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.

     

The effect of wilting on the selection of leaves by the leaf-cutting ant Atta laevigata

Using field assays of leaf preference, we tested the hypothesis that wilting affects the selection of leaves by the leaf-cutting ant Atta laevigata (Fr. Smith). Detached leaves were left to air-dry until noticeably wilted. The area removed by the ants from wilted leaves was significantly greater than the area removed from fresh leaves, this effect being observed in several plant species, in leaves of different age, and in assays with different ant colonies. Leaves collected from water-stressed plants were also preferred to leaves from non-stressed plants. A. laevigata was found to employ a two-stage, size-related, strategy when cutting plants. Larger workers climbed the plant stem and dropped whole leaves to the ground by severing their petioles; smaller workers cut the lamina of the dropped leaves. The ants frequently left dropped leaves on the ground, until the next foraging day or even later, when they were harvested in a wilted condition in preference to newly-dropped leaves.It is possible that during wilting some repellent substances evaporate or become less effective, thus enhancing leaf palatability. Alternatively or in addition, changes in nutrient and water content may have rendered wilted leaves more palatable to leaf-cutting ants.     

Effect of Wilting on Carbohydrates during Incubation of Excised Bean Leaves in the Dark

Excised bean (Phaseolus vulgaris) leaves were used to compare the changes in various carbohydrates during dark incubation in a wilted and turgid condition. After a rapid (less than 1 hour) wilt, leaves were incubated at a constant water content (75% of original fresh weight).     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure     

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.     

Effect of volatiles emitted from wilted Populus nigra L. leaves on the mating and oviposition of Helicoverpa armigera (Hübner)

Wilted black poplar, Populus nigra ‘Italica’ L., leaves are very attractive to a vast number of noctuid moth species. This provides an opportunity for the development of effective trapping methods for the integrated management of pest species, such as Helicoverpa armigera, a major global and economically important insect pest.In the present study, we investigated the (1) nocturnal attraction patterns of H. armigera males and females to wilted P. nigra leaves; (2) effects of P. nigra volatiles on the mate-searching behavior of males through laboratory serial-chamber bioassays and field trapping; and (3) effects of P. nigra volatiles on the ovipositional choice and reproductive performance of females. Females and males, when tested alone, could be attracted by wilted P. nigra leaves, and the time periods of the first two attraction peaks were largely overlapped between sexes. Streams consisting of wilted P. nigra leaves and virgin females were not more attractive than virgin females alone, regardless of the stream sequence in a serial chamber. However, a stream of virgin females passed through wilted P. nigra leaves was more attractive than wilted P. nigra leaves alone. The addition of P. nigra extracts and its major aromatic components to the sex lure of H. armigera did not attract more moths than the sex lure alone. The volatiles from wilted P. nigra leaves were significantly more attractive to ovipositing females than those from cotton, tomato, and corn leaves, but equally attractive to tobacco leaves. Females exposed to volatiles from different leaves (P. nigra, cotton, and tobacco) showed similar fecundities. In summary, the attraction of moths to wilted P. nigra leaves may be attributable to multiple mechanisms, including the adsorption of sex pheromones, ovipostional attraction, and possible feeding attraction.     

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.

     

Abscisic Acid Metabolism in Water-stressed Bean Leaves

Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-¹⁴C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of ¹⁴C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 μg to approximately 0.5 μg/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 μg to 1.0 μg and DPA from 0.8 μg to over 3 μg/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 μg/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal.     

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

Barley (Hordeum vulgare cv Prior) leaves converted l-U-(14)C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We conclude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves.     

Water relations of turgor recovery and restiffening of wilted cabbage leaves in the absence of water uptake

A novel phenomenon in which wilted cabbage leaves appeared to regain positive turgor pressures without additional water uptake has been previously reported (J Levitt [1986] Plant Physiol 82: 147-153). These experiments were replicated and the biophysical nature of turgor recovery characterized. Leaf water potential and its components were assayed in hydrated, wilted, and desiccated leaves which appeared to regain turgor after wilting. The hypotheses that turgor recovery was due to an increased volumetric elastic modulus (ε), or alternatively the result of solute redistribution were tested. Quantitative evidence that turgor recovery occurs in excised leaves was found. Leaf turgor pressure in hydrated leaves (~0.6 megapascal) decreased to zero upon wilting. After continued desiccation, turgor pressure returned to approximately 0.3 megapascal even though leaf relative water content declined. The ε of hydrated leaves was large and there was no evidence of an increased ε in the turgor-recovered leaves. Solute mobilization occurred during desiccation. The apoplastic osmotic potential decreased from −0.15 to −0.44 megapascal in hydrated and turgor-recovered leaves, respectively, and solutes were transported from the lamina to the midrib tissue. Solute redistribution coupled with the high ε may have resulted in localized turgor recovery in specific cells in the desiccated leaves.     

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.     

Reduction of turgor induces rapid changes in leaf translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23°C air for 20 min. Poly(A)⁺ RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)⁺ RNAs increased in wilted plants. Most of the poly(A)⁺ RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A λgt10 cDNA library was constructed using poly(A)⁺ RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)⁺ RNAs which are induced in wilted shoots.     

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.     

Changes in Amino Acid Content of Excised Leaves During Incubation. III. Role of Sugar in the Accumulation of Proline in Wilted Leaves

A previously reported accumulation of proline in wilted turnip leaves has been observed in 6 additional species representing 5 different families. The results of experiments on the conditions affecting the behavior of proline in wilted leaves were interpreted to mean that the supply and metabolism of available carbohydrate was essential for proline accumulation.     

Water Stress in Plants

The abscisic acid (ABA) content was determined quantitatively in the leaves from wilted and unwilted tomato plants (Lycopersicon esculentum Mill. CV. Revermun) by the use of the wheat coleoptile test and gas-liquid chromatography (GLC). Plants which have received an insufficient daily water supply for 18 days showed adaptation to wilting conditions. The plants adjust to the added amount of water by regulating their water loss through transpiration. The concentration of ABA was not higher in the leaves of plants adapted to water stress than in plants that were watered abundantly. Wilted detached leaves and leaves from rapidly wilted intact plants showed the well-known reaction by increasing the ABA level. A possible role of ABA in the early stages of the adaptation process is discussed.     

The Water Balance of Cut Rose Flowers

A sharp decline in water potential of petal tissue associated with wilting of cut rose flowers is described. Such a decline did not develop in senescing intact flowers. A circadian rhythm in water absorption by cut flowers was observed. The decline in fresh weight observed in the last phase of the vase life of cut flowers occurred earlier in a short-lived cultivar than in a longer-lived one. A decline in potential conductivity to water was observed with time in stems of cut roses. Concomitantly cellulase activity increased after cutting. Flowers held in cellulase solution wilted earlier than the controls. However, no difference was found in these two parameters between two cultivars differing in their longevity. The difference in longevity between the two cultivars was large especially under conditions promoting high transpiration rates, and was narrowed when flowers were either held in mild conditions, or the leaves were stripped off. Although stomates were equally open in intact flowers of the two cultivars, in cut flower shoots of the short-lived cultivar stomates were more widely open. In accordance transpiration rates were higher, and wilting occurred earlier in the short-lived cultivar than in the long-lived one. It is concluded that the earlier wilting of cut flowers of the short-lived cultivar is mainly due to lower ability to close stomates in response to water stress conditions, and not to earlier formation of vascular blockage.     

Development of redox potential-controlled schemes for very-high-gravity ethanol fermentation

Fermentation redox potential reflects the momentary physiological status of organisms. Controlling redox potential can modulate the redistribution of intracellular metabolic flux to favor the formation of the desired metabolite. Accordingly, we have developed three redox potential-controlled schemes to maximize their effects on the very-high-gravity (VHG) ethanol fermentation. They are aeration-controlled scheme (ACS), glucose-controlled feeding scheme (GCFS), and combined chemostat and aeration-controlled scheme (CCACS). These schemes can maintain fermentation redox potential at a prescribed level (i.e., -50, -100, and -150 mV) by supplementing sterile air, fresh glucose media, or a combination of sterile air and fresh glucose media into a fermenter to counteract the decline of redox potential due to yeast growth. When ACS was employed, the fermentation efficiency at -150 mV is superior to the other two redox potential levels especially when the initial glucose concentration is higher than 250 g/l. The redox potential-controlled period for ACS, GCFS, and CCACS at -150 mV under the same 200 g glucose/l condition was 2.5, 21.7 and 64.6h and the corresponding fermentation efficiency was 85.9,89.3 and 92.7%, respectively.     

Abscisic Acid accumulation in spinach leaf slices in the presence of penetrating and nonpenetrating solutes

Abscisic acid (ABA) accumulated in detached, wilted leaves of spinach (Spinacia oleracea L. cv Savoy Hybrid 612) and reached a maximum level within 3 to 4 hours. The increase in ABA over that found in detached turgid leaves was approximately 10-fold. The effects of water stress could be mimicked by the use of thin slices of spinach leaves incubated in the presence of 0.6 molar mannitol, a compound which causes plasmolysis (loss of turgor). About equal amounts of ABA were found both in the leaf slices and in detached leaves, whereas 2 to 4 times more ABA accumulated in the medium than in the slices. When spinach leaf slices were incubated with ethylene glycol, a compound which rapidly penetrates the cell membrane causing a decrease in the osmotic potential of the tissue and only transient loss of turgor, no ABA accumulated. Ethylene glycol was not inhibitory with respect to ABA accumulation. Spinach leaf slices incubated in both ethylene glycol and mannitol had ABA levels similar to those found when slices were incubated with mannitol alone. Increases similar to those found with mannitol also occurred when Aquacide III, a highly purified form of polyethylene glycol, was used. Aquacide III causes cytorrhysis, a situation similar to that found in wilted leaves. Thus, it appears that loss of turgor is essential for ABA accumulation.

When spinach leaf slices were incubated with solutes which are supposed to disturb membrane integrity (KHSO₃, 2-propanol, or KCl) no increase in ABA was observed. These data indicate that, with respect to the accumulation of ABA, mannitol caused a physical stress (loss of turgor) rather than a chemical stress (membrane damage).

     

Behavior of Corn and Sorghum under Water Stress and during Recovery

Corn (Zea mays L.) and sorghum (Sorghum vulgare, Pers.) plants were grown in a vermiculite-gravel mixture in controlled environment chambers until they were 40 days old. Water was withheld until they were severely wilted, and they were then rewatered. During drying and after rewatering stomatal resistance was measured with a diffusion porometer each morning, and water saturation deficit and water potential were measured on leaf samples. The average resistance of the lower epidermis of well watered plants was lower for corn than for sorghum. When water stress developed, the stomata began to close at a higher water potential in corn than in sorghum. The stomata of both species began to reopen normally soon after the wilted plants were rewatered, and on the 2nd day the leaf resistances were nearly as low as those of the controls. The average leaf water potential of well watered corn was −4.5 bars; that of sorghum, −6.4 bars. The lowest leaf water potential in stressed corn was −12.8 bars at a water saturation deficit of 45%. The lowest leaf water potential in stressed sorghum was −15.7 bars, but the water saturation deficit was only 29%. At these values the leaves of both species were tightly rolled or folded and some injury was apparent. Thus, although the average leaf resistance of corn is little lower than that of sorghum, corn loses much more of its water before the stomata are fully closed than does sorghum. The smaller reduction in water content of sorghum for a given reduction in leaf water potential is characteristic of drought-resistant species.     

Chlorophyll-type compounds in soil

Summary The amounts of chlorophyll-type compounds in materials commonly deposited on or in soil were measured and the processes that destroy them in materials on the soil surface, and the ways they may enter the soil, were studied. Of plant material commonly deposited on the soil, freshly-cut ryegrass and lucerne contained most of such compounds and cereal straw least. Faeces from grazing cattle and sheep contained nearly as mush as grass; farmyard manure contained only five per cent as much as fresh faeces.Nine-tenths of the chlorophyll in chopped-up, fresh ryegrass leaves was decomposed in six days; this decomposition was attributed to tissue enzymes and was prevented by boiling, drying, water-logging or freezing. Microorganisms decomposed about sixty per cent of chlorophyll in ryegrass leaves in 90 days.A large amount of chlorophyll-type compounds in faeces on soil leached 4 inches deep into the soil during 90 days in the autumn. Soil under 100-year-old, grazed pasture contained more of these compounds than under grassland that was cut for hay each year.