Effect of Water Stress on Proline Synthesis from Radioactive Precursors

Abstract

Barley (ffordeum vulgare L. var. Prior) leaves converted more '4C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (A1-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of 14C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.Although several plants have been reported to accumulate free proline during periods of water deficit (2,12,15,16,18) or when subjected to other stress (7, 8), the biochemical changes linking water stress and proline accumulation are not well understood. Barnett and Naylor (2) and Morris et al. (13) have observed increased incorporation of 14C-glutamic acid into proline in wilted leaves as compared to turgid leaves of Bermuda grass and turnip. This increase in radiotracer incorporation might reflect a stimulation of proline biosynthesis by water stress, but the data presented did not fully eliminate the possibility that inhibited protein synthesis might account for the results.The Bermuda grass experiment was done after a long period of drought, so that the large amount of free proline already present during "4C-glutamate feeding could have acted as a trapping pool for radioactive proline, accounting for the results in the absence of an actual increase in the rate of conversion of glutamate to proline. In this paper we describe 14C-glutamate feeding experiments designed to avoid these difficulties in interpretation, and to localize the point in the proline biosynthetic pathway at which a stress-induced stimulation would most likely occur.

Removal of Feedback Inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress

“…This is contradictory to the fact that plants under stress continue to synthesize Pro and build up the Pro pool. To explain this paradox, it has been assumed that the P5CS enzyme may undergo a conformational change and lose its feedback regulation property (Boggess et al, 1976a(Boggess et al, , 1976b. We reasoned that if the feedback regulation of the wild-type P5CS is completely lost during stress, then expression of the mutant P5CS, i.e.…”

“…Earlier experiments suggested that Pro accumulation in plants under stress may involve the loss of feedback regulation due to a conformational change in the P5CS protein (Boggess et al, 1976a(Boggess et al, , 1976b. In bacteria, Pro biosynthesis has been shown to be regulated by the end product inhibition of ␥-GK activity (Smith et al, 1984).…”

Contribution of Arginine to Proline Accumulation in Water-stressed Barley Leaves

“…Experimental procedures were as previously described (3). Briefly, second leaves of 2-week-old barley plants (Hordeum vulgare cv Prior) were excised and allowed to take up radioactive precursor (L-U-_4C-arginine, 50 mCi/mol, obtained from Amersham/Searle and purified by elution from Dowex 50 in the ammonium form) in 4 ,1l aqueous solution.…”

“…Glutamic acid and arginine are likely precursors for water stress-induced proline synthesis, as either may be converted to P5C (Al-pyrroline-5-carboxylic acid) which is the immediate precursor of proline. While glutamate is clearly involved in proline synthesis during water stress (1,3,6), the contribution of arginine has not been evaluated. Although leaf tissues typically contain little free arginine, the inexplicably low protein-arginine content of wilted Bermuda grass leaves (1) suggests that in at least some cases, arginine might contribute significantly to the build up of free proline that occurs during water stress.…”

Rapid proline uptake in cultured tobacco cells and inhibition by a proline analog

“…High intracellular proline concentrations may protect against water stress, perhaps by an osmolar effect [l&19]. Proline accumulation probably results from an increased rate of synthesis, though transport may be important in some cases [20]. Studies on proline transport have been reported for algae, plant tissues and organelles [21-251. cultured embryos [30] and cells [29,3 1,321.…”