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.

     

Proline accumulation and its implication in cold tolerance of regenerable maize callus

Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99³H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19°C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the medium to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to the culture media also increased the tolerance of callus to 4°C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4°C for 90 days which is more than double the typical survival time of callus. These studies show that proline and conditions which induce proline accumulation increase the cold tolerance of regenerable maize callus.     

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 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.     

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.     

The Mechanism of Abscisic Acid-induced Proline Accumulation in Barley Leaves

When leaf blades of fully expanded second leaves of barley (cv. Prior) were excised and incubated with the cut end in a 20 milligram per liter solution of abscisic acid, they accumulated proline at the rate of about 1 micromole per hour per gram fresh weight after a 3- to 4-hour lag. This accumulation occurred reproducibly only if leaves were pretreated by placing the cut end in a solution consisting of 50 millimolar sucrose and 1 millimolar glutamate. Treated leaves were taken from plants which had been in the light for 24 hours.     

Proline level is partly under the control of abscisic acid in canola leaf discs during recovery from hyper-osmotic stress

The relationships between the changes induced in abscisic acid (ABA) and proline contents were investigated in canola ( Brassica napus L.) leaf discs (CLDs) subjected to sequential hyper- and hypo-osmotic treatments. Changes in ABA content were found to precede that of proline, suggesting that ABA could exert regulatory effects on both osmo-induced proline accumulation and its subsequent mobilization during recovery. When exogenously supplied in the light during recovery, ABA caused inhibition of the apparent rate of proline mobilization with an I50 of about 20 μ M . Under dark conditions, the ABA effect was not observed. This is in accordance with the fact that this effect could be suppressed in the light while using photosynthesis inhibitors. The ABA effect was partly restored under dark conditions when the CLDs were supplied with NADPH at high concentration. Under both conditions, ABA exerted its effect when supplied together with cycloheximide, suggesting that it does not rely on protein synthesis. When turgid CLDs, which heavily absorbed exogenously supplied l -proline, were incubated in the light on the reference medium with ABA, proline mobilization was also restricted, provided ABA was supplied during the period of proline uptake. Surprisingly, in CLDs that had recovered their full turgor after application of successive hyper- and hypo-osmotic treatments, application of ABA led to a very high accumulation of proline. In contrast, in freshly cut turgid CLDs incubated in presence of ABA, the basic proline level was only weakly enhanced. Thus, the apparent inhibitory effect of ABA on proline mobilization could be mediated through both an activation of biosynthesis and an inhibition of catabolism of this amino acid via light-dependent processes that remain to be elucidated.     

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.     

Proline accumulation and the adaptation of cultured plant cells to water stress

The transfer of cultured tomato cells (Lycopersicon esculentum cv VFNT-Cherry) to a low water potential environment resulted in an increased dry weight to fresh weight ratio accompanied by a rapid accumulation of proline. Proline content continued to increase as osmotic adjustment and growth occurred. The initial increase in proline concentration was accompanied by a drop in turgor. However, proline levels continued to increase with a gain in turgor during osmotic adjustment. Thus, the accumulation of proline depended not only on cell water potential, or on the initial loss of turgor but more closely on cell osmotic potential. The ultimate level of proline depended on the level of adaptation. Proline levels remained high after more than 100 cell generations in low water potential media, but declined rapidly after transfer to media with a less negative water potential. Addition of exogenous proline to the medium during water stress and during osmotic downshock alleviated the normally resulting inhibition of growth. The results suggest a positive role for proline accumulation in adaptation of cells to changing external water potentials.     

The effect of sodium chloride on solute potential and proline accumulation in soybean leaves

Two cultivars of soybean (Glycine max [L.] Merr.) were grown in solution with up to 100 millimolar NaCl. Leaf solute potential was −1.1 to −1.2 megapascals in both cultivars without NaCl. At 100 millimolar NaCl leaf solute potential was −3.1 to −3.5 megapascals in Bragg and −1.7 megapascals in Ransom. The decrease in solute potential was essentially proportional to the concentration of NaCl. In both salt susceptible Bragg and salt semitolerant Ransom, leaf proline was no more than 0.4 micromole per gram fresh weight at or below 20 millimolar NaCl. At 40 and 60 millimolar NaCl, Bragg leaf proline levels were near 1.2 and 1.9 micromoles per gram fresh weight, respectively. Proline did not exceed 0.5 micromole per gram fresh weight in Ransom even at 100 millimolar NaCl. Proline accumulated in Bragg only after stress was severe enough to induce injury; therefore proline accumulation is not a sensitive indicator of salt stress in soybean plants.     

Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.     

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola)

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na₂SO₄. In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na₂SO₄. Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na₂SO₄ concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na₂SO₄. Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.     

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).

     

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.     

The Relation between Accumulation of Abscisic Acid and Proline in Detached Rice Leaves

The relation between abscisic acid (ABA) and proline accumulation was investigated in detached rice (Oryza sativa L.) leaves. In darkness, proline content increased about 2-, 2,5- and 6-fold after 24, 48 and 72 h. ABA content reached maximum after 48 h. In the light, proline content remained almost unchanged until 48 h and subsequently increased slightly. ABA content in the light was lower than in darkness, but the maximum was also after 48 h. During 12-h exposure to decreased air humidity, proline content gradually increased, but ABA content increased about 25-fold after 4 h and declined thereafter. Exogenous application of ABA resulted in an increase in proline content in detached rice leaves under both light and darkness.     

Physiology of rice tungro virus disease: Proline accumulation due to infection

Tungro virus infection stimulates proline accumulation in leaves of rice ( Oryza sativa L.), especially in a sensitive cultivar, Taichung Native 1. Disease-induced proline accumulation increases with the severity of the disease. Proline also accumulates in senescing, detached healthy rice leaves. The magnitude of proline accumulation in these leaves was further accentuated by ABA and retarded by kinetin. In the absence of drought stress, virus infection induces severe symptoms (stunting) in a drought tolerant cultivar (Lalnakanda 41) when compared to cultivars with intermediate (MW 10) and high sensitivity (Cauvery) to drought. Thus tungro virus mimics water stress in inducing proline accumulation in rice leaves. In both cases a common factor, ABA, may mediate proline accumulation. In drought stress, proline accumulation is associated with tolerance, while in virus stress, proline accumulation is connected with sensitivity. It is, therefore, clear that proline cannot always act to relieve physiological stress.     

Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves

Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than −10 bars. At −20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24-hour period.     

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.

     

Proline Metablism During Water Stress in Mulberry

The influence of water stress on proline metabolism was studiedin 3-month-old mulberry plants at four levels of water stress.Leaf water potential was drastically decreased in all treatments.Though leaf area and relative water content were decreased,drastic decrease was observed only in very severe stress treatments.Proline accumulation was observed both in roots and leaves instress treatments; but accumulation was greater in roots thanin leaves. The enzymes, proline dehydrogenase and proline oxidase,were inhibited under stress conditions. Proline oxidase wasmore inhibited in roots than in leaves. The significance ofthe relative activities of these two enzymes is discussed. Key words: Water stress, proline dehydrogenase, proline oxidase     

Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans

It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues.