A regulatory role for proline metabolism in Arabidopsis thaliana (L.) Heynh.
Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Of these, proline is the most extensively studied. Conclusive demonstration that this imino acid acts as a compatible solute which mediates osmotic adjustment has yet to be achieved, although a causal relationship between increased proline synthesis and plant tolerance of hyperosmotic stresses has previously been demonstrated. It is proposed that in many plants, the metabolic implications of the regulated increase in proline synthesis and/or a decline in proline degradation during stress may play a more important role in acclimation to adverse conditions than the simple accumulation of the end-product of these adjustments. In particular, the stress-induced increase in the transfer of reducing equivalents into proline by Δ¹-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) may be a protective mechanism whereby many species ameliorate shifts in cellular redox potential which accompany all biotic and abiotic stresses which cause proline accumulation, including those that do not cause cellular dehydration. The presence of several putative stress-regulated promoter elements in the AtP5CS1, AtP5CS2 and AtP5CR genes of Arabidopsis thaliana strongly implicates an adaptive role for stress-induced increases in proline synthesis in this species. Sequence homologies of several regions within the 5' untranslated regions of these genes to promoter elements which have been shown to participate in redox control of gene expression, the actions of phytochrome and hormones, and tissue-specific regulation of gene expression are also identified. These provide useful indicators both of the mechanisms by which proline synthesis is regulated and how these may relate to its importance in maintaining metabolic homeostasis. In an attempt to resolve the functionality of proline accumulation under stress, chimeric antisense genes comprising 1050 bp and 999 bp fragments of Arabidopsis cDNAs encoding AtP5CS1 and AtP5CR respectively were inserted in the reverse orientation between the CaMV 35S promoter and the GUS gene (encodes β-glucuronidase) in the plant transformation vector pBI121. These constructs were introduced separately into Arabidopsis by cocultivation with Agrobacterium tumefaciens strains carrying the pBI-P5CS1 (AS) and pBI-P5CR(AS) plasmids. Transgenic plants, which were selected on the basis of kanamycin resistance, regenerated at a low frequency in the presence of 1 mM proline. Transformation of 13 pBI-P5CS1(AS) and 7 pBI-P5CR(AS) lines was confirmed by PCR-mediated amplification of gene fragments within the introduced T-DNA. Segregation ratios for kanamycin resistance indicated that most of the lines have multiple T-DNA insertions. Transformants were characterised with respect to their growth rates and free proline content. In at least two pBI-P5CS1(AS) transformants and two pBI-P5CR(AS) transformants, a reduction in root growth rates in the presence of inhibitory concentrations of NaCI correlated with reduced β-glucuronidase activity relative to transgenic lines that were no more sensitive to NaCI than were controls. A reduction in root growth rate both in the absence and presence of hyperosmotic stress was noted in two pBI-P5CS1(AS) transformants, designated A5 and B12. In 14 day-old plants of the T₂ generation of both A5 and B12, free proline levels were significantly lower than in wild-type plants both in the absence of stress and following 24 h incubation in either 250 mM NaCI or 550 mM sorbitol or at 5 °C. In both lines, reduced growth rates in the absence of osmotic stress could be restored by exogenous proline, but not by exogenous glutamate. When used at isosmotic concentrations, sorbitol caused a larger reduction in free proline levels in both A5 and B12 than did NaCI. This observation may relate to an ABM-mediated post-transcriptional effect on AtP5CS1 gene expression which affects NaCI-, but not sorbitol-mediated proline accumulation in Arabidopsis. Post-transcriptional regulation of the expression of the genes involved in proline biosynthesis may account, at least partly, for the absence of dramatic phenotypic effects in any of the pBI-P5CS1(AS) or pBI-P5CR(AS) lines. Under the premise that regulation of shifts in proline metabolism regulate cellular redox potential under conditions of stress may be mirrored by the involvement of proline metabolism in modulating metabolism during normal growth and development, the effects of exogenous proline on Arabidopsis seed germination, seedling growth and in vitro shoot organogenesis were investigated. A dose-dependent inhibition of radicle emergence by millimolar concentrations of proline could be overcome by the artificial oxidants methylene blue and phenazine ethosulphate. Assays of the rate-limiting dehydrogenases of the oxidative pentose phosphate pathway (OPPP), as well as changes in the contributions of ¹⁴C₁ - and ¹⁴C₆ -labelled glucose to respired CO₂ during germination, are consistent with activation of the OPPP during Arabidopsis seed germination. An approximately four-fold increase in free proline, which peaked at the time of radical emergence, was not parallelled by changes in other amino acids and could not be ascribed to degradation of seed storage proteins. Delayed radical emergence in T₂ generation seeds of the pBI-P5CS1(AS) lines A5 and B12 correlated with an approximately 35% reduction in the maximal concentration of proline accumulated during germination. Millimolar concentrations of exogenous proline had a dose-dependent inhibitory effect on Arabidopsis seedling growth both in the light and in darkness. This reduction in growth arises at least in part from a decline in cell elongation. Accordingly, exogenous proline increased total extractable peroxidase activity in Arabidopsis seedlings through the selective induction of peroxidase isoforms. Histochemical analysis of the hypocotyls of plants grown in the presence of exogenous proline suggested that proline increased the levels of lignin and/or the phenolic precursors thereof. A dose-dependent decrease in extractable chlorophyll and damage to chloroplastic and mitochondrial ultrastructure was observed in 21 day-old Arabidopsis seedlings grown in the presence of millimolar concentrations of exogenous proline. In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mM proline, and to a lesser extent by 5 mM proline, but inhibited by inclusion of 10 mM proline in the hormonallysupplemented regeneration media. The ability of low concentrations of proline analogues (azetidine-2-carboxylate and thioproline) to overcome the stimulatory effect of 1 mM proline, and a slight increase in the stimulative effects of 1 mM proline by D-proline, are consistent with an important role for the interconversions of proline and its precursors in regulating cell division and differentiation. Together, these data strongly support an important role for the interconversions of proline and its precursors in the regulation of intermediary metabolism under both normal and stressful conditions. These findings draw into question the widely accepted, although poorly investigated, hypothesis that proline is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular metabolism. The novel proposal that stress-induced changes in proline metabolism exert a regulatory effect though an influence on the level of reduction of the cellular NADP pool is discussed in relation to recent evidence that a signal related to proline synthesis and/or degradation selectively increases the expression of stress-induced plant genes.