Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.

Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.

Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

     

Effect of water stress on proline synthesis from radioactive precursors

Barley (Hordeum vulgare L. var. Prior) leaves converted more ¹⁴C-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 (Δ¹-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.     

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.     

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.

     

Comparative Study of the Metabolism of 1-Aminocyclopropane-1-carboxylic Acid and Senescence of Water-Stressed and ABA-Treated Excised Rice Leaves

Changes in the metabolism of 1-aminocyclopropane-l-carboxylicacid (ACC) during senescence in the light in turgid, water-stressed,and ABA-treated, excised rice leaves were examined. The decreasesin levels of Chl and protein were more rapid in the water-stressedand in the ABA-treated leaves than in the turgid leaves. Inturgid leaves, levels of proline remained very low, but theyincreased considerably as a result of water stress or treatmentwith ABA. The production of ethylene was strongly inhibitedby water stress and by ABA through the inhibition of the synthesisof ACC and/or the conversion of ACC to ethylene. In turgid leaves,the level of 1-(malonylamino)cyclopropane-l-carboxylic acid(MACC) increased with time during incubation in the light. Waterstress resulted in a pattern of accumulation of MACC similarto that in the turgid control. However, ABA blocked the malonylationof ACC. (Received July 27, 1989; Accepted March 12, 1990)     

Water Stress in Leaves of Pbaseolus vulgaris Infected with Xanthomonas campestris pv. phaseoli

Infection of bean leaves ( Phaseolus vulgaris ) by Xanthomonas campestris pv. phaseoli in the field frequently resulted in the appearance of isolated flaccid areas in green leaf tissue adjacent to necrotic and chlorotic lesions. The flaccid leaf areas had significantly higher stomatal resistances compared to nearby turgid areas on the same leaf, and the turgid areas had stomatal resistances that were the same or only moderately elevated compared to those of healthy leaves. The flaccid tissues also had significantly lower relative water contents than turgid tissues on the same leaf demonstrating that pathogeninduced water stress was localized. The levels of free proline, another indicator of water stress, were directly correlated (r²= 0.556) with disease severity. The change in free proline content implied that water stress increased in direct proportion with the amount of tissue infected. Water stress may be due to the disruption of xylem elements by the invasion of X. c. phaseoli from nearby lesions. One result of xylem invasion could have been severe water deficits which were sufficient to cause stomatal closure and leaf flaccidity; however, this effect was highly localized and the remainder of the diseased leaf was either significantly less water stressed or not affected.     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

Senescence of Rice Leaves VI. Comparative Study of the Metabolic Changes of Senescing Turgid and Water-Stressed Excised leaves

Some metabolic changes of senescing turgid and water-stressedexcised rice leaves were compared under incubation in the dark.The decrease of the chlorophyll and protein level and the increaseof the -amino nitrogen were faster in the water-stressed leavesthan in the turgid leaves during the first two or three daysof incubation. However, the changes in levels of chlorophyll,protein and a-amino nitrogen were later retarded by water stress.The rate of decline in soluble sugar was more rapid in the turgidthan in the water-stressed leaves. In turgid leaves, there wasa slight but significant increase in the proline content inthe first day of incubation; subsequently, proline accumulatedrelatively rapidly, yet at a slower rate than in the stressedleaves. The activity of acid inorganic pyrophosphatase increased,but that of alkaline inorganic pyrophosphatase decreased duringthe senescence of both turgid and water-stressed leaves. Theenzyme activities are, therefore, due to two separate enzymeproteins. Water stress enhanced the increase of acid inorganicpyrophosphatase activity only during the first two days; itretarded the decrease of alkaline inorganic pyrophosphataseactivity at later stage of incubation. It is concluded that water stress does not accelerate all themetabolic changes associated with the senescence of excisedleaves. (Received January 24, 1981; Accepted March 27, 1981)     

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.

During the time proline was accumulating, [¹⁴C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [¹⁴C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.

These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

     

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.     

Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants

Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K⁺ + Na⁺) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Effect of water stress on proline catabolism in tobacco leaves

Proline-[¹⁴C] infiltrated into leaf disks of tobacco (Nicotiana tabacum cv BY-4) in the dark was converted to glutamic acid and then metabolized through the TCA cycle. A smaller amount of proline-[¹⁴C] was metabolized when the leaf disks were wilted than when turgid. During a 6 hr period following rehydration, disks converted a larger amount of proline-[¹⁴C] to oxidized products than when wilted, although the proline content of rehydrated disks had not declined. These results indicate that proline oxidation is inhibited by water stress.     

Changes in Free Amino Acids in White and Green Tissues of Variegated Tobacco Leaves during Water Stress

Variegated tobacco leaves, white on one side of the midrib andgreen on the other, were detached from the stem and incubatedunder water stress or turgid conditions for 4 days to determineany changes in the levels of free amino acids. Drastic changes in the free amino acid composition occurredin the green tissue during the water stress period, but onlyvery small changes in the white tissue. During that time, themost striking difference between the two tissues was the largeamount of proline accumulated in the green tissue, but not inthe white. An exogenous supply of sucrose increased the prolinecontent in both tissues during water stress. An exogenous supplyof glutamic acid increased the proline and asparagine contentsin the green tissue, but it increased only the asparagine contentin the white tissue during water stress. (Received May 4, 1982; Accepted August 12, 1982)     

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.

     

Biochemical Changes in Excised Leaves of Oryza sativa Subjected to Water Stress

Excised rice (Oryza sativa L. cv. Ratna) leaves were used to compare the changes in the levels of various biochemical intermediates and enzyme activities during senescence in turgid and water-stressed conditions. Chlorophyll, total protein and soluble protein content decreased but α-amino nitrogen content increased during the senescence of turgid leaves. In the leaves subjected to water stress, these changes were accelerated, the acceleration being greater with higher degree of water stress. Starch, soluble sugars, total carbohydrates and non-reducing sugar content decreased during senescence of turgid leaves. Water stress accelerated the changes in the levels of starch and non-reducing sugar, but the changes in the levels of soluble sugars and total carbohydrates were retarded. Reducing sugar content increased at first and then decreased in the turgid leaves, and water stress accelerated the change. The decline in the catalase activity and the increase in the peroxidase activity with time was faster in the water-stressed leaves than in the turgid leaves. Acid inorganic pyrophosphatase activity increased, but alkaline inorganic pyrophosphatase activity decreased during the senescence of turgid leaves, and such changes were accelerated by water stress. The results suggest that water stress does not accelerate all the processes connected with leaf senescence.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

Leaf developmental stage modulates metabolite accumulation and photosynthesis contributing to acclimation of Arabidopsis thaliana to water deficit

We examined whether young and mature leaves of Arabidopsis thaliana in their response to mild water deficit (MiWD) and moderate water deficit (MoWD), behave differentially, and whether photosynthetic acclimation to water deficit correlates with increased proline and sugar accumulation. We observed that with increasing water deficit, leaf relative water content decreased, while proline and sugar accumulation increased in both leaf-developmental stages. Under both MiWD and MoWD, young leaves showed less water loss and accumulated higher level of metabolites compared to mature leaves. This, leaf age-related increase in metabolite accumulation that was significantly higher under MoWD, allowed young leaves to cope with oxidative damage by maintaining their base levels of lipid peroxidation. Thus, acclimation of young leaves to MoWD, involves a better homeostasis of reactive oxygen species (ROS), that was achieved among others by (1) increased sugar accumulation and (2) either increased proline synthesis and/or decreased proline catabolism, that decrease the NADPH/NADP⁺ ratio, resulting in a higher level of oxidized state of quinone A and thus in a reduced excitation pressure, and by (3) stimulation of the photoprotective mechanism of non-photochemical quenching, that reflects the dissipation of excess excitation energy in the form of harmless heat, thus protecting the plant from the damaging effects of ROS.     

Cell Shape in Leaves of Drought-stressed Barley Examined by Low Temperature Scanning Electron Microscopy

Cells in leaves of well-watered and slowly drought-stressedbarley seedlings were examined by low temperature scanning electronmicroscopy, when the leaves were turgid, when just wilting,and when sufficiently stressed to prevent either regain of turgor(leaf blades) or regrowth (leaf sheath bases) after rewatering.Deformation of the cell surface was a major response to cellvolume reduction during stress. Folds occurred in the wallsof cells in leaf blades which were just wilting. In severelystressed and damaged plants a range of cell shapes and deformationsoccurred characteristic of a particular cell type and oftenunlike the control cell shape. Cell shape, drought, frost, barley, Hordeum