Biology: Photosynthesis and Stomatal Opening

Structure of Stoma and Mechanism of Stomatal Opening and Closing

A stoma is a minute pore on the epidermis of aerial parts of plants through which exchange of gases and transpiration takes place. Each stoma is surrounded by a pair of kidney shaped guard cells.

Structure of Stoma

A stoma is a minute pore on the epidermis of aerial parts of plants through which exchange of gases and transpiration takes place.

Each stoma is surrounded by a pair of kidney shaped guard cells. Each guard cell is a modified epidermal cell showing a prominent nucleus, cytoplasm and plastids. The wall of the guard cell is differentially thickened. The inner wall of each guard cell facing the stoma is concave and is thick and rigid. The outer wall is convex and is thin and elastic.

The guard cells are surrounded by a variable number of epidermal cells called subsidiary cells.

Mechanism of Stomatal Opening and Closing

Opening and closing of stomata takes place due to changes in turgor of guard cells. Generally stomata are open during the day and close at night.

The turgor changes in the guard cells are due to entry and exit of water into and out of the guard cells. During the day, water from subsidiary cells enters the guard cells making the guard cells fully turgid. As a result, the thin elastic convex outer walls are bulged out causing the thick and rigid concave inner walls to curve away from each other causing the stoma to open.

During night time, water from guard cells enters the subsidiary cells and as a result, the guard cells become flaccid due to decrease in turgor pressure. This causes the inner concave walls to straighten up and the stoma closes.

The actual mechanism responsible for entry and exit of water to and from the guard cells has been explained by several theories.

The Starch - Sugar interconversion Theory

Steward (1964) holds that during the day the enzyme phosphorylase converts starch to sugar, thus increasing osmotic potential of guard cells causing entry of water. The reverse reaction occurs at night bringing about closure.

ii. Proton(H+) - Potassium Pump Hypothesis

Levit in 1974 combined the points in Scarth's and Steward's hypothesis and gave a modified version of the mechanism of stomatal movement which was called the proton - potassium pump hypothesis.

According to this hypothesis K+ ions are transported into the guard cells in the presence of light. The sequence of events taking place is

i. Under the influence of light, protons formed by dissociation of malic acid move from cytoplasm in to the chloroplasts of guard cells.

ii. To counter the exit of protons, K+ ions enter the guard cells from the surrounding mesophyll cells.

iii. K+ ions react with the malate ions present in the guard cells to form potassium malate.

iv. Potassium malate causes increase in the osmotic potential of guard cells causing entry of water into the guard cells as a result of which the stoma opens.

v. At night the dissociation of potassium malate takes place and K+ ions exit out of guard cells causing loss of water from guard cells and so the stoma closes

This theory is the widely accepted one as Levitt was able to demonstrate rise in K+ ion level during the day and the formation of organic acids like malic acid with the unused CO2 present in the guard cells.

Factors Affecting Stomatal Movement

There are a number of factors which influence stomatal movements. These include light, temperature, potassium chloride, organic acid, carbondioxide concentration, water and abscissic acid.

Light:Light greatly influences the opening and closing of stomata as it stimulates production of malic acid due to conversion of starch to sugar. Stomata do not open in U-V light and green light but remain opened in the blue and red regions of the spectrum.

Temperature-Stomata open with rise in temperature and close at lower temperature as light and temperature are directly related. But higher temperatures also cause stomatal closure.

Potassium Chloride-Accumulation of potassium chloride causes opening of stomata.

Organic Acid-The increase of organic acid content in the guard cells causes the stomata to open.

Carbondioxide Concentration-Stomatal movement is influenced by the concentration of carbondioxide. At low concentrations of CO₂, the stomata open. With increase in the concentration of CO₂, the stomata begin to close and when CO₂ concentration of cells is higher than its concentration in the air, the stomata completely close.

Stomatal movement is always influenced by the CO₂ concentration of the intercellular spaces of the leaf and not the concentration of the air.

Water-Water is responsible for causing changes in the turgor of the guard cells. Guard cells become flaccid on losing water and so the stomata close. Similarly the guard cells become fully turgid on gaining water and the stomata open. Under conditions of water scarcity also, the stomata close.

Abscissic Acid-Abscissic acid accumulates in the leaves when the plants experience water stress or water deficit. It has been observed, that ABA (Abscissic acid) stimulates closure of stomata under these conditions.

IV. Light Absorption in Chloroplasts

A. Chloroplasts in plant & algal cells absorb light energy from the sun during the light dependent reactions

B. Photosynthetic cells may have thousands of chloroplasts

C. Chloroplasts are double membrane organelles with the an inner membrane folded into disc-shaped sacs called thylakoids

D. Thylakoids, containing chlorophyll and other accessory pigments, are in stacks called granum (grana, plural)

E. Grana are connected to each other & surrounded by a gel-like material called stroma

F. Light-capturing pigments in the grana are organized into photosystems

Thylakoids contain a variety of pigments ( green red, orange, yellow…)
Chlorophyll (C55H70MgN4O6) is the most common pigment in plants & algae
Chlorophyll a & chlorophyll b are the 2 most common types of chlorophyll in autotrophs
Chlorophyll absorbs only red, blue, & violet light
Chlorophyll b absorbs colors or light energy NOT absorbed by chlorophyll a
The light energy absorbed by chlorophyll b is transferred to chlorophyll a in the light reactions

Carotenoids are accessory pigments in the thylakoids & include yellow, orange, & red

Each turn of the Calvin cycle fixes One CO2 molecule so it takes six turns to make one molecule of glucose

IX. Photosystems & Electron Transport Chain

Only 1 in 250 chlorophyll molecules (chlorophyll a) actually converts light energy into usable energy
These molecules are called reaction-center chlorophyll
The other molecules (chlorophyll b, c, & d and carotenoids) absorb light energy and deliver it to the reaction-center molecule
These chlorophyll molecules are known as antenna pigments
A unit of several hundred antenna pigment molecules plus a reaction center is called a photosynthetic unit or photosystem
There are 2 types of photosystems — Photosystem I & Photosystem II
Light is absorbed by the antenna pigments of photosystems II and I
The absorbed energy is transferred to the reaction center pigment, P680 in photosystem II, P700 in photosystem I
P680 in Photosystem II loses an electron and becomes positively charged so it can now split water & release electrons (2H2O 4H+ + 4e- + O2)
Electrons from water are transferred to the cytochrome complex of Photosystem I
These excited electrons activate P700 in photosystem I which helps reduce NADP+ to NADPH
NADPH is used in the Calvin cycle
Electrons from Photosystem II replace the electrons that leave chlorophyll molecules in Photosystem I

PHOTOSYNTHESIS IN HIGHER PLANTS

· Photosynthesis: Photosynthesis is an enzyme regulated anabolic process of manufacture of organic compounds inside the chlorophyll containing cells from carbon dioxide and water with the help of sunlight as a source of energy.

Site for photosynthesis:

· Photosynthesis takes place only in green parts of the plant, mostly in leaves.

· Within a leaf, photosynthesis occurs in mesophyll cells which contain the chloroplasts.

· Chloroplasts are the actual sites for photosynthesis.

· The thylakoids in chloroplast contain most of pigments required for capturing solar energy to initiate photosynthesis.

· The membrane system (grana) is responsible for trapping the light energy and for the synthesis of ATP and NADPH. Biosynthetic phase (dark reaction) is carried in stroma.

Pigments involved in photosynthesis:

· Chlorophyll a : (Bright or blue green in chromatograph). Major pigment, act as reaction centre, involved in trapping and converting light into chemical energy.

· Chlorophyll b : (Yellow green)

· Xanthophylls : (Yellow)

· Carotenoid : (Yellow to yellow-orange)

· In the blue and red regions of spectrum shows higher rate of photosynthesis.

What is light reaction?

· Light reactions or the ‘Photochemical ‘phase includes light absorption, splitting of water, evolution of oxygen and formation of high energy compound like ATP and NADPH.

· Light Harvesting Complexes (LHC) : The light harvesting complexes are made up of hundreds of pigment molecules bound to protein within the photosystem I (PSI) and photosystem II (PSII).

· Each photosystem has all the pigments except one molecule of chlorophyll ‘a’ forming a light harvesting system (antennae).

· The reaction centre (chlorophyll a) is different in both the photosystems.

· Photosystem I (PSI) : Chlorophyll ‘a’ has an absorption peak at 700 nm (P700).

· Photosystem II (PSII) : Chlorophyll ‘a’ has absorption peak at 680 nm (P680).

Process of photosynthesis :

· It includes two phases - Photochemical phase and biosynthetic phase.

· Photochemical phase (Light reaction) : This phase includes - light absorption, splitting of water, oxygen release and formation of ATP and NADPH.

· Biosynthetic phase (Dark reaction) : It is light independent phase, synthesis of food material (sugars).

Photolysis of water :

· PS-II loose electrons continuously, filled up by electrons released due to photolysis of water.

· Water is split into H⁺, (O) and electrons in presence of light and Mn²⁺ and Cl^-.

· This also creates O₂ the bi-product of photosynthesis.

· Photolysis takes place in the vicinity of the PS-II.

· 2H₂O → 4H⁺ + O₂ + 4e^-.

Photophosphorylation :

· The process of formation of high-energy chemicals (ATP and NADPH).

Non Cyclic photophosphorylation :

· Two photosystems work in series – First PSII and then PSI.

· These two photosystems are connected through an electron transport chain (Z. Scheme).

The electron transport :

· In photosystem centre chlorophyll a absorbs 680 nm wavelength of red light causing electrons to become excited and release two electrons from the atomic nucleus.

· These electrons are accepted by primary electron acceptor i.e. ferredoxin.

· The electron from the ferredoxin passed to electron transport system consisting cytochromes.

· The electron moved in down hill in terms of redox potential by oxidation-reduction reactions.

· Finally the electron reached photosystem-I.

· Simultaneously electron released from photosystem-I is accepted by electron acceptor.

· Electron hole created in PS-I is filled up by the electron from PS-II.

· Electron from PS-I passed down hill and reduce NADP into NADPH⁺ + H⁺.

ATP and NADPH + H⁺ are synthesized by this process. PSI and PSII are found in lamellae of grana, hence this process is carried here.

Cyclic photophosphorylation :

· Only PS-I works, the electron circulates within the photosystem.

· It happens in the stroma lamellae (possible location) because in this region PS-II and NADP reductase enzyme are absent. Hence only ATP molecules are synthesize

Light Reactions: Photosystem I & II

When photosystem II absorbs light, an electron excited to a higher energy level in the reaction center chlorophyll (P680) is captured by the primary electron acceptor. The oxidized chlorophyll is now a very strong oxidizing agent; its electron “hole” must be filled.
An enzyme extracts electrons from water and supplies them to P680, replacing the electrons that the chlorophyll molecule lost when it absorbed light energy. This reaction splits a water molecule into two hydrogen ions and an oxygen atom, which immediately combines with another oxygen atom to form O₂. This splitting of water is responsible for the release of O₂ into the air.
Each photoexcited electron (energized by light) passes from the primary electron acceptor in photosystem II to photosystem I via an electron transport chain. This electron transport chain is very similar to the one in cellular respiration; however, the carrier proteins in the chloroplast ETC are different from those in the mitochondrial ETC.
As electron move down the chain, their exergonic “fall”to a lower energy level is harnessed by the thylakoid membrane to produce ATP (by chemiosmosis). The production of ATP in the chloroplast is called photophosphorylation because the energy harnessed in the process originally came from light. This process of ATP production is called non-cyclic photophosphorylation. The ATP generated in this process will provide the energy for the synthesis of glucose during the Calvin cycle (light independent reactions).
When an electron reaches the “bottom” of the electron transport chain, it fills an electron “hole” in the chlorophyll a molecule in the reaction center of photosystem I (P700). The hole was created when light energy drives an electron from P700 to the primary electron acceptor of photosystem I.
The primary electron acceptor of photosystem I passes the excited electrons to a second electron transport chain which transmits them to an iron-containing protein. An enzyme reaction transfers the electrons from the protein to NADP⁺ that forms NADPH (which has high chemical energy due to the energy of the electrons). NADPH is the reducing agent needed for the synthesis of glucose in the Calvin cycle.

Cyclic vs. Non-cyclic Electron Flow

Under certain conditions, the photoexcited electrons take an alternative path called cyclic electron flow, which uses photosystem I (P700) but not photosystem II (P680). This process produces no NADPH and no O_2,but it does make ATP. This is called cyclic photophosphorylation. The chloroplast shifts to this process when the ATP supply drops and the level of NADPH rises. Often the amount of ATP needed to drive the Calvin cycle exceeds what is produced in non-cyclic photophosphorylation. Without sufficient ATP, the Calvin cycle will slow or even stop. The chloroplast will continue cyclic photophosphorylation until the ATP supply has been replenished. ATP is produced through chemiosmosis in both cyclic and non-cyclic photophosphorylation.

Biosynthetic phase in C3 plants :

· ATP and NADH, the products of light reaction are used in synthesis of food. The first CO₂ fixation product in C3 plant is 3-phosphoglyceric acid or PGA.

· In some other plants the first stable product is an organic acid called oxaloacetic acid a 4-C compound hence is called C4 plants.

The Calvin cycle :

· The CO₂ acceptor molecule is RuBP (Ribulose bisphosphate).

· The cyclic path of sugar formation is called Calvin cycle on the name of Melvin Calvin, the discoverer of this pathway. Calvin cycle proceeds in three stages:

· Carboxylation :

o Carboxylation is the fixation of CO₂ into a stable organic intermediate.

o CO₂ combines with Ribulose 1, 5 bisphosphate to form 3 PGA in the presence of RuBisCo enzyme.

· Reduction :

o These are a series of reactions that lead to the formation of glucose.

o 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO₂ molecule fixed.

o The fixation of six molecules of CO₂ and 6 turns of the cycle are required for the formation of one molecule of glucose.

· Regeneration of RuBP :

o Regeneration of the CO₂ acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted.

o Regeneration steps required one ATP for phosphorylation to form RuBP.

· Hence for every CO₂ molecule entering the Calvin cycle, 3 molecules of ATP and 2 molecules of NADPH are required.

The C₄ pathway :

· Plants that are adapted to dry tropical regions have the C₄ pathway.

· C₄ oxaloacetic acid is the first CO₂ fixation product.

· These plants have special type of leaf anatomy, they tolerate higher temperatures.

· The leaf has two types of cells: mesophyll cells and Bundle sheath cells (Kranz anatomy).

· Initially CO₂ is taken up by phosphoenol pyruvate (PEP) in mesophyll cells and changed to oxaloacetic acid (OAA) in the presence of PEP carboxylase.

· Oxaloacetate is reduced to malate/asparate that reaches into bundle sheath cells.

· In the bundle sheath cells these C₄ acids are broken down to release CO₂ and a 3-carbon molecule i.e. pyruvic acid.

· The CO₂ released in the bundle sheath cell enters the C3 cycle because these cells are rich in enzyme Ribulose bisphosphate carboxylase-oxygenase (RuBisCO).

· The pyruvate formed in the bundle sheath cell transported back to the mesophyll cell, get phosphorylated to form phosphoenol pyruvate.

Photorespiration: It has been observed that a high concentration of oxygen, inhibits photosynthesis. This is due to the reason that the CO₂ fixing enzyme RUBP carboxylase not only accepts CO₂ but can also combine with O₂. Since the reaction is an oxygenation reaction, the same enzyme is called RUBP oxygenase.

· In C3 plants some O₂ binds with RuBisCo and hence CO₂ fixation is decreased.

· In this process RuBP instead of being converted to 2 molecules of PGA, binds with O₂ to form one molecule of PGA and phosphoglycolate.

· In the photorespiratory pathway there is neither synthesis of sugar, nor of ATP. Rather it results in the release of CO₂ with utilization of ATP.

· In the photorespiratory pathway there is no synthesis of ATP or NADPH.

· Therefore photorespiration is a wasteful process.

In the chloroplast, phosphoglycolate is unstable and is converted to glycolate. PGA is used up in the calvin cycle. The peroxisomes present in the cell convert the glycolate into glyoxylate and then into glycine. Glycine is converted to serine and CO₂ in the mitochondria. The serine thus formed is converted to glycerate, through a series of reactions which occurs in the peroxisome.This process is known as photorespiration
Photorespiration is defined as a light dependent uptake of O₂ and output of CO₂. In C₄ plant photorespiration does not occur:

o RuBisCO enzyme is present in the bundle sheath cells.

o Primary carboxylation is takes place in the mesophyll cell by PEP carboxylase.

o CO₂ supplied to bundle sheath cell by C₄ acid intermediate.

o Hence C₄ plants are photosynthetically more efficient than C3 plant.

C3, C4 and CAM Plants

C3, C4 and CAM are the three different processes that plants use to fix carbon during the process of photosynthesis. Fixing carbon is the way plants remove the carbon from atmospheric carbon dioxide and turn it into organic molecules like carbohydrates.

C3 Plants

The C3 pathway gets its name from the first molecule produced in the cycle (a 3-carbon molecule) called 3-phosphoglyceric acid. About 85% of the plants on Earth use the C3 pathway to fix carbon via the Calvin Cycle. During the one-step process, the enzyme RuBisCO (ribulose bisphosphate carboxylase/oxygenase) causes an oxidation reaction in which some of the energy used in photosynthesis is lost in a process known as photorespiration. The result is about a 25% reduction in the amount of carbon that is fixed by the plant and released back into the atmosphere as carbon dioxide. The carbon fixation pathways used by C4 and CAM plants have added steps to help concentrate and reduce the loss of carbon during the process. Some common C3 plant species are spinach, peanuts, cotton, wheat, rice, barley and most trees and grasses.

The image above shows the C3 carbon fixation pathway also known as the Calvin Cycle, used my many types of plants.

C4 Plants

The C4 process is also known as the Hatch-Slack pathway and is named for the 4-carbon intermediate molecules that are produced, malic acid or aspartic acid. It wasn’t until the 1960s that scientists discovered the C4 pathway while studying sugar cane. C4 has one step in the pathway before the Calvin Cycle which reduces the amount of carbon that is lost in the overall process. The carbon dioxide that is taken in by the plant is moved to bundle sheath cells by the malic acid or aspartic acid molecules (at this point the molecules are called malate and aspartate). The oxygen content inside bundle sheath cells is very low, so the RuBisCO enzymes are less likely to catalyze oxidation reactions and waste carbon molecules. The malate and aspartate molecules release the carbon dioxide in the chloroplasts of the bundle sheath cells and the Calvin Cycle begins. Bundle sheath cells are part of the Kranz leaf anatomy that is characteristic of C4 plants.

About 3% or 7,600 species of plants use the C4 pathway, about 85% of which are angiosperms (flowering plants). C4 plants include corn, sugar cane, millet, sorghum, pineapple, daisies and cabbage.

The image above shows the C4 carbon fixation pathway.

CAM Plants

Plants that use crassulacean acid metabolism, also known as CAM plants, are succulents that are efficient at storing water due to the dry and arid climates they live in. The word crassulacean comes from the Latin word crassus which means “thick.” There are over 16,000 species of CAM plants on Earth including cacti, sedum, jade, orchids and agave. Succulent plants like cacti have leaves that are thick and full of moisture and can also have a waxy coating to reduce evaporation.

CAM plants keep their stoma close during the day to prevent water loss. Instead, the stoma are opened at night to take in carbon dioxide from the atmosphere. The carbon dioxide is converted to a molecule called malate which is stored until the daylight returns and photosynthesis begins via the Calvin Cycle.

The image above shows the CAM carbon fixation pathway used by plants that live in dry and arid environments.

Questions

Question 1: By looking at a plant externally can you tell whether a plant is C3 or C4? Why and how?
Answer One cannot distinguish whether a plant is C3 or C4 by observing its leaves and other morphological features externally. Unlike C3 plants, the leaves of C4 plants have a special anatomy called Kranz anatomy and this difference can only be observed at the cellular level. For example, although wheat and maize are grasses, wheat is a C3 plant, while maize is a C4 plant.

Question 2: By looking at which internal structure of a plant can you tell whether a plant is C3 or C4? Explain.
Answer The leaves of C4 plants have a special anatomy called Kranz anatomy. This makes them different from C3 plants. Special cells, known as bundle-sheath cells, surround the vascular bundles. These cells have a large number of chloroplasts. They are thick-walled and have no intercellular spaces. They are also impervious to gaseous exchange. All these anatomical features help prevent photorespiration in C4 plants, thereby increasing their ability to photosynthesise.

Question 3: Even though a very few cells in a C4 plant carry out the biosynthetic – Calvin pathway, yet they are highly productive. Can you discuss why?
Answer The productivity of a plant is measured by the rate at which it photosynthesises. The amount of carbon dioxide present in a plant is directly proportional to the rate of photosynthesis. C4 plants have a mechanism for increasing the concentration of carbon dioxide. In C4 plants, the Calvin cycle occurs in the bundle-sheath cells. The C4 compound (malic acid) from the mesophyll cells is broken down in the bundle-
sheath cells. As a result, CO2 is released. The increase in CO2 ensures that the enzyme RuBisCo does not act as an oxygenase, but as a carboxylase. This prevents photorespiration and increases the rate of photosynthesis. Thus, C4 plants are highly productive.

Question 4: RuBisCo is an enzyme that acts both as a carboxylase and oxygenase. Why do you think RuBisCo carries out more carboxylation in C4 plants?
Answer The enzyme RuBisCo is absent from the mesophyll cells of C4 plants. It is present in the bundle-sheath cells surrounding the vascular bundles. In C4 plants, the Calvin cycle occurs in the bundle-sheath cells. The primary CO2 acceptor in the mesophyll cells is phosphoenol pyruvate – a three-carbon compound. It is converted into the four-carbon compound oxaloacetic acid (OAA). OAA is further converted into malic acid. Malic acid is transported to the bundle-sheath cells, where it undergoes decarboxylation and CO2 fixation occurs by the Calvin cycle. This prevents the enzyme RuBisCo from acting as an oxygenase.

Question 5: Suppose there were plants that had a high concentration of Chlorophyll-b, but lacked chlorophyll-a, would it carry out photosynthesis? Then why do plants have chlorophyll-b and other accessory pigments?
Answer Chlorophyll-a molecules act as antenna molecules. They get excited by absorbing light and emit electrons during cyclic and non-cyclic photophosphorylations. They form the reaction centres for both photosystems I and II. Chlorophyll-b and other photosynthetic pigments such as carotenoids and xanthophylls act as accessory pigments. Their role is to absorb energy and transfer it to chlorophyll-a. Carotenoids and xanthophylls also protect the chlorophyll molecule from photo-oxidation. Therefore, chlorophyll-a is essential for photosynthesis.
If any plant were to lack chlorophyll-a and contain a high concentration of chlorophyll-b, then this plant would not undergo photosynthesis.

Question 6: Why is the colour of a leaf kept in the dark frequently yellow, or pale green? Which pigment do you think is more stable?
Answer Since leaves require light to perform photosynthesis, the colour of a leaf kept in the dark changes from a darker to a lighter shade of green. Sometimes, it also turns yellow. The production of the chlorophyll pigment essential for photosynthesis is directly proportional to the amount of light available. In the absence of light, the production of chlorophyll-a molecules stops and they get broken slowly. This changes the colour of the leaf gradually to light green. During this process, the xanthophyll and carotenoid pigments become predominant, causing the leaf to become yellow. These pigments are more stable as light is not essential for their production. They are always present in plants.

Question 7: Look at leaves of the same plant on the shady side and compare it with the leaves on the sunny side. Or, compare the potted plants kept in the sunlight with those in the shade. Which of them has leaves that are darker green? Why?
Answer Light is a limiting factor for photosynthesis. Leaves get lesser light for photosynthesis when they are in shade. Therefore, the leaves or plants in shade perform lesser photosynthesis as compared to the leaves or plants kept in sunlight.
In order to increase the rate of photosynthesis, the leaves present in shade have more chlorophyll pigments. This increase in chlorophyll content increases the amount of light absorbed by the leaves, which in turn increases the rate of photosynthesis. Therefore, the leaves or plants in shade are greener than the leaves or plants kept in the sun.