Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Role of carbohydrates in proline accumulation in wilted barley leaves

The effect of wilting on proline synthesis, proline oxidation, and protein synthesis—all of which contribute to proline accumulation—was determined in nonstarved barley (Hordeum vulgare L.) leaves. Nonstarved leaves were from plants previously in the light for 24 hours and starved leaves were from plants previously in the dark for 48 hours. Wilted leaves from nonstarved plants accumulated proline at the rate of about 1 μmole per hour per gram of fresh weight whereas wilted leaves from starved plants accumulated very little proline. Wilting caused a 40-fold stimulation of proline synthesis from glutamate in nonstarved leaves but had very little effect in starved leaves. Proline oxidation and protein synthesis, on the other hand, were inhibited by wilting in both nonstarved and starved leaves. Thus, the role of carbohydrates in proline accumulation is to supply precursors for the stimulated proline synthesis. These results further indicate that the main metabolic response causing proline to accumulate in wilted barley leaves is the stimulation of proline synthesis from glutamate. The difference between these results and those obtained with beans is discussed.

Wilting caused an increased conversion of glutamate to other products. In nonstarved leaves, conversion to organic acids as well as to proline was increased. In starved leaves, wilting caused an increase in the conversion of glutamate to glutamine, aspartate, asparagine, and organic acids.

     

The process of abscisic acid-induced proline accumulation and the levels of polyamines and quaternary ammonium compounds in hydrated barley leaves

The contents of polyamines and quaternary ammonium compounds (QAC), substances involved in several kinds of stress phenomena, were tested in abscisk acid (ABA)-treated barley leaves (Hordeum vulgare L. cv. Georgie) accumulating pro-line. The characteristic parameters of the accumulation of proline induced by ABA [i.e. kinetics of accumulation, synergistic interaction hormone-K(Na)Cl, enhancing effect of Cl-, inhibiting effect of tetraethylammonium chloride (TEA), D-mannose and glucosamine] were tacking as far as polyamine and QAC content was concerned. Moreover: i) ABA slightly decreased the level of spermine and spermidine, slightly increased that of putrescine but did not influence the level of QAC; ii) the content of polyamines was reduced by KCl; iii) treatment with sorbitol increased the level of polyamines and prevented proline accumulation induced by ABA. These results indicate that there is no relationship between ABA-induced proline accumulation, polyamine level and QAC level; furthermore, accumulation of proline by ABA treatment is possible without increasing the levels of polyamines and QAC.     

Metabolism of [5-h]proline by barley leaves and its use in measuring the effects of water stress on proline oxidation

The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H₂O as a measure of proline oxidation. When [5-³H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H₂O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and γ-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the ³H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring ³H₂O recovery from [5-³H]proline were only slightly lower than rates estimated by incorporation of ¹⁴C into oxidized products and loss of ¹⁴C from total proline. Therefore, ³H₂O recovery from [5-³H]proline is useful in assessing the effects of stress on proline metabolism.

Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation.

     

Thioproline-induced accumulation of proline in barley leaf sections

Abstract Treatment of barley leaf sections with 0.1 mol m^?3 thioproline (L-thiazolidine-4-carboxylic acid) was found to induce a marked increase in proline together with some decrease in glutamate, whereas the levels of other andno acids were not influenced. This result is discussed in relation to the significance of the increase in proline in tissues treated with abscisic acid or subjected to water stress.     

The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.     

Effect of light on abscisic acid‐induced proline accumulation in leaves: comparison between barley and wheat

Light enhanced the abscisic acid‐induced accumulation of proline in barley ( Hordeum vulgare L. cv. Georgie) and wheat ( Triticum durum L. cv. Valnova). In wheat ABA is ineffective in the dark. In both barley and wheat, the accumulation of proline in the light showed the same characteristics as those of the process that occurs in barley in the dark, namely a synergistic interaction between the hormone and K(Na)Cl, an enhancing effect of Cl⁻ anion in excess over K⁺ cation in the incubation medium, and an inhibiting effect of D ‐mannose and monensine. In wheat, furthermore, light is needed during treatment with ABA if proline is to accumulate. Light was effective in both wheat and barley during the second or accumulation phase of the hormonal process, whereas the events occurring in the first (or lag) phase did not require light. The results suggest that in wheat light induces a putative factor(s) involved in the proline accumulation pathway that is lost in the dark, whereas in barley it is present in the dark.     

Effects of weak acids on proline accumulation in barley leaves: a comparison between abscisic acid and isobutyric acid

Abstract. When isobutyric acid (IBA) or abscisic acid (ABA) are supplied to leaf sections a similar rapid and marked decrease in the intracellular pH is observed. This acidification is accompanied by an increase in proline level which is about the same for both 3 mol m⁻³ IBA and 1 mol m⁻³ ABA treatments.
Fusicoccin (FC), known to act at the proton pump level, almost completely suppresses the ABA-induced acidification of the cell sap, whereas it only partially counteracts the acidifying effect of IBA, in particular during short periods of treatment. This effect of FC is paralleled by a similar inhibition of the induced proline accumulation: in fact, FC completely suppresses the ABA-induced increase in proline during short treatment periods, whereas it is only effective in inhibiting the IBA-induced proline accumulation after long treatment periods.
These data seem to suggest that the ABA- and IBA-induced changes in proline level might be mediated by changes in the intracellular pH.     

Changes in foliar proline concentration of osmotically stressed barley

The amino acid proline is accumulated in plant tissues in response to a variety of stresses. The existence of two routes for its biosynthesis is well documented. However, little is known about the contribution of each pathway to the accumulation of free proline under stress conditions. In the present study young barley plants were subjected to osmotic stress by treating their roots with 25% polyethylene glycol. Prior to stress imposition roots were incubated for 24 h in nutrient solution containing proline or one of its metabolic precursors: glutamate and ornithine. Free proline quantity in the leaves was measured before and after stress. Relative water content (RWC) was used as a measure of the plant water status. Foliar proline levels showed a significant increase in ornithine- and proline-pretreated plants compared to the control. Nevertheless, no considerable changes in leaf RWC were observed. It was shown that before stress application only ornithine but not glutamate was immediately metabolized to proline. Under stress conditions, however, both precursors were converted into proline. The possible role of this amino acid in the processes of post stress recovery is discussed.     

Regulation of sucrose-sucrose-fructosyltransferase in barley leaves

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

Salicylic acid decreases the levels of dehydrin-like proteins in Tibetan hulless barley leaves under water stress

The effects of salicylic acid (SA) on the accumulation of dehydrins in leaves of Tibetan hulless barley seedlings under water stress were investigated. The results indicated that SA decreased the levels of the four dehydrin-like proteins induced by water stress. The concentrations of these dehydrin-like proteins increased under water stress. However, their levels in SA-pretreated seedlings were always lower than in those receiving only water stress. Our results also indicated that the levels of dehydrin-like proteins decreased as the SA concentration increased. In SA-pretreated seedlings, electrolyte leakage, MDA and H2O2 content were rather higher than in seedlings receiving only water stress. By these results, we suggest that lower levels of dehydrin-like proteins in seedlings with SA treatment may be due to the greater accumulation of H2O2 induced by SA, which causes more oxidative injury under water stress.