Mineral Nutrition

 

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Mineral Nutrition and Agricultural Yields

The study of how plants obtain and use mineral nutrients is called mineral nutrition.

High agricultural yields are directly dependent on the use of mineral fertilizers. In fact, the yields of most crops show a direct, linear increase with the amount of fertilizer they absorb. This is because crops need essential nutrients like nitrogen (N), phosphorus (P), and potassium (K) to grow and thrive.

Global consumption of these primary fertilizer elements has risen dramatically over the past several decades. From 1960 to 1990, annual consumption surged from 30 million to 143 million metric tons. After a decade of more careful use due to rising costs, consumption has again climbed, reaching 180 million metric tons per year in 2010.

Environmental Consequences of Fertilizer Use

While fertilizers are crucial for food production, their use comes with significant environmental costs.

• Energy Consumption: Over half of the energy used in agriculture is dedicated to producing, distributing, and applying nitrogen fertilizers.

• Nonrenewable Resources: The production of phosphorus fertilizers relies on nonrenewable resources, which are projected to reach peak production this century.

• Inefficient Use: Plants typically use less than half of the fertilizer applied to the soil. The rest can leach into surface water or groundwater, associate with soil particles, or contribute to air pollution.

• Water Contamination: Fertilizer runoff, particularly from nitrate, has led to widespread water contamination. Many wells in the United States and other agricultural regions now exceed federal safety standards for nitrate concentrations in drinking water.

• Atmospheric Nitrogen Deposition: Human activities release nitrogen (as nitrate and ammonium) into the environment, which is then deposited in the soil by rain. This process, known as atmospheric nitrogen deposition, is changing ecosystems globally.

Defining Essential Elements for Plants

An essential element is a chemical element that a plant must have to complete its life cycle. Without it, the plant will show severe abnormalities in its growth, development, or reproduction. 

These elements are a fundamental part of the plant's structure or metabolism.

Plants can synthesize all the necessary compounds for normal growth if they have access to these essential elements, along with water and sunlight. 

Classification of Essential Mineral Elements

Essential mineral elements are classified into two groups based on their concentration in plant tissue:

• Macronutrients: needed in large quantities.
• Micronutrients: needed in very small quantities, often called trace elements.

The first three elements—hydrogen (H), carbon (C), and oxygen (O)—are crucial but are not classified as mineral nutrients because plants primarily get them from water (H2​O) and carbon dioxide (CO2​).

Element	Chemical symbol	Concentration in dry matter (% or ppm)
Obtained from water or carbon dioxide
Hydrogen	H	6
Carbon	C	45
Oxygen	O	45
Obtained from the soil
Macronutrients
Nitrogen	N	1.5
Potassium	K	1
Calcium	Ca	0.5
Magnesium	Mg	0.2
Phosphorus	P	0.2
Sulfur	S	0.1
Silicon	Si	0.1
Micronutrients
Chlorine	Cl	100
Iron	Fe	100
Boron	B	20
Manganese	Mn	50
Sodium	Na	10
Zinc	Zn	20
Copper	Cu	6
Nickel	Ni	0.1
Molybdenum	Mo	0.1

A more functional classification system, based on the biochemical role of the elements, divides essential elements into four groups:

• Group 1: Building Blocks of Organic Compounds

These elements, nitrogen (N) and sulfur (S), are assimilated by plants and used to create essential organic molecules like amino acids, nucleic acids, and proteins.

• Group 2: Energy Storage and Structural Integrity

This group includes elements like phosphorus (P), boron (B), and silicon (Si). They are often found in the plant as esters, playing a vital role in energy storage reactions and maintaining the structural integrity of the plant.

• Group 3: Osmotic and Enzymatic Regulation 

Elements in this group, such as potassium (K), calcium (Ca), and magnesium (Mg), exist as free or bound ions in plant tissue. They act as enzyme cofactors, regulate the plant's osmotic potential, and control membrane permeability.

• Group 4: Electron Transfer 

This group is made up of metals like iron (Fe), which are crucial for reactions involving the transfer of electrons, such as those that occur during photosynthesis and respiration.

 

Mineral nutrient	Functions
Group 1: Nutrients that are part of carbon compounds
N (Nitrogen)	Constituent of amino acids, amides, proteins, nucleic acids, nucleotides, coenzymes, hexosamines, etc.
S (Sulfur)	Component of cysteine, cystine, methionine. Constituent of lipoic acid, coenzyme A, thiamine pyrophosphate, glutathione, biotin, 5'-adenylylsulfate, and 3'-phosphoadenosine.
Group 2: Nutrients that are important in energy storage or structural integrity
P (Phosphorus)	Component of sugar phosphates, nucleic acids, nucleotides, coenzymes, phospholipids, phytic acid, etc. Has a key role in reactions that involve ATP.
Si (Silicon)	Deposited as amorphous silica in cell walls. Contributes to cell wall mechanical properties, including rigidity and elasticity.
B (Boron)	Complexes with mannitol, mannan, polymannuronic acid, and other constituents of cell walls. Involved in cell elongation and nucleic acid metabolism.
Group 3: Nutrients that remain in ionic form
K (Potassium)	Required as a cofactor for more than 40 enzymes. Principal cation in establishing cell turgor and maintaining cell electroneutrality.
Ca (Calcium)	Constituent of the middle lamella of cell walls. Required as a cofactor by some enzymes involved in the hydrolysis of ATP and phospholipids. Acts as a second messenger in metabolic regulation.
Mg (Magnesium)	Required by many enzymes involved in phosphate transfer. Constituent of the chlorophyll molecule.
Cl (Chlorine)	Required for the photosynthetic reactions involved in O2​ evolution.
Zn (Zinc)	Constituent of alcohol dehydrogenase, glutamic dehydrogenase, carbonic anhydrase, etc.
Na (Sodium)	Involved with the regeneration of phosphoenolpyruvate in C4​ and CAM plants. Substitutes for potassium in some functions.
Group 4: Nutrients that are involved in redox reactions
Fe (Iron)	Constituent of cytochromes and nonheme iron proteins involved in photosynthesis, N2​ fixation, and respiration.
Mn (Manganese)	Required for activity of some dehydrogenases, decarboxylases, kinases, oxidases, and peroxidases. Involved with other cation-activated enzymes and photosynthetic O2​ evolution.
Cu (Copper)	Component of ascorbic acid oxidase, tyrosinase, monoamine oxidase, uricase, cytochrome oxidase, phenolase, laccase, and plastocyanin.
Ni (Nickel)	Constituent of urease. In N2​-fixing bacteria, constituent of hydrogenases.
Mo (Molybdenum)	Constituent of nitrogenase, nitrate reductase, and xanthine dehydrogenase.

Non-Essential but Beneficial Elements

Some elements, while not considered essential for all plants, can still accumulate in plant tissues and may even be beneficial. For example:

• Aluminum (Al): Although not essential, some plants accumulate it, and small amounts can even stimulate growth.

• Selenium (Se): Some plant species can accumulate large amounts of this element.

• Cobalt (Co): It's not required by most plants, but it is essential for the function of nitrogen-fixing microorganisms that live in symbiosis with certain plants, as it is part of vitamin B12. Without cobalt, these nitrogen-fixing nodules cannot develop properly.

Studying Plant Nutrition: Beyond the Soil

To figure out if an element is essential for a plant, scientists need a way to grow the plant with all nutrients present except for the one being tested. This is incredibly difficult to do with soil, which is a complex medium full of various minerals.

In the 19th century, pioneering botanists like Julius von Sachs and Wilhelm Knop found a solution: they grew plants with their roots in a solution of inorganic salts and water, completely without soil. This technique, called solution culture or hydroponics, proved that plants could get all the nutrients they need from mineral elements, water, air, and sunlight alone.

What Is Hydroponics?

Hydroponics is the method of growing plants in a nutrient-rich solution without soil.

For this technique to work, the nutrient solution must be in a large enough volume or be adjusted frequently to prevent the plant's roots from drastically changing the concentration of minerals and the pH. 

It's also critical to provide the roots with enough oxygen, which is often done by bubbling air vigorously through the solution.

Hydroponics is used commercially for growing many crops, including tomatoes, cucumbers, and cannabis, in greenhouses and indoors. 

Types of hydroponic systems

Standard hydroponic system: In standard hydroponic culture, plants are suspended with their stems just above a tank filled with a nutrient solution. An air stone—a porous device that creates a stream of fine bubbles—is used to pump air into the tank, ensuring the solution is fully saturated with oxygen. It is also called deep water culture (DWC).

Deep water culture

Substrate-based systems: In these systems, plants are supported by inert materials like sand, gravel, or rockwool. The nutrient solution is then flushed through this material. If the nutrient solution is delivered directly to the base of each plant via a drip line, it is called drip system. Excess solution is either collected and recycled (closed system) or allowed to run off (open system).

Nutrient Film Technique (NFT): In the nutrient film technique (NFT), a pump circulates nutrient solution from a main reservoir. The solution flows in a thin, continuous layer along the bottom of a tilted channel or trough, where it bathes the plants' roots before returning to the reservoir. This method ensures that the roots receive a constant and ample supply of both water and oxygen.

Nutrient film technique

Ebb-and-Flow Systems: Also known as flood and drain systems, this method involves periodically flooding the plant roots with a nutrient solution, which then recedes to expose the roots to a moist, oxygen-rich environment. Similar to aeroponics, these systems typically require a higher concentration of nutrients.

Ebb-and-flow system

Aeroponics: This technique suspends plant roots in the air and continuously sprays them with a nutrient solution. This allows for easy control of the gaseous environment around the roots. However, it requires a higher concentration of nutrients and is not as widely used due to technical challenges.

Aeroponics

Wick System: One of the simplest types of hydroponics, a wick system uses a wick (like a string or felt strip) to draw nutrient solution from a reservoir up into a growing medium where the plant's roots are located. This is a passive system with no moving parts.

Wick system

Nutrient Solution for Hydroponics

For a plant to grow rapidly in a soil-free environment, it needs a carefully balanced nutrient solution. Early scientists like Knop tried to create these solutions, but this formulation, called Knop's solution, contained only a few elements. Their success was often accidental because the chemicals they used were contaminated with other essential elements they didn't know about. Today, we use more sophisticated formulas, like the modified Hoagland solution, to provide all the necessary minerals for fast, healthy plant growth.

Knop's solution

Knop's solution was an early and influential nutrient solution used for hydroponics. It contained five key mineral salts, which were initially thought to be all a plant needed to grow. The composition of Knop's solution included:

• Calcium nitrate (Ca(NO3​)2​)

• Potassium nitrate (KNO3​)

• Monopotassium phosphate (KH2​PO4​)

• Magnesium sulfate (MgSO4​)

• Iron salt (typically ferrous sulfate, FeSO4​)

 

Hoagland's Medium

Hoagland's medium is a widely used nutrient solution for growing plants in hydroponic culture. It was originally formulated by Dennis R. Hoagland and his colleagues in 1938 at the University of California, Berkeley. The original formulation was a precise mixture of inorganic salts that supplied all the essential macro- and micronutrients known at the time for optimal plant growth.

The key components of the original Hoagland's solution included:

• Macronutrients: Provided by potassium nitrate (KNO3​), calcium nitrate (Ca(NO3​)2​), monopotassium phosphate (KH2​PO4​), and magnesium sulfate (MgSO4​).

• Micronutrients: Supplied as a trace element mixture containing iron sulfate (FeSO4​), boric acid (H3​BO3​), manganese chloride (MnCl2​), zinc sulfate (ZnSO4​), copper sulfate (CuSO4​), and molybdic acid (H2​MoO4​). A key innovation was the use of an iron salt that was kept soluble by the addition of tartaric acid.

Modifications to Hoagland's Medium

Over time, researchers made several modifications to the original Hoagland's medium to improve its stability, effectiveness, and applicability for different research needs. These modifications address some of the limitations of the original formula.

1. Nitrogen Source: The original formula primarily used nitrate (NO3−​) as the nitrogen source. Modified versions often add a small amount of ammonium ($N{H}_4^{+}$). This balanced nitrogen supply helps to stabilize the solution's pH, as the plant's uptake of both cation (NH4+​) and anion (NO3−​) forms of nitrogen prevents the large pH shifts that can occur with nitrate-only solutions.

2. Iron Chelation: A major modification was the shift from tartaric acid to synthetic chelating agents like EDTA (ethylenediaminetetraacetic acid). EDTA is much more effective at keeping iron soluble and available to the plant over a wider range of pH conditions, preventing the iron from precipitating out of the solution and becoming unavailable.

3. Nutrient Concentration: Many modified solutions are more concentrated than the original. This allows researchers to dilute them to specific levels, making them adaptable for different plant species and growth stages. It also allows for longer periods between solution changes.

4. Additional Elements: Some modified versions include additional elements, such as silicon (Si), which have been shown to be beneficial for the growth and health of certain plants, even if they are not considered essential for all species.

Key Features of a Modern Nutrient Solution

• High Concentrations: Contain high concentrations of nutrients to ensure the plants have a steady supply. These levels can be much higher than those found in soil. While this allows for longer periods between changes, it can be too intense for young plants, so researchers often dilute the solution and replenish it more frequently.

• Balanced Nitrogen: A balanced mix of both ammonium (NH4+​) and nitrate (NO3−​) is ideal for providing nitrogen. Using both forms helps prevent rapid changes in the solution's pH and promotes a better balance of positively and negatively charged ions within the plant.

• Maintaining Iron Availability: A significant challenge is keeping iron available to the plants. Iron can easily precipitate out of the solution, especially in alkaline conditions or when phosphate is present, making it inaccessible to the roots. To solve this, scientists use chelating agents (or chelators).

What is a chelator?

A chelator is a molecule that binds to metal ions, like iron, forming a stable, soluble complex. This "cages" the iron, preventing it from precipitating out of the solution while still allowing the plant to absorb it.

Early researchers used simple chelators like citric acid. Modern solutions often use more advanced chemicals like ethylenediaminetetraacetic acid (EDTA) or diethylene triaminepentaacetic acid (DTPA, or pentetic acid) to keep iron and other metal ions available to the plant. After the plant absorbs the iron, the chelator can diffuse back into the solution to bind with more metal ions.

Mineral Deficiencies and Their Impact on Plants

When a plant doesn't get enough of an essential element, it develops a nutritional disorder with specific deficiency symptoms. In a controlled hydroponic environment, it's easy to link a missing element to a particular set of symptoms, like a change in leaf color. However, diagnosing deficiencies in soil-grown plants is more complex due to several factors:

• Multiple Deficiencies: A plant might be lacking several elements at once.

• Element Interactions: Too little or too much of one element can cause a deficiency of another.

• Disease Mimicry: Symptoms of some viral diseases can look very similar to those of a nutrient deficiency.

These symptoms are the visible signs of metabolic disruptions caused by an insufficient supply of an essential element. The specific symptoms are directly related to the element's function in the plant.

The Mobility of Essential Elements

A key factor in diagnosing a nutrient deficiency is understanding how mobile an element is within the plant. The location of the symptoms—whether they appear in older or younger leaves first—provides a crucial clue.

• Mobile Elements: Some elements, such as nitrogen (N), phosphorus (P), and potassium (K), can be easily moved from older, mature leaves to younger, actively growing leaves. If the supply of a mobile element is cut off, the plant will relocate it from the older leaves, causing deficiency symptoms to appear first in the older leaves.

• Immobile Elements: Other elements, like boron (B), iron (Fe), and calcium (Ca), cannot be easily moved from older leaves. As a result, when the supply is inadequate, the younger leaves are the first to show symptoms because they are unable to receive the element from the older parts of the plant.

The specific symptoms and functions of each essential element are discussed in detail based on their biochemical roles, but it's important to remember that these symptoms can vary depending on the plant species.

The Roles and Deficiency Symptoms of Essential Elements

An insufficient supply of an essential element leads to a nutritional disorder, resulting in specific deficiency symptoms. The location of these symptoms—whether they first appear on older or younger leaves—is a crucial clue, as it depends on the element's mobility within the plant.

Group 1: Elements that are Part of Carbon Compounds

This group includes nitrogen (N) and sulfur (S). They are essential for building core organic molecules like proteins and nucleic acids.

• Nitrogen (N): Plants need more nitrogen than any other mineral. It's a key component of chlorophyll, amino acids, and DNA. A nitrogen deficiency quickly stunts growth and causes chlorosis (yellowing of leaves), starting with the older leaves because nitrogen is highly mobile and is relocated to newer leaves. Severe deficiency can cause older leaves to turn tan and fall off. Plants may also develop slender, woody stems and a purple coloration in some species due to a buildup of other pigments.

• Sulfur (S): Sulfur is a building block for important amino acids like cysteine and methionine, as well as several essential coenzymes. Symptoms of sulfur deficiency are similar to nitrogen deficiency—chlorosis, stunting, and purple coloration—because both are used to build proteins. However, sulfur is less mobile than nitrogen, so chlorosis usually appears in younger leaves first.

Group 2: Elements for Energy Storage and Structural Integrity

This group includes phosphorus (P), silicon (Si), and boron (B), which are often found in plants as ester linkages.

• Phosphorus (P): Phosphorus (as phosphate) is a vital part of ATP (the plant's energy currency), DNA, and cell membranes. Deficiency symptoms include stunted growth, dark green leaves (which may be malformed and have dead spots), and sometimes a purple coloration due to excess pigment production. Unlike nitrogen deficiency, the purple color is not accompanied by chlorosis.

• Silicon (Si): While only a few plant families absolutely require silicon to complete their life cycle, many species benefit from it. Silicon helps reinforce cell walls, making plants more resistant to falling over (lodging) and fungal infections.

• Boron (B): Boron is critical for cell wall structure, cell elongation, and hormone regulation. A boron deficiency can cause black necrosis of young leaves and terminal buds, making stems brittle and causing the plant to lose apical dominance and become highly branched.

Group 3: Elements that Remain in Ionic Form

These elements, including potassium (K), calcium (Ca), and magnesium (Mg), are present as ions and are crucial for a variety of cellular processes.

• Potassium (K): Potassium is essential for regulating osmotic potential and activating many enzymes. Since it's mobile, deficiency symptoms—like mottled or marginal chlorosis that turns into necrosis—start in older leaves. Stems can also become weak, and in some plants, root systems may become more susceptible to disease.

• Calcium (Ca): Calcium has a structural role in cell walls and acts as a signal to regulate cellular processes. Because calcium is immobile, deficiency symptoms appear in younger leaves and meristematic regions (where cells divide rapidly), causing necrosis of root tips and young leaves.

• Magnesium (Mg): Magnesium is the central atom in chlorophyll and is required to activate many enzymes. Like potassium, it is mobile, so its deficiency symptoms—interveinal chlorosis (yellowing between the veins)—first appear in older leaves.

Group 4: Elements Involved in Redox Reactions

This group consists of metals that can undergo reversible oxidation and reduction, making them essential for electron transfer reactions.

• Iron (Fe): Iron is a component of enzymes involved in electron transfer and is crucial for chlorophyll synthesis. Unlike magnesium, iron is immobile, so its deficiency causes interveinal chlorosis that starts in younger leaves. In severe cases, the entire leaf can turn white.

• Manganese (Mn): Manganese activates several enzymes and is essential for the splitting of water during photosynthesis. Deficiency symptoms include interveinal chlorosis and small necrotic spots on the leaves.

• Copper (Cu): Copper is also involved in redox reactions, particularly in photosynthesis. A deficiency often results in dark green leaves with necrotic spots that appear at the tips of younger leaves.

• Nickel (Ni): Nickel is required by only one known enzyme in higher plants, urease. Deficiency causes a buildup of urea and leaf tip necrosis.

• Molybdenum (Mo): Molybdenum is a component of several important enzymes, including those for nitrogen fixation and nitrate reduction. A deficiency leads to general chlorosis and necrosis of older leaves. In some plants, it can cause "whiptail disease," where leaves are twisted and eventually die.

Photosynthesis: Regulation of Carbon fixation reactions

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In photosynthetic organisms, the carbon fixation reactions are regulated by light to ensure that the production of carbohydrates is synchronized with the availability of light energy. This regulation involves several mechanisms, including the activation of key enzymes, changes in the concentration of metabolic intermediates, and the formation of supramolecular complexes.

Induction Period in Carbon Fixation

After the onset of illumination, there is an induction period during which the rate of CO2​ fixation increases gradually before reaching a steady state. This initial lag is because the photosynthetic enzymes need time to be activated by light, and the concentration of Calvin-Benson cycle intermediates needs to increase.

• During the induction period, six triose phosphates produced in the Calvin-Benson cycle are used for the regeneration of ribulose 1,5-bisphosphate (RuBP).

• At the steady state, five of the six triose phosphates regenerate RuBP, while the remaining one is either used for starch formation in the chloroplast or sucrose synthesis in the cytosol.

Regulation of the Calvin-Benson Cycle

The regulation of the Calvin-Benson cycle involves both long-term and short-term mechanisms.

Long-term regulation involves changes in the amount of enzymes, which is controlled by the expression of nuclear and chloroplast genes. Regulatory signaling between nucleus and plastids is mostly anterograde — that is, the products of nuclear genes control the transcription and translation of plastid genes. 

Short-term regulation involves post-translational modifications that can rapidly alter the activity of enzymes.

Light-dependent mechanisms quickly regulate the specific activity of five key enzymes:

1. Rubisco

2. Fructose 1,6-bisphosphatase

3. Sedoheptulose 1,7-bisphosphatase

4. Phosphoribulokinase

5. NADP–glyceraldehyde-3-phosphate dehydrogenase

Rubisco: Activation and Catalysis

Rubisco, a crucial enzyme for carbon fixation, is regulated by Rubisco-activase. 

A CO2​ molecule has a dual role in Rubisco's activity: it acts as an activator and a substrate.

• Activation: The activation process begins when an activator CO2​ molecule forms a lysyl-carbamate with the enzyme. The subsequent binding of Mg2+ stabilizes this complex, making Rubisco catalytically active. In the light, an increase in both the stroma's pH and Mg2+ concentration facilitates this activation.

• Catalysis: A separate CO2​ molecule, the substrate CO2​, then reacts with RuBP at the active site, yielding two molecules of 3-phosphoglycerate.

Activation of RuBisCo by CO2 and Mg2+

Rubisco-activase overcomes several types of inhibition. Sugar phosphates like xylulose 1,5-bisphosphate and 2-carboxyarabinitol 1-phosphate (naturally occurring inhibitor) can bind tightly to uncarbamylated Rubisco, preventing its activation and inhibiting catalysis. The substrate RuBP can also bind to the carbamylated enzyme, preventing activation. Rubisco-activase removes these inhibitors, allowing the enzyme to be activated by activator CO2 and Mg2+. 

Formation of the Dead-End Complex

• A Rubisco active site that has lost its activating CO2​ molecule (a process called decarbamylation) can bind to RuBP.

• This binding leads to a conformational change in Rubisco, which creates an inactive, dead-end complex where RuBP is tightly bound to the enzyme.

How Rubisco-Activase Restores Activity

Rubisco-activase removes the tightly bound RuBP in an ATP dependent process. The steps are as follows: 

1. Activase Priming: ATP hydrolysis by Rubisco-activase is required for the conformational changes that prime the activase.

2. Physical Interaction: The primed Rubisco-activase physically interacts with the Rubisco-RuBP dead-end complex.

3. Conformational Change: This interaction changes the conformation of Rubisco, reducing its affinity for RuBP.

4. RuBP Dissociation: With a lower affinity, the tightly bound RuBP dissociates from the active site of Rubisco.

5. Reactivation: Once the active site is free, Rubisco-activase likely dissociates, allowing the site to be reactivated through carbamylation by CO2​. This allows the enzyme to resume its role in carbon fixation.

At high temperatures, Rubisco deactivates faster than Rubisco-activase can reactivate it, leading to a loss of function.

Activation of RuBisCo by RuBisCo activase (adapted from Jensen (2000) PNAS)

The Ferredoxin-Thioredoxin System

The ferredoxin-thioredoxin system links the light-dependent reactions of photosynthesis to the regulation of Calvin-Benson cycle enzymes. Light transfers electrons from water to ferredoxin. This reduced ferredoxin is then used to reduce ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin (Trx). The reduced thioredoxin activates target enzymes by reducing their regulatory disulfide bonds.

This system regulates the activity of several enzymes, including:

• Fructose 1,6-bisphosphatase

• Sedoheptulose 1,7-bisphosphatase

• Phosphoribulokinase

• NADP–glyceraldehyde-3-phosphate dehydrogenase

When it's dark, electron flow from ferredoxin stops, and thioredoxin becomes oxidized, ultimately inactivating the target enzymes.

Regulation via ferredoxin-thioredoxin system

Light-Dependent Ion Movements and Enzyme Activity

Light also regulates Calvin-Benson cycle enzymes through the movement of ions. When illuminated, protons flow from the stroma into the thylakoid lumen, while Mg2+ is released from the thylakoid space into the stroma. This increases the stromal pH and Mg2+ concentration, which activates enzymes like Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and phosphoribulokinase. These ionic changes are reversed in the dark, deactivating the enzymes.

Regulation of Chloroplast Enzymes via Supramolecular Complexes

Another mechanism for light-dependent regulation of the Calvin cycle involves the formation of supramolecular complexes. This process primarily centers on a small regulatory protein called CP12 and involves regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK).

A2B2 and A4 Forms of GAPDH

The regulation of GAPDH is complex, involving two major isoforms in higher plants: 

• the heterotetramer A2B2
• the homotetramer A4

Both are assembled from different subunits and play distinct roles in the light-dark regulation of photosynthesis. 

A4 Homotetramer

• Composition: The A4 form consists of four identical GapA subunits. It is the catalytically active homotetramer.

• Regulation: The A4 homotetramer is regulated by its interaction with the small protein CP12.

A2B2 Heterotetramer

• Composition: The A2B2 form is made of two GapA subunits and two GapB subunits. This is the most abundant and active form of GAPDH in land plants.

• Regulation: Its regulation is largely autonomous, primarily controlled by the C-terminal extension (CTE) of the GapB subunit. 

A4-GAPDH Regulation

The CP12-Mediated Complex:

In the dark, when there is no need for active carbon fixation, the regulatory protein CP12, which is oxidized by the absence of light, binds to two key Calvin cycle enzymes:

• A4 isoform of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

• Phosphoribulokinase (PRK)

This binding forms a ternary supramolecular complex (GAPDH/CP12/PRK). The formation of this complex effectively sequesters and inactivates both GAPDH and PRK, switching off the Calvin-Benson cycle and preventing the wasteful consumption of ATP and NADPH that would otherwise occur in the dark.

Dissociation in the Light:

When light becomes available, the thylakoid electron transport chain produces reducing power in the form of NADPH. This is used to reduce ferredoxin, which then reduces thioredoxin. The reduced thioredoxin acts on the CP12-mediated complex.

Reduced thioredoxin cleaves the disulfide bonds within the oxidized CP12. This reduction of CP12 causes the entire supramolecular complex to dissociate. The dissociation releases the A4 isoform of GAPDH and PRK, which become catalytically active.

This mechanism ensures that the Calvin cycle is tightly coupled to the availability of light, providing a quick and efficient way to regulate carbon fixation in response to light-dark transitions.

Regulation by CP12-Mediated Complex (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

A2B2-GAPDH Regulation

The A2B2 isoform is a heterotetramer made of A and B polypeptides. The B subunit contains a C-terminal extension with two cysteines that can form a disulfide bridge. The A2B2 form can also assemble into a larger, inactive A8B8 oligomer.

• In Darkness: The A2B2-GAPDH forms the inactive A8B8 oligomer.

• In Light: Reduced thioredoxin (Trx) cleaves the disulfide bond in the B subunit of the A8B8 oligomer. This reduction converts the inactive oligomer into the active A2B2-GAPDH. This autonomous redox-modulation is particularly suited for conditions of very active photosynthesis.

Regulation by C-terminal extension (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

GAPDH Isoform	Composition	Regulation Mechanism	Active State	Inactive State
A4	A4 homotetramer	Forms a ternary complex with CP12 and PRK in the dark.	Free, active enzymes.	Inactive ternary complex.
A2B2	A2B2 heterotetramer	Forms an A8B8 oligomer in the dark; regulated by disulfide bond in subunit B.	Active A2B2 tetramer.	Inactive A8B8 oligomer.

Regulation of C4 Enzymes

In C4 plants, light also regulates key enzymes through thiol-disulfide exchange and phosphorylation-dephosphorylation.

• NADP–malate dehydrogenase (MDH) is activated in the light by the ferredoxin-thioredoxin system (reduced by thioredoxin) and deactivated in the dark (oxidized).

Regulation of MDH (Carr et al. 1999, Cell)

• Phosphoenolpyruvate (PEP) carboxylase is regulated by diurnal phosphorylation. Phosphorylation makes it active at night and insensitive to inhibition by malate. Dephosphorylation by a protein phosphatase in the day brings the enzyme to low activity.

Regulation of PEP

• Pyruvate–phosphate dikinase is regulated by a bifunctional kinase-phosphatase enzyme. In the dark, ADP-dependent phosphorylation deactivates the enzyme, while in the light, Pi-dependent dephosphorylation restores its activity.