Content:1.1 Introduction 1.5 Regulation of Activity of Enzyme1.2 Kranz Anatomy 1.6 Significance1.3 Hatch and Slack Pathway 1.7 Conclusion1.4 Energy Requirement 1.8 References

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1.1 Introduction:

Carbon fixation is basically the process by which plants assimilate carbon dioxide in the atmosphere to form metabolically active compounds. At first it was thought that Calvin Cycle or C3 cycle accounted for CO2 assimilation in all plants. In Calvin cycle enzyme ribulose - 1,5 - bis-phosphate carboxylase (RUBISO) to fix CO2 into a three carbon compound 3-phosphoglycerate. Radioactive labeling of 14CO2 experiments revealed that in maize (Zea mays) and sugarcane (Saccharum officinarum) four carbon compounds malate and aspartate were the earliest labeled products (Kortschak, Hartt and Burr, 1965) It was M.D. Hatch and C.R.Slack that proposed a model for the C4 dicarboxylic acid pathway, wherein CO2 is at first fixed to a four carbon compound, decarboxylated and then again re-fixed into a three carbon compound. This lead to an alternative form of CO 2

fixation, which is know as Hatch and Slack Cycle or C4 cycle since a four carbon compound is first stable product. This pathway was first reported in members of the family Graminaeae (grasses) like sugarcane, maize, sorghum, etc. the cycle is now known to occur in 16 families of both monocotyledons and dicotyledons and is particularly prominent is as mentioned earlier; in Gramineae, Chenopodiaceae( Atriplex) and Cyperaceae. Plants that fix carbon through C4 pathway have a peculiar characteristic feature in the anatomy of their leaves. This special character is known as Kranz anatomy. Kranz in German means a halo, ring or wreath. The essential difference between the Calvin cycle and the Hatch and Slack Pathway is that the efficiency of CO2 fixation is improved vastly. C4 pathway is used as a mechanism to concentrate the amount of CO2

by means of the modification in anatomy as well as avoid the counter-react the oxygenase activity of RUBISCO by PEP Carboxylase. Thus make Hatch and Slack pathway a more efficient metabolism as compared to Calvin cycle.

1.2 Kranz Anatomy:

The leaves of C4 plants differ from that of C3 plants because they have specialized structural and biochemical features different from the latter. The leaves of C4 plants are composed of two types of photosynthetic cells, namely the mesophyll cells and the Kranz or bundle sheath cells. Bundle sheath may be single layered as in maize or double layered as in sugarcane. If two layered, the outer one is green and the inner one is thick walled and called mestomal sheath. Here two types of chloroplast are present. The bundle sheath chloroplasts which are larger in size and lack grana and contain starch grains are also known as agranal chloroplasts. Mesophyll chloroplast which are also similar in size but

they contain grana and lack starch grains. These are also known as granal chloroplast. This feature of having two different types of chloroplast is called as Chloroplast Dimorphism. These two chloroplasts show division of labor. No mesophyll cell of a C4

plant is more than two or three cells away from the nearest bundle sheath cell. In addition, an extensive network of plasmodesmata connects mesophyll and bundle sheath cells, thus providing a pathway for the flow of metabolites between the cell types.

1.3 Hatch and Slack Pathway:

The Hatch-Slack cycle or C4 cycle can be simplified into four major steps:a. In the mesophyll region atmospheric CO2 is fixed by carboxylation of

phosphoenol-pyruvate to form a C4 acid usually malate or aspartate.b. Transport o this produced C4 acid into the bundle sheath cells.c. In the bundle sheath cells, C4 acids undergo decarboxylation to generate CO2

which is the reduced to sugar via the Calvin cycle.d. The C3 acid formed as a result of decarboxylation is transported back to the

mesophyll cells and CO2 acceptor phosphoenol-pyruvate is regenerated.

First reaction shows that the three carbon compound of mesophyll cell chloroplast stroma known as Phosphoenol-pyruvate accepts a molecule of CO2 in the form of bicarbonate ions (HCO3

-) in the presence of PEP (phosphoenol-pyruvate) carboxylase to form the first stable four carbon compound which is Oxalo-Acetate

CO2 + Phosphoenol-Pyruvate → Oxaloacetate + H3PO4 + H2O[In presence of PEP Carboxylase]

Then this Oxalo Acetate is converted into Malate by Malate dehydrogenase. Malate dehydrogenase being from the class Oxidoreductase usually requires NADP or NAD+ as a cofactor. In this case malate dehydrogenase utilizes NADP + H-

Oxalocetate + NADPH + H+→ Malate + NADP+

[In presence of malate dehydrogenase]

Malate formed in mesophyll cell is transported to bundle sheath cells through plasmodesmata. This acid then undergoes decarboxylation to Pyruvate which is a three carbon compound catalyzed by malate dehydrogenase and regenerates NADPH + H+ in the previous reaction.

Malate + NADP+→ Pyruvate + NADPH + H+ + CO2

Since it is a decarboxylation reaction, the liberated CO2 is then accepted by RuBP of bundle sheath cell chloroplast stroma to run Calvin cycle during which ATP and NADPH2 are utilized. The pyruvate formed by malate is transported back to mesophyll cell where it is phosphorylated to phosphoenol-pyruvate by pyruvate dikinase which requires utilization of 2 ATP for each molecule conversion.

Pyruvate + 2 ATP + 2 Pi → Phosphoenol-pyruvic acid + 2 AMP + 2 PPi

In this way PEP is regenerated. In some C4 plants aspartate is formed from oxaloacetate, instead of malate by transamination reaction. There are two carboxylation reactions in this pathway. One in mesophyll cells and one in bundle sheath cells, hence it can also be known as Dicarboxylic pathway.

1.4 Energy Requirement:

An interesting feature of the cycle is that regeneration of the primary acceptor – phosphoenol-pyruvate consumes two ‘high energy’ phosphate bonds, one in the reaction catalyzed by pyruvate-orthophosphate dikinase and another in the conversion of PPi to 2Pi which is catalyzed by pyrophosphatase. Therefore actual requirement is equal to two molecules of ATP. This energy is in addition to three ATP required for fixation of one molecule of CO2 through Calvin cycle. Therefore, C4 plants consume 5ATP molecules per molecule of CO2 fixed instead of 3 ATP molecules as witnessed in the C3 plants. For the formation of a glucose molecule, C4 plants require 30 ATP while C3 plants utilize only 18ATP. This use of additional 12 ATP molecules is basically used for the sole purpose of concentrating CO2 at the site of RUBISCO so as the reduce photorespiration.

The net effect of C4 cycle is to basically convert a dilute solution of CO2 in the mesophyll region into a concentrated CO2 solution in the bundle sheath cells. The cycle efficiently shuttles CO2 from the atmosphere into the bundle sheath cells. This transport process generates a much higher concentration of CO2 from the atmosphere in the bindle sheath cells than would occur in equilibrium with the external atmosphere. Since the affinity of RUBISCO in the bundle sheath is more toward CO2, the transported CO2 at the carboxylation site of RUBISCO results in the suppression of oxygenation of ribulose 1, 5-bisphosphate and hence photorespiration is reduced.

1.5 Regulation of Activity of Enzyme:

The key role in regulation of several specific enzymes is that of Light. These enzymes are namely – PEP carboxylase, NADP-malate dehydrogenase and pyruvate-orthophosphate dikinase. The activity of these enzymes depends upon the variation of photon flux density. This can majorly be achieved in these enzymes by two different processes i.e. reduction-oxidation of thiol groups and phosphorylation – dephosphorylation.

PEP carboxylase is activated by light dependent phosphorylation and dephosphorylation mechanism that is not yet characterized. PEP carboxylase has another system that carries out ‘fine control’ of carboxylase activity which mainly comprises of metabolites and pH. This control is of feed-back regulation type. Since the activity of PEP carboxylase increase with a decrease in pH, when the C4 acids or metabolites are formed there is an increase in pH which renders the activity of the enzyme.

NADP-malate dehydrogenase is activated by light via the thioredoxin system of chloroplast.The third regulatory enzyme pyruvate-orthophosphate dikinase is rapidly inactivated by an unusual ADP-dependent phosphorylation of the enzyme when the photon flux density drops. Activation is then accomplished by cleavage of this phosphorylized group. Both of the reactions, phosphorylation and dephosphorylation, appear to be catalyzed by a single regulatory protein.

1.6 Significance:

C4 plants are more efficient than the C3 plants in CO2 fixation because at high O2

concentration and high light intensity the enzyme RUBISCO in C3 plants perform oxygenase activity which leads to photo-respiration. Photorespiration reduces the efficiency of photosynthesis by removing the carbon molecules from C3 cycle. Hence in C4 pathway the concentration of CO2 itself is being concentrated when it reaches the bundle sheath region to enter the Calvin cycle thereby reducing the chances of photo-respiration and increase the efficiency of photosynthesis by the C4 plants.

Since C4 plants have a higher demand for energy they require more quanta of light per CO2 molecule as compared to that of C3 plants. In normal air the quantum requirement of a C3 plant changes the factors that affect the balance between photosynthesis and photorespiration, such as the temperature. In contrast, because of the mechanisms built in C4 plants to avoid photorespiration, the quantum requirement of C4 plants remains relatively constant under different environment conditions.Another plus point is the use of enzyme PEP carboxylase. PEP carboxylase utilizes HCO3

- as its subtrate. The affinity of PEP carboxylase is sufficiently high that the enzyme is saturated by HCO3

-which makes it to an equilibrium with air level of CO2. Also since the substrate is HCO3

-, oxygen is not a competitor in the reaction. This high activity of PEP carboxylase enables C4 plants to reduce stomatal apertures thereby conserving water as well as fixing CO2 at rates equal to or higher than the C3 plants.

1.7 Conclusion:

As C4 plants are able to assimilate over twice as much as carbon as C3 plants for each unit of water transpired, this adaptation makes C4 plants suitable for regions of periodic drought such as tropical savannas. The interest in C4 biology results from the increase in global awareness to the difficulty we face trying to provide food and fuel for a growing population. One way to increase yields could be to introduce C4 traits into C3 plants. Another reason for current C4 research is the global focus on biofuels. Two of the current major biofuel crops, sugarcane and maize are both C4 species. Whereas the future of sugarcane as a fuel crop is almost certain, the use of maize has certain points to be worked out on. Another important area for developing studies with C4 plants is that of herbicides. A great number of weeds that are usually found in soyabean, wheat and other C3 species are indeed C4 plants. Compounds that specifically inhibit the Hatch-Slack

cycle could be used as herbicides with a high degree of efficiency. Hence, more studies are still required in fields of plant physiology, biochemistry and molecular biology to evaluate these possibilities

Fig.1.1 T.S. of a C4 leaf showing Kranz anatomy

Fig 1.2 Difference between C3 plant and C4 plant anatomy

Fig 1.3 Comparison between C3 and C4 plant

Fig 1.4 Hatch and Slack Pathway

Fig 1.5 CO2 Fixation In C3 and C4 pathway

1.8 References: Fundamentals of Botany by N.K.Soni Introduction to Biotechnology by Dr, B.L. Saini The World of the Cell by Becker http://www.photosynthesisinfo.com/hatch-and-slack-cycle/ http://edudel.nic.in/PAHAL/biology_260309/biology_ch_3_dt_030409.pdf http://plantphys.info/plant_physiology/c4cam.shtml