Amino Acid and Protein Metabolism - Plants and Crops Science

Amino Acid and Protein Metabolism

INTRODUCTION

Amino acid metabolism is one of the most important biochemical processes in plants; similar to other topics in biochemistry, it has been affected by tremendous developments in science. Following are four actively studied aspects in the field of amino acid metabolism:

1) the identification of new transporters of amino acids and other N-forms like nitrate and ammonium;

2) the characterization of factors controlling the metabolism in situ in distinct cells or subcellular compartments;

3) the regulation of the multiple isoenzymes of amino acid metabolism in the context of a single plant, and

4) the role of amino acids as signaling molecules.

Studies on protein metabolism, on the other hand, have focused on the processes of protein synthesis. The complex regulation of protein degradation is today attracting more attention, particularly because proteolysis is involved in cellular processes such as programmed cell death, circadian rhythm, and the defense response in plants. 

This chapter summarizes the latest insights in the studies of amino acid metabolism and protein degradation. The focus of this review is to examine the regulation of amino acid metabolism and protein processing in the context of a single plant. Particular emphasis is given to the enzymes involved in NH4 + assimilation, which are often oligomers located in different subcellular compartments. The mechanism op.

AMINO ACID METABOLISM

Ammonium is the inorganic N-form to be incorporated into carbon skeletons for the production of amino acids. Several transporters have been identified for NO3, NH4 +, and amino acids that contribute to a wide array of physiological activities. N-forms assimilated into glutamine or glutamate disseminate into plant metabolism because they are N-donors to other amino acids, nucleotides, chlorophylls, polyamines, and alkaloids. 

Amino acids are well known as building blocks of proteins, and they are essential in both primary and secondary plant metabolism. Various amino acids also perform other important roles, as signaling molecules or precursors of stress-related compounds under adverse environmental conditions, in particular glutamate, which is found in the intersection of several metabolic pathways. In the chloroplast, one net glutamate molecule is produced by the concerted action of glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT), which form the GS-GOGAT cycle, responsible for the prevalent NH4 + assimilation in plants. Additionally, two molecules of glutamate are generated as the end product of lysine catabolism by the saccharopine pathway in seeds of cereals and dicots.

This pathway may be involved in the transient synthesis of glutamate, which then functions as a messenger between cells during organ development or in response to environmental changes. In fact, glutamate could be converted into g-aminobutyric acid, a stress-related signaling molecule; proline, an osmolyte providing drought tolerance under water stress; and arginine, a precursor of polyamines and nitric oxide generated during stress.

In the era of functional genomics and metabolic engineering, new experimental approaches are appearing that help us understand the regulation of amino acid metabolism. Among the analytical methods, nuclear magnetic resonance (NMR) spectroscopy is a promising technique that yields insight into the integration and regulation of plant metabolism through nondestructive and noninvasive measurements. Through in vivo NMR methods and under certain experimental conditions, it was possible to differentiate amino acid pools from cytosolic or vacuolar compartments.

Certainly, by inducing alkalization in sycamore cells and leaves of Kerguelen cabbage, it was demonstrated that the concentration of amino acids in the cytosol was much higher than in the vacuole and that proline accumulated to a concentration 2–3 times greater in the cytosol than in the vacuole. The enzymes involved in NH4 + assimilation are generally isoenzymes of different oligomeric arrangements, which are often located in particular subcellular compartments or within different organs and tissues.

Whether these enzymes play overlapping (redundant) or distinct (nonredundant) roles, the factors controlling this process during plant growth and development are still a matter of discussion. The strategies used to study these topics are the production of either mutant plants defective in a particular isoenzyme or transgenic plants overexpressing one gene member of a small gene family regulated differentially during the life span of the cells. To illustrate the application of these technologies, studies were selected showing the role of enzymes involved in glutamate metabolism. All plants contained two types of GOGAT, an NADH-dependent enzyme and an Fd-GOGAT, unique to photosynthetic organisms.[3] Thus, while Fd-GOGAT accounts for 96% of the total GOGAT activity in leaves, NADHGOGAT constitutes the predominant isoenzyme in roots.

To assess the in vivo role of GOGAT in primary nitrogen assimilation and in photorespiration, an Arabidopsis mutant deficient in Fd-GOGAT was studied. Gene expression combined with Fd-GOGAT-deficient mutant analyses demonstrated that Arabidopsis contains two expressed genes (GLU1 and GLU2) encoding two distinct Fd-GOGAT isoforms. GLU1 gene product plays a major role in photorespiration as well as in primary nitrogen assimilation in leaves. The Fd-GOGAT isoenzyme encoded by GLU2 is proposed to be involved in primary nitrogen assimilation in roots. These contrasting patterns of gene expression suggest nonoverlapping roles for GLU1 and GLU2. As an example of the second approach is the constitutive overexpression of the cytosolic GS in alfalfa.

GS isoenzymes can be localized in the chloroplast (GS2) or in the cytosol (GS1), and they have distinct in vivo functions. Plants appear to possess a single nuclear gene encoding GS2 and multiple GS1 genes, which are members of small gene families and differentially regulated. In this study, a GS1 gene was constitutively expressed in all cell types of alfalfa driven by the cauliflower mosaic virus promoter to bypass the transcriptional regulation component. 

The GS1 gene was transcribed in these transgenic plants, but GS1 was unstable and did not accumulate. The results suggested that GS is regulated at multiple steps, besides being regulated at the transcriptional level. One step of regulation is mRNA stability which may be controlled by the glutamine/glutamate ratio, the ATP/ADP ratio, or the redox balance. Another level of regulation would be protein turnover and would involve the inactivation of GS by oxygen radicals generated by a redox reaction.

PROTEIN DEGRADATION

Protein degradation is an important aspect of the cell cycle that occurs in the normal life of the plant. A process of protein selection occurs to specifically degrade proteins; some proteins are degraded when they become damaged. The proteases found in plants can be identified as matrix metalloproteases, processing proteases, and proteases involved in the mobilization of storage-protein reserves. Proteolysis also takes place during photoinhibition in the chloroplast, programmed cell death, and photomorphogenesis in the developing seedling involving several subcellular compartments.

In the chloroplast, for example, and due to its endosymbiotic origin, each protease is related to a bacterial counterpart. The FtsH1 protein, which is involved in the D1 protein degradation, is related to Escherichia coli FtsH1 protein, a metalloproteinase, and chaperone. Another example is the ClpP, which is responsible for the regulated degradation of the cytochrome b6f complex. Among the mechanisms operating in protein degradation, the conjugation of proteins by ubiquitin has been implicated.

A protein with a chain of at least four ubiquitin subunits is recognized by the proteasome and degraded. The ubiquitin subunits are removed from the substrate by a ubiquitin-specific protease and recycled. Protein structures are also affected by the state of oxidation of some amino acid residues, which may be the principal parameter affecting the in vivo and in vitro stability of proteins.

Transition metals, ozone, nitric oxides, and metal ions are involved in these oxidative modifications of proteins and free amino acids. Protein oxidation was rigorously tested and verified in E. coli GS. In plants, GS2 is extremely prone to oxidative cleavage, and reduced transition metals—presumably resulting from the destruction of iron-sulfur clusters during photoinhibition—play a crucial role in the degradation process. In this process, proteolytic enzymes may not be involved because protease inhibitors provide little or no protection to light-induced GS degradation.

CONCLUSION

In the past few years the study of amino acid and protein metabolism has diverged enormously, due to the advances of emerging tools in genomics. Understanding this complex regulated network requires the identification of the factors controlling gene expression in response to changes in N status. Data are now emerging to provide evidence that NO3, NH4 +, or amino acids may also serve as signaling molecules in plants, aside from their role as building blocks. There is also increasing evidence that glutamate may function in a manner that is analogous to its signaling function in the animal nervous system.

Several N-metabolizing enzymes have been at least partially characterized and much more information is emerging regarding the organization and control of the genes encoding these enzymes, as well as the modulation of the activity of the enzymes themselves once synthesized. These studies will bring new perspectives to improve the level of essential amino acids in plant seeds. Regarding protein metabolism, our focus will change in the future to the study of the complex regulation of protein degradation, which still needs more attention.