Pyrroline-5-carboxylate reductase (P5CR) has been identified and characterized in several plant species (e.g. Kreuger et al, 1986; Treichel, 1986; LaRosa et al, 1991). A chloroplast localization of this enzyme has been reported in pea (Rayapati et al, 1989).

However, there have been no reports of GK or GPR activities in higher plants since the first report of the occurrence of in vitro synthesis of ¹⁴C-GSA from ¹⁴C-glutamate, Mg²⁺, ATP and NADPH in cell free extracts of beet (Morris et al, 1969). The enzyme activity described by Morris et al (1969) is similar to that reported for the pyrroline-5-carboxylate synthase (P5CS) activity of mammalian cells (Lodato et al, 1981; Wakabayashi and Jones, 1983), where a bifunctional enzyme catalyzes the first two steps of proline biosynthesis (Aral et al, 1996).

Substantial progress has been made in recent years in the cloning of cDNAs encoding the proline biosynthetic enzymes from higher plants by complementation of proline-requiring mutants of Escherichia coli (Verma et al, 1992).

A cDNA encoding P5CR was cloned by direct complementation of an E. coli proC proline auxotroph with a soybean nodule cDNA expression library (Delauney and Verma, 1990), facilitating the isolation of P5CR homologs from Pisum sativum (Williamson and Slocum, 1992) and Arabidopsis thaliana (Verbruggen et al, 1993).

P5CR transcripts increase in abundance in response to osmotic stress, indicating that P5CR gene transcription is under osmotic stress control (Delauney and Verma, 1990; Williamson and Slocum, 1992; Verbruggen et al, 1993). The P5CR gene of Arabidopsis is developmentally regulated. The P5CR promoter directs strong GUS expression in root tips, the shoot meristem, guard cells, pollen grains, ovules and developing seeds (Hua et al, 1997).

The P5CR cDNA from soybean, when over-expressed in tobacco, did not result in a significantly increased proline level in the transgenic plants, despite a 100-fold greater P5CR activity than wild-type (Szoke et al, 1992). Thus, P5CR may not be the rate-limiting step in proline accumulation (LaRosa et al, 1991; Delauney and Verma, 1993).

A cDNA encoding both GK and GPR was isolated from a mothbean cDNA expression library employing E. coli proA, proB and proBA proline auxotrophs, and screening for cDNAs which permit growth in the absence of proline. This cDNA was found to encode a bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) (Hu et al, 1992). The single major open-reading frame of this cDNA encodes a polypeptide of 73.2 kDa which has two distinct domains exhibiting 55.3% overall similarity to E. coli GK and 57.9% similarity to E. coli GPR, respectively (Hu et al, 1992) [note that to find plant P5CS clones in the ExPASy Enzyme database consult: gamma-glutamyl kinase (GK) and glutamyl phosphate reductase (GPR) (glutamate-5-semialdehyde dehydrogenase)].

The GK of the bifunctional P5CS of Vigna is inhibited by proline (50% inhibition with 6 mM proline) (Hu et al, 1992). This enzyme appears to be much less sensitive to feedback inhibition than the wild-type GK of E. coli (Hu et al, 1992).

Northern analyses indicate that the P5CS gene is induced (particularly in leaves) by treatment of Vigna plants with 200 mM NaCl (Hu et al, 1992). Desiccation, salinity stress and ABA dramatically induce P5CS mRNA transcript abundance in Arabidopsis (Yoshiba et al, 1995). Savoure et al (1997) suggest that the expression of the proline biosynthetic genes are dependent upon at least two signal transduction cascades; one triggered by exogenously applied ABA in the absence of stress, and the other triggered by cold and osmotic stress independently of exogenously applied ABA.

Recently transgenic tobacco plants over-expressing P5CS have been obtained, and these appear to have increased resistance to both water deficits and salinity stress (Kishor et al, 1995). However, insufficient data is available to conclude with certainty that proline accumulation in these transgenic plants contributes to their enhanced stress resistance via osmotic adjustment, or by some other mechanism(s) (Sharp et al, 1996). As discussed by Hare and Cress (1997), the controversial data reported for the water relations of these transgenic plants emphasize the need to investigate non-osmotic explanations for the phenotypes observed.

Hare and Cress (1997) argue that the different subcellular localizations of proline biosynthesis (cytoplasm) and oxidation (mitochondrion), the NADPH cofactor preference of the biosynthetic enzymes, and NADH cofactor preference for the proline oxidation pathway, would enable proline biosynthesis to enhance activity of the cytoplasmic oxidative pentose phosphate pathway, and provide a mechanism of interconversion of the phosphorylated and non-phosphorylated pools of pyridine nucleotide cofactors. They suggest that the osmoprotective effects of proline accumulation may be of secondary importance to the associated metabolic implications of proline synthesis and degradation. Further investigations of transgenic plants enginereed for altered proline synthesis and/or catabolism clearly need to consider not only the consequences on absolute proline level and the biophysical effects exerted by elevated cytoplasmic proline concentrations, but also the fluxes to and from the proline pool (Hare and Cress, 1997).

In growing tissue, the proline deposition rate will be equal to the combined rates of proline synthesis, proline release from protein, and proline import, minus the combined rates of proline catabolism, proline export, proline utilization in protein synthesis, and the rate of pool dilution caused by water uptake during growth (Rhodes and Handa, 1989; Voetberg and Sharp, 1991). The latter will in turn be determined by fundamental plant water relations of the growing region, including water potential, solute potential, yield threshold, turgor, and potential difference between the xylem and growing cells (Nonami et al, 1997), as well as the levels of growth inhibitory metabolites such as ABA, whose synthesis or compartmentation may respond rapidly to changes in turgor and/or metabolism (e.g. intracellular pH) (Rhodes, 1987). The feedback loops in this system are clearly complex, with ABA and osmotic signals altering expression of genes encoding proline biosynthesis enzymes, proline and osmotic signals altering expression of PDH, proline feedback inhibiting its own synthesis, and proline per se (if accumulated to sufficiently high levels) contributing to solute potential, and hence turgor and growth maintenance. The full promise of transgenic plants engineered for proline metabolism in understanding of proline’s role(s) in stress resistance will likely not be realized until attention is given to the growing regions as well as mature tissues (cf. Kishor et al, 1995).

The consideration of growing and non-growing tissues and the fluxes between them will be essential in testing the intriguing scheme recently proposed by Hare and Cress (1997) in which proline and P5C might act as an intercellular signalling system. In this scheme, proline produced from P5C in an "effector" tissue is transported via the phloem to a "target" tissue characterized by a high energy requirement, where proline degradation generates reducing equivalents needed to drive TCA cycle activity. The P5C or glutamate generated may then be translocated back to the effector tissue where conversion to proline regenerates NADP⁺ needed to prime the oxidative pentose phosphate pathway.

P5C or glutamic-5-semialdehyde, or a molecule derived from them, is implicated as a regulator of other osmotic stress responsive genes, including dehydrins and salT (a NaCl, drought, ABA and proline-inducible gene that it a sensitive indicator of abiotic stress) in rice (Iyer and Caplan, 1998). The proline metabolites appear to be more potent inducers of salT than proline (Iyer and Caplan, 1998).

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