LS3121 Plant Physiology

Lec 9 Respiration and Lipid Metabolism

I. Cellular Respiration

1. Breakdown of large molecules

(1). Starch Degradation

a. -Amylase: attacks -1,4 linkages anywhere in a starch molecule.

b. -Amylase: attacks -1,4 linkages from nonreducing end.

c. Starch phosphorylase

d. R enzyme (debranching enzyme)

e. a-Glucosidase

(2). Sucrose Degradation

(3). Respiration is a multistep process in which glucose is oxidized.

a. Three steps of respiration

(a). Glycolysis: glucose 2 pyruvate

(b). Tricarboxylic cycle: pyruvate CO₂

(c). Electron transport chain

b. The overall reaction of respiration

C₆H₁₂O₆ + 6 O₂ + 6 H₂O + 32 ADP + 32 Pi 6 CO₂ + 12 H₂O + 32 ATP

2. Glycolysis and fermentation

(1). Compartment: cytosol

(2). Substrate-level phosphorylation: a direct transfer of a phosphate moiety from a substrate molecule to ATP.

(3). In the absence of O₂, fermentation allows regeneration of NAD⁺ needed for glycolysis.

3. Gluconeogenesis:

(1). Gluconeogenesis operates in the germination of seeds in higher plants.

(2). Plants use glycolytic pathway in reverse direction to synthesize glucose.

(3). The interconversion between F-6-P and F-1,6-BP

a. Phosphofructokinase (in glycolysis)

Fructose-6-P + ATP fructose-1,6-BP + ADP + Pi

b. Fructose-1,6-bisphosphatase (in gluconeogenesis)

Fructose-1,6-BP + ADP + Pi fructose-6-P + ATP

II. Oxidative Respiration

1. The plant mitochondria

2. TCA cycle in the mitochondrion

(1). Compartment: mitochondrial matrix.

(2). The TCA cycle

2 pyruvate + 8 NAD⁺ + 2 ubiquinone + 2 ADP^2- + 2 H₂PO₄^- + 4 H₂O

6 CO₂ + 2 ATP^3- + 8 NADH + 8 H⁺ + 2 ubiquinol

(3) The TCA cycle of plant cells has some unique features

a. The succinyl-CoA synthetase produces ATP in plants, GTP in animals.

b. The significant NAD⁺ malic enzyme activity.

(a). Malate can be synthesized from PEP in the cytosol

PEP carboxylase

PEP + CO₂ OAA

Malate dehydrogenase

OAA + NADH malate + NAD⁺

(b). Malate is then transported into the mitochondrial matrix by a dicarboxylate transporter on the inner membrane.

3. The electron transport chain of mitochondrion

(1). Reduced compounds produced by mitochondrial respiration:

a. From glycolysis pathway: 2 NADH

b. From TCA cycle pathway: 8 NADH, 2 FADH₂

(2). The electron carriers unique in plant mitochondria

a. A NADH dehydrogenase complex (external NADH dehydrogenase) that faces the intermembrane space.

b. A rotenone-resistant NADH dehydrogenase.

c. The cyanide-resistant pathway (an alternative oxidase).

(3). ATP synthesis in the mitochondrion is coupled to electron transport.

a. ADP:O ratios: the number of ATPs synthesized per two electrons transferred to oxygen.

b. Oxidative phosphorylation: chemiosmotic hypothesis.

(a). The proton driving force is contributed by both a membrane electrical potential component (DEm) and a chemical potential component (DpH).

(b). The proton driving force is coupled to the synthesis of ATP by the F₀F₁-ATP synthase, which is associated with the inner membrane.

(c). Uncouplers such as FCCP make the inner membrane leaky to protons.

c. The respiratory control ratio (RCR)

state 3 rate

RC ratio =

state 4 rate

(5). Energy production

4. Plants have a unique cyanide-resistant respiration pathway

(1). In the presence of 1 mM cyanide, < 1% respiration exists in animals.

(2). Cyanide-resistant respiration represents 10-25%.

(3). Cyanide-resistant terminal oxidase.

(4). Inhibitor: salicylhydroxamic acid (SHAM).

(5). An energetically wasteful process?

a. The floral apex of the Araceae and pollination.

b. Increase in alternative respiration and temperature.

c. Salicylic acid is the chemical signal for initiating the thermogenic event.

5. Respiration is regulated by energy demand and the concentration of key metabolites

(1). The cytoplasmic ATP/ADP ratio.

(2). The fructose-2,6-BP in the cytoplasm.

(3). The energy charge indicates the cell's energy status.

(4). The matrix NADH:NAD⁺ ratio and ATP concentration.

6. The pentose phosphate pathway oxidizes glucose to ribulose-5-phosphate and reduces NADP⁺ to NADPH.

(1). It produces 2 NADPH, ribose-5-phosphate and erythrose-4-phosphate.

(2). PPP is as essential as the glycolysis and the Kreb's cycle to plants.

(3). Regulation: Glucose-6-P dehydrogenase is inhibited in the light by high NADPH:NADP ratio in the chloroplast and a reductive inactivation involving the ferredoxin: thioredoxin system.

7. Respiration is tightly coupled to other metabolic pathways in the cell.

III. Whole-Plant Respiration

1. Internal factors

(1). Plant species and plant growth habits

(2). Plant tissue type

a. Developing buds show high rates of respiration.

b. The climacteric rise in respiration at the onset of fruit ripening.

2. Environmental factors

(1). External oxygen concentration

(2). Temperature, light, plant water status, organic ions and injury

IV. Lipid Metabolism

1. Triacylglycerols in most seeds are stored in the cytoplasm of either cotyledon or endosperm cells in spherosomes (oleosomes, lipid bodies).

(1). A single layer of phospholipids surrounds the spherosome.

(2). Several proteins, e.g. oleosins.

2. Triacylglycerol biosynthesis

(1). It is energetically expensive.

(2). It takes place in several cell organelles.

(3). Biosynthesis of fatty acids and triacylglycerol (TAG) in higher plants

a. Acetyl CoA carboxylase.

b. Fatty acyl synthetase complex.

c. Acyl carrier protein.

d. Require 1 ATP and 2 NADPH for the addition of each 2-C unit.

3. Phospholipid biosynthesis occurs in the ER and mitochondrial membranes

(1). Mitochondria are able to synthesize phosphatidic acid, CDP-diacylglycerol, phosphatidylglycerol and cardiolipin (diphosphatidylglycerol, a unique mitochondrial inner membrane lipid).

(2). Glactolipids are synthesized at inner membrane of chloroplast envelope.

4. In germinating seeds, lipids are converted into carbohydrates

(1). Glyoxysomes are found in the oil-rich storage tissues of seeds.

(2). Acetyl CoA is metabolized in glyoxysome via glyoxylate cycle to produce succinate. Succinate is converted into malate in mitochondria.

(3). In cytosol, malate is converted into OAA and then to glucose via gluconeogenesis.

5. Oleosins

(1). Alkaline proteins (15-26 kD) that are unique to oil bodies.

(2). Oleosins serve a structural role and provide specific binding site for lipase during germination.

Lecture 10 Assimilation of Mineral Nutrients

I. Nutrient assimilation

1. Nitrate assimilation

2. Biological nitrogen fixation

3. Sulfate assimilation

II. The Nitrogen Cycle

1. Ammonification, nitrification and denitrification

(1). Ammonification: amino acids are returned to the soil and converted to ammonia by soil microorganisms.

(2). Nitrification and the nitrifying bacteria

Nitrosomonas, Nitrococcus

NH₃ NO₂^-

Nitrobacter

NO₂^- NO₃^-

(3). Denitrification

Thiobacillus denitrificans

NO₃^- N₂

2. Nitrogen fixation (chemical and biological reactions).

(1). Lightening HNO₃

(2). Biological nitrogen fixation: leguminous crops

III. The Nitrate Assimilation Pathway

1. Nitrate uptake

(1). Metabolic energy is required.

(2). Nitrate induces its own specific transport system.

(3). Excess nitrate may be taken up by the vacuole.

2. Nitrate is reduced to nitrite by plant nitrate reductase.

(1). This occurs in the cytosol.

Nitrate reductase (NR)

NO₃^- + NAD(P)H + 2 H⁺ + 2 e^- NO₂^- + NAD(P)⁺ + H₂O

a. The NADH nitrate reductase may be homodimers (each of 100 kDa).

b. Each contains 3 prosthetic groups: FAD, heme and a Mo complex (pterin).

(2). Nitrate reductase is an inducible enzyme

a. Nitrate reductase activity is substrate inducible.

b. Nitrate, light and carbohydrates regulate nitrate reductase via a protein phosphatase that dephosphorylates several serine residues on the NR.

3. Nitrite is reduced to ammonium by nitrite reductase.

(1). The nitrite is rapidly transported into plastids.

Nitrite reductase (NiR)

NO₂^- + 6 Fd_red + 6 e^- + 8 H⁺ NH₄⁺ + 6 Fd_ox + 2 H₂O

a. NiR in chloroplasts and root plastid consists of a polypeptide (63 kDa), two prosthetic groups, an iron-sulfur cluster (Fe₄S₄) and a specialized heme.

b. NO₃^- and light induce the transcription of NiR mRNA.

4. Plant can assimilate nitrate in both roots and shoots.

IV. Ammonium assimilation

1. The glutamate synthase cycle: ammonia is rapidly incorporated into organic compounds.

(1). Glutamine synthetase (GS)

Glutamate + NH₄⁺ + ATP glutamine + ADP + Pi

a. GS has a MM of 350 kDa and is composed of 8 nearly identical subunits.

b. GS in root plastids generates amide nitrogen for local consumption, GS in the shoot chloroplast reassimilates photorespiratory NH₄⁺.

c. The cytosolic forms are expressed in germinating seeds or in the vascular bundles of roots and shoots to produce glutamine.

(2). Glutamate synthase (GOGAT)

Glutamine + 2-oxoglutarate + NADH + H⁺ 2 glutamate + NAD⁺

Glutamine + 2-oxoglutarate + Fd_red 2 glutamate + Fe_ox

a. The NADH type is located in the plastids of the nonphotosynthetic tissues.

b. The ferrodoxin-dependent type is found in chloroplasts (MM 165 kDa).

(3). Glutamate dehydrogenase (GDH)

2-Oxoglutarate + NH₄⁺ + NAD(P)H glutamate + NAD(P)⁺ + H₂O

a. GDH is localized in the chloroplast (NADP form) and the mitochondria.

b. GDH has a relatively high Km for ammonia.

(4). Nitrogen is incorporated into other amino acids through transamination.

a. Aspartate aminotransferase

Glutamate + oxaloacetate aspartate + 2-oxoglutarate

pyridoxal phosphate (vitamin B6) is a cofactor.

b. Asparagine synthetase

Glutamine + asparate + ATP glutamate + asparagine + ADP + Pi

V. Biological Nitrogen Fixation

1. Free-living nitrogen fixers (nonsymbiotic)

(1). Cyanobacteria: Anabaena, Nostoc, Calothrix

(2). Other bacteria:

a. Aerobic: Azotobacter, Beijerinckia

b. Facultative: Bacillus, Klebsiella

c. Anaerobic (nonphotosynthetic: Clostridium, photosynthetic: Chromatium, Rhodospirillum)

2. The Symbiotic nitrogen fixers

(1). The host root produces nodules upon infection by Rhizobium.

(2). Rhizobium species exhibit host preference, the microsymbiont

a. Legume rhizobia: Rhizobium, Bradyrhizobium, Azorhizobium

b. Ulmaceae: Parasponia; Alder: Flankia

(3). Infection and nodule development

a. The early stage-colonization and nodule initiation

(a). Root excretes flavonoids, rhizobia bind to emerging root hairs.

(b). In response to NOD D, rhizobia induce root hair curling, trapping rhizobia within the coils.

(c). Rhizobia-host interactions: lectins and complex polysaccharides.

b. Invasion of the root hair and the infection thread

(a). Forming the infection thread by the fusion of Golgi secretory vesicles.

(b). The infection thread reaches the end of the cell, and its membrane fuses with the plasma membrane of the host root hair cell.

c. The release of bacteria

(a). Rhizobia are released into the apoplast and penetrate to the subepidermal cell plasma membrane, leading to the initiation of a new infection thread.

(b). The infection thread extends and branches until it reaches target cells.

(c). The bacteroids remain surrounded by the peribacteroid membrane.

3. Establishing symbiosis requires an exchange of signals

(1). Plant nodulin genes (Nod).

(2). Rhizobial nodulation genes (nod)

a. nifD and nifK MoFe protein; nifH and nifF Fe protein

b. NodA (N-acetyltransferase), NodB (chitin-ologosaccharide deacetylase), NodC (chitin-ologosaccharide synthase) for nodulation

c. NodD regulatory gene, lipochitin oligosaccahride signal molecules

4. The biochemical process of nitrogen fixation

(1). The overall reaction for nitrogen fixation

Nitrogenase

N₂ + 8 e^- + 8 H⁺ + 16(Mg)ATP 2 NH₃ + H₂ + 16 (Mg)ADP + 16 Pi

(2). The nitrogenase can be separated into two components.

a. The MoFe protein

(a). A tetramer (total mass of 180-235 kDa).

(b). Two Mo atoms per molecule (two Mo-Fe-S clusters).

(c). It is inactivated by oxygen.

b. The Fe protein

(a). A dimer (each of 30 to 72 kDa) contains 4Fe-4S^2- iron-sulfur cluster.

(b). The Fe protein is extremely sensitive to oxygen.

(3). Energy cost of nitrogen fixation

(4). The nitrogenase is irreversibly inactivated by oxygen.

a. In the filamentous cyanobacteria- the free-living bacteria.

(a). Heterocysts form in the anaerobic conditions.

(b). Heterocysts differentiate when NH4+ is deprived.

(c). PSII is absent from heterocysts.

b. In symbiotic nitrogen fixation, bacteria are associated with roots of legumes. The amount of free oxygen is reduced by the high respiration of the symbionts and by the presence of leghemoglobin.

(5). Uptake hydrogenase

5. Export of fixed nitrogen from nodules

(1). Amide (asparagine and glutamine) exporters: temperate-region legumes such as pea, clover and broad bean.

(2). Ureide (allantoin, allantoic acid and citrulline) exporters: tropical-region legumes such as soybean, cowpea, kidney bean and peanut.

VI. Nitrogen Nutrition

1. Crop growth and environmental factors

2. The application of nitrogen fertilizers overcomes environmentally imposed nitrogen limitation.

VII. Others

1. Sulfur assimilation

(1). Sulfate assimilation requires the reduction of sulfate to cysteine.

(2). Sulfate assimilation occurs mostly in leaves.

(3). Methionine is synthesized from cysteine.

2. Phosphate assimilation: phosphate is absorbed by plant roots and incorporated into a variety of organic compounds.

Lec 11 Carbon Assimilation and Productivity

I. Productivity

1. Gross primary productivity (GPP): total carbon assimilation.

2. Net primary productivity (NPP): correct the energy loss due to respiration.

II. Respiration and Carbon Economy

1. Growth and maintenance

(1). Growth respiration includes the carbon actually incorporated plus the carbon respired to produce the energy for biosynthesis and growth.

(2). Maintenance respiration provides energy for processes that do not result in net increase in dry matter, such as turnover of organic molecules, maintenance of membrane structure, turgor, and solute exchange.

2. Growth rate and relative growth rate

III. Factors Influencing Photosynthesis and Productivity

1. Light

(1). Rate of CO₂ uptake versus fluence rate

(2). Light compensation point and light saturation

(3). C3 versus C4 plants

(4). Shade leaves and sun leaves

(5). Long-term carbon gain is dependent on cumulative irradiance over the growing season.

2. Available carbon dioxide

(1). The availability of CO₂ is often a limiting factor in photosynthesis.

(2). CO₂ compensation point and CO₂ saturation

(3). Limitation of photosynthesis (PS) rate as a function of CO₂ concentration

a. At low [CO₂], PS is limited by carboxylation capacity of Rubisco.

b. At high [CO₂], PS is limited by the rate of regeneration of RuBP.

(4). The most efficient use is to maintain intercellular CO₂ levels in the transition zone.

3. Temperature

(1). Rate of the reaction at two temperatures 10°C apart

RT+10

Q10 =

RT

(2). Photosynthetic reactions in the thylakoid membranes are largely independent of temperature.

4. Soil water potential

(1). The rate of PS declines under conditions of water stress.

(2). C4 plants enjoy some advantage over C3 plants with respect to PS and water stress because of their higher water use efficiency.

5. Nutrient supply, pathology, and pollutants

6. Leaf factors

(1). Leaf development and aging

(2). Canopy structure (age, morphology, angle and spacing)

(3). Leaf area index (LAI): the ratio of PS leaf area to cover ground.

(4). Heliotropism or solar tracking: the leaf blades move in such a way that surfaces remain perpendicular to the sun's direct rays.

IV. Primary Productivity on a Global Scale

1. Terrestrial ecosystems retain a much larger nutrient capital in the soil and litter.

2. The amount of land available for cultivation is limited.