B.Sc (H) Botany Semester 6

Core Paper 13 - Metabolism

E- lesson on C4 and Crassulacean Acid Metabolism By P. Chitralekha Associate professor Department of Botany Dyal Singh College C4 Pathway of Photosynthesis

• 30-50% yield is lost due to oxygenase activity of Rubisco.

• Must reduce oxygenase activity, either by reducing oxygen concentration or increasing carbon dioxide concentration around Rubisco.

• C4 pathway is a carbon dioxide concentration mechanism to reduce yield loss.

• Evolved many times in different families.

• Mostly found in growing in tropics where plants face high temperatures and light intensity, and water stress, which keep stomata closed most of the time limiting carbon dioxide diffusion into leaves.

• More than 8000 carryout C4 photosynthesis; 40% monocots belonging to just 3 families, Poaceae, Cyperaceae and Hydrocharitaceae, and about 4,5% dicots belonging to Chenopodiaceae, Amaranthaceae, Portulaccaceae, Asteraceae, Brassicaceae, Euphorbiacea, Aizoaceae, Nyctaginaceae, Zygophyllacea etc.

• In 1965, Kortschak, Hartt and Burr from Hawaii discovered that the first product of carbon dioxide fixation is not a C3 compound (phosphoglyceric acid) but C4 compounds (malic acid or aspartic acid). Hence the name - C4 pathway of photosynthesis. In 1966, Hatch and Slack from Australia confirmed the findings and subsequently, workout the entire pathway in corn, sugar cane, sorghum etc. • In C4 pathway, there are two events i) Initial fixation of CO2 in C4 organic acids ii) Fixation of CO2 in carbohydrates via Calvin cycle or C3 pathway

• The two events are spatially separated. In one region, generally the outer, CO2 is fixed in C4 acids, and light reactions occur releasing oxygen. The acids are then transported to the inner region where they breakdown to release CO2 and maintain a significantly high concentration of CO2. The inner region contains Rubisco and other Calvin cycle enzymes which fix the released CO2 into carbohydrates. This way Rubisco remains surrounded by CO2 and away from oxygen, preventing the oxygenase activity of Rubisco and subsequent photorespiration.

• Majority of C4 plants posses a characteristic leaf anatomy known as kranz anatomy (German word kranz meaning wreath). In this type of anatomy, vascular bundles are surrounded by a ring of bundle sheath cells, large chlorenchymatous cells containing chloroplasts without grana. Outer to the bundle sheath cells are the mesophyll cells with normal chloroplasts. The bundle sheath cells are connected with mesophyll cells through plasmodesmata, but are often surrounded by a gas impermeable outer suberin wall layer.

• The mesophyll cells i) have chloroplasts with grana and storm and carryout light reactions producing NADPH and ATP. ii) contain an abundance of carbonic anhydrase in the cytoplasm which converts CO2 into bicarbonate ion (HCO3-) which is more soluble in water than CO2. iii) contain large amounts of cytosolic enzyme, phosphoenolpyruvate carboxylase (PEPC) which catalyses the carboxylation of phosphoenolpyruvate with the help of HCO3- and produces C4 acid, oxaloacetate. PEPC has a high affinity for HCO3- and can fix efficiently very small amounts of CO2. Unlike Rubisco, PEPC cannot bind to oxygen, so cannot carryout oxygenase activity. iv) lack Rubisco enzyme • Bundle sheath cells i) contain chloroplasts without grana, so lack PSII, and do not produce NADPH but produce ATP probably through cyclic photophosphorylation. ii) contain large amount of Rubisco and enzymes of Calvin cycle which fix carbon dioxide released from C4 acids into carbohydrates. iii) contain a suberin wall layer which prevents diffusion of released CO2 back into mesophyll cells. iv) lacks carbonic anhydrase enzyme and has low concentration of PEPC.

• C4 pathway:

1. CO2 enters mesophyll cells. 2. CO2 is converted to bicarbonate by carbonic anhydrase in the cytoplasm. 3. Bicarbonate reacts with phosphoenolpyruvate to form oxaloacetate (OAA), catalysed by PEP carboxylase in the cytoplasm. 4. OAA is transported from mesophyll cells to bundle sheath cells. 5. OAA is broken down to C3 acid and CO2 . 6. CO2 in the bundle sheath cells is fixed into sugars with the help of Rubisco and other Calvin cycle enzymes. 7. C3 acid is transported to mesophyll cells and converted to pyruvate. 8. Pyruvate is converted to phosphoenolpyruvate with the consumption of two energy-rich phosphates of ATP, which is converted to AMP.

CO2

Mesophyll CO2 Carbonic AMP ATP Cell anhydrase Pyruvate HCO3- PEP PEPC C3 acid C4 acid

C3 acid C4 acid

CO2 Calvin Bundle sheath cycle Sugar Cell

• In C4 metabolism, in comparison to C3, two energy-rich phosphates of ATP i.e., effectively 2 extra ATP molecules are used. ATP AMP AMP + ATP 2 ADP

2 ATP 2 ADP

• Therefore, to fix one molecule of CO2, 2 NADPH and 5 ATP are required or to synthesise one glucose molecule, 12 NADPH and 30 ATP are required. There are three major variants of C4 metabolism:

NADP-Malic enzyme type e.g. Maize, Sugarcane

NAD-Malic enzyme type PEP-Carboxykinase type e.g. Pennisetum, Setaria e.g. Panicum maximum • Some plants show C3-C4 intermediate photosynthesis: - leaves possess kranz anatomy - have reduced levels of photorespiration - both mesophyll and bundle sheath cells have chloroplasts with grana and starch grains e.g. Alternanthera ficoidea, Parthenium hysterophorus

• Single cell C4 photosynthesis is found in some Chenopodiaceae and Amaranthaceae species. - compartmentalisation of cytoplasm within each mesophyll cell present - peripheral cytoplasm has chloroplasts without grana, no Rubisco, abundance of PEPC, no mitochondria = mesophyll cells - central cytoplasm has chloroplasts with grana, Rubisco present, abundance of mitochondria = bundle sheath cells e.g. Bienertia cycloptera, Suaeda aralocaspica Crassulacean Acid Metabolism (CAM)

• Type of metabolism first discovered in members of .

• Similar to C4 metabolism with two events i) Initial fixation of CO2 in C4 organic acids ii) Fixation of CO2 in carbohydrates via Calvin cycle or C3 pathway

• But unlike C4 metabolism where the two events are separated in space and occur in different cells, in CAM, the two events are separated in time; the first one occurring at night and the second in the day.

• Found in more than 16,000 species belonging to 40 families and 300 genera, majority of which are succulent xerophytes growing in dry arid regions or regions with extreme diurnal temperature changes such as the Alpine regions where nights are freezing and day has high light intensity, or are epiphytes without direct access to water.

• e.g. Kalanchoe (Crassulaceae), Opuntia (Cactaceae), Vanilla (Orchidaceae), Agave (Agavaceae), Pedilanthus (Euphorbiaceae), Isoetes (pteridophyte) Welwitschia (gymnosperm).

• The metabolism has evolved many times in unrelated families, mostly as an adaptation to conserve water. Stomata remain closed during day time when water loss through transpiration is high and open during night to allow fixation of carbon dioxide.

Since photosynthesis occurs with closed stomata, CAM requires only 5%-10% of water required for C3 photosynthesis. • Some plants are facultative CAM plants, switching to CAM under water stress conditions. e.g. Mesembryanthemum, a halophyte, and Portulacaria afra (jade plant) are C3 plants but under salt or drought stress undertake CAM. Similarly, Portulaca oleracea is a C4 plant but under drought stress switches to CAM.

• Some aquatic plants such as Isoetes, aquatic, Sagittaria have evolved CAM photosynthesis not because of water stress but because of CO2 stress. Diffusion of CO2 in water is 1000 times slower than in air, and during the day photosynthesis by other aquatic plants reduces availability of CO2 further. To overcome this, these plants fix CO2 at night.

• Mechanism of CO2 fixation At night 1. CO2 diffuses into mesophyll cells through open stomata. 2. CO2 is converted to bicarbonate by carbonic anhydrase in the cytoplasm. 3. Bicarbonate reacts with phosphoenolpyruvate to form oxaloacetate (OAA) catalysed by PEP carboxylase in the cytoplasm. Phosphoenolpyruvate is produced from starch or sugars like sucrose. 4. OAA is reduced to malate by NAD-malate dehydrogenase. 5. Malate is transported into vacuole and stored.

During the day 6. Stored malate is released from the vacuole into the cytoplasm. 7. CO2 is released from malate by different plants in three different ways, as in C4 metabolism, via NADP-malic enzyme, NAD-malic enzyme or phosphoenolpyruvate carboxykinase. • CO2 released is fixed into sugars by Rubisco and other Calvin cycle enzymes using the ATP and NADPH produced by light reaction.

• The pyruvate formed from malate after release of CO2 is converted to phosphoenolpyruvate and then to 3 phosphoglycerate which enters Calvin cycle and is fixed into sugar or starch to be reused at night to generate PEP for CO2 fixation.

• As in C4 metabolism, 2 extra ATP molecules are used up to fix 1 CO2 molecule, i.e., to fix 1 CO2 molecule a total of 5 ATP and 2 NADPH molecules are required in CAM. • As malate is stored in the vacuole in the form of malic acid, the pH of the vacuole decreases to as low as 3, at which point malic acid is largely unionised and therefore, osmotically inactive. So large amounts of malic acid can be stored in vacuoles.

• Since the capacity of vacuoles to store malate is limited, biomass increase in CAM plants is usually low, and thus, plants grow slowly.

• As long as malate is available, CO2 is released constantly and its concentration is maintained high. During this period photorespiration is negligible. But towards the end of the day, when malate reserves are used up, photorespiration can become high and limit growth of CAM plants.

• PEP carboxylase can exist in two forms, an inactive day-form and an active night-form which is activated through phosphorylation by PEP carboxykinase enzyme. The activity of the enzyme is under circadian control.

• PEP carboxylase does not function during the day as i) it is converted to the inactive day-form which has less affinity for PEP, ii) it is inhibited by high levels of malate which flood the cytoplasm during the day (the night form of the enzyme is insensitive to malate concentration). 1. How is photorespiration eliminated in CAM and C4 metabolism?

2. Write down the similarities and differences between CAM and C4 metabolism.

3. Why have some aquatic plants evolved Crassulacean acid metabolism?

4. Write down the differences between mesophyll and bundle sheath cells in C4 plants.

5. C4 pathway/metabolism and CAM are CO2 concentration mechanisms. Comment.

6. C4 pathway/metabolism and CAM are water conserving mechanisms. Comment.

7. Differentiate between Rubisco and PEP carboxylase.