Various transaminases transfer the reduced amino group to 2-oxo organic acids to form amino acids. Besides 20 proteinogenic amino acids which are used as building blocks of proteins, a vast number of non-proteinogenic amino acids are formed, either as intermediates of amino acid biosynthetic pathways, e.g. homoserine or homocysteine, or as products of certain pathways where these amino acids serve special functions in metabolism, such as ornithine and citrulline as part of the urea cycle or canavanine A as insect antifeedants.Furthermore, a huge body of the amino acids are precursors for metabolic pathways (see, Buchanan et al., 2000). Glutamine is the precursor of heme groups and chlorophylls and methionine, respectively its direct activated product, S-adenosylmethionine (SAM), is precursor of the vitamin biotin, or is used directly as a major methyl group donor of numerous reactions of the C1 metabolism in cells, to synthesise the ‘aging’ hormone ethylene responsible for fruit ripening as well as the precursor of polyamines which are involved in stabilising and regulating DNA. The aromatic amino acids are beside other compounds precursors of pigments, phytoalexins, structural compounds as lignins, (here methionine is involved again in methylation reactions) as well as precursor of the plant hormone auxin and an almost innumerable number of secondary metabolites plants being able to synthesise. Furthermore, amino acids are involved in stress responses such as scavenging of active oxygen species, either directly or through regeneration of ascorbic acid by glutathione, a tripeptide (γγ-glutamyl-cysteinyl-glycine) or metal detoxification by phytochelatins (poly-glutathione). Thioredoxins are involved in controlling the redox status of a cell; they control photosynthesis by transmitting the dark-to-light switching signal. These examples are just a glimpse of the role of amino acids and peptides; several more are likely to emerge in the coming years.
Nutrient Uptake and Assimilation
Among the various minerals essential for plant growth the most limiting macronutrients are nitrogen, phosphorus and sulphur. A limitation of any of these minerals limits biomass formation in natural ecosystems and, in particular, plant production in agriculture.
Nitrogen Assimilation and Reduction
Nitrogen represents the mineral nutrient required in the largest quantities by plants and is most limiting where maximal biomass production is desired (Stitt, 1999; Tischner, 2000). The uptake and metabolism of nitrate and ammonia have been extensively investigated and analysed (Figure 27.1). It is now established that all steps of primary nitrogen assimilation are targets of several signal transduction cascades that integrate external stimuli and internal conditions of the plant. Within this regulatory network, nitrate reductase (NR) catalyses one of the most controlled reactions in plants, receiving input from light, photosynthesis, CO2, oxygen availability and nutrient status at the transcriptional and post-translational level. Nitrate uptake systems across membranes exist as high- and low-affinity forms. High-affinity nitrate transporters are either encoded by nitrate inducible or constitutive genes and have Michaelis-Menten constants for nitrate of 6–100 μμM. They are mostly expressed in the outer layers of roots to mediate increased uptake when the external nitrate supply is low. A constitutive low-affinity uptake system operates at nitrate concentrations above ~0.25 mM. Whereas the role of high-affinity transport systems is evident, the function of low-affinity transport systems is less clear. Optimal plant growth is achieved with balanced ratios of nitrate and ammonia but not with nitrogen source alone. The relative contributions of nitrate and ammonia to total nitrogen uptake differ considerably between plant species and ambient availability in soil. Ammonia uptake is mediated by families of active membrane transporters (Km <0.5–40 μμM), which exhibit differential expression in response to light, tissue and nutrient status.
The conversion of nitrate to nitrite is performed by NR, and of nitrite to ammonia by nitrite reductase (NiR). NR is cytosolic, mainly located in root epidermis and cortical cells and leaf mesophyll cells. NiR is chloroplast localised and encoded in the nuclear genome. In photosynthetic tissues reducing equivalents for the reduction of nitrate to ammonia are derived directly from photosynthetic electron transport. Reduced ferredoxin (Fd) is the electron donor that fuels the catalytic activity of NiR. In non-photosynthetic tissue, NADPH derived from the oxidative pentose phosphate pathway can be used to generate ammonia from nitrate. Experimental supplementation with nitrate induces genes of nitrate assimilation and carbon metabolism, mainly to provide carbon skeletons as acceptors for reduced nitrogen. Cross talk between assimilation pathways is further indicated by the finding that low sugar levels are able to repress NR expression, even in the presence of otherwise inductive nitrate concentrations and a down-regulation of NR expression is even observed under conditions of sulphate limitation. However, neither the chemical nature of the signal compounds nor the sensors responsible for these reactions are precisely known. Nitrate and ammonia could be sensed as free ions by extra cellular or intracellular receptors, but downstream products or the carbon could also exert regulation as well as the nitrogen to carbon ratio.
Though the picture is far from being resolved a number of nitrogen sensing and regulatory systems have been described such as a MADS-box like transcription factor responding to local nitrate supply, a transcription factor of the myb structural family and a putative protein kinase gene up regulated by nitrogen deficiency. Furthermore, an extra cellular nitrate reduction system which might act as a nitrate sensor via the release of nitric oxide (NO), GLB1 which is a structural PII homologue of the bacterial glutamine synthetase regulator and up regulated by light and sucrose, respectively repressed by glutamine and glutamate, and 14-3-3 protein family members that reversibly bind to phosphopeptide motifs in diverse target proteins in plant, fungi and animals, resulting in altered activities of enzymes and regulatory proteins.
Sulphur Assimilation and Reduction
Sulphate uptake and assimilation are carried out in plants by a unique pathway that is distinct from that in bacteria and fungi (Anderson, 1990; Leustek and Saito, 1999; Hawkesford, 2000; Hawkesford and Wray, 2000; Leustek et al., 2000; Saito, 2000; Grossman and Takahashi, 2001; Hawkesford, 2002) (Figure 27.2). Beside the most regulatory step, the sulphate uptake by roots which enables the plant to achieve the inner-cellular homeostasis, significant regulatory steps of sulphur incorporation into organic compounds are catalysed by adenosine 5′-phosphosulphate reductase (APR). APR controls the flux of intermediates to yield sufficient reduced sulphur (Suter et al., 2000; Tsakraklides et al., 2002). A further step of control is exerted by the cysteine synthase complex providing the carbon/nitrogen backbone for cysteine formation (Blaszczyk et al., 1999; Harms et al., 2000). Alterations to any of these three processes can have profound effects on cysteine biosynthesis and on the capacity of plants to grow in soils in which nutrient resources are limiting. Essentially most of the reduced sulphur is channelled from cysteine into methionine, Fe/S clusters, vitamin cofactors and proteins required to carry out crucial structural, catalytic and regulatory functions in the cell. Despite this importance for plant biochemistry, plant sulphur metabolism has been much less thoroughly investigated than that of nitrogen. It has, however, gained much more attention in recent years after the unexpected observation of sulphur limitation in agricultural production at least in Europe due to reduced aerial pollution.
Although the uptake and transport of sulphate probably take the same combined apoplastic/symplastic route as, for example, nitrate and phosphate, the sulphate ion seems to be much less mobile after deposition in vacuoles of the source tissue. Sulphate transporters have been cloned and functionally characterised from several species. They can be grouped into high (Km 0.1–1 μμM) and low (Km 1–10 μμM) affinity proton/co-transporters. Expression analysis of sulphate transporters demonstrated that they are present in root hairs and epidermis for sulphate acquisition and in vascular bundles of root and leaf for the allocation of sulphate. Several sulphate transporter genes are induced within hours of sulphate deficiency and are rapidly repressed upon renewed supply of sulphate. Once the sulphate enters the cell it is activated by ATP sulphurylase to form adenosine 5′-phosphosulphate (APS). ATP sulphurylase (ATP-S) isoforms in plants are located either in plastids or in the cytosol. The cDNAs for these isoforms were first isolated from potato. In Arabidopsis there appear to be at least three plastidic and one putatively cytosolic ATP-S. The APS generated by ATP-S can serve as a substrate for sulphate reduction or can be phosphorylated by APS kinase to yield 3′-phosphoadenosine 5′-phosphosulphate (PAPS). PAPS is the substrate of various sulphotransferases to catalyse the sulphatation of a range of metabolites including flavanols, choline, and glucosides.
The sulphate of APS is reduced to sulphite by the plastid-localised APS sulphotransferase, also termed APS reductase (APR). The reductant used by the enzyme is probably reduced glutathione; a domain of the enzyme resembles a glutathione-dependent reductase. APR transcript accumulates during S-starvation, suggesting that a key juncture for controlling assimilatory processes occurs at the point at which APS interacts with either APS kinase or APR. The sulphite generated in the APR catalysed reaction is reduced to sulphide by plastidial sulphite reductase (SiR), the gene of which has been recently identified. Electrons used for sulphite reduction are donated by reduced ferredoxin. Sulphide is finally transferred to activated serine, O-acetyl serine, yielding cysteine through the activity of the enzyme O-acetyl serine-(thiol) lyase (OASTL). Cysteine is the first organic compound carrying reduced sulphur and the precursor of all following metabolic steps carrying a sulphur or thiol moiety.
The allocation of reduced sulphur proceeds via the phloem. Glutathione (GSH) and S-methylmethionine (SMM) appear to have a role in transport as well as being an interorgan signal for the sulphur status from the shoot to the root. In plants such as wheat, substantial amounts of reduced sulphur are transported as SMM from source leaves to sink tissues. Similar to nitrate assimilation, either sulphate or its downstream metabolites are suspected to trigger changes in the mRNA levels of the sulphate transporter and APR genes. An activating effect on uptake and APR activity has been demonstrated for O-acetylserine (OAS), an intermediate in cysteine biosynthesis (for review see Buchanan et al., 2000). An investigation of changes in OAS levels may suggest a link between sulphur, nitrogen and carbon metabolism, because external supplies of these macronutrients mutually affect at least single steps within the assimilatory activities of each pathway and photosynthesis.
As a number of amino acid related topics will be covered through other sections of the handbook, this chapter will highlight examples in which combined molecular, biochemical and genetic approaches have helped to define the pathways and uncover regulatory mechanisms of amino acid biosynthesis in plants. Especially, we will focus on biotechnology driven research and its implications for both basic and applied research. Rational engineering of amino acid biosynthesis is exploited for as diverse aspects as herbicide design and quality improvement of crop plants. A comprehensive review of the general biochemistry of amino acid synthesis can be found elsewhere (Miflin and Lea, 1990; Singh et al., 1992; Singh, 1999; Buchanan et al., 2000).
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