Nitrogen fixation and nitrogen assimilation

Education

1. TOPIC 9 Nitrogen fixation & N itrate assimilation PLANT BIOCHEMISTRY (BAPS1321) Assoc. Prof. Dr Maizatul Akma Ibrahim Department of Plant Science, Kulliyyah of Science, International Islamic University Malaysia [email protected]

2. Legume, Symbiosis, Nodule-inducing bacteria A legume is a plant in the family Leguminosae, or the fruit or seed of such a plant. Legume includes 20, 000 species : alfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soybeans, peanuts, tamarind. Symbiosis : is close and often long-term interaction between two or more different biological species Nodule-inducing bacteria/ n 2 -fixing bacteria : capable of transforming atmospheric nitrogen into fixed nitrogen, inorganic compounds usable by plants. L e g u m e s :

3. Nodule-inducing bacteria: Rhizobium, Bradyrhizobium and Azorhizobium are collectively called Rhizobia. Rhizobia are strictly aerobic gram-negative rods, which live in the soil and grow heterotrophically in the presence of organic compounds. Commonly Rhizobium forms nodule with peas Bradyrhizobium forms nodule with soybean Azorhizobium forms nodule with sesbania

4. n 2 -fixing bacteria are able to synthesize ammonia from atmospheric nitrogen, which supply the plant with organic nitrogen. Plants provide metabolites for their nutrition Symbiosis of legumes with n 2 -fixing bacteria is important for agriculture In temperate climates, cultivation of legumes can lead to N 2 fixation of 100-400 kg N 2 /ha/year

5. How does legume form a nodule/symbiosis with nodule-inducing bacteria? The uptake of rhizobia into the host plant is a controlled infection. The rhizobia form species-specific nodulation factors (signaling molecule), eg. lipochito-oligosaccharides. After the root hair has been invaded by the rhizobia, an infection thread forms, which extends into the cortex of the roots, branches there and infects the cells of the nodule primordium. A nodule thus develops from the infection thread.

6. The nodules are connected with the root via vascular tissues, which supply them substrates produced by photosynthesis. The bacteria incorporated into the plant cell are enclosed by a peribacteroid membrane, which derives from the plasma membrane of the infected plant cell . Fig. Controlled infection of a host cell by rhizobia

7. Nodule formation relies on a balanced interplay of bacterial & plant gene expression The host plant signals its readiness to form nodules b y e x c r e t i n g s e v e r a l f l a v o n o i d s a s s i g n a l compounds for the chemo-attraction of rhizobia. These flavonoids bind to a bacterial nod gene protein which activates the transcription of the other nod, nol and noe genes. Proteins, which are required especially for the formation of nodules, and which are synthesized by the host plant in the course of nodule formation, are called nodulins. The plant genes encoding these proteins are called nodulin genes.

8. Metabolic products are exchanged between bacteroids & host cells The main substrate provided by the host cells to the bacteroids is malate , synthesized from sucrose, which is delivered by the sieve tubes. Malate taken up into the bacteroids is oxidized by the citrate cycle (Fig). The reducing equivalents thus generated are the fuel for the fixation of N 2 NH 4 is delivered as a product of N 2 fixation via a specific transporter to the host cell, where it is subsequently converted mainly into glutamine and asparagine and then transported via the xylem vessels to the other parts of the plant. The nodules of some plants (e.g., soybean) export the fixed nitrogen as ureides (urea degradation products), especially allantoin and allantoic acid

9. Fig. Metabolism of infected cells in a root nodule. Glutamine and asparagine are synthesized as the main products of N 2 fixation

10. Plants improve their nutrition by symbiosis with fungi Plant growth is limited not only by the supply of nitrate but also with phosphate. Because of its low solubility, the extraction of phosphate by the roots from the soil requires very efficient uptake systems. In order to increase the uptake of phosphate, most plants enter a symbiosis with fungi. Fungi are able to form a mycelium with hyphae that are well suited to penetrate soil particles, thereby mobilizing the nutrients. The symbiotic fungi (microsymbionts) deliver these nutrients to the plant root (macrosymbiont) and are in turn supplied by the plant with carbon metabolites for maintaining their own metabolism.

11. Arbuscular mycorrhiza A mycorrhiza is a symbiotic association between a fungus & the roots of a vascular plant The arbuscular mycorrhiza has been detected in more than 80% of all terrestrial plant species. In this symbiosis the hyphae of fungus penetrates the cortex of plant roots and forms a treelike invaginations, termed arbuscul (Fig.) The arbuscul form a large surface, enabling an efficient exchange of compounds between the fungus and the host. The fungus delivers phosphate, nitrate, K + ions, and water, and the host delivers carbohydrates. Lifetime is less than two weeks, therefore the maintenance of symbiosis requires a constant formation of new arbuscules.

12. Fig. Schematic representation of an arbuscul. The hypha of a symbiotic fungus spreads into the intercellular space of the root cortex. From there tree-like invaginations into the inner layer of the cortex are formed. The large contact area between the host and the microsymbiont enables an efficient exchange of compounds.

13. Nitrate assimilation: reduction of NO 3 to NH 3 Nitrate is assimilated in the leaves and also in the roots The nitrate is taken up from the soil by the root. It can be stored in the vacuoles of the roots cells or assimilated in the cells of root epidermis and the cortex. Nitrate is reduced to NH 4 in the epidermal and cortical cells of the root. + This NH 4 is used mainly for the synthesis of glutamine and asparagine + (amide). These two amino acids can be transported to the leaves via the xylem vessels. Surplus nitrate is carried via the xylem vessels to the mesophyll cells, where nitrate can be stored temporarily in the vacuole. Nitrate is reduced to nitrite in the cytosol by nitrate reductase and then nitrite is further reduced NH 4 by nitritie reductase in the chloroplasts , + from which amino acids are formed. H + transport out of the cells of the root and the mesophyll proceeds via an H + -P-ATPase.

14. Fig. Nitrate assimilation in the roots and leaves of plant

15. Controlled of nitrate assimilation Nitrate assimilation must be regulated in such a way that the production of amino acids does not exceed the demand. It is important that nitrate reduction does not proceed faster than nitrite reduction, to prevent the accumulation of toxic levels of nitrite. For example, dangerous levels of nitrite may accumulate under anaerobic soil conditions in the case of excessive moisture. Flooded roots are able to release nitrite into the soil water, avoiding the buildup of toxic levels of nitrite. This escape route, however, does not function in leaves, and therefore the strict control of nitrate reduction is especially important.

16. Nitrate assimilation is strictly controlled by - The synthesis of the nitrate reductase protein is regulated at the level of gene expression Various factors control the synthesis of the enzyme at the level of gene expression. The synthesis of the nitrate reductase protein is stimulated by glucose and other carbohydrates generated by photosynthesis, and is inhibited by NH4 + , glutamine and other amino acids Thus, by regulating its synthesis, the activity of nitrate reductase in the tissue can be altered within hours.

17. 2. Nitrate reductase is also regulated by reversible covalent modification The regulation of de novo synthesis of nitrate reductase (NR) allows regulation of the enzyme activity within a time span of hours. This would not be sufficient to prevent an acute accumulation of nitrite in the plants during darkening or sudden shading of the plant. Synthesis of the NR protein is stimulated by carbohydrates and light [+], and inhibited by glutamine or other amino acids [–]. There is a dynamic equilibrium between the active and inactive form of the enzyme. Okadaic acid , an inhibitor of protein phosphatases, counteracts the activation of nitrate reductase.

18. 3. 14-3-3 proteins are important metabolic regulators The nitrate reductase inhibitor protein belongs to a family of highly conserved regulatory proteins called 14-3-3 proteins 14-3-3 proteins bind to a variety of proteins and change their activity. Thus 14-3-3 proteins regulate in plants the activity of the H + -P-ATPase of the plasma membrane. 14-3-3 proteins regulate the function of transcription factors and protein transport into chloroplasts.

19. The end product of nitrate assimilation Amino acid → Nitrate assimilation product which has no special transport forms. All amino acids present in the mesophyll cells are exported via the sieve tubes. Synthesis of these amino acids takes place mainly in the chloroplasts. Glutamate and glutamine represent the major portion of the synthesized amino acids. Also, serine, glycine and alanine are often formed . CO 2 assimilation provides the carbon skeletons required for the synthesis of the various amino acids.

20. Fig. Origin of carbon skeletons of individual amino acid

21. Storage proteins Products of nitrate assimilation are deposited in plants as storage proteins, which have no enzymatic activity and are often deposited in the cell within protein bodies. Protein bodies are enclosed by a single membrane that derived from the endoplasmic reticulum and the golgi apparatus or the vacuoles. Storage proteins can be deposited in various plant organs, such as leaves, stems, and roots . They are stored in seeds and tubers and also in the cambium of tree trunks during winter to enable the rapid formation of leaves during seed germination and sprouting. In cereals the protein content amounts to 10% to 15% of the dry weight but in some legumes (e.g., soybean) it is as high as 40% to 50%. About 85% of these proteins are storage proteins.

22. Globulins are the most abundant storage proteins Prolamins are formed as storage proteins in grasses, such as cereals 2S-Proteins are present in seeds of dicot plants