Dark Reactions and Photorespiration

Dark phase : The pathway by which all photosynthetic eukaryotic organisms ultimately incorporate CO₂ into carbohydrate is known as carbon fixation or photosynthetic carbon reduction (PCR) cycle or dark reactions.

The dark reactions are sensitive to temperature changes, but are independent of light hence it is called dark reaction, however it depends upon the products of light reaction of photosynthesis, i.e., NADP .2H and ATP.

The carbon dioxide fixation takes place in the stroma of chloroplasts because it has enzymes essential for fixation of CO₂ and synthesis of sugar.

The techniques used for studying different steps were Radioactive tracer technique using ¹⁴C (Half life - 5720 years), Chromatography and Autoradiography and the material used was Chlorella (Cloacal alga) and Scenedesmus (these are microscopic, unicellular algae and can be easily maintained in laboratory).

The assimilation and reduction of CO₂ takes place in this reaction by which carbohydrate is synthesized through following three pathways :

(1) Calvin cycle : Calvin and Benson discovered the path of carbon in this process. This is known as C₃ cycle because CO₂ reduction is cyclic process and first stable product in this cycle is a 3-C compound (i.e., 3-Phosphoglyceric acid or 3-PGA).

Calvin cycle is divided into three distinct phases : Carboxylation, Glycolytic reversal, regeneration of RuBP.

In this cycle, CO₂ acceptor molecule is RuBP or RuDP (i.e., Ribulose 1, 5-biphosphate or Ribulose 1, 5-diphosphate). There occurs covalent bonding of CO₂ to RuBP and the enzyme catalyzing this reaction is RuBP-carboxylase/oxygenase (Rubisco).

As calvin cycle takes in only one carbon (as CO₂) at a time, so it takes six turns of the cycle to produce a net gain of six carbons (i.e., hexose or glucose).

In this cycle, for formation of one mole of hexose sugar (Glucose), 18 ATP and 12 NADPH₂ are used.

The plants in which this pathway of CO₂ reduction occurs, are called C-3 plants.

About 85% of plant species are C-3 plants, including cereals (e.g., barley, rice, oat, wheat), groundnut, sugarbeet, cotton, tobacco, spinach, soybean most trees and lawn grasses etc.

(2) Hatch and Slack cycle (C₄ cycle) : Kortschak and Hart supplied CO₂ to the leaves of sugarcane, they found that the first stable product is a four carbon (C₄) compound oxalo acetic acid instead of 3-carbon atom compound. The detailed study of this cycle has introduced by M.D. Hatch and C.R. Slack (1966). So it is called as “Hatch and Slack cycle”. The stable product in C₄ plant is a dicarboxylic substance. Hence it is called dicarboxylic acid cycle or DCA-cycle. C₄ plants are true xerophytic plants.

The important C₄ plants are sugarcane, maize, Sorghum, Cyperus rotundus, Digitaria brownii, Amaranthus, etc. These plants have “Kranz” (German term meaning halo or wreath) type of leaf anatomy. The vascular bundles, in C₄ leaves are surrounded by a layer of bundle sheath cells that contain large number of chloroplasts. The chloroplasts in C₄ leaves are dimorphic (Two morphologically distinct types). The chloroplasts of bundle sheath cells are larger in size and arranged centripetally. They contain starch grains but lack grana. The mesophyll cells, on the other hand, contain normal types of chloroplasts. Mesophyll and bundle sheath cells are connected by plasmodesmata. The mesophyll cells perform C₄ cycle and the cells of bundle sheath perform C₃ cycle.

CO₂ taken from the atmosphere is accepted by phosphoenolpyruvic acid (PEP) present in the chloroplasts of mesophyll cells of these leaves, leading to the formation of a 4-C compound, oxaloacetic acid (OAA). This acid is converted to another 4-C acid, the malic acid which enters into the chloroplasts of bundle sheath cells and there undergoes oxidative decarboxylation yielding pyruvic acid (a 3-C compound) and CO₂. CO₂ released in bundle sheath cells reacts with Ribulose-1,5-biphosphate (RuBP) already present in the chloroplasts of bundle sheath cells and thus Calvin cycle starts from here. Pyruvic acid re-enters mesophyll cells and regenerates phosphoenol pyruvic acid. CO₂ after reacting with RuBP gives rise to sugars and other carbohydrates. In C₄ plants, there are 2 carboxylation reactions, first in mesophyll chloroplast and second in bundle sheath chloroplast.

C₄ plants are better photosynthesizers. There is no photorespiration in these plants. In C₄ plants, for formation of one molecule of hexose (glucose) 30 ATP and 12 NADPH₂ are required.

Characteristics of C₄ cycle

(1) C₄ species have greater rate of CO₂ assimilation than C₃ species. This is on account of the fact that

(i) PEP carboxylase has great affinity for CO₂.

(ii) C₄ plants show little photorespiration as compared to C₃ plants, resulting in higher production of dry matter.

(2) C₄ plants are more adapted to environmental stresses than C₃ plants.

(3) CO₂ fixation by C₄ plants requires more ATP than that by C₃ plants. This additional ATP is needed for conversion of pyruvic acid to phosphoenol pyruvic acid and its transport.

(4) CO₂ acceptor molecule in C₄ plants is PEP. Further, PEP-carboxylase (PEPCO) is the key enzyme (RuBP-carboxylase enzyme is negligible or absent in mesophyll chloroplast, but is present in bundle sheath chloroplast).

(3) Crassulacean acid metabolism (CAM) : This dark CO₂ fixation pathway proposed by Ting (1971). It operates in succulent or fleshy plants e.g., Cactus, Sedum, Kalanchoe, Opuntia, Agave, Orchid, Pineapple and Bryophyllum helping them to continue photosynthesis under extremely dry condition.

The stomata of succulent plants remain closed during day and open during night to avoid water loss (Scotactive stomata). They store CO₂ during night in the form of malic acid in presence of enzyme PEP carboxylase. The CO₂ stored during night is used in Calvin cycle during day time. Succulents refix CO₂ released during respiration and use it during photosynthesis.

This diurnal change in acidity was first discovered in crassulacean plants e.g., Bryophyllum. So it is called as crassulacean acid metabolism.

Formation of malic acid during dark is called acidification or phase-I. Release of $C{O}_2$ for actual photosynthesis during day is called deacidification or phase-II.

Characteristics of CAM pathway

(1) There is decrease in pH during the night and increase in pH during the day.

(2) CAM plants have enzymes of both C₃ and C₄ cycle in mesophyll cells. This metabolism enable CAM plants to survive under xeric habitats. These plants have also the capability of fixing the CO₂ lost in respiration.

(3) Malic acid is stored in the vacuoles during the night which is decarboxylated to release $C{O}_2$ during the day.

Photorespiration

Decker and Tio (1959) reported that light induces oxidation of photosynthetic intermediates with the help of oxygen in tobacco. It is called as photorespiration. The photorespiration is defined by Krotkov (1963) as an extra input of O₂ and extra release of CO₂ by green plants is light.

Photorespiration is the uptake of O₂ and release of CO₂ in light and results from the biosynthesis of glycolate in chloroplasts and subsequent metabolism of glycolate acid in the same leaf cell. Biochemical mechanism for photorespiration is also called glycolate metabolism.

Loss of energy occurs during this process. The process of photorespiration involves the involvement of chloroplasts, peroxisomes and mitochondria. RuBP carboxylase also catalyses another reaction which interferes with the successful functioning of Calvin cycle.

Biochemical mechanism

(1) Ribulose-1, 5-biphosphate $\stackrel{O_2}{\to }$ 2 Phosphoglycolic acid +3 Phosphoglyceric acid

(2) 2 Phosphoglycolic acid + H₂O $\stackrel{\text{Phosphatase}}{\to }$ Glycolic acid + Phosphoric acid.

(3) Glycolic acid + O₂ $\underset{\text{Oxidase}}{\stackrel{\text{Glycolic}\text{acid}}{\to }}$ Glyoxylic acid + H₂O₂$2H_2{O}_2\stackrel{\text{Catalase}}{\to }2H_{2}O+O_2$

(4) Glyoxylic acid + Glutamic acid $\underset{\text{transaminase}}{\stackrel{\text{Glutamate}-\text{glyoxylate}}{\to }}$ Glycine + a-keto glutaric acid

(5) 2 Glycine + H₂O + NAD⁺ $\to$ Serine + CO₂ + NH₃ + NADH

(6) Serine + Glyoxylic acid $\to$ Hydroxypyruvic acid + Glycine Hydroxypyruvic acid $\to$ Glyceric acid

(7) Glyceric acid + ATP $\to$ 3 phosphoglyceric acid + ADP + phosphate

Importance of photorespiration : Photorespiration is quite different from respiration as no ATP or NADH are produced. Moreover, the process is harmful to plants because as much as half the photosynthetically fixed carbon dioxide (in the form of RuBP) may be lost into the atmosphere through this process.

Any increase in O₂ concentration would favour the uptake of O₂ rather than CO₂ and thus, inhibit photosynthesis for this rubisco functions as RuBP oxygenase. Photorespiration is closely related to CO₂ compensation point and occurs only in those plants which have high CO₂ compensation point such as C₃ plants.

Photorespiration generally occurs in temperate plants. Few photorespiring plants are : Rice, bean, wheat, barley etc. Inhibitors of glycolic acid oxidase such as hydroxy sulphonates inhibit the process of photorespiration. Unlike usual mitochondria respiration neither reduced coenzymes are generated in photorespiration nor the oxidation of glycolate is coupled with the formation of ATP molecules. Photorespiration (C₂ cycle) is enhanced by bright light, high temperature, high oxygen and low CO₂ concentration.