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Researclh inito the physiological effects of gibberellic acid (GA) and other gibberellins has been confined largely to descriptive accounts of stimulatory (3) and inhibitory (18) actions, while few studies have attempted to integrate the action of GA with the normal physiological processes of the plant. One of the most clearly recognized stimulatory actions of GA is its ability to break dormancy (3), or conversely, its germination-promoting effect. Hayashi (7) demonstratedI that treatment with gibberellins hastened the germination of barley and rice and, more recently, other wvorkers have reported similar successes also with barley (8,12,19 22, 1959. increases, however, as germination progresses, and it is enhanced by compounds which stimulate germination. In addition, maltose is a compound commonly found in germinating grain and, with the exception of D-enzyme, the amylases are the only enzymes in higher plants known to be capable of its formation. It is difficult to conceive of starch hydrolysis in the endosperm taking place in the absence of amylase activity, and any indication of the release of sugar coincident with, and possibly even dlependent upon amylase activation would be useful in estimating the endogenous role of this enzyme. MATERIALS AND METHODSAll water used in these experiments was doubly glass distilled, and all GA solutions were freshly prepared the day of the experiment.RESPIRATION. Hordeurn distichumii var. Baku (a naked seed) and Prior (a husked seed) from the 1956 harvest were used. The husk of the Prior seeds was removed by hand and seeds of both varieties were cut in half transversely before treatment was commenced. The endosperm halves were incubated in the dark at 250 C for 18 hours. Petri dishes (4 in) containing eight endosperm halves and two sheets of filter paper, moistened with either 8 ml water or 8 ml water plus 200 gammas GA, were used. Following incubation, each sample of endosperm was rinsed thoroughly with water, blotted and weighed. The samples were then placed in Warburg flasks containing solutions similar to those of the initial soaking period (i.e., either 2 ml water or 2 ml water plus 200 gammas GA). The center wells contained 0.2 ml 10 % NaOH and the temperature was maintained at 250 C. STORAGE CONDITIONS. As noted by Merry and Goddard (10), seeds with a constant initial moisture content evidence much less variability than seeds with differing moisture contents. Consequently, Merry and Goddard's storage technique was adopted for all seeds other than those used in the respiration experi-

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The conversion of proline to glutamic acid and hence to other soluble compounds (prolne oxidation) proceeds readily in turgid barley (Hordeum vulgare) leaves and is stimulated by higher concentrations of proline. This suggests that proline oxidation could function as a control mechanism for maintain*ag low ceilular levels of proline in turgid tissue.In water-stressed tissue, however, proline oxidation is reduced to negligible rates. These results are consistent with the idea that proline accumulation results from inactivation by water stress of normal control mechanisms. It seems likely that inhibition of proline oxidation is necessary in maintaining the high levels of proline found in stressed barley leaves.Radiotracer experiments have implied that the synthesis of proline from glutamic acid is stimulated by water stress (1, 10). We have confirmed and strengthened these earlier results, and presented evidence that the stimulatory effect of stress is likely to be on P5C2 formation (6), consistent with the implication of other experiments that loss of feedback control of proline biosynthesis occurs in barley leaves during water stress (4). These studies also produced evidence that proline oxidation may occur rapidly in barley leaves (4) and that water stress may reduce its rate (3,6). In this paper, we present more detailed information concerning proline oxidation and its response to proline concentration and water stress. MATERIALS AND METHODSGrowth of barley (Hordeum vulgare cv. Prior) plants and methods of wilting, feeding of radioactive precursors, and sample analysis have been described (6). Two-week-old plants were wilted intact by flooding the rooting medium with polyethylene glycol-4000 (30 g/100 ml) 1 hr before beginning the experiment; alternatively, second leaves were excised and wilted to 75% of initial fresh weight in a lighted fume hood. The latter wilting procedure (which also took about 1 hr) was preferred as it gave more reproducible rates of water loss. [14C]proline, radioactivity was recovered about 60% as soluble products and 40% as protein. After acid hydrolysis, the proteinbound radioactivity was found to be 85 to 90% proline, with the rest as glutamic and aspartic acids. In the soluble fraction, measurable radioactivity was recovered in glutamate, glutamine, aspartate, y-amino-butyrate, alanine, and several ninhydrinnegative spots. Traces of 14C were detected in glycine, serine, asparagine, citrulline, and arginine. Although not measured in this experiment, 14CO2 has constituted less than 1.5% of the total radioactivity under comparable conditions. Table I (column 1) shows the distribution of radioactivity in these metabolites as a percentage of the total soluble radioactivity. Glutamate was always the most heavily labeled proline metabolite, and in short experiments (10-15 min), was the only labeled compound detected autoradiographically. This suggests that metabolism of proline consists of proline oxidation (conversion to glutamate) followed by further metabolism of glutamate. In ...

Glutamine synthetase from barley (Hordeum disticeaim L.) is precipitated by polyethylene glycol (PEG). Proline, in a concentration-dependent manner, reduces the amount of enzyme precipitated by PEG, although the effect of the imino acid can be counteracted by raising the level of PEG. The effect of PEG is a function of mer number and concentration and the influence of both elements can be ameliorated by proline. PEGinduced enzyme precipitation is a function of pH, as is its interaction with both proline and betaine in the reaction. The lack of effect of amount of enzyme on the proline and PEG effects supports the conclusion that, in this system, proline and PEG do not function through interaction with the protein. Other compounds, such as glycine, glucose, and sucrose, can decrease the PEG-induced precipitation of the enzyme, although glycerol was not active under the conditions employed.The results are consistent with the proposition that a protein-containing system in which high concentrations of proline and/or betaine are present, is better 'protected' against the biologically unfavorable consequences of dehydration-induced thermodynamic perturbation.The accumulation of the imino acid, proline, in the free uncombined form is a characteristic response of many plants to many types of stress (3). The interpretation of this response has varied from its description as a useful criterion for the selection of varieties suited to arid areas (27) to nothing more than a measure of the rate of senescence (9). Choosing between these extreme interpretations is difficult since plants cannot be deprived of proline, and external applications are confounded by hydration and penetration problems on the one hand, and metabolism and compartmentation of the applied compound on the other.Recent attempts to demonstrate a conceptually useful role for the accumulated proline (which may reach submaximum levels as high as 0.1-0.3 M in the wheat apex [17] or 5-10% of the dry weight of halophytic tissue [281) have centered on the amelioration of deleterious effects of heat, pH, salt, and chemicals on enzyme activity in in vitro and organelle systems (1,4,19,21,22,35). In all cases reported, significant and, indeed, important proline concentration-dependent protection against a range of perturbing or stressful conditions was afforded to several enzymes. among, if not the most prominent, and is clearly one which causes large scale proline accumulation. No in vitro system has been described with which deleterious effects of dehydration on enzyme activity can be measured, and, thus, it has not been possible to directly test the hypothesis that proline may diminish the effect of dehydration on enzymes and/or enzyme activity. Nonetheless, solvation effects do influence thermodynamic activation parameters ofenzymes (8, 13) and any component which has the ability to effect enzyme solvation may also be expected to influence enzyme activity.A system with which to test the ability of proline to effect enzyme solvation was developed from the...

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.

Barley (cv.Prior) and radish (cv.White Icicle) plants were subjected to heat stress (39�C) for 1–5 days when growing in either a high (90–95 %) or low (50%) relative humidity environment. Although the plants were watered daily, leaf water potential (ψw) in heat-stressed plants at low relative humidity fell to a low level (– 30 bars, day 5) whereas there was no such decrease in the leaves of those maintained at a high humidity (– 4.6 bars). Growth in height was inhibited and leaf chlorophyll concentration decreased by high temperature, these effects being accentuated by accompanying water-stress in the leaves. On the other hand, proline only accumulated in the leaves when (ψw) fell, there being apparently no direct response to elevated temperature. These effects were confirmed for a range of temperatures extending to a maximum of 41�C. Proline accumulated rapidly, after an initial lag phase, in both barley and radish plants exposed to a low temperature (4�C). This accumulation was unrelated to any changes in the leaf water relationships. Neither leaf water potential, osmotic potential nor turgor demonstrated changes with low temperature sufficient to account for the accumulation of proline. The characteristics of this cold- induced accumulation of proline are compared with those of water stress-induced accumulation. * Part V, Aust. J. Biol. Sci., 1973, 26, 319–27.