using a spectrometer with a leaf clip (UniSpec, PP systems, Amesbury, MA, United States). Light absorptance was calculated as 1− r e f l e c t a n c e − t r a n s m i t t a n c e . We verified that this method results in similar absorptance spectra as the use of an integrating sphere. Absorptance of each of the nine light spectra used in this study were calculated from the overall leaf absorptance spectrum and the spectra of the red, green, and blue LEDs.

Leaf Photosynthesis Measurements

All gas exchange measurements were made with a leaf gas exchange system (CIRAS-3, PP Systems). Light was provided by the LEDs built into the chlorophyll fluorescence module (CFM-3, PP Systems). This module has dimmable LED arrays of different colors, with peaks at 653 nm [red, full width at half maximum (FWHM) of 17 nm], 523 nm (green, FWHM of 36 nm), and 446 nm (blue, FWHM of 16 nm). Nine different combinations of red, green, and blue light were used in this study ( Table 1 ). Throughout the measurements, the environmental conditions inside the cuvette were controlled by the leaf gas exchange system. Leaf temperature was 23.0 ± 0.1°C, CO 2 concentration was 400.5 ± 4.1 μmol⋅mol –1 , and the VPD of air in the leaf cuvette was 1.8 ± 0.3 kPa (mean ± SD).

List of light spectrum abbreviations and their spectral composition.

Photosynthesis – Light Response Curves

To explore photosynthetic efficiency of light with different spectra, we constructed light response curves for lettuce plants using each light spectrum. Lettuce plants were exposed to 10 PPFD levels ranging from 30 to 1,300 μmol⋅m –2 ⋅s –1 (30, 60, 90, 120, 200, 350, 500, 700, 1,000, and 1,300 μmol⋅m –2 ⋅s –1 ) in ascending orders for light response curves. Photosynthetic measurements were taken on 40–66 days old lettuce plants. Lettuce plants were taken out of the growth chamber and dark-adapted for 30 min. Starting from the lowest PPFD , one newly expanded leaf was exposed to all nine spectra. Net CO 2 assimilation rate ( A n ) of the leaf was measured using the leaf gas exchange system. Under each light spectrum, three A n readings were recorded at 10 s intervals after readings were stable (about 4–20 min depending on PPFD after changing PPFD and spectrum). The three A n readings were averaged for analysis. After A n measurements under all nine light spectra were taken, the leaf was exposed to the next PPFD level and A n measurements were taken with the light spectra in the same order, until measurements were completed at all PPFD levels. Throughout the light response curves, C i decreased with increasing PPFD , from 396 ± 10 μmol⋅mol –1 at a PPFD of 30 μmol⋅m –2 ⋅s –1 to 242 ± 44 μmol⋅mol –1 at a PPFD of 1,300 μmol⋅m –2 ⋅s –1 . To account for the potential effect of plants and the order of the spectra on assimilation rates, the order of the different spectra was re-randomized for each light response curve, using a Latin square design with plant and spectrum as the blocking factors. Data were collected on nine different plants.

Regression curves (exponential rise to maximum) were fitted to the data for each light spectrum and replication (plant):

where R d is the dark respiration rate, QY m,inc is the maximum quantum yield of CO 2 assimilation (initial slope of light response curve, mol of CO 2 fixed per mol of incident photons) and A g,max is the light-saturated gross assimilation rate. The A n,max is the light-saturated net assimilation rate and was calculated as A n , m a x = A g , m a x - R d . The maximum quantum yield of CO 2 assimilation was also calculated on absorbed light basis as Q ⁢ Y m , a ⁢ b ⁢ s = Q ⁢ Y m , i ⁢ n ⁢ c l ⁢ i ⁢ g ⁢ h ⁢ t ⁢ a ⁢ b ⁢ s ⁢ o ⁢ r ⁢ p ⁢ t ⁢ a ⁢ n ⁢ c ⁢ e .

The instantaneous quantum yield of CO 2 assimilation based on incident PPFD ( QY inc ) was calculated as A g P ⁢ P ⁢ F ⁢ D for each PPFD at which A n was measured, where the gross CO 2 assimilation rate ( A g ) was calculated as A g = A n + R d . To account for differences in absorptance among the different light spectra, the quantum yield of CO 2 assimilation was also calculated based on absorbed light base, as Q ⁢ Y a ⁢ b ⁢ s = A g P ⁢ P ⁢ F ⁢ D × l ⁢ i ⁢ g ⁢ h ⁢ t ⁢ a ⁢ b ⁢ s ⁢ o ⁢ r ⁢ p ⁢ t ⁢ a ⁢ n ⁢ c ⁢ e , where light absorptance is the absorptance of lettuce leaves for each specific light spectrum. The differential QY , the increase in assimilation rate per unit of additional incident PPFD , was calculated as the derivative of Eq. 1:

Photosynthesis – Internal CO 2 Response ( A/C i ) Curves

To explore the underlying physiological mechanisms of assimilation responses to different light spectra, we constructed A/C i curves. Typically, A/C i curves are collected under saturating PPFD . We collected A/C i curves at two PPFD s (200 and 1,000 μmol⋅m –2 ⋅s –1 ) to explore interactive effects of light spectrum and PPFD on the assimilation rate. At a PPFD of 200 μmol⋅m –2 ⋅s –1 , red light has the highest A n and green light the lowest A n , while at PPFD of 1,000 μmol⋅m –2 ⋅s –1 , red and green light resulted in the highest A n and blue light in the lowest A n .

We used the rapid A/C i response (RACiR) technique that greatly accelerates the process of constructing A/C i curves ( Stinziano et al., 2017 ). We used a Latin square design, similar to the light response curves. A/C i curves were measured under the same nine spectra used for the light response curves. Nine lettuce plants were used as replicates. For each A/C i curve, CO 2 concentration in the leaf cuvette started from 0 μmol⋅mol –1 , steadily ramping to 1,200 μmol⋅mol –1 over 6 min. A reference measurement was also taken at the beginning of each replication with an empty cuvette to correct for the reaction time of the leaf gas exchange system. Post-ramp data processing was used to calculate the real A and C i with the spreadsheet provided by PP systems, which yielded the actual A/C i curves with C i range of about 100–950 μmol mol –1 . Throughout the data collection, leaf temperature was 24.4 ± 1.3°C and VPD in the cuvette was 1.4 ± 0.2 kPa.

Curve fitting for A/C i curves was done by minimizing the residual sum of squares, following the protocol developed by Sharkey et al. (2007) . Among our nine replicates, four plants did not show clear Rubisco limitations at low PPFD and for those plants Rubisco limitation ( V c,max ) was not included in the model ( Sharkey et al., 2007 ). We therefore report V c,max values for high PPFD only. The J was determined for all light spectra at both PPFD s. We therefore report V c,max was determined for all light spectra only at high PPFD . The quantum yield of electron transport [ QY(J) ] was calculated on both incident and absorbed PPFD basis as Q ⁢ Y ⁢ ( J ) i ⁢ n ⁢ c = J P ⁢ P ⁢ F ⁢ D and Q ⁢ Y ⁢ ( J ) a ⁢ b ⁢ s = Q ⁢ Y ⁢ ( J ) i ⁢ n ⁢ c l ⁢ i ⁢ g ⁢ h ⁢ t ⁢ a ⁢ b ⁢ s ⁢ o ⁢ r ⁢ p ⁢ t ⁢ a ⁢ n ⁢ c ⁢ e , respectively. We did not estimate triose phosphate utilization, because the A/C i curves often did not show a clear plateau.

Data Analysis

The QY m,inc , QY m,abs , and A g,max were analyzed with ANOVA to determine the effects of light spectrum using SAS (SAS University Edition; SAS Institute, Cary, NC, United States). A n , QY inc , and QY abs at each PPFD level and V c,max and J estimated from A/C i curves were similarly analyzed with ANOVA using SAS. A n at different PPFD levels were analyzed with regression analysis to detect interactive effect of blue, green, and red light on leaf assimilation rates using the fractions of red, blue, and green light as explanatory variables (JMP Pro 15, SAS Institute).

Leaf Absorptance

A representative spectrum of light absorptance, reflectance and transmittance of a newly fully expanded lettuce leaf is shown in Figure 2 . In the blue region, 400–500 nm, the absorptance by “Green Towers” lettuce leaves was high and fairly constant, averaging 91.6%. The leaf absorptance decreased as the wavelength increased from 500 to 551 nm where the absorptance minimum was 69.8%. Absorptance increased again at longer wavelengths, with a second peak at 666 nm (92.6%). Above 675 nm, the absorptance decreased steadily to <5% at 747 nm ( Figure 2 ). The absorptance spectrum of our lettuce leaves is similar to what McCree (1971) obtained for growth chamber-grown lettuce, with the exception of slightly higher absorptance in the green part of the spectrum in our lettuce plants. Using this spectrum, the absorptance of the blue, green, and red LED lights were calculated to be 93.2 ± 1.0%, 81.1 ± 1.9% and 91.6 ± 1.1%, respectively. Absorptance of all nine spectra was calculated based on their ratios of red, green, and blue light ( Table 2 ).

Light absorptance, reflectance, and transmittance spectrum of a newly fully expanded “Green Towers” lettuce leaf.

Light absorptance and transmittance of new fully expanded “Green towers” lettuce leaves under nine light spectra.

Light Quality and Intensity Effects on Photosynthetic Parameters

Light response curves of lettuce under all nine spectra are shown in Figure 3 , with regression coefficients in Supplementary Table 1 . It is worth noting that a few plants showed photoinhibition under 100B (decrease in A n with PPFD > 1,000 μmol⋅m –2 ⋅s –1 ). Those data were excluded in curve fitting for light response curves to better estimate asymptotes. Photoinhibition was not observed under other spectra.

Net assimilation ( A n ) – light response curves of “Green Towers” lettuce under nine light spectra. Error bars represent the standard deviation ( n = 9). Inserts show A n against PPFD of 30-90 μmol⋅m –2 ⋅s –1 s to better show the initial slopes of curves. The composition of the nine light spectra is shown in Table 1 . The light spectra in the graphs are (A) 100B, 100G, and 100R; (B) 100B, 80B20G, 20B80G, and 100G; (C) 100G, 80G20R, 20G80R, and 100R; and (D) 20B80R, 16B20G64R, and 100G.

The QY m,inc of lettuce plants was 22 and 27% higher under red light (74.3 mmol⋅mol –1 ) than under either 100G (60.8 mmol⋅mol –1 ) or 100B (58.4 mmol⋅mol –1 ), respectively ( Figure 4A and Supplementary Table 1 ). Spectra with a high fraction of red light (64% or more) resulted in a high QY m,inc ( Figure 4A ), while 80G20R resulted in an intermediate QY m,inc ( Figure 4A ). To determine whether differences in QY m,inc were due to differences in absorptance or in the ability of plants to use the absorbed photons for CO 2 assimilation, we also calculated QY m,abs . On an absorbed light basis, 100B light still resulted in the lowest QY m,abs (62.7 mmol⋅mol –1 ) and red light resulted in the highest QY m,abs (81.1 mmol⋅mol –1 ) among narrow waveband lights ( Figure 4B ). Green light resulted in a QY m,abs (74.9 mmol⋅mol –1 ) similar to that under red light, but significantly higher than that of blue light ( Figure 4B ). We did not find any interactions (synergism or antagonism) between lights of different colors, with all physiological responses under mixed spectra being similar to the weighted average of responses under single colors. Thus, for the rest of the results we focus on the three narrow waveband spectra.

Maximum quantum yield of CO 2 assimilation of “Green Towers” lettuce based on incident ( QY m,inc ) (A) and absorbed light ( QY m,abs ) (B) under nine different light spectra. Values are calculated as the initial slope of the light response curves of corresponding light spectra (see Figure 3 ). Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent the standard deviation ( n = 9). The composition of the nine light spectra is shown in Table 1 .

Among the three narrow waveband lights, 100G resulted in the highest A g,max (20.0 μmol⋅m –2 ⋅s –1 ), followed by red (18.9 μmol⋅m –2 ⋅s –1 ), and blue light (17.0 μmol⋅m –2 ⋅s –1 ) ( Figure 5 and Supplementary Table 1 ). As with QY m,inc and QY m,abs , combining two or three colors of light resulted in an A g,max similar to the weighted averages of individual light colors.

Maximum gross assimilation rate ( A g,max ) of “Green Towers” lettuce under different light spectra, calculated from the light response curves. Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent standard deviation ( n = 9). The composition of the nine light spectra is shown in Table 1 .

QY inc initially increased with increasing PPFD and peaked at 90–200 μmol⋅m –2 ⋅s –1 , then decreased at higher PPFDs ( Figure 6A ). The QY inc under 100R was higher than under either green or blue light at low PPFD (≤300 μmol⋅m –2 ⋅s –1 ). Although 100G resulted in lower QY inc than 100B at low PPFD (≤300 μmol⋅m –2 ⋅s –1 ), the decrease in QY inc under 100G with increasing PPFD was slower than that with 100B or 100R. Above 500 μmol m –2 s –1 , the QY inc with 100G was comparable to the QY inc with 100R, and higher than with 100B ( Figure 6A ). The QY abs with 100R was higher than that with either 100G or 100B at PPFDs from 60 to 120 μmol⋅m –2 ⋅s –1 ( p < 0.05). The QY abs with 100G was similar to 100B at low PPFD , but decreased slower than that with either 100R or 100B as PPFD increased. At PPFD ≥ 500 μmol⋅m –2 ⋅s –1 , QY abs was lowest under 100B among the three monochromatic lights ( p < 0.05) ( Figure 6B ).

The quantum yield of CO 2 assimilation of “Green Towers” lettuce as a function of incident ( QY inc ) (A) and absorbed PPFD ( QY abs ) (B) under blue, green, and red LED light. Error bars represent the standard deviation ( n = 9).

The differential QY , which quantifies the increase in CO 2 assimilation per unit of additional PPFD , decreased with increasing PPFD . The differential QY with 100R was higher than those with 100B and 100G at low PPFD . At a PPFD of 30 μmol⋅m –2 ⋅s –1 , the differential QY was 70.5 mmol⋅mol –1 for 100R, 59.4 mmol⋅mol –1 for 100G, and 55.8 mmol⋅mol –1 for 100B ( Figure 7 ). However, the differential QY with 100R decreased rapidly with increasing PPFD and was lower than the differential QY with 100G at high PPFD ( Figure 7 ). At high PPFD , the differential QY with 100G was highest among three monochromatic light ( Figure 7 ). For instance, at a PPFD of 1,300 μmol⋅m –2 ⋅s –1 , the differential QY with 100G was 1.09 mmol⋅mol –1 , while those with 100B and 100R were 0.64 mmol⋅mol –1 and 0.46 mmol⋅mol –1 , respectively ( Figure 7 ).

The differential quantum yield of CO 2 assimilation ( differential QY ) of “Green Towers” lettuce under blue, green, and red LED light as a function of the PPFD . The differential QY is the increase in net assimilation per unit additional PPFD and was calculated as the first derivate of the light response curves ( Figure 3 ). The insert shows the differential quantum yield plotted at PPFDs of 1,000–1,300 μmol m –2 s –1 s to better show differences at high PPFD (note the different y -axis scale).

Effect of Light Spectrum and Intensity on J and V c,max

J of lettuce leaves at low PPFD was lowest under 100G (47.4 μmol⋅m –2 ⋅s –1 ), followed by 100B (56.1 μmol⋅m –2 ⋅s –1 ), and highest under 100R (64.1 μmol⋅m –2 ⋅s –1 ) ( Figure 8A ). At high PPFD , on the other hand, J of leaves exposed to 100G (115.3 μmol⋅m –2 ⋅s –1 ) and 100R (112.1 μmol⋅m –2 ⋅s –1 ) were among the highest, while J of leaves under 100B was the lowest (97.0 μmol⋅m –2 ⋅s –1 ) ( Figure 8A ). At high PPFD , V c,max of leaves under blue light (59.3 μmol⋅m –2 ⋅s –1 ) was lower than V c,max of leaves under 16B20G64R light (72.1 μmol⋅m –2 ⋅s –1 ), but none of the other treatments differed significantly ( Figure 8 ). When PPFD increased from 200 to 1,000 μmol⋅m –2 ⋅s –1 , J under green light increased by 143%, while J under blue and red light increased by 73% and 75%, respectively ( Figure 8A ). J and V c,max at high PPFD were strongly correlated ( R 2 = 0.82) ( Supplementary Figure 3 ).

Electron transport rate ( J ) at PPFD s of 200 (left bars) and 1,000 μmol m –2 s –1 (right bars) (A) and maximum Rubisco carboxylation rate ( V c,max ) at a PPFD of 1,000 μmol m –2 s –1 (B) of “Green Towers” lettuce, as estimated from A/C i curves under different light spectra. Bars with the same letter are not statistically different ( p ≤ 0.05). Error bars represent the standard deviation ( n = 9). The light composition of the nine light spectra is shown in Table 1 .

Interactive Effect of Light Spectrum and PPFD on Photosynthesis

There was an interactive effect of light spectrum and PPFD on photosynthetic properties of lettuce. Under low light conditions (≤200 μmol⋅m –2 ⋅s –1 ), the QY inc of lettuce leaves under green light was lowest among blue, green, and red light ( Figure 6A ), due to its lower absorptance by lettuce leaves. After accounting for absorptance, green photons were used at similar efficiency as blue photons, while red photons were used most efficiently ( Figure 6B ). The QY m,abs under green and red light were higher than under blue light ( Figure 4B ). At high PPFD , green and red light had similar quantum yield, higher than that of blue light, both on an absorbed and incident light basis ( Figure 6A ). Multiple factors contributed to the interactive effect of light spectrum and PPFD on quantum yield and photosynthesis.

Light Absorptance and Non-Photosynthetic Pigments Determine Assimilation at Low PPFD

QY m,inc with blue and green light was lower than with red light ( Figure 4A ), consistent with McCree’s action spectrum ( McCree, 1971 ). But when taking leaf absorptance into account, QY m,abs was similar under green and red light and lower under blue light ( Figure 4B ). Similarly, at low PPFD (≤200 μmol⋅m –2 ⋅s –1 ), QY inc of lettuce leaves was highest under red, intermediate under blue, and lowest under green light. When accounting for leaf absorptance, QY abs under red light remained highest and QY abs under both green and blue light were similar at low PPFD ( Figure 6A ). Consistent with our data, previous studies also documented that, once absorbed, green light can drive photosynthesis efficiently at low PPFD ( Balegh and Biddulph, 1970 ; McCree, 1971 ; Evans, 1987 ; Sun et al., 1998 ; Nishio, 2000 ; Terashima et al., 2009 ; Hogewoning et al., 2012 ; Vogelmann and Gorton, 2014 ). For example, the QY m,abs of spinach ( Spinacia oleracea ) and cabbage ( Brassica oleracea L. ) was highest under red light, followed by that under green light and lowest with blue light. But on incident light basis, QY m,inc of under green light was lower than under red or blue light ( Sun et al., 1998 ).

Both our data ( Figure 4B ) and those of Sun et al. (1998) show that QY m,abs with blue light is lower than that with red and green light, indicating that blue light is used intrinsically less efficiently by lettuce. Blue light, and, to a lesser extent, green light is absorbed not just by chlorophyll, but also by flavonoids and carotenoids ( Sun et al., 1998 ). Those pigments can divert energy away from photochemistry and thus reduce the QY abs under blue light. Flavonoids (e.g., anthocyanins) are primarily located in the vacuole and cannot transfer absorbed light energy to photosynthetic pigments ( Sun et al., 1998 ). Likewise, free carotenoids do not contribute to photochemistry ( Hogewoning et al., 2012 ). Carotenoids in light-harvesting antennae and reaction centers channel light energy to photochemistry, but with lower transfer efficiency than chlorophylls ( Croce et al., 2001 ; de Weerd et al., 2003a , b ; Wientjes et al., 2011 ; Hogewoning et al., 2012 ). Therefore, absorption of blue light by flavonoids and carotenoids reduces the quantum yield of CO 2 assimilation. Thus, even with the high absorptance of blue light by green leaves, QY m,abs of leaves under blue light was the lowest among the three monochromatic lights ( Figure 4B ). It is likely that the lower QY abs under green light than that under red light was also due to absorption of green light by carotenoids and flavonoids ( Hogewoning et al., 2012 ). At high PPFD , absorption of blue light by flavonoids and carotenoids still occurs, but this is less of a limiting factor for photosynthesis, since light availability is not limiting under high PPFD .

Light Dependence of Respiration and Rubisco Activity May Reduce the Quantum Yield at Low PPFD

At PPFD s below 200 μmol⋅m –2 ⋅s –1 , the QY inc and QY abs of lettuce showed an unexpected pattern in response to PPFD ( Figure 6 ). Unlike the quantum yield of PSII, which decreases exponentially with increasing PPFD ( Weaver and van Iersel, 2019 ), QY inc and QY abs increased initially with increasing PPFD ( Figure 6 ). A similar pattern was previously observed by Craver et al. (2020) in petunia ( Petunia × hybrida ) seedlings. This pattern could result from light-dependent regulation of respiration ( Croce et al., 2001 ), alternative electron sinks such as nitrate reduction ( Skillman, 2008 ; Nunes-Nesi et al., 2010 ), or Rubisco activity ( Campbell and Ogren, 1992 ; Zhang and Portis, 1999 ). In our calculations, we assumed that the leaf respiration in the light was the same as R d . However, leaf respiration in the light is lower than in the dark, in a PPFD -dependent manner ( Brooks and Farquhar, 1985 ; Atkin et al., 1997 ), which can lead to overestimation of A g with increasing PPFD . When we accounted for this down-regulation of respiration, using the model by Müller et al. (2005) to correct A g , QY inc , and QY abs , we found that depression of respiration by light did not explain the initial increase in QY inc and QY abs we observed ( Supplementary Figure 4 ). Alternative electron sinks in the chloroplasts that are upregulated in response to light can explain the low QY inc , and QY abs at low PPFD , because they compete with the Calvin cycle for reducing power (ferredoxin/NADPH). Such processes include photorespiration ( Krall and Edwards, 1992 ), nitrate assimilation ( Nunes-Nesi et al., 2010 ), sulfate assimilation ( Takahashi et al., 2011 ) and the Mehler reaction ( Badger et al., 2000 ) and their effect on QY inc , and QY abs would be especially notable under low PPFD ( Supplementary Figure 5 ).

Upregulation of Rubisco activity by Rubisco activase in the light may also have contributed to the increase in QY inc and QY abs at low PPFD ( Campbell and Ogren, 1992 ; Zhang and Portis, 1999 ). In the dark, 2-carboxy-D-arabinitol-1-phosphate (CA1P) or RuBP binds strongly to the active sites of Rubisco, preventing carboxylation activity. In the light, Rubisco activase releases the inhibitory CA1P or RuBP from the catalytic site of Rubisco, in a light-dependent manner ( Campbell and Ogren, 1992 ; Zhang and Portis, 1999 ; Parry et al., 2008 ). At PPFD < 120 μmol⋅m –2 ⋅s –1 , low Rubisco activity may have limited photosynthesis.

Light Distribution Within Leaves Affects QY at High PPFD

Except for the initial increase at low PPFD , both QY inc and QY abs decreased with increasing PPFD . QY inc decreased slower under green than under red or blue light ( Figure 6A ). At a PPFD ≥ 500 μmol⋅m –2 ⋅s –1 , QY inc under green light was higher than that under blue light ( Figure 6A ). Accordingly, A n under blue light was lower than under green and red light at PPFD s above 500 μmol⋅m –2 ⋅s –1 ( Figure 3A ). The lower QY inc under blue light than under green and red light at high PPFD can be explained by disparities in the light distribution within leaves.

Blue and red light were strongly absorbed by lettuce leaves (93.2 and 91.6%, respectively), while green light was absorbed less (81.1%) ( Table 2 ). Similar low green absorptance was found in sunflower ( Helianthus annuus L.), snapdragon ( Antirrhínum majus L.) ( Brodersen and Vogelmann, 2010 ), and spinach ( Vogelmann and Han, 2000 ). In leaves of those species, absorption of red and blue light peaked in the upper 20% of leaves, and declined sharply further into the leaf. Absorption of red light decreased slower with increasing depth than that of blue light ( Vogelmann and Han, 2000 ; Brodersen and Vogelmann, 2010 ). Green light absorption peaked deeper into leaves, and was more evenly distributed throughout leaves, because of low absorption of green light by chlorophyll ( Vogelmann and Han, 2000 ; Brodersen and Vogelmann, 2010 ). The more even distribution of green light within leaves, as compared to red and blue light, can explain the interactive effects between PPFD and light spectrum on leaf photosynthesis. It was estimated that less than 10% of blue light traveled through the palisade mesophyll and reached the spongy mesophyll in spinach, while about 35% of green light and 25% of red light did so ( Vogelmann and Evans, 2002 ). It was also estimated that chlorophyll in the lowermost chloroplasts of spinach leaves absorbed about 10% of green and <2% of blue light, compared to chlorophyll in the uppermost chloroplasts ( Vogelmann and Evans, 2002 ; Terashima et al., 2009 ).

The more uniform green light distribution within leaves may be a key contributor to higher leaf level QY inc under high PPFD because less heat dissipation of excess light energy is needed ( Nishio, 2000 ; Terashima et al., 2009 ). Reaction centers near the adaxial leaf surface receive more excitation energy under blue, and to a lesser extent under red light, than under green light, because of the differences in absorptance. Consequently, under high intensity blue light, NPQ is up-regulated in the chloroplasts near the adaxial leaf surface to dissipate some of the excitation energy ( Sun et al., 1998 ; Nishio, 2000 ), lowering the QY inc under blue light. Since less green light is absorbed near the adaxial surface, less heat dissipation is required. When incident light increased from 150 to 600 μmol⋅m –2 ⋅s –1 , the fraction of whole leaf CO 2 assimilation that occurred in the top half of spinach leaves remained the same under green light (58%), but decreased from 87 to 73% under blue light. This indicates more upregulation of heat dissipation in the top of the leaves under blue, than under green light ( Evans and Vogelmann, 2003 ). On the other hand, the bottom half of the leaves can still utilize the available light with relatively high QY inc , since the amount of light reaching the bottom half is relatively low, even under high PPFD ( Nishio, 2000 ). By channeling more light to the under-utilized bottom part of leaves, leaves could achieve higher QY inc even under high intensity green light. In our study, high QY inc under green light and low QY inc under blue light at high PPFD ( Figure 6 ) can be thus explained by the large disparities in the light environment in chloroplasts from the adaxial to the abaxial side of leaves due to differences in leaf absorptance. Similarly, differential QY of lettuce leaves was highest under green light and lower under blue and red light at high PPFD (>300 μmol⋅m –2 ⋅s –1 ) ( Figure 7 ), also potentially because of the more uniform distribution of green light and the uneven distribution of blue and red light in leaves.

Along the same line, A n of lettuce leaves was the lowest under blue light at PPFD > 500 μmol⋅m –2 ⋅s –1 ( Figure 3 ). Also, A n of lettuce leaves approached light saturation at lower PPFD s under blue and red light, than under green light ( Figure 3A ). Under blue, green, and red light, lettuce leaves reached 95% of A n,max at PPFD s of 954, 1,110 and 856 μmol⋅m –2 ⋅s –1 , respectively. This can be seen more clearly in the differential QY at high PPFD ( Figure 7 ). At a PPFD of 1,300 μmol⋅m –2 ⋅s –1 , green light had a differential QY of 1.09 mmol⋅mol –1 , while that of red and blue light was only 0.46 and 0.69 mmol⋅mol –1 , respectively ( Figure 7 ). Green light also resulted in a higher A g,max (22.9 μmol⋅m –2 ⋅s –1 ) than red and blue light (21.8 and 19.3 μmol⋅m –2 ⋅s –1 , respectively) ( Figure 5 ). As discussed before, the high A g,max under green light resulted from the more uniform light distribution under green light, allowing deeper cell layers to photosynthesize more. Previous research similarly found that at high PPFD (>500 μmol⋅m –2 ⋅s –1 ), A n of both spinach and cabbage were lower under blue light than under white, red and green light ( Sun et al., 1998 ). Overall, under high PPFD , the differences in light distribution throughout a leaf are important to quantum yield and assimilation rate, since it affects NPQ up-regulation ( Sun et al., 1998 ; Nishio, 2000 ). However, light distribution within a leaf is less important at low than at high PPFD , because upregulation of NPQ increases with increasing PPFD ( Zhen and van Iersel, 2017 ).

Light Spectrum Affects J , but Not V c,max

We examined the effect of light quality and intensity on J and V c,max ( Figure 8 ). For the light-dependent reactions, the interactive effect between light spectra and PPFD found for CO 2 assimilation and quantum yield was also observed for J ( Figure 8A ). At low PPFD (200 μmol⋅m –2 ⋅s –1 ), green light resulted in the lowest J and red light in the highest J among single waveband spectra. But at a PPFD of 1,000 μmol⋅m –2 ⋅s –1 , red and green light resulted in the highest J and blue light in the lowest J ( Figure 8A ), similar to the differences in A g .

There was no clear evidence of Rubisco limitations to photosynthesis at a PPFD of 200 μmol⋅m –2 ⋅s –1 , so the rate of the light-dependent reactions likely limited photosynthesis. This is corroborated by the strong correlation between A g and J at a PPFD of 200 μmol⋅m –2 ⋅s –1 . Although Rubisco limitations to photosynthesis were observed at a PPFD of 1,000 μmol⋅m –2 ⋅s –1 , there were no meaningful differences in V c,max in response to light spectrum, in contrast to J ( Figure 8 ).

When PPFD increased 5×, from 200 to 1,000 μmol⋅m –2 ⋅s –1 , there was only a 1.7 to 2.4× increase in J , indicating a lower QY(J) inc at higher PPFD . This matches the lower QY inc and the asymptotic increase in A n in response to increasing PPFD ( Figure 3 ). The relative increase of J under green light (143%) was greater than that under both blue and red light (73 and 75%, respectively) as PPFD increased. This similarly can be attributed to a more uniform energy distribution of green light among reaction centers throughout a leaf and weaker upregulation of non-photochemical quenching with increasing green light intensity ( Sun et al., 1998 ; Nishio, 2000 ; Evans and Vogelmann, 2003 ), as discussed before.

There was a strong correlation between J and A g under the nine light spectra at both PPFD levels ( Figure 9A ). QY abs and QY(J) abs are similarly strongly correlated ( Figure 9B ). Unlike J , V c,max was largely unaffected by light spectra ( Figure 8B ) and was not correlated with A g (data not shown). There was, however, a strong correlation between J and V c,max at a PPFD of 1,000 μmol⋅m –2 ⋅s –1 ( R 2 = 0.82, Supplementary Figure 3 ), suggesting that J and V c,max are co-regulated. Similarly, Wullschleger (1993) noted a strong linear relationship between J and V c,max across 109 C 3 species. The ratio between J and V c,max in our study (1.5–2.0) similar to the ratio found by Wullschleger (1993) . These results suggest that the interactive effect of light spectra and PPFD resulted from effects on J , which is associated with light energy harvesting by reaction centers, rather than from V c,max .

The correlation between gross CO 2 assimilation rate ( A g ) estimated from light response curves and electron transport rate ( J ) estimated from A/C i curves (A) , and between the quantum yield of CO 2 assimilation ( QY abs ) and the quantum yield of electron transport on an absorbed light basis [ QY(J) abs ] (B) , under low PPFD (200 μmol m –2 s –1 ) and high PPFD (1,000 μmol m –2 s –1 ) under nine light spectra averaged over nine “Green Towers” lettuce plants. The color scheme representing the nine spectra is the same as Figure 8 .

No Interactive Effects Among Blue, Green, and Red Light

The Emerson enhancement effect describes a synergistic effect between lights of different wavebands (red and far-red) on photosynthesis ( Emerson, 1957 ). McCree (1971) attempted to account for interactions between light with different spectra when developing photosynthetic action spectra and applied low intensity monochromatic lights from 350 to 725 nm with white background light to plants. His results showed no interactive effect between those monochromatic lights and white light ( McCree, 1971 ). We tested different ratios of blue, green, and red light and different PPFD s, and similarly did not find any synergistic or antagonistic effect of different wavebands on any physiological parameters measured or calculated.

Importance of Interactions Between PPFD and Light Quality and Its Applications

The interactive effect between PPFD and light quality demonstrates a remarkable adaptation of plants to different light intensities. By not absorbing green light strongly, plants open up a “green window,” as Terashima et al. (2009) called it, to excite chloroplasts deeper into leaves, and thus facilitating CO 2 assimilation throughout the leaf. While red light resulted in relatively high QY inc , QY abs and A n at both high and low PPFD ( Figures 3 , ​ ,6), 6 ), it is still mainly absorbed in the upper part of leaves ( Sun et al., 1998 ; Brodersen and Vogelmann, 2010 ). Green light can penetrate deeper into leaves ( Brodersen and Vogelmann, 2010 ) and help plants drive efficient CO 2 assimilation at high PPFD ( Figures 3 , ​ , 5 5 ).

Many early photosynthesis studies investigated the absorptance and action spectrum of photosynthesis of green algae, e.g., Haxo and Blinks (1950) or chlorophyll or chloroplasts extracts, e.g., Chen (1952) . Extrapolating light absorptance of green algae and suspension of chlorophyll or chloroplast to whole leaves from can lead to an underestimation of absorptance of green light by whole leaves and the belief that green light has little photosynthetic activity ( Moss and Loomis, 1952 ; Smith et al., 2017 ). Photosynthetic action spectra developed on whole leaves of higher plants, however, have long shown that green light effectively contributes to CO 2 assimilation, although with lower QY inc than red light ( Hoover, 1937 ; McCree, 1971 ; Inada, 1976 ; Evans, 1987 ). The importance of green light for photosynthesis was clearly established in more recent studies, emphasizing its role in more uniformly exciting all chloroplasts, which especially important under high PPFD ( Sun et al., 1998 ; Nishio, 2000 ; Terashima et al., 2009 ; Hogewoning et al., 2012 ; Smith et al., 2017 ). The idea that red and blue light are more efficient at driving photosynthesis, unfortunately, still lingers, e.g., Singh et al. (2015) .

Light-emitting diodes (LEDs) have received wide attention in recent years for use in controlled environment agriculture, as they now have superior efficacy over traditional lighting technologies ( Pattison et al., 2018 ). LEDs can have a narrow spectrum and great controllability. This provides unprecedented opportunities to fine tune light spectra and PPFD to manipulate crop growth and development. Blue and red LEDs have higher efficacy than white and green LEDs ( Kusuma et al., 2020 ). By coincidence, McCree’s action spectrum ( Figure 1 ; McCree, 1971 ) also has peaks in the red and blue region, although the peak in the blue region is substantially lower than the one in the red region. Therefore, red and blue LEDs are sometimes considered optimal for driving photosynthesis. This claim holds true only under low PPFD . Green light plays an important role in photosynthesis, as it helps plants to adapt to different light intensities. The wavelength-dependent absorptance of chlorophylls channels green light deeper into leaves, resulting in more uniform light absorption throughout leaves and providing excitation energy to cells further from the adaxial surface. Under high PPFD , this can increase leaf photosynthesis. Plant evolved under sunlight for hundreds of millions of years, and it seems likely that the relatively low absorptance of green light contributes to the overall photosynthetic efficiency of plants ( Nishio, 2000 ).

There was an interactive effect of light spectrum and PPFD on leaf photosynthesis. Under low PPFD , QY inc was lowest under green and highest under red light. The low QY inc under green light at low PPFD was due to low absorptance. In contrast, at high PPFD , green and red light achieved similar QY inc , higher than that of blue light. The strong absorption of blue light by chlorophyll creates a large light gradient from the top to the bottom of leaves. The large amount of excitation energy near the adaxial side of a leaf results in upregulation of nonphotochemical quenching, while chloroplasts near the bottom of a leaf receive little excitation energy under blue light. The more uniform distribution of green light absorption within leaves reduces the need for nonphotochemical quenching near the top of the leaf, while providing more excitation energy to cells near the bottom of the leaf. We also found that the interactive effect of light spectrum and PPFD on photosynthesis was a result of the light-dependent reactions; gross assimilation and J were strongly correlated. We detected no synergistic or antagonistic interactions between blue, green, and red light.

Data Availability Statement

Author contributions.

JL and MI designed the experiment, discussed the data, and revised the manuscript. JL performed the experiment, analyzed data, and prepared the first draft. Both authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

Funding. This study was funded by the USDA-NIFA-SCRI award number 2018-51181-28365, project Lighting Approaches to Maximize Profits.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021.619987/full#supplementary-material

Supplementary Figure 1

(Related to Figure 6 ) Quantum yield of CO 2 assimilation of “Green Towers” lettuce as a function of incident ( QY inc ) (A,C,E,G) and absorbed PPFD ( QY abs ) (B,D,F,H) under nine light spectra (see Table 1 ). Error bars represent standard deviation ( n = 9).

Supplementary Figure 2

(Related to Figure 7 ) Differential quantum yield of CO 2 assimilation ( differential QY ) of “Green Towers” lettuce under nine light spectra as a function of the PPFD . Inserts show differential QY at PPFD s of 1,000–1,300 μmol⋅m –2 s –1 s to better show differences at high PPFD (note the different y -axis scale). The composition of the nine light spectra is shown in Table 1 . The light spectra in the graphs are (A) 100B, 100G and 100R; (B) 100B, 80B20G, 20B80G and 100G; (C) 100G, 80G20R, 20G80R and 100R; and (D) 20B80R, 16B20G64R and 100G.

Supplementary Figure 3

(Related to Figure 6 ) The correlation between electron transport ( J ) and maximum Rubisco carboxylation rate ( V c,max ) of “Green Towers” lettuce estimated from A/C i curves under PPFD (1000 μmol m –2 s –1 ) under nine light spectra ( p < 0.001).

Supplementary Figure 4

(Related to Figure 6 ) The comparison between QY inc before (A) and after (B) correcting for light-suppression of respiration under blue, green, and red LED light. Note that the initial increase in QY inc became more pronounced after correction of light suppressed respiration.

Supplementary Figure 5

The comparison between QY abs before (A) and after (B) correcting for alternative electron sinks under blue, green, and red LED light. Assuming a simplified electron sink that diverts energy of 15 μmol m –2 s –1 of absorbed photons (an arbitrary value used for illustrative purposes only) away from the Calvin cycle under all PPFD s, the corrected QY abs was calculated based on remaining photons available to support Calvin cycle processes (B) . Note that the pattern of QY inc after correcting of alternative electron sink (B) is similar to quantum yield of PSII measured by chlorophyll fluorescence by Weaver and van Iersel (2019) .

Supplementary Table 1

Dark respiration rate (R d ), maximum quantum yield of CO 2 assimilation (QY m,inc ) and maximum gross assimilation rate (A g,max ) of “Green towers” lettuce derived from the light response curves for nine different spectra using Eq. 1. The light response curves are shown in Figure 3 . *See light composition of nine lights presented here in Table 1 .

• Atkin O. K., Westbeek M., Cambridge M. L., Lambers H., Pons T. L. (1997). Leaf respiration in light and darkness (A comparison of slow- and fast-growing Poa species). Plant Physiol. 113 961–965. 10.1104/pp.113.3.961 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Badger M. R., von Caemmerer S., Ruuska S., Nakano H. (2000). Electron flow to oxygen in higher plants and algae: rates and control of direct photoreduction (Mehler reaction) and rubisco oxygenase. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355 1433–1446. 10.1098/rstb.2000.0704 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Balegh S. E., Biddulph O. (1970). The photosynthetic action spectrum of the bean plant. Plant Physiol. 46 1–5. 10.1104/pp.46.1.1 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Brodersen C. R., Vogelmann T. C. (2010). Do changes in light direction affect absorption profiles in leaves? Funct. Plant Biol. 37 403–412. 10.1071/fp09262 [ CrossRef ] [ Google Scholar ]
• Brooks A., Farquhar G. D. (1985). Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light. Planta 165 397–406. 10.1007/BF00392238 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Campbell W. J., Ogren W. L. (1992). Light activation of rubisco by rubisco activase and thylakoid membranes. Plant Cell Physiol. 33 751–756. 10.1093/oxfordjournals.pcp.a078314 [ CrossRef ] [ Google Scholar ]
• Chen S. L. (1952). The action spectrum for the photochemical evolution of oxygen by isolated chloroplasts. Plant Physiol. 27 35–48. 10.1104/pp.27.1.35 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Craver J. K., Nemali K. S., Lopez R. G. (2020). Acclimation of growth and photosynthesis in Petunia seedlings exposed to high-intensity blue radiation. J. Am. Soc. Hortic. Sci. 145 152–161. 10.21273/jashs04799-19 [ CrossRef ] [ Google Scholar ]
• Croce R., Müller M. G., Bassi R., Holzwarth A. R. (2001). Carotenoid-to-chlorophyll energy transfer in recombinant major light-harvesting complex (LHCII) of higher plants. I. Femtosecond transient absorption measurements. Biophys. J. 80 901–915. 10.1016/S0006-3495(01)76069-9 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• de Weerd F. L., Dekker J. P., van Grondelle R. (2003a). Dynamics of β-carotene-to-chlorophyll singlet energy transfer in the core of photosystem II. J. Phys. Chem. B 107 6214–6220. 10.1021/jp027737q [ CrossRef ] [ Google Scholar ]
• de Weerd F. L., Kennis J. T., Dekker J. P., van Grondelle R. (2003b). β-Carotene to chlorophyll singlet energy transfer in the photosystem I core of Synechococcus elongatus proceeds via the β-carotene S2 and S1 states. J. Phys. Chem. B 107 5995–6002. 10.1021/jp027758k [ CrossRef ] [ Google Scholar ]
• Emerson R. (1957). Dependence of yield of photosynthesis in long-wave red on wavelength and intensity of supplementary light. Science 125 746–746. 10.1126/science.125.3251.746 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Evans J. (1987). The dependence of quantum yield on wavelength and growth irradiance. Funct. Plant Biol. 14 69–79. 10.1071/PP9870069 [ CrossRef ] [ Google Scholar ]
• Evans J., Vogelmann T. C. (2003). Profiles of 14C fixation through spinach leaves in relation to light absorption and photosynthetic capacity. Plant Cell Environ. 26 547–560. 10.1046/j.1365-3040.2003.00985.x [ CrossRef ] [ Google Scholar ]
• Haxo F. T., Blinks L. (1950). Photosynthetic action spectra of marine algae. J. Gen. Physiol. 33 389–422. 10.1085/jgp.33.4.389 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hogewoning S. W., Wientjes E., Douwstra P., Trouwborst G., Van Ieperen W., Croce R., et al. (2012). Photosynthetic quantum yield dynamics: from photosystems to leaves. Plant Cell 24 1921–1935. 10.1105/tpc.112.097972 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hoover W. H. (1937). The dependence of carbon dioxide assimilation in a higher plant on wave length of radiation. Smithson. Misc. Collect. 95 1–13. [ Google Scholar ]
• Inada K. (1976). Action spectra for photosynthesis in higher plants. Plant Cell Physiol. 17 355–365. 10.1093/oxfordjournals.pcp.a075288 [ CrossRef ] [ Google Scholar ]
• Krall J. P., Edwards G. E. (1992). Relationship between photosystem II activity and CO2 fixation in leaves. Physiol. Plant. 86 180–187. 10.1111/j.1399-3054.1992.tb01328.x [ CrossRef ] [ Google Scholar ]
• Kusuma P., Pattison P. M., Bugbee B. (2020). From physics to fixtures to food: current and potential LED efficacy. Hortic. Res. 7 : 56 . 10.1038/s41438-020-0283-7 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• McCree K. J. (1971). The action spectrum, absorptance and quantum yield of photosynthesis in crop plants. Agric. Meteorol. 9 191–216. 10.1016/0002-1571(71)90022-7 [ CrossRef ] [ Google Scholar ]
• McCree K. J. (1972). Test of current definitions of photosynthetically active radiation against leaf photosynthesis data. Agric. Meteorol. 10 443–453. 10.1016/0002-1571(72)90045-3 [ CrossRef ] [ Google Scholar ]
• Moss R. A., Loomis W. E. (1952). Absorption spectra of leaves. I. the visible spectrum. Plant Physiol. 27 370–391. 10.1104/pp.27.2.370 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Müller J., Wernecke P., Diepenbrock W. (2005). LEAFC3-N: a nitrogen-sensitive extension of the CO 2 and H 2 O gas exchange model LEAFC3 parameterised and tested for winter wheat (Triticum aestivum L.). Ecol. Modell. 183 183–210. 10.1016/j.ecolmodel.2004.07.025 [ CrossRef ] [ Google Scholar ]
• Nishio J. (2000). Why are higher plants green? Evolution of the higher plant photosynthetic pigment complement. Plant Cell Environ. 23 539–548. 10.1046/j.1365-3040.2000.00563.x [ CrossRef ] [ Google Scholar ]
• Nunes-Nesi A., Fernie A. R., Stitt M. (2010). Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol. plant 3 973–996. [ PubMed ] [ Google Scholar ]
• Parry M. A., Keys A. J., Madgwick P. J., Carmo-Silva A. E., Andralojc P. J. (2008). Rubisco regulation: a role for inhibitors. J. Exp. Bot. 59 1569–1580. 10.1093/jxb/ern084 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Pattison P., Lee K., Stober K., Yamada M. (2018). Energy Savings Potential of SSL in Horticultural Applications. Washington, DC: U.S. Department of Energy, Office of Scientific and Technical Information. [ Google Scholar ]
• Sharkey T. D., Bernacchi C. J., Farquhar G. D., Singsaas E. L. (2007). Fitting photosynthetic carbon dioxide response curves for C3 leaves. Plant Cell Environ. 30 1035–1040. 10.1111/j.1365-3040.2007.01710.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Singh D., Basu C., Meinhardt-Wollweber M., Roth B. (2015). LEDs for energy efficient greenhouse lighting. Renew. Sustain. Energy Rev. 49 139–147. [ Google Scholar ]
• Skillman J. B. (2008). Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark. J. Exp. Bot. 59 1647–1661. 10.1093/jxb/ern029 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Smith H. L., McAusland L., Murchie E. H. (2017). Don’t ignore the green light: exploring diverse roles in plant processes. J. Exp. Bot. 68 2099–2110. 10.1093/jxb/erx098 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Stinziano J. R., Morgan P. B., Lynch D. J., Saathoff A. J., McDermitt D. K., Hanson D. T. (2017). The rapid A–Ci response: photosynthesis in the phenomic era. Plant Cell Environ. 40 1256–1262. 10.1111/pce.12911 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Sun J., Nishio J. N., Vogelmann T. C. (1998). Green light drives CO2 fixation deep within leaves. Plant Cell Physiol. 39 1020–1026. 10.1093/oxfordjournals.pcp.a029298 [ CrossRef ] [ Google Scholar ]
• Takahashi H., Kopriva S., Giordano M., Saito K., Hell R. (2011). Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes. Annu. Rev. Plant Biol. 62 157–184. 10.1146/annurev-arplant-042110-103921 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Terashima I., Fujita T., Inoue T., Chow W. S., Oguchi R. (2009). Green light drives leaf photosynthesis more efficiently than red light in strong white light: revisiting the enigmatic question of why leaves are green. Plant cell physiol. 50 684–697. 10.1093/pcp/pcp034 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Vogelmann T., Han T. (2000). Measurement of gradients of absorbed light in spinach leaves from chlorophyll fluorescence profiles. Plant Cell Environ. 23 1303–1311. 10.1046/j.1365-3040.2000.00649.x [ CrossRef ] [ Google Scholar ]
• Vogelmann T. C., Evans J. (2002). Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence. Plant Cell Environ. 25 1313–1323. 10.1046/j.1365-3040.2002.00910.x [ CrossRef ] [ Google Scholar ]
• Vogelmann T. C., Gorton H. L. (2014). “ Leaf: light capture in the photosynthetic organ ,” in The Structural Basis of Biological Energy Generation , ed. Hohmann-Marriott M. F. (Dordrecht: Springer Netherlands; ), 363–377. [ Google Scholar ]
• Weaver G., van Iersel M. W. (2019). Photochemical characterization of greenhouse-grown Lettuce ( Lactuca sativa L. ‘Green Towers’) with applications for supplemental lighting control. HortScience 54 317–322. 10.21273/hortsci13553-18 [ CrossRef ] [ Google Scholar ]
• Wientjes E., van Stokkum I. H., van Amerongen H., Croce R. (2011). The role of the individual Lhcas in photosystem I excitation energy trapping. Biophys. J. 101 745–754. 10.1016/j.bpj.2011.06.045 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wullschleger S. D. (1993). Biochemical limitations to carbon assimilation in C3 Plants—a retrospective analysis of the A/Ci curves from 109 species. J. Exp. Bot. 44 907–920. 10.1093/jxb/44.5.907 [ CrossRef ] [ Google Scholar ]
• Zhang N., Portis A. R. (1999). Mechanism of light regulation of rubisco: a specific role for the larger rubisco activase isoform involving reductive activation by thioredoxin-f. Proc. Natl. Acad. Sci. U.S.A. 96 9438–9443. 10.1073/pnas.96.16.9438 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhen S., Haidekker M., van Iersel M. W. (2019). Far−red light enhances photochemical efficiency in a wavelength−dependent manner. Physiol. Plant. 167 21–33. 10.1111/ppl.12834 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zhen S., van Iersel M. W. (2017). Photochemical acclimation of three contrasting species to different light levels: implications for optimizing supplemental lighting. J. Am. Soc. Hortic. Sci. 142 346–354. 10.21273/jashs04188-17 [ CrossRef ] [ Google Scholar ]

• Open access
• Published: 17 October 2001

The role of chlorophyll b in photosynthesis: Hypothesis

• Laura L Eggink 1 ,
• Hyoungshin Park 1 , 2 &
• J Kenneth Hoober 1  

BMC Plant Biology volume  1 , Article number:  2 ( 2001 ) Cite this article

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The physico-chemical properties of chlorophylls b and c have been known for decades. Yet the mechanisms by which these secondary chlorophylls support assembly and accumulation of light-harvesting complexes in vivo have not been resolved.

Presentation

Biosynthetic modifications that introduce electronegative groups on the periphery of the chlorophyll molecule withdraw electrons from the pyrrole nitrogens and thus reduce their basicity. Consequently, the tendency of the central Mg to form coordination bonds with electron pairs in exogenous ligands, a reflection of its Lewis acid properties, is increased. Our hypothesis states that the stronger coordination bonds between the Mg atom in chlorophyll b and chlorophyll c and amino acid sidechain ligands in chlorophyll a/b - and a/c -binding apoproteins, respectively, enhance their import into the chloroplast and assembly of light-harvesting complexes.

Several apoproteins of light-harvesting complexes, in particular, the major protein Lhcb1, are not detectable in leaves of chlorophyll b -less plants. A direct test of the hypothesis – with positive selection – is expression, in mutant plants that synthesize only chlorophyll a, of forms of Lhcb1 in which weak ligands are replaced with stronger Lewis bases.

Implications

The mechanistic explanation for the effects of deficiencies in chlorophyll b or c points to the need for further research on manipulation of coordination bonds between these chlorophylls and chlorophyll-binding proteins. Understanding these interactions will possibly lead to engineering plants to expand their light-harvesting antenna and ultimately their productivity.

In plants and algae, the reaction centers of photosystem I and II are enclosed within core complexes that contain a precisely defined set of proteins – essentially all encoded in the chloroplast genome. The primary cofactor for the photochemical reactions in these complexes, chlorophyll (Chl) a, is also required for assembly of these complexes. The end-product of the Chl biosynthetic pathway in plants in the dark, protochlorophyllide (Pchlide), is unable to support the assembly processes, which suggests that the light-dependent reduction of the double bond between C17 and C18 of Pchlide (see legend to Fig. 1 ) has a profound effect on the properties of the molecule. Plants and green algae (Chlorophyta) contain in addition Chl b, an accessory Chl found only in peripheral light-harvesting complexes (LHCs). These complexes usually contain three xanthophyll molecules, two luteins and one neoxanthin, and nearly equal amounts of Chl a and Chl b (7 or 8 Chl a and 5 or 6 Chl b molecules for the major LHCII, with an a/b ratio of 1.4) bound to proteins (LHCPs) that are encoded in the nuclear genome and imported into the plastid after synthesis in the cytosol. Chl b -less mutant plants are deficient in Chl and, although containing fully functional reaction centers, have a relatively low photosynthetic capacity and greater sensitivity to high-intensity light because of a deficiency in LHCs [ 1 ]. Algal species in the family Chromophyta contain Chl c (Fig. 1 ) instead of Chl b, which is also restricted to LHCs and seems to serve the same function in these organisms that Chl b provides in the green plants [ 2 ]. A large volume of data exists in the literature on these Chl derivatives. In this article we propose a mechanism for the important auxiliary roles these Chls play in photosynthesis.

Structures of Chls. (a) Stereochemistry and numbering system in monovinyl-Chl a. Variations of Chl include (b) Chl b (7-formyl, R = phytyl); and (c) Chl c 1 (17 1 -dehydro-Pchlide, R 1 = methyl; R 2 = ethyl or vinyl; R = H). Pchlide is similar to Chl but contains a saturated propionic rather than acrylic acid group on C17. (Structures as in [ 49 ]).

Presentation of the hypothesis

Etioplasts, the form of the plastid that develops in dark-grown plants, were unable to insert LHCPs into membranes unless Chl was added [ 3 ]. In these experiments, in which the Zn derivatives were used because of their increased chemical stability over the Mg-containing molecules, Zn-pheophytin b was more effective in insertion than Zn-pheophytin a. An important role of Chl b was further revealed by experiments in which newly synthesized LHCPs were detected by pulse-labeling in Chl b -less mutant plants but the proteins were not recovered in chloroplasts isolated from these plants [ 4 ]. These Chl b -less plants did not accumulate several of the LHCPs, in particular Lhcb1, Lhcb6 and Lhca4 [ 5 ]. Chl b was not detected in plants exposed to intermittent light (cycles of 2 min of light and 98 min of darkness), which accumulated only small amounts of Chl a and thylakoid membranes [ 6 , 7 ]. Wild-type plants treated in this way accumulated only one LHCP (Lhcb5), while Chl b -less mutants exposed to intermittent light lacked all LHCPs [ 8 ]. In complementary fashion, Chl b did not accumulate when synthesis of LHCPs was inhibited [ 9 ]. When bean plants exposed to intermittent light were treated with chloramphenicol to inhibit synthesis of proteins on chloroplast ribosomes, Chl b and LHCPs accumulated in parallel with no increase in synthesis of total Chl [ 10 ]. These results indicate that photosystem I and II core complex proteins, which are synthesized in the chloroplast, compete effectively with LHCP for small amounts of Chl a made under these conditions, and that Chl b does not accumulate until sufficient Chl a is made to satisfy core complexes.

Experiments with the model alga Chlamydomonas reinhardtii [ 11 ] showed that LHCPs were not detectably imported into the chloroplast in the absence of Chl synthesis and instead accumulated outside of the chloroplast in the cytosol and in vacuoles [ 12 ]. High concentrations of chloramphenicol caused strong suppression of total Chl synthesis when dark-grown algal cells were exposed to light, possibly by inhibition of Mg-chelatase [ 13 ] in addition to chloroplast protein synthesis. Synthesis of LHCPs on cytoplasmic ribosomes was not inhibited by chloramphenicol, and the proteins accumulated to the same level as in untreated cells [ 14 , 15 ]. However, because of the low rate of Chl synthesis, only a small fraction of the proteins were imported into the chloroplast and remained at the initial site of integration. As illustrated in Fig. 2b , immunoelectron microscopy detected LHCPs along the chloroplast envelope. LHCPs were not detected in the interior of the chloroplast, although cell fractionation recovered a substantial amount in a soluble form [ 14 ]. In control cells, the amount of Chl and thylakoid membranes increased rapidly when cells were illuminated, and LHCPs were detected in thylakoid membranes throughout the chloroplast (Fig. 2a ). This result, obtained with cells incubated at 25°C, was consistent with localization of LHCPs on envelope membranes in cells immediately after initiation of thylakoid biogenesis at 38°C [ 16 ]. At the higher temperature, not all the newly synthesized LHCPs were incorporated into envelope membranes, and the excess accumulated in cytosolic vacuoles [ 16 , 17 ]. This evidence for the envelope as the site of initial interaction of LHCPs with Chl was also supported by proliferation of envelope-derived vesicles in dark-grown Chlamydomonas cells exposed to only a few minutes of light [ 18 ] and the lack of thylakoids in a mutant of Arabidopsis deficient in a protein apparently required for formation of vesicles from the inner membrane of the envelope [ 19 ].

Immunoelectron microscopic localization of LHCPs in dark-grown cells of C. reinhardtii exposed to light for 6 h at 25°C (a) without or (b) in the presence of 200 μg chloramphenicol ml -1 . The experimental conditions were described previously [ 14 ]. Bound antibodies were detected with protein A conjugated to 10-nm gold particles [ 12 ]. c, chloroplast; G, Golgi; m, mitochondrion; n, nucleus; v, vacuoles. The bar = 0.5 μm.

These experiments with in vivo systems demonstrated that Chl b provides a function in LHC assembly that is not served by Chl a. Association of Chl with proteins occurs through coordination bonds between the Mg of Chl, as the Lewis acid, and amino acid sidechains as Lewis bases. The availability of an unshared pair of electrons in the Lewis base (the ligand) varies widely and is the primary factor in the strength of the coordination bond. The chemical properties of the central Mg in Chl also influence the strength of the resulting coordination bond. Biosynthetic modifications to the periphery of the tetrapyrrole ring progressively cause withdrawal of electrons from the pyrrole nitrogens, thereby decreasing their basicity [ 20 , 21 ]. For example, oxidation of the 7-methyl group in Chl a to the electronegative aldehyde of Chl b reduces the pK of the pyrrole nitrogens by 2 pH units. Similarly, oxidation of the propionyl sidechain on Pchlide to the acrylate group in Chl c brings its electronegative carboxyl group into conjugation with the π system of the macrocycle, with the same effect [ 22 ]. As a consequence, the central Mg atom of Chls b and c has a greater affinity for exogenous electrons, thus is a stronger Lewis acid. These considerations point to the possibility that proteins form stronger coordination bonds with Chls b and c than with Chl a, which may be particularly critical with ligands that are weak Lewis bases. The lack of an aldehyde group on the periphery of the macrocycle of Chl c, which replaces Chl b in homologous complexes in chromophytic algae, indicates that the primary interaction between Chls and the proteins does not involve such substituents. Whether phytylation of Chl is important for binding to proteins is not clear, because Chl c is incorporated into Chl a/c -protein complexes without esterification.

Testing the hypothesis

Tamiaki et al. [ 23 ] demonstrated that introduction of an oxygen atom to the periphery of a Zn-tetrapyrrole macrocycle, as occurs in the conversion of Chl a to Chl b, increased about two-fold the equilibrium constant for formation of the coordination complex with pyridine in benzene. Consistent with this observation, studies of detergent-induced dissociation of LHCs suggested that Chl b is held by the proteins approximately two-times more tightly than Chl a [ 24 ]. Tighter binding of Chl b is apparently responsible for the well-known stability of light-harvesting complexes during mildly denaturing gel electrophoresis. The initial accumulation of LHCPs in the chloroplast envelope implies that Chl interacts with these proteins, likely by binding to the conserved motif in the first membrane-spanning region (helix-1) [ 25 , 26 ] when transit through the envelope is initiated. Molecular modeling suggested that this 'retention' motif – ExxHxR in the first and ExxNxR in the third membrane-spanning region – within all LHCPs and related proteins provides two ligands for Chl, an ion-pair between Glu (E) and Arg (R) and the sidechain of either His (H) or Asn (N). Binding of Chl a to a 16-mer synthetic peptide was reduced by one-half when His within the motif sequence was replaced with Ala [ 27 ]. Replacement in addition of the Glu or Arg with Ala eliminated binding to the synthetic peptide. Import of a mutant LHCP into isolated chloroplasts was nearly abolished when His within the motif was substituted with Ala [ 28 ]. Association of Chl with this motif, therefore, appears essential for continuation of the proteins on the pathway of assembly of an LHC.

An illustration of the effect of binding two molecules of Chl with enhanced affinity to a retention motif is shown in Fig. 3 . Assuming a relative equilibrium constant of 3.0 for Chl a and 5.0 for Chl b binding to a ligand in LHCP (numerals approximated from data obtained by Tamiaki et al. [ 23 ]), the increase in affinity of Chl b with the protein leads to a nearly three-fold increase in stability of the complex over that with Chl a when two molecules are bound. This conclusion is derived from the equations: R + Chl ↔ R·Chl; R·Chl + Chl↔ R·Chl 2 ; R + 2Chl ↔ R·Chl 2 ; K eq = [R·Chl 2 ]/ [R] [Chl] 2 . The additional molecules of Chl b in LHCII would further enhance this effect by shifting the equilibria toward complex formation.

Graphical illustration of the relative equilibrium constants for complexes of Chl with retention motifs when one (blue) or two (magenta) molecules of Pchlide, Chl a or Chl b are bound.

The most electronegative ligand in LHCPs is the sidechain of His. Less strong Lewis bases are the charge-compensated Glu in an ion-pair with Arg, the amide group of Gln and Asn, and finally the carbonyl of the peptide backbone as the weakest [ 29 ]. The importance of the ligand was demonstrated by substitution of His with the weaker Lewis base Asn in the apoprotein of the bacterial light-harvesting complex LH1, which eliminated assembly of the complex in vivo and reconstitution in vitro [ 30 ]. Formation of a stable coordination bond with a weaker Lewis base is expected to require a stronger Lewis acid. Consistent with this prediction, a position in CP29, a minor LHCII, was preferentially filled during reconstitution by Chl a when the amino acid residue was the normal Glu, in an ion-pair with a bound Ca ++ ion, but occupancy was shifted toward Chl b when the ligand was a weaker base, the amide group of Gln [ 31 ]. Although the on-rate for Chl b may be slower than that for Chl a, because binding may be impeded by a water molecule more strongly coordinated to the central Mg atom of Chl b, the greater Lewis acid strength of Chl b allows more stable bonds with the weaker ligands.

Our hypothesis on the biological role of Chl b should be reflected in the binding sites of Chl in LHCII. Resolution of the structure of native LHCII at 3.4 Å [ 29 ] revealed locations of individual Chls but did not provide identification of the Chl in each site or whether any site in the complex has mixed occupancy. The model developed from this work suggested that binding sites in the core of the complex, near the central lutein molecules, were occupied by Chl a, whereas Chl b was more peripheral. From measurements of ultrafast energy transfer kinetics within native LHCII, Gradinaru et al. [ 32 ] suggested that indeed lutein transferred excitation energy entirely to Chl a while neoxanthin, a xanthophyll bound near helix-2 (see Fig. 4 ), transferred energy to Chl b. With similar techniques, however, Croce et al. [ 33 ] presented evidence for detectable transfer of energy from lutein to Chl b, which suggested close contact of Chl b molecules with the central luteins. Several groups developed a more direct approach for determining occupancy by analyzing effects on the composition of the final complex, after in vitro reconstitution, when amino acid residues in LHCPs were replaced with substitutes that are unable to serve as a ligand. For example, steric hindrance caused by substitution of bulky Phe for Gly78 (residue numbers are given with reference to Lhcbl) in the position designated a 6 [ 29 ] prevented this peptide carbonyl, non-H-bonded because of Pro82 one helical turn further, from serving as a ligand (see Fig. 4 ). This change resulted in loss of one Chl b after reconstitution [ 34 ]. Gln 131 ( b 6) and Glu 139 (in an ion-pair with Arg142) ( b 5) were also identified as ligands to Chl b [ 31 , 34 – 36 ]. Remelli et al. [ 35 ] found that substitution of ligand Gln197 ( a 3) or His212 ( b3 ) with Leu or Val, respectively, led to sub-integral loss of Chl a and Chl b, which indicated mixed occupancy in each site. Studies by Rogl and Kühlbrandt [ 34 ], on the other hand, suggested that both sites were filled with Chl a. Assignments after in vitro reconstitution may have a degree of uncertainty, because the composition of the final complex varies as a function of the Chl a:b ratio in the reconstitution mixture [ 37 ]. Mixed sites, even when Chl a was present in excess in the mixture [ 35 ], probably reflected a preference for binding of Chl b to the protein. Based on ligand strength, and the likelihood that occupancy is unambiguous in vivo [ 34 ], His212 may serve as a ligand for Chl a and Gln197 for Chl b.

Model of the association of Chl with Lhcb1. The arrangement of the protein in thylakoid membranes is illustrated according to ref. 50. The "core" Chls ( a 1, a 2, a 4 and a 5) are shown as Chl a according to ref. [ 35 ]. The green color marks positions of Chl b as proposed in the text. Sites a 3 and b 3, although mixed in occupancy after reconstitution [ 35 ], were assigned as shown based on ligand strength. At least four of the five Chl b molecules are coordinated directly to the protein. The biological requirement of Chl b for accumulation of Lhcb1 (see text) suggests an alternate assignment for a 4, as also proposed in ref. [ 34 ].

Mutation of Glu65 ( a 4) or Asn183 ( a 2) each resulted in loss of one Chl a and one Chl b [ 35 ]. Rogl and Kühlbrandt [ 34 ] suggested that Glu65 (in an ion-pair with Arg185) may be a ligand for Chl b, with another site, occupied by Chl a, affected by loss of the protein-bound Chl. Chl b in site a4 would be consistent with the biological necessity of association of Chl b with helix-1 for retention of the protein in the chloroplast. However, based on similarity to results from reconstitution of the more simple CP29 (Lhcb4) [ 31 ], Remelli et al. [ 35 ] suggested that Glu65 ( a 4) and Asn183 ( a 2) are occupied by Chl a . Loss of the latter Chl apparently resulted in loss of 'out-lying' Chl b in site b2, which is near a 2 in the 3-dimensional structure. These assignments thus account for the five Chl b molecules in the complex (Fig. 4 ). Site b 1 must consequently be filled with a Chl a molecule [ 38 ]. The orientation of the transition moments of Chl b in sites b 5 and b 6 [ 38 ] suggest that an 'out-lying' Chl a molecule could coordinate with the formyl group of Chl b, a sterically more favorable arrangement than coordination to the 13 1 -carbonyl oxygen because of the opposing orientations of the 13 2 -carboxymethyl and 17-propionyl group (Fig. 1 ). Although coordination of an 'out-lying' Chl to a protein-bound Chl would enhance Lewis acid strength of the latter, the distances between Chls [ 29 , 35 ] suggest that interaction would require mediation by water molecules. Alternatively, these Chls may coordinate with peptide carbonyl groups.

Site a 6, considered to be filled with Chl b [ 34 , 38 ], may play a role in retention of LHCPs in the chloroplast. Lhcb6, a minor LHCP, contains Gly instead of Pro at the position analogous to 82 in Lhcb1, thus eliminating the peptide bond carbonyl of Gly as a ligand, but Lhcb6 has a potential ligand for Chl b in Gln83 [ 39 , 40 ]. These two LHCP sub-species, along with Lhca4 are most affected by the lack of Chl b in vivo [ 5 ]. Lhcb4 (apoprotein of CP29) has Val instead of Pro at 'position 82', and the absence of site a 6 in Lhcb4 may contribute to its drastic reduction in Chl b -less mutants [ 5 , 8 ]. However, Lhcb2, Lhcb3, Lhca1, Lhca2 and Lhca3 contain the Gly peptide carbonyl as a ligand (each has Pro at 'position 82' [ 40 ]) but are reduced only slightly, if any, in amount by the lack of Chl b. Site a 6 may therefore not be essential to accumulation of the protein but serve in concert with initial involvement of Chl b, directly or indirectly, with the completely conserved retention motif. Because interactions that develop during import may be altered as the result of conformational changes as the complex assembles, in particular, as the retention motif loop [ 27 ] is stretched into a helical structure, the final occupancy in each site in the final complex may not reflect the initial associations. Understanding the constraints on assembly of the complex in vivo – including retraction into the cytosol when the amount of Chl is insufficient [ 12 ] – and the order in which Chls are bound, will require new experimental design. We expect that synthesis of Chl b by Chl(ide) a oxidase [ 41 ] will be determined by the local environment around specific Chl a molecules, created by the assembly process. It is interesting to note that the retention motif in all LHCs that contain Chl b is followed by a Trp residue, which may be involved in synthesis of Chl b.

A converse mutagenesis approach would provide a rigorous test of the hypothesis. A stable complex should be achieved with only Chl a, in a Chl b -less plant or by in vitro reconstitution, when weak ligands in LHCPs are replaced with stronger Lewis bases. Increased strength of the engineered coordination bonds with Chl a should compensate for the lack of Chl b. In particular, a stable complex should accumulate after Gln131, Glu139, Asn183 and Gln197 in Lhcb1 are replaced with His. A stronger ligand could also be introduced in the position of Gly78, which seems to be the weakest ligand in the complex. Substitution of these amino acids in the sequence of Lhcb1, a major LHCP that can not be detected in Chl b -less plants [ 5 , 8 ], would be expected to restore accumulation of the protein with only Chl a. This experiment provides a positive in vivo selection for validation of the hypothesis, in contrast to the dramatic decrease in accumulation of the proteins when ligands are removed by substitution with non-ligand amino acids [ 42 ]. Furthermore, whereas stable complexes can be achieved by reconstitution with wild-type Lhcb1 and only Chl b but not only Chl a [ 37 , 43 ], the hypothesis predicts that stable complexes can be reconstituted with the mutant protein containing these substitutions and Chl a.

Implications of the hypothesis

An extensive amount of evidence in the literature supports the hypothesis presented in this article on the role of Chl b. It should be noted, however, that several LHCPs accumulate in chloroplasts in the absence of Chl b [ 5 , 8 ], perhaps because they integrate more easily into membranes, which implies that other features of the proteins are involved. The work already done has established that several LHCPs are imported into the chloroplast at a substantial rate only when sufficient Chl b is available and they accumulate initially in the envelope membrane. Results from in vivo experiments have shown that interaction of Chl b with the first membrane-spanning region, including the retention motif, is critical for progression of import of these proteins. The initial steps in assembly also require the abundant xanthophyll lutein [ 26 ], which has not been the focus of this article. The availability of Chl b thus strongly regulates import of LHCPs as well as assembly and eventual accumulation of light-harvesting complexes. The resulting dramatic enhancement in the efficiency of light capture for photosynthesis apparently provided a strong evolutionary pressure for development of the ability of photosynthetic organisms to synthesize Chl b or Chl c [ 44 ].

The structure of LHCs has been extensively studied and linkage of the complexes to reaction centers, physically and functionally, is well understood. Further understanding of LHC assembly requires a better knowledge of the characteristics of the reaction catalyzed by Chl(ide) a oxidase and whether Chl b is restricted to these complexes because LHCP serves as a specific effector of the oxidation of Chl(ide) a or whether the protein simply provides binding sites for Chl b and prevents its conversion back to Chl a [ 45 ]. The latter appears less likely as a specific effect, because similar ligands should occur in other proteins. In particular, the early-light induced proteins are homologous to LHCPs but bind little if any Chl b [ 46 ]. The mechanism of Chl b synthesis, an oxidation of the methyl group at position 7 [ 41 ], will be an area of active research in the future, now that the gene for Chl(ide) a oxidase has been identified [ 47 , 48 ]. Moreover, it is not known whether a pool of free Chl b exists in a local environment in chloroplast membranes that is mimicked by the amount of Chl b in reconstitution experiments. Attempts to understand assembly of the complex in vivo will provide ample opportunity for additional experimental work.

Abbreviations

chlorophyll

chlorophyllide

protochlorophyllide

light-harvesting complex

LHC associated primarily with photosystem II

LHC apoprotein

apoproteins of LHCs associated with photosystem II or I, respectively.

Leverenz JW, Öquist G, Wingsle G: Photosynthesis and photoinhibition in leaves of chlorophyll b -less barley in relation to absorbed light. Physiol Plant. 1992, 85: 495-502. 10.1034/j.1399-3054.1992.850313.x10.1034/j.1399-3054.1992.850313.x.

Article   CAS   Google Scholar  

Durnford DG, Deane JA, Tan S, McFadden GI, Gant E, Green BR: A plylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution. J Mol Evol. 1999, 48: 59-68.

Article   PubMed   CAS   Google Scholar  

Kuttkat A, Edhofer I, Eichacker LA, Paulsen H: Light-harvesting chlorophyll a/b -binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b. J Biol Chem. 1997, 272: 20451-20455. 10.1074/jbc.272.33.20451.

Preiss S, Thornber JP: Stability of the apoproteins of light-harvesting complex I and II during biogenesis of thylakoids in the chlorophyll b -less barley mutant chlorina f2. Plant Physiol. 1995, 107: 709-717.

PubMed   CAS   PubMed Central   Google Scholar  

Bossmann B, Grimme LH, Knoetzel J: Protease-stable integration of Lhcb1 into thylakoid membranes is dependent on chlorophyll b in allelic chlorina-f2 mutants of barley ( Hordeum vulgare L.). Planta. 1999, 207: 551-558. 10.1007/s00425005051710.1007/s004250050517.

Akoyunoglou G, Argyroudi-Akoyunoglou JH: Effects of intermittent light and continuous light on the Chl formation in etiolated plants at various ages. Physiol Plant. 1969, 22: 228-295.

Article   Google Scholar  

Akoyunoglou G: Development of the photosystem II unit in plastids of bean leaves greened in periodic light. Arch Biochem Biophys. 1977, 183: 571-580.

Król M, Spangfort MD, Huner NPA, Öquist G, Gustafsson P, Jansson S: Chlorophyll a/b -binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b -less barley mutant. Plant Physiol. 1995, 107: 873-883. 10.1104/pp.107.3.873.

Article   PubMed   PubMed Central   Google Scholar  

Maloney MA, Hoober JK, Marks DB: Kinetics of chlorophyll accumulation and formation of chlorophyll-protein complexes during greening of Chlamydomonas reinhardtii y-1 at 38°C. Plant Physiol. 1989, 91: 1100-1106.

Article   PubMed   CAS   PubMed Central   Google Scholar  

Tzinas G, Argyroudi-Akoyunoglou JH: Chloramphenicol-induced stabilization of light-harvesting complexes in thylakoids during development. FEBS Lett. 1988, 229: 134-141. 10.1016/0014-5793(88)80813-5.

Harris EH: Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol. 2001, 52: 363-406. 10.1146/annurev.arplant.52.1.363.

Park H, Hoober JK: Chlorophyll synthesis modulates retention of apoproteins of light-harvesting complex II by the chloroplast in Chlamydomonas reinhardtii. Physiol Plant. 1997, 101: 135-142. 10.1034/j.1399-3054.1997.1010118.x10.1034/j.1399-3054.1997.1010118.x.

Kannangara CG, Vothknecht UC, Hansson M, von Wettstein D: Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit xantha-G as a functional counterpart of Rhodobacter subunit BchD. Mol Gen Genet. 1997, 254: 85-92. 10.1007/s004380050394.

Hoober JK: A major polypeptide of chloroplast membranes of Chlamydomonas reinhardi. Evidence for synthesis in the cytoplasm as a soluble component. J Cell Biol. 1972, 52: 84-96. 10.1083/jcb.52.1.84.

Hoober JK, Stegeman WJ: Kinetics and regulation of synthesis of the major polypeptides of thylakoid membranes in Chlamydomonas reinhardtii y-1 at elevated temperatures. J Cell Biol. 1976, 70: 326-337. 10.1083/jcb.70.2.326.

White RA, Wolfe GR, Komine Y, Hoober JK: Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening of Chlamydomonas reinhardtii at 38°C. Photosynth Res. 1996, 47: 267-280.

Wolfe GR, Park H, Sharp WP, Hoober JK: Light-harvesting complex apoproteins in cytoplasmic vacuoles in Chlamydomonas reinhardtii (Chlorophyta). J Phycol. 1997, 33: 377-386.

Hoober JK, Boyd CO, Paavola LG: Origin of thylakoid membranes in Chlamydomonas reinhardtii y-1 at 38°C. Plant Physiol. 1991, 96: 1321-1328.

Kroll D, Meierhoff K, Bechtold N, Kinoshita M, Westphal S, Vothknecht U, Soll J, Westhoff P: VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation. Proc Natl Acad Sci USA. 2001, 98: 4238-4242. 10.1073/pnas.061500998.

Phillips JN: Physico-chemical properties of porphyrins. In: Comprehensive Biochemistry, Vol 9 (edited by Florkin M, Stotz EH), Amsterdam, Elsevier. 1963, 34-72.

Google Scholar  

Smith KM: General features of the structure and chemistry of porphyrin compounds. In: Porphyrins and Metalloporphyrins (Edited by Smith KM) Amsterdam, Elsevier Scientific. 1975, 1-58.

Dougherty RC, Strain HH, Svec WA, Uphaus RA, Katz JJ: The structure, properties and distribution of chlorophyll c. J Am Chem Soc. 1970, 92: 2826-2833.

Tamiaki H, Yagai S, Miyatake T: Synthetic zinc tetrapyrroles complex with pyridine as a single axial ligand. Bioorg Med Chem. 1998, 6: 2171-2178. 10.1016/S0968-0896(98)00154-0.

Ruban AV, Lee PJ, Wentworth M, Young AJ, Horton P: Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complex. J Biol Chem. 1999, 274: 10458-10465. 10.1074/jbc.274.15.10458.

Green BR, Pichersky E: Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors. Photosynth Res. 1994, 39: 149-162.

Hoober JK, Eggink LL: Assembly of light-harvesting complex II and biogenesis of thylakoid membranes in chloroplasts. Photosynth Res. 1999, 61: 197-215. 10.1023/A:1006313703640.

Eggink LL, Hoober JK: Chlorophyll binding to peptide maquettes containing a retention motif. J Biol Chem. 2000, 275: 9087-9090. 10.1074/jbc.275.13.9087.

Kohorn BD: Replacement of histidines of light harvesting chlorophyll a/b binding protein II disrupts chlorophyll-protein complex assembly. Plant Physiol. 1990, 93: 330-342.

Kühlbrandt W, Wang DN, Fujiyoshi Y: Atomic model of plant light-harvesting complex by electron crystallography. Nature. 1994, 367: 614-621. 10.1038/367614a0.

Article   PubMed   Google Scholar  

Davis CM, Bustamante PL, Todd JB, Parkes-Loach PS, McGlynn P, Olsen JD, McMaster L, Hunter CN, Loach PA: Evaluation of structure-function relationships in the core light-harvesting comlex of photosynthetic bacteria by reconstitution with mutant polypeptides. Biochemistry. 1997, 36: 3671-3679. 10.1021/bi962386p.

Bassi R, Croce R, Cugini D, Sandona D: Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites. Proc Natl Acad Sci USA. 1999, 96: 10056-10061. 10.1073/pnas.96.18.10056.

Gradinaru CC, Stokkum van IHM, Pascal AA, van Grondelle R, van Amerongen H: Identifying the pathways of energy transfer between carotenoids and chlorophylls in LHCII and CP29. A multicolor, femtosecond pump-probe study. J Phys Chem B. 2000, 104: 9330-9342. 10.1021/jp001752i10.1021/jp001752i.

Croce R, Müller MG, Bassi R, Holzwarth AR: Carotenoid-to-chlorophyll energy transfer in recombinant major light-harvesting complex (LHCII) of higher plants. I. Femtosecond transient absorption measurements. Biophys J. 2001, 80: 901-915.

Rogl H, Külbrandt W: Mutant trimers of light-harvesting complex II exhibit altered pigment content and spectroscopic features. Biochemistry. 1999, 38: 16214-16222. 10.1021/bi990739p.

Remelli R, Varotto C, Sandoná D, Croce R, Bassi R: Chlorophyll binding to monomeric light-harvesting complex: a mutational analysis of chromophore-binding residues. J Biol Chem. 1999, 274: 33510-33521. 10.1074/jbc.274.47.33510.

Yang C, Kosemund K, Comet C, Paulsen H: Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein. Biochemistry. 1999, 38: 16205-16213. 10.1021/bi990738x.

Kleima FJ, Hobe S, Calkoen F, Urbanus ML, Peterman EJG, van Grondelle R, Paulsen H, van Amerongen H: Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences. Biochemistry. 1999, 38: 6587-6596. 10.1021/bi982823v.

van Amerongen H, van Grondelle R: Understanding the energy transfer function of LHCII, the major light-harvesting complex of green plants. J Phys Chem B. 2001, 105: 604-617. 10.1021/jp002840610.1021/jp0028406.

Jansson S: The light-harvesting chlorophyll a/b -binding proteins. Biochim Biophys Acta. 1994, 1184: 1-19. 10.1016/0005-2728(94)90148-1.

Jansson S: A guide to the Lhc genes and their relatives in Arabidopsis. Trends Plant Sci. 1999, 4: 236-240. 10.1016/S1360-1385(99)01419-3.

Oster U, Tanaka R, Tanaka A, Rüdiger W: Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. Plant J. 2000, 21: 305-310. 10.1046/j.1365-313X.2000.00672.x.

Flachmann R, Kühlbrandt W: Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants. Proc Natl Acad Sci USA. 1996, 93: 14966-14971. 10.1073/pnas.93.25.14966.

Schmid VHR, Thomé P, Rühle W, Paulsen H, Kühlbrandt W, Rogl H: Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II. FEBS Lett. 2001, 499: 27-31. 10.1016/S0014-5793(01)02509-1.

Tomitani A, Okada K, Miyashita H, Matthijs HCP, Ohno T, Tanaka A: Chlorophyll b and phycobilins in the common ancestor of cyanobacteria and chloroplasts. Nature. 1999, 400: 159-162. 10.1038/22101.

Ohtsuka T, Ito H, Tanaka A: Conversion of chlorophyll b to chlorophyll a and the assembly of chlorophyll with apoproteins by isolated chloroplasts. Plant Physiol. 1997, 113: 137-147.

Adamska I, Kruse E, Kloppstech K: Stable insertion of the early light-induced proteins into etioplast membranes requires chlorophyll a. J Biol Chem. 2001, 276: 8582-8587. 10.1074/jbc.M010447200.

Tanaka A, Ito H, Tanaka R, Tanaka NK, Yoshida K: Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proc Natl Acad Sci USA. 1998, 95: 12719-12723. 10.1073/pnas.95.21.12719.

Espineda CE, Linford AL, Devine D, Brusslan JA: The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana . Proc Natl Acad Sci USA. 1999, 96: 10507-10511. 10.1073/pnas.96.18.10507.

Scheer H: Structure and occurrence of chlorophylls. In: Chlorophylls (Edited by Scheer H) Boca Raton, CRC Press. 1991, 3-30.

Green BR, Durnford DG: The chlorophyll-carotenoid proteins of oxygenic photosynthesis. Annu Rev Plant Physiol Plant Mol Biol. 1996, 47: 685-714. 10.1146/annurev.arplant.47.1.685.

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Acknowledgements

L.L.E was supported by Graduate Training Grant DGE9553456 from the National Science Foundation. This is publication number 498 from the Center for the Study of Early Events in Photosynthesis at Arizona State University.

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Eggink, L.L., Park, H. & Hoober, J.K. The role of chlorophyll b in photosynthesis: Hypothesis. BMC Plant Biol 1 , 2 (2001). https://doi.org/10.1186/1471-2229-1-2

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DOI : https://doi.org/10.1186/1471-2229-1-2

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Photosynthesis

Photosynthesis is a process by which phototrophs convert light energy into chemical energy, which is later used to fuel cellular activities. The chemical energy is stored in the form of sugars, which are created from water and carbon dioxide.

Table of Contents

• What is Photosynthesis?
• Site of photosynthesis

What Is Photosynthesis in Biology?

The word “ photosynthesis ” is derived from the Greek words  phōs  (pronounced: “fos”) and σύνθεσις (pronounced: “synthesis “) Phōs means “light” and σύνθεσις   means, “combining together.” This means “ combining together with the help of light .”

Photosynthesis also applies to other organisms besides green plants. These include several prokaryotes such as cyanobacteria, purple bacteria and green sulfur bacteria. These organisms exhibit photosynthesis just like green plants.The glucose produced during photosynthesis is then used to fuel various cellular activities. The by-product of this physio-chemical process is oxygen.

A visual representation of the photosynthesis reaction

• Photosynthesis is also used by algae to convert solar energy into chemical energy. Oxygen is liberated as a by-product and light is considered as a major factor to complete the process of photosynthesis.
• Photosynthesis occurs when plants use light energy to convert carbon dioxide and water into glucose and oxygen. Leaves contain microscopic cellular organelles known as chloroplasts.
• Each chloroplast contains a green-coloured pigment called chlorophyll. Light energy is absorbed by chlorophyll molecules whereas carbon dioxide and oxygen enter through the tiny pores of stomata located in the epidermis of leaves.
• Another by-product of photosynthesis is sugars such as glucose and fructose.
• These sugars are then sent to the roots, stems, leaves, fruits, flowers and seeds. In other words, these sugars are used by the plants as an energy source, which helps them to grow. These sugar molecules then combine with each other to form more complex carbohydrates like cellulose and starch. The cellulose is considered as the structural material that is used in plant cell walls.

Where Does This Process Occur?

Chloroplasts are the sites of photosynthesis in plants and blue-green algae.  All green parts of a plant, including the green stems, green leaves,  and sepals – floral parts comprise of chloroplasts – green colour plastids. These cell organelles are present only in plant cells and are located within the mesophyll cells of leaves.

Also Read:  Photosynthesis Early Experiments

Photosynthesis Equation

Photosynthesis reaction involves two reactants, carbon dioxide and water. These two reactants yield two products, namely, oxygen and glucose. Hence, the photosynthesis reaction is considered to be an endothermic reaction. Following is the photosynthesis formula:

Unlike plants, certain bacteria that perform photosynthesis do not produce oxygen as the by-product of photosynthesis. Such bacteria are called anoxygenic photosynthetic bacteria. The bacteria that do produce oxygen as a by-product of photosynthesis are called oxygenic photosynthetic bacteria.

Structure Of Chlorophyll

The structure of Chlorophyll consists of 4 nitrogen atoms that surround a magnesium atom. A hydrocarbon tail is also present. Pictured above is chlorophyll- f,  which is more effective in near-infrared light than chlorophyll- a

Chlorophyll is a green pigment found in the chloroplasts of the  plant cell   and in the mesosomes of cyanobacteria. This green colour pigment plays a vital role in the process of photosynthesis by permitting plants to absorb energy from sunlight. Chlorophyll is a mixture of chlorophyll- a  and chlorophyll- b .Besides green plants, other organisms that perform photosynthesis contain various other forms of chlorophyll such as chlorophyll- c1 ,  chlorophyll- c2 ,  chlorophyll- d and chlorophyll- f .

Also Read:   Biological Pigments

Process Of Photosynthesis

At the cellular level,  the photosynthesis process takes place in cell organelles called chloroplasts. These organelles contain a green-coloured pigment called chlorophyll, which is responsible for the characteristic green colouration of the leaves.

As already stated, photosynthesis occurs in the leaves and the specialized cell organelles responsible for this process is called the chloroplast. Structurally, a leaf comprises a petiole, epidermis and a lamina. The lamina is used for absorption of sunlight and carbon dioxide during photosynthesis.

Structure of Chloroplast. Note the presence of the thylakoid

“Photosynthesis Steps:”

• During the process of photosynthesis, carbon dioxide enters through the stomata, water is absorbed by the root hairs from the soil and is carried to the leaves through the xylem vessels. Chlorophyll absorbs the light energy from the sun to split water molecules into hydrogen and oxygen.
• The hydrogen from water molecules and carbon dioxide absorbed from the air are used in the production of glucose. Furthermore, oxygen is liberated out into the atmosphere through the leaves as a waste product.
• Glucose is a source of food for plants that provide energy for  growth and development , while the rest is stored in the roots, leaves and fruits, for their later use.
• Pigments are other fundamental cellular components of photosynthesis. They are the molecules that impart colour and they absorb light at some specific wavelength and reflect back the unabsorbed light. All green plants mainly contain chlorophyll a, chlorophyll b and carotenoids which are present in the thylakoids of chloroplasts. It is primarily used to capture light energy. Chlorophyll-a is the main pigment.

The process of photosynthesis occurs in two stages:

• Light-dependent reaction or light reaction
• Light independent reaction or dark reaction

Stages of Photosynthesis in Plants depicting the two phases – Light reaction and Dark reaction

Light Reaction of Photosynthesis (or) Light-dependent Reaction

• Photosynthesis begins with the light reaction which is carried out only during the day in the presence of sunlight. In plants, the light-dependent reaction takes place in the thylakoid membranes of chloroplasts.
• The Grana, membrane-bound sacs like structures present inside the thylakoid functions by gathering light and is called photosystems.
• These photosystems have large complexes of pigment and proteins molecules present within the plant cells, which play the primary role during the process of light reactions of photosynthesis.
• There are two types of photosystems: photosystem I and photosystem II.
• Under the light-dependent reactions, the light energy is converted to ATP and NADPH, which are used in the second phase of photosynthesis.
• During the light reactions, ATP and NADPH are generated by two electron-transport chains, water is used and oxygen is produced.

The chemical equation in the light reaction of photosynthesis can be reduced to:

2H 2 O + 2NADP+ + 3ADP + 3Pi → O 2 + 2NADPH + 3ATP

Dark Reaction of Photosynthesis (or) Light-independent Reaction

• Dark reaction is also called carbon-fixing reaction.
• It is a light-independent process in which sugar molecules are formed from the water and carbon dioxide molecules.
• The dark reaction occurs in the stroma of the chloroplast where they utilize the NADPH and ATP products of the light reaction.
• Plants capture the carbon dioxide from the atmosphere through stomata and proceed to the Calvin photosynthesis cycle.
• In the Calvin cycle , the ATP and NADPH formed during light reaction drive the reaction and convert 6 molecules of carbon dioxide into one sugar molecule or glucose.

The chemical equation for the dark reaction can be reduced to:

3CO 2 + 6 NADPH + 5H 2 O + 9ATP → G3P + 2H+ + 6 NADP+ + 9 ADP + 8 Pi

* G3P – glyceraldehyde-3-phosphate

Calvin photosynthesis Cycle (Dark Reaction)

Also Read:  Cyclic And Non-Cyclic Photophosphorylation

Importance of Photosynthesis

• Photosynthesis is essential for the existence of all life on earth. It serves a crucial role in the food chain – the plants create their food using this process, thereby, forming the primary producers.
• Photosynthesis is also responsible for the production of oxygen – which is needed by most organisms for their survival.

Frequently Asked Questions

1. what is photosynthesis explain the process of photosynthesis., 2. what is the significance of photosynthesis, 3. list out the factors influencing photosynthesis., 4. what are the different stages of photosynthesis, 5. what is the calvin cycle, 6. write down the photosynthesis equation..

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Please What Is Meant By 300-400 PPM

PPM stands for Parts-Per-Million. It corresponds to saying that 300 PPM of carbon dioxide indicates that if one million gas molecules are counted, 300 out of them would be carbon dioxide. The remaining nine hundred ninety-nine thousand seven hundred are other gas molecules.

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scientific hypothesis

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• National Center for Biotechnology Information - PubMed Central - On the scope of scientific hypotheses
• LiveScience - What is a scientific hypothesis?
• The Royal Society - Open Science - On the scope of scientific hypotheses

scientific hypothesis , an idea that proposes a tentative explanation about a phenomenon or a narrow set of phenomena observed in the natural world. The two primary features of a scientific hypothesis are falsifiability and testability, which are reflected in an “If…then” statement summarizing the idea and in the ability to be supported or refuted through observation and experimentation. The notion of the scientific hypothesis as both falsifiable and testable was advanced in the mid-20th century by Austrian-born British philosopher Karl Popper .

The formulation and testing of a hypothesis is part of the scientific method , the approach scientists use when attempting to understand and test ideas about natural phenomena. The generation of a hypothesis frequently is described as a creative process and is based on existing scientific knowledge, intuition , or experience. Therefore, although scientific hypotheses commonly are described as educated guesses, they actually are more informed than a guess. In addition, scientists generally strive to develop simple hypotheses, since these are easier to test relative to hypotheses that involve many different variables and potential outcomes. Such complex hypotheses may be developed as scientific models ( see scientific modeling ).

Depending on the results of scientific evaluation, a hypothesis typically is either rejected as false or accepted as true. However, because a hypothesis inherently is falsifiable, even hypotheses supported by scientific evidence and accepted as true are susceptible to rejection later, when new evidence has become available. In some instances, rather than rejecting a hypothesis because it has been falsified by new evidence, scientists simply adapt the existing idea to accommodate the new information. In this sense a hypothesis is never incorrect but only incomplete.

The investigation of scientific hypotheses is an important component in the development of scientific theory . Hence, hypotheses differ fundamentally from theories; whereas the former is a specific tentative explanation and serves as the main tool by which scientists gather data, the latter is a broad general explanation that incorporates data from many different scientific investigations undertaken to explore hypotheses.

Countless hypotheses have been developed and tested throughout the history of science . Several examples include the idea that living organisms develop from nonliving matter, which formed the basis of spontaneous generation , a hypothesis that ultimately was disproved (first in 1668, with the experiments of Italian physician Francesco Redi , and later in 1859, with the experiments of French chemist and microbiologist Louis Pasteur ); the concept proposed in the late 19th century that microorganisms cause certain diseases (now known as germ theory ); and the notion that oceanic crust forms along submarine mountain zones and spreads laterally away from them ( seafloor spreading hypothesis ).

Hypothesis n., plural: hypotheses [/haɪˈpɑːθəsɪs/] Definition: Testable scientific prediction

Table of Contents

What Is Hypothesis?

A scientific hypothesis is a foundational element of the scientific method . It’s a testable statement proposing a potential explanation for natural phenomena. The term hypothesis means “little theory” . A hypothesis is a short statement that can be tested and gives a possible reason for a phenomenon or a possible link between two variables . In the setting of scientific research, a hypothesis is a tentative explanation or statement that can be proven wrong and is used to guide experiments and empirical research.

It is an important part of the scientific method because it gives a basis for planning tests, gathering data, and judging evidence to see if it is true and could help us understand how natural things work. Several hypotheses can be tested in the real world, and the results of careful and systematic observation and analysis can be used to support, reject, or improve them.

Researchers and scientists often use the word hypothesis to refer to this educated guess . These hypotheses are firmly established based on scientific principles and the rigorous testing of new technology and experiments .

For example, in astrophysics, the Big Bang Theory is a working hypothesis that explains the origins of the universe and considers it as a natural phenomenon. It is among the most prominent scientific hypotheses in the field.

“The scientific method: steps, terms, and examples” by Scishow:

Biology definition: A hypothesis  is a supposition or tentative explanation for (a group of) phenomena, (a set of) facts, or a scientific inquiry that may be tested, verified or answered by further investigation or methodological experiment. It is like a scientific guess . It’s an idea or prediction that scientists make before they do experiments. They use it to guess what might happen and then test it to see if they were right. It’s like a smart guess that helps them learn new things. A scientific hypothesis that has been verified through scientific experiment and research may well be considered a scientific theory .

Etymology: The word “hypothesis” comes from the Greek word “hupothesis,” which means “a basis” or “a supposition.” It combines “hupo” (under) and “thesis” (placing). Synonym:   proposition; assumption; conjecture; postulate Compare:   theory See also: null hypothesis

Characteristics Of Hypothesis

A useful hypothesis must have the following qualities:

• It should never be written as a question.
• You should be able to test it in the real world to see if it’s right or wrong.
• It needs to be clear and exact.
• It should list the factors that will be used to figure out the relationship.
• It should only talk about one thing. You can make a theory in either a descriptive or form of relationship.
• It shouldn’t go against any natural rule that everyone knows is true. Verification will be done well with the tools and methods that are available.
• It should be written in as simple a way as possible so that everyone can understand it.
• It must explain what happened to make an answer necessary.
• It should be testable in a fair amount of time.
• It shouldn’t say different things.

Sources Of Hypothesis

Sources of hypothesis are:

• Patterns of similarity between the phenomenon under investigation and existing hypotheses.
• Insights derived from prior research, concurrent observations, and insights from opposing perspectives.
• The formulations are derived from accepted scientific theories and proposed by researchers.
• In research, it’s essential to consider hypothesis as different subject areas may require various hypotheses (plural form of hypothesis). Researchers also establish a significance level to determine the strength of evidence supporting a hypothesis.
• Individual cognitive processes also contribute to the formation of hypotheses.

One hypothesis is a tentative explanation for an observation or phenomenon. It is based on prior knowledge and understanding of the world, and it can be tested by gathering and analyzing data. Observed facts are the data that are collected to test a hypothesis. They can support or refute the hypothesis.

For example, the hypothesis that “eating more fruits and vegetables will improve your health” can be tested by gathering data on the health of people who eat different amounts of fruits and vegetables. If the people who eat more fruits and vegetables are healthier than those who eat less fruits and vegetables, then the hypothesis is supported.

Hypotheses are essential for scientific inquiry. They help scientists to focus their research, to design experiments, and to interpret their results. They are also essential for the development of scientific theories.

Types Of Hypothesis

In research, you typically encounter two types of hypothesis: the alternative hypothesis (which proposes a relationship between variables) and the null hypothesis (which suggests no relationship).

Simple Hypothesis

It illustrates the association between one dependent variable and one independent variable. For instance, if you consume more vegetables, you will lose weight more quickly. Here, increasing vegetable consumption is the independent variable, while weight loss is the dependent variable.

Complex Hypothesis

It exhibits the relationship between at least two dependent variables and at least two independent variables. Eating more vegetables and fruits results in weight loss, radiant skin, and a decreased risk of numerous diseases, including heart disease.

Directional Hypothesis

It shows that a researcher wants to reach a certain goal. The way the factors are related can also tell us about their nature. For example, four-year-old children who eat well over a time of five years have a higher IQ than children who don’t eat well. This shows what happened and how it happened.

Non-directional Hypothesis

When there is no theory involved, it is used. It is a statement that there is a connection between two variables, but it doesn’t say what that relationship is or which way it goes.

Null Hypothesis

It says something that goes against the theory. It’s a statement that says something is not true, and there is no link between the independent and dependent factors. “H 0 ” represents the null hypothesis.

Associative and Causal Hypothesis

When a change in one variable causes a change in the other variable, this is called the associative hypothesis . The causal hypothesis, on the other hand, says that there is a cause-and-effect relationship between two or more factors.

Examples Of Hypothesis

Examples of simple hypotheses:

• Students who consume breakfast before taking a math test will have a better overall performance than students who do not consume breakfast.
• Students who experience test anxiety before an English examination will get lower scores than students who do not experience test anxiety.
• Motorists who talk on the phone while driving will be more likely to make errors on a driving course than those who do not talk on the phone, is a statement that suggests that drivers who talk on the phone while driving are more likely to make mistakes.

Examples of a complex hypothesis:

• Individuals who consume a lot of sugar and don’t get much exercise are at an increased risk of developing depression.
• Younger people who are routinely exposed to green, outdoor areas have better subjective well-being than older adults who have limited exposure to green spaces, according to a new study.
• Increased levels of air pollution led to higher rates of respiratory illnesses, which in turn resulted in increased costs for healthcare for the affected communities.

Examples of Directional Hypothesis:

• The crop yield will go up a lot if the amount of fertilizer is increased.
• Patients who have surgery and are exposed to more stress will need more time to get better.
• Increasing the frequency of brand advertising on social media will lead to a significant increase in brand awareness among the target audience.

Examples of Non-Directional Hypothesis (or Two-Tailed Hypothesis):

• The test scores of two groups of students are very different from each other.
• There is a link between gender and being happy at work.
• There is a correlation between the amount of caffeine an individual consumes and the speed with which they react.

Examples of a null hypothesis:

• Children who receive a new reading intervention will have scores that are different than students who do not receive the intervention.
• The results of a memory recall test will not reveal any significant gap in performance between children and adults.
• There is not a significant relationship between the number of hours spent playing video games and academic performance.

Examples of Associative Hypothesis:

• There is a link between how many hours you spend studying and how well you do in school.
• Drinking sugary drinks is bad for your health as a whole.
• There is an association between socioeconomic status and access to quality healthcare services in urban neighborhoods.

Functions Of Hypothesis

The research issue can be understood better with the help of a hypothesis, which is why developing one is crucial. The following are some of the specific roles that a hypothesis plays: (Rashid, Apr 20, 2022)

• A hypothesis gives a study a point of concentration. It enlightens us as to the specific characteristics of a study subject we need to look into.
• It instructs us on what data to acquire as well as what data we should not collect, giving the study a focal point .
• The development of a hypothesis improves objectivity since it enables the establishment of a focal point.
• A hypothesis makes it possible for us to contribute to the development of the theory. Because of this, we are in a position to definitively determine what is true and what is untrue .

How will Hypothesis help in the Scientific Method?

• The scientific method begins with observation and inquiry about the natural world when formulating research questions. Researchers can refine their observations and queries into specific, testable research questions with the aid of hypothesis. They provide an investigation with a focused starting point.
• Hypothesis generate specific predictions regarding the expected outcomes of experiments or observations. These forecasts are founded on the researcher’s current knowledge of the subject. They elucidate what researchers anticipate observing if the hypothesis is true.
• Hypothesis direct the design of experiments and data collection techniques. Researchers can use them to determine which variables to measure or manipulate, which data to obtain, and how to conduct systematic and controlled research.
• Following the formulation of a hypothesis and the design of an experiment, researchers collect data through observation, measurement, or experimentation. The collected data is used to verify the hypothesis’s predictions.
• Hypothesis establish the criteria for evaluating experiment results. The observed data are compared to the predictions generated by the hypothesis. This analysis helps determine whether empirical evidence supports or refutes the hypothesis.
• The results of experiments or observations are used to derive conclusions regarding the hypothesis. If the data support the predictions, then the hypothesis is supported. If this is not the case, the hypothesis may be revised or rejected, leading to the formulation of new queries and hypothesis.
• The scientific approach is iterative, resulting in new hypothesis and research issues from previous trials. This cycle of hypothesis generation, testing, and refining drives scientific progress.

Importance Of Hypothesis

• Hypothesis are testable statements that enable scientists to determine if their predictions are accurate. This assessment is essential to the scientific method, which is based on empirical evidence.
• Hypothesis serve as the foundation for designing experiments or data collection techniques. They can be used by researchers to develop protocols and procedures that will produce meaningful results.
• Hypothesis hold scientists accountable for their assertions. They establish expectations for what the research should reveal and enable others to assess the validity of the findings.
• Hypothesis aid in identifying the most important variables of a study. The variables can then be measured, manipulated, or analyzed to determine their relationships.
• Hypothesis assist researchers in allocating their resources efficiently. They ensure that time, money, and effort are spent investigating specific concerns, as opposed to exploring random concepts.
• Testing hypothesis contribute to the scientific body of knowledge. Whether or not a hypothesis is supported, the results contribute to our understanding of a phenomenon.
• Hypothesis can result in the creation of theories. When supported by substantive evidence, hypothesis can serve as the foundation for larger theoretical frameworks that explain complex phenomena.
• Beyond scientific research, hypothesis play a role in the solution of problems in a variety of domains. They enable professionals to make educated assumptions about the causes of problems and to devise solutions.

Research Hypotheses: Did you know that a hypothesis refers to an educated guess or prediction about the outcome of a research study?

It’s like a roadmap guiding researchers towards their destination of knowledge. Just like a compass points north, a well-crafted hypothesis points the way to valuable discoveries in the world of science and inquiry.

Choose the best answer. 

Send Your Results (Optional)

Further reading.

• RNA-DNA World Hypothesis
• BYJU’S. (2023). Hypothesis. Retrieved 01 Septermber 2023, from https://byjus.com/physics/hypothesis/#sources-of-hypothesis
• Collegedunia. (2023). Hypothesis. Retrieved 1 September 2023, from https://collegedunia.com/exams/hypothesis-science-articleid-7026#d
• Hussain, D. J. (2022). Hypothesis. Retrieved 01 September 2023, from https://mmhapu.ac.in/doc/eContent/Management/JamesHusain/Research%20Hypothesis%20-Meaning,%20Nature%20&%20Importance-Characteristics%20of%20Good%20%20Hypothesis%20Sem2.pdf
• Media, D. (2023). Hypothesis in the Scientific Method. Retrieved 01 September 2023, from https://www.verywellmind.com/what-is-a-hypothesis-2795239#toc-hypotheses-examples
• Rashid, M. H. A. (Apr 20, 2022). Research Methodology. Retrieved 01 September 2023, from https://limbd.org/hypothesis-definitions-functions-characteristics-types-errors-the-process-of-testing-a-hypothesis-hypotheses-in-qualitative-research/#:~:text=Functions%20of%20a%20Hypothesis%3A&text=Specifically%2C%20a%20hypothesis%20serves%20the,providing%20focus%20to%20the%20study.

©BiologyOnline.com. Content provided and moderated by Biology Online Editors.

Last updated on September 8th, 2023

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October 31, 2020

Multi-Hypothesis Modeling of Photosynthesis

Novel method to compare models shows surprising dominance of empirical assumptions in mechanistic simulations of photosynthesis.

Diagram of photosynthesis model to calculate net carbon assimilation (A, μmol m−2 s−1, grey rectangle). Inputs (diamonds), parameters (triangles), functions (ellipses) and target state variable (rectangle) are shown, as is the breakdown on the model into the four processes (colors; limiting‐rate selection, carboxylation, electron transport and triose phosphate use). Arrows represent the flow of information from distal parts of the model (inputs and parameters), through intermediate functions to the end goal.

[Reprinted under a CC BY 4.0 License from Walker, A.P. et al . "Multi-Hypothesis Comparison of Farquhar and Collatz Photosynthesis Models Reveals the Unexpected Influence of Empirical Assumptions at Leaf and Global Scales." Global Change Biology 27 , 804–822 (2020). DOI: 10.1111/gcb.15366 ]

The Science

Scientific hypotheses describe how processes might work in the natural world. Computer models are built from mathematical descriptions of these hypotheses. Alternative hypotheses are common, especially in environmental sciences, yet most computer models cannot easily switch among these alternative hypotheses. With funding from the U.S. Department of Energy, scientists have developed a new model that can switch between hypotheses and prioritize which process to study further, a “multi-hypothesis model.” Using the model to study common leaf photosynthesis models, scientists found the surprising importance of a process that has previously received little attention. New data were then collected to discriminate among the alternative hypotheses, finding support for the more traditional approach.

Leaf photosynthesis models simulate the rhythms of carbon dioxide (CO 2 ) transfer from the atmosphere to plants. This study highlights a key shortfall in photosynthesis modeling and in the general approach to developing and using predictive models of the terrestrial biosphere. Models can reach the same endpoint in multiple ways, leading to models “getting it right for the wrong reasons.” The multi-hypothesis approach will help to identify key processes causing model differences and evaluate alternative hypotheses to describe those processes, ultimately leading to more robust predictions of terrestrial ecosystems.

Leaf photosynthesis models are the beating heart of global carbon cycle models. These photosynthesis models simulate the rhythms of CO 2 transfer from the atmosphere into plants and terrestrial ecosystems. The reigning king of photosynthesis models is the Farquhar model published in 1980. However, despite its almost ubiquitous use, there are a number of variations in how the mechanics of various component processes are mathematically described (i.e., there are various mathematical hypotheses that describe some of the sub-processes within the overarching Farquhar model). The consequences of these alternative choices have never been formally investigated, in part because methods to formally investigate model sensitivity to variation in how processes are represented have only recently been developed. Novel multi-hypothesis modeling methods were applied to investigate the influence of 14 parameters and four processes with alternative representations in photosynthesis models, finding the surprising dominance of a process that has not been extensively evaluated with data. Running the alternatives of this dominant process in global models resulted in a difference in photosynthesis equivalent to annual human CO 2 emissions. This multi-hypothesis model evaluation identified as important two alternative hypotheses for photosynthetic limiting rate selection. Novel, high-resolution photosynthesis measurements were designed and undertaken to discriminate among these hypotheses. General support for the original Farquhar implementation was found and is recommended for use to reduce uncertainty in global photosynthesis simulations.

Principal Investigator

Anthony Walker Oak Ridge National Laboratory [email protected]

Program Manager

Daniel Stover U.S. Department of Energy, Biological and Environmental Research (SC-33) Environmental System Science [email protected]

U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research: Next Generation Ecosystem Experiments-Tropics, ORNL Terrestrial Ecosystem Science SFA, university grant DE-SC0019438. National Science Foundation support for National Center for Atmospheric Research.

Related Links

• Related publication

Walker, A.P., et al. "Multi-Hypothesis Comparison of Farquhar and Collatz Photosynthesis Models Reveals the Unexpected Influence of Empirical Assumptions at Leaf and Global Scales." Global Change Biology 27 804–822  (2020). https://doi.org/10.1111/gcb.15366 .

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• Knowledge Base

Methodology

• How to Write a Strong Hypothesis | Steps & Examples

How to Write a Strong Hypothesis | Steps & Examples

Published on May 6, 2022 by Shona McCombes . Revised on November 20, 2023.

A hypothesis is a statement that can be tested by scientific research. If you want to test a relationship between two or more variables, you need to write hypotheses before you start your experiment or data collection .

Example: Hypothesis

Daily apple consumption leads to fewer doctor’s visits.

Table of contents

What is a hypothesis, developing a hypothesis (with example), hypothesis examples, other interesting articles, frequently asked questions about writing hypotheses.

A hypothesis states your predictions about what your research will find. It is a tentative answer to your research question that has not yet been tested. For some research projects, you might have to write several hypotheses that address different aspects of your research question.

A hypothesis is not just a guess – it should be based on existing theories and knowledge. It also has to be testable, which means you can support or refute it through scientific research methods (such as experiments, observations and statistical analysis of data).

Variables in hypotheses

Hypotheses propose a relationship between two or more types of variables .

• An independent variable is something the researcher changes or controls.
• A dependent variable is something the researcher observes and measures.

If there are any control variables , extraneous variables , or confounding variables , be sure to jot those down as you go to minimize the chances that research bias  will affect your results.

In this example, the independent variable is exposure to the sun – the assumed cause . The dependent variable is the level of happiness – the assumed effect .

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Step 1. ask a question.

Writing a hypothesis begins with a research question that you want to answer. The question should be focused, specific, and researchable within the constraints of your project.

Step 2. Do some preliminary research

Your initial answer to the question should be based on what is already known about the topic. Look for theories and previous studies to help you form educated assumptions about what your research will find.

At this stage, you might construct a conceptual framework to ensure that you’re embarking on a relevant topic . This can also help you identify which variables you will study and what you think the relationships are between them. Sometimes, you’ll have to operationalize more complex constructs.

Step 3. Formulate your hypothesis

Now you should have some idea of what you expect to find. Write your initial answer to the question in a clear, concise sentence.

4. Refine your hypothesis

You need to make sure your hypothesis is specific and testable. There are various ways of phrasing a hypothesis, but all the terms you use should have clear definitions, and the hypothesis should contain:

• The relevant variables
• The specific group being studied
• The predicted outcome of the experiment or analysis

5. Phrase your hypothesis in three ways

To identify the variables, you can write a simple prediction in  if…then form. The first part of the sentence states the independent variable and the second part states the dependent variable.

In academic research, hypotheses are more commonly phrased in terms of correlations or effects, where you directly state the predicted relationship between variables.

If you are comparing two groups, the hypothesis can state what difference you expect to find between them.

6. Write a null hypothesis

If your research involves statistical hypothesis testing , you will also have to write a null hypothesis . The null hypothesis is the default position that there is no association between the variables. The null hypothesis is written as H 0 , while the alternative hypothesis is H 1 or H a .

• H 0 : The number of lectures attended by first-year students has no effect on their final exam scores.
• H 1 : The number of lectures attended by first-year students has a positive effect on their final exam scores.

If you want to know more about the research process , methodology , research bias , or statistics , make sure to check out some of our other articles with explanations and examples.

• Sampling methods
• Simple random sampling
• Stratified sampling
• Cluster sampling
• Likert scales
• Reproducibility

 Statistics

• Null hypothesis
• Statistical power
• Probability distribution
• Effect size
• Poisson distribution

Research bias

• Optimism bias
• Cognitive bias
• Implicit bias
• Hawthorne effect
• Anchoring bias
• Explicit bias

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A hypothesis is not just a guess — it should be based on existing theories and knowledge. It also has to be testable, which means you can support or refute it through scientific research methods (such as experiments, observations and statistical analysis of data).

Null and alternative hypotheses are used in statistical hypothesis testing . The null hypothesis of a test always predicts no effect or no relationship between variables, while the alternative hypothesis states your research prediction of an effect or relationship.

Hypothesis testing is a formal procedure for investigating our ideas about the world using statistics. It is used by scientists to test specific predictions, called hypotheses , by calculating how likely it is that a pattern or relationship between variables could have arisen by chance.

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McCombes, S. (2023, November 20). How to Write a Strong Hypothesis | Steps & Examples. Scribbr. Retrieved September 3, 2024, from https://www.scribbr.com/methodology/hypothesis/

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