During photosynthesis, plants incorporate atmospheric CO₂ into carbohydrates using the energy of sunlight. CO₂ enters the leaf through pore-like openings located predominately in the lower epidermis of the leaf called stomata. The sizes of the stomata are directly controlled through regulating the turgor pressure of the surrounding cells, known as guard cells. By inflating and deflating the guard cells, the plant can optimize the balance between CO₂ uptake and water loss in real time as environmental conditions change.

Stomatal Opening

Stomata open when the pair of guard cells is fully turgid and pressing firmly against each other. Although this seems counterintuitive, the pressurized condition results in open stomata because of the unique anatomy of these specialized cells. In particular, the interior walls of the guard cells are heavily reinforced with cellulose, making them bow open when stressed by the influx of water. But what causes this influx of water? As usual, water flows in response to the water potential gradient.

In sunny conditions, blue light stimulates a H+-ATPase in the guard cell plasma membrane, which hydrolyzes ATP and pumps H+ out of the cell. The outward flow of H+ results in the inside of the cell becoming more and more negatively charged. This hyperpolarization leads to the activation of an inward-rectifying K+ channel, resulting in the influx of K+ ions. This influx of K+ activates a H+/Cl- symporter, which imports Cl- ions. The net uptake of both K+ and Cl- significantly reduces the water potential, resulting in the influx of water from the apoplast.

Stomatal Closing

Stomata close in response to a number of different signals. One such signal is water stress, usually signaled through the presence of the drought hormone, abscisic acid (ABA). The perception of ABA in the guard cell leads to the release of intracellular Ca++, which triggers the opening of a plasma membrane anion channel. This allows the efflux of Cl- ions, leading to the depolarization of the membrane. Upon membrane depolarization, an outward-rectifying K+ channel opens and allows the escape of K+. The loss of both K+ and Cl- increases the water potential of the guard cell, leading to water efflux.

One important question is, where are the large supplies of ions and water coming from that lead to stomatal opening, and where do these go when stomata close? Guard cells do not exist in isolation, but are found situated in the epidermis next to companion cells. As the concentration of ions is increasing in guard cells, those ions are being supplied by companion cells. Likewise, the ions that flow out of guard cells undergoing stomatal closing are taken up by the companion cells.

The major export product from photosynthesis is glyceraldehyde-3-phosphate (G3P), a triose phosphate carbohydrate, which can enter either the starch or sucrose biosynthesis pathway depending on conditions in the cell. During the daytime, much of the carbon that is fixed by photosynthesis remains in the chloroplast and enters the starch biosynthesis pathway. At night, carbon stored in the form of starch is mobilized by conversion to sucrose, which is synthesized in the cytoplasm.

Starch Biosynthesis

Starch, formally known as α-amylose, is a long-chain polysaccharide made of α 1→4 linked glucose, where the chain length numbers in the hundreds or thousands. α-amylose forms a single helix structure because of its regular repeating pattern, and this secondary structure readily crystallizes. The first step in the synthesis of α-amylose is the formation of hexose phosphates, including fructose 6-phosphate, glucose 6-phosphate, and glucose 1-phosphate. Glucose 1-phosphate is further ‘activated’ by reacting with the sugar nucleoside ATP to produce ADP-glucose. This form of glucose is highly reactive and readily joins an elongating chain of α-amylose at the 4-carbon position to give the characteristic α 1→4 linkage.

While α-amylose represents about 30% of the total starch in most plants, the rest of the starch is in a highly branched form called amylopectin. Rather than forming straight chain helices that readily crystallize, amylopectin does not crystallize. Amylopectins are formed by starch branching enzymes that form branches among short α-amylose chains that are α 1→6 glycosidic bonds.

Sucrose Biosynthesis

When triose phosphates are exported from the chloroplast, they enter the sucrose biosynthetic pathway in a similar manner as the start of the starch pathway — by condensation to form a pool of hexose phosphates. Also like starch biosynthesis, glucose 1-phosphate reacts with a sugar nucleoside, in this case UTP instead of ATP, to form UDP-glucose. Sucrose is the result of the condensation reaction between this UDP-glucose and fructose 6-phosphate. This sucrose serves as the major form of transportable carbohydrate within the plant.

Regulation

What determines whether the triose phosphates formed by photosynthesis enter the starch or sucrose pathways is the activity of a chloroplast envelope transporter. This transporter, called the triose phosphate-Pi antiporter, exchanges triose phosphates for Pi (inorganic phosphate) between the stroma and cytoplasm. When concentrations of Pi are high in the cyctoplasm, the antiporter is activated and exports triose phosphates in exchange for the uptake of Pi, with the cytoplasmic triose phosphate entering sucrose synthesis. On the other hand, when cytoplasmic Pi is low, no exchange happens and triose phosphates remain in the chloroplast to enter the starch synthesis pathway.

Despite some apparent benefits of photorespiration, several taxa of plants have evolved strategies that minimize photorespiration. These additional pathways for carbon fixation evolved in plants growing under high light conditions with limited water resources and took advantage of proteins from other primary metabolic processes, co-opting them for a new purpose. These pathways exist in addition to the process of photosynthetic carbon reduction already described, serving to concentrate CO₂ for incorporation by Rubisco rather than operating instead of Rubsico.

C4 Pathway

One additional pathway that concentrates CO₂ for Rubisco is known as the C4 pathway, and it is found in some members of the grass (Poaceae) family such as maize, sorghum, and sugar cane. These grass species display a specialized anatomical adaptation consisting of a ring of cells surrounding leaf vascular bundles called the bundle-sheath cells. The expression of Rubisco and other genes encoding enzymes of the Calvin-Benson pathway are limited to the bundle-sheath cells, resulting in the spatial separation of initial carbon fixation and Rubisco activity. The initial fixation of CO₂ happens in the mesophyll cells of the leaf, and is carried out by an enzyme usually associated with glycolysis called phosphoenolpyruvate (PEP) carboxylase. Following carbon fixation, the resulting 4-carbon molecule (malate) is transported into a bundle-sheath cell and decarboxylated, giving off a CO₂ that will be incorporated by Rubisco into the Calvin-Benson cycle.

The formation of a gradient never occurs without the input of energy, and this process of concentrating CO₂ in the bundle-sheath is no exception. C4 plants invest heavily in the form of ATP and NADPH to sustain the gradient, but because this pathway is found in plants in high light environments, this cost is easily absorbed. Ha.

CAM Pathway

Unlike C4 plants, CAM plants do not have any particular anatomical adaptations related to carbon fixation. CAM plants employ the same alternate enzymes as the C4 plants for initial carbon fixation, but place them under the control of circadian regulation, restricting carbon fixation to the night, when stomata are open. This arrangement allows the plant to exchange gas with the environment when temperatures are much lower, resulting in significant water savings. As CO₂ is fixed by PEP carboxylase, the product (malate) is imported to the vacuole and stored as malic acid. During the daytime, when stomata are closed, this malate is exported from the vacuole and decarboxylated, increasing CO₂ concentrations and favoring its use over O₂ by Rubisco.

Regulation

PEP carboxylase activity is regulated by two main mechanisms. High concentrations of malate cause a form of enzyme inhibition known as feedback inhibition, in which the product of a pathway exerts an influence over an enzyme upstream of its production. Levels of malate are kept low in the vicinity of PEP carboxylase by exporting it to the bundle-sheath cells in the case of C4 pathway plants or importing it into the vacuole in the case of CAM plants. Another means of regulation over these alternative carbon fixation pathways involves the phosphorylation of PEP carboxylase, which occurs in the light. Upon phosphorylation, the affinity of PEP carboxylase for the substrate phosphoenolpyruvate increases. The affinity of an enzyme for its substrate, known as the Michaelis constant (K_m), can be empirically determined in a carefully-controlled in vitro experiment. A decreasing K_m is indicative of greater substrate binding at a lower substrate concentration. Conversely, the phosphorylation of PEP carboxylase causes an increase in the inhibition constant (K_i) for malate, meaning that the same amount of malate would inhibit the enzyme less under phosphorylated conditions.

The enzyme responsible for incorporating atmospheric CO₂ in the reactions of photosynthesis is called Rubisco, an ancient, large, multi-subunit enzyme with a major apparent weakness. In addition to incorporating CO₂, Rubisco can also bind O₂ and perform an oxygenation reaction with the substrate ribulose bis-phosphate. The results of this oxygenation reaction are one molecule of phopshoglycerate and one molecule of a 2-carbon product called phosphoglycolate. While the PGA can enter the reduction pathway in the chloroplast, the phosphoglycolate cannot and instead enters a recovery pathway spread across two other organelles, the peroxisome and mitochondria. The oxygenation reaction together with the various recovery steps are known as the photorespiratory pathway and represent a significant energetic cost to the plant.

There are probably many explanations for the existence of this costly pathway, beginning with a consideration of the conditions under which Rubisco evolved. Photosynthetic carbon reduction probably began over 1 billion years ago, and is itself thought to have been responsible for the formation of most of the O₂ in the atmosphere. In other words, in the earliest cases, there was little to no competition between CO₂ and O₂ for the active site of Rubisco. So the simplest explanation for why oxygenation happens is that there was not a selective pressure to discriminate between these two substrates. Another explanation is that, given the integration of the recovery pathway with various metabolic pathways such as amino acid biosynthesis, it represents a source of inputs to these pathways. A third explanation is that photorespiration is a strategy for the plant to cope with excess light energy under conditions in which CO₂ is limiting. Such conditions arise when the plant is water-stressed and closes stomata to conserve water, which results in a build-up of O₂ and draw-down of CO₂. Evidence for photorespiration as an important energy-shunting pathway comes from experiments in which mutants for various steps in the recovery pathway were isolated and grown under various conditions. Mutants grow normally under low-light conditions, but when challenged with light levels similar to full sun the mutants show symptoms consistent with damage due to photoinhibition.

Below is a flow diagram of the Calvin cycle, found in the chloroplasts of photosynthetic organisms. Each substrate and enzyme is clickable, and leads to a site with detailed information on the participant. A PDF of this flowchart is also available for printing.