A regulatory role for proline metabolism in Arabidopsis thaliana (L.) Heynh.
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Date
1998
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Abstract
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.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.
Keywords
Arabidopsis thaliana., Plants, Effect of stress on., Proline., Plant molecular genetics., Theses--Botany.