Does proline accumulated in leaves of water deficit stressed barley plants confine cell membrane injury? I. Free proline accumulation and membrane injury index in drought and osmotically stressed plants

The aim of this work was to examine the relationship between proline accumulation and membrane injury in barley leaves suffering from the effects of water deficit. Water deficit stress was induced by water withholding or by immersing the roots in polyethylene glycol (PEG 6000) solution of osmotic potential −1.5 MPa. The effect of water stress on proline accumulation and on membrane injury was evaluated in leaf blades of several barley genotypes. Substantial differences in proline accumulation and membrane injury indices among most of the genotypes investigated were observed. It was found that in drought stressed plants a higher ability to accumulate proline positively correlates with lower membrane injury. Whereas, in osmotically stressed plants the highest proline accumulation in the leaves was noticed in genotype with the largest membrane injury. The possible role of proline in membrane protection under conditions of slow-acting drought or shock-acting osmotic stress is discussed.     

Does proline accumulated in leaves of water deficit stressed barley plants confine cell membrane injuries? II. Proline accumulation during hardening and its involvement in reducing membrane injuries in leaves subjected to severe osmotic stress

The purpose of this research was to examine whether proline accumulation in leaves of barley under conditions of mild water deficit (PEG — 0.75 MPa imposed on roots) may modify membrane injuries caused by subsequent severe osmotic stress (PEG — 1.6 MPa imposed on leaves). Six-day-old seedlings of four barley genotypes were used in the experiments. Substantial and different proline accumulation was found in the leaves of mild water deficit-stressed plants of the most investigated genotypes. This stress factor caused rather a small decrease in RWC and did not lead to membrane injuries. Severe osmotic stress imposed on leaves caused considerable membrane injuries in all the genotypes investigated. Leaves of plants pre-stressed with mild water deficit and then subjected to severe osmotic stress exhibited about a 50% lower membrane injury than those of not pre-stressed plants. A possible role of proline accumulated in the leaves of pre-stressed plants in the process of alleviating cell membrane injuries in the leaves subsequently exposed to severe water deficit is discussed.     

The effect of salicylic acid on barley response to water deficit

The effect of a moderate (PEG −0.75 MPa) and severe (PEG −1.5 MPa) water deficit on SA content in leaves and roots as well as the effect of pre-treatment with SA on reaction to water stress were evaluated in two barley genotypes — the modern cv. Maresi and a wild form of Hordeum spontaneum. Water deficit increased SA content in roots, whereas SA content in leaves did not change. The level of SA in the roots of control plants was about twofold higher in ‘Maresi’ than in H. spontaneum. After 6 hours of a moderate stress the level of SA increased about twofold in H. spontaneum and about two and a half-fold in ‘Maresi’. Under severe stress conditions the level of SA increased about twofold in the both genotypes, but not before 24 hrs of the stress. Plant treatment with SA before stress reduced a damaging action of water deficit on cell membrane in leaves. A protective effect was more noticeable in H. spontaneum than in ‘Maresi’. SA treatment increased ABA content in the leaves of the studied genotypes. An increase of proline level was observed only in H. spontaneum. The obtained results suggest that ABA and proline can contribute to the development of antistress reactions induced by SA.     

Water Stress Induced Proline Accumulation in Contrasting Wheat Genotypes as Affected by Calcium and Abscisic Acid

Proline accumulation and mobilization in roots of 7-d-old seedlings of wheat genotypes varying in sensitivity towards water stress were compared. Water stress was induced by polyethylene glycol (PEG-6000; osmotic potential −1.5 MPa) in the presence of 0.1 mM abscisic acid (ABA), 1 mM calcium chloride, 0.5 mM verapamil (Ca²⁺ channel blocker), 0.5 mM fluridone (inhibitor of ABA biosynthesis). While both the genotypes did not differ in total proline accumulation, rate of proline accumulation and utilization was higher in tolerant genotype C 306 as compared to susceptible genotype HD 2380. The treatment with ABA and CaCl₂ caused further increase in proline accumulation during stress and reduced its mobilization during recovery. The membrane stability and elongation rate of roots was observed to be higher at ABA and calcium treatment in both the genotypes under stress. As was evident from inhibitor studies, the tolerant genotype was more responsive to ABA and the susceptible one to calcium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical responses to drought stress in mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.): evaluation of proline,glycine betaine and abscisic acid accumulation in five cultivars

Five popularly grown mulberry cultivars (K-2, MR-2, TR-10, BC2-59 and S-13) were subjected to drought stress by withholding irrigation, to obtain leaf water potentials (Ψ_w) ranging from −0.75, −1.50 and −2.25 MPa. Accumulation of proline, glycine betaine and abscisic acid (ABA) were quantified in control and water stressed mulberry leaves. The activities of enzymes involved in proline accumulation including glutamate dehydrogenase (EC1.4.1.2-4), pyrroline-5-carboxylate synthetase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC1.5.1.2), ornithine transaminase (EC 2.6.1.13) were significantly enhanced in the leaves of all the cultivars with decreasing leaf water potentials, while the activities of proline dehydrogenase (EC 1.5.1.2) were reduced with progressive increase in water stress. Accumulation of proline, glycine betaine and abscisic acid was relatively higher in S-13 and BC2-59 compared to K-2, MR-2 and TR-10 under water deficit conditions. Our results demonstrate that S-13 and BC2-59 have superior osmoprotectant mechanisms under water-limited growth regimes.     

Carbendazim alleviates effects of water stress on chickpea seedlings

Carbendazim (methyl-2-benzimidazole carbamate) promoted root growth of chickpea (Cicer arietinum L.) seedlings subjected to polyethylene glycol (PEG, osmotic potential −0.5 MPa) induced water stress. The relative water content, membrane stability index, 2,3,5-triphenyltetrazolium chloride reduction and contents of some osmolytes (proline, sucrose, glucose and fructose) enhanced significantly while the contents of lipid peroxides and hydrogen peroxide diminished effectively by addition of 0.05 % carbendazim into PEG solution. This revised version was published online in September 2005 with the corrected author information.     

Osmotic adjustment inSorghum bicolor (L. Moench) grown under moisture stress in soil and osmotically modified solution cultures

Sorghum (Sorghum bicolor L. Moench) plants were grown in solution culture and stressed at three rates of decreasing leaf water potential (−0.123, −0.068 and −0.029 MPa day⁻¹) achieved by the incremental addition of an osmoticum, polyethylene glycol (PEG) 6000 to the solutions. Plants were also grown in soil and given different amounts of water which resulted in rates of decreasing leaf water potentials of −0.130 and −0.073 MPa day⁻¹. The rate of stress and the culture system influenced the accumulation of solutes in the cell, but not cell volume. A rapid stress (−0.123 and −0.130 MPa day⁻¹) to approximately −1.6 MPa leaf water potential resulted in 0.75 and 0.16 MPa of osmotic adjustment in the PEG and soil culture respectively. At moderate stress (−0.068 and −0.073 MPa day⁻¹) respective values were 1.68 and 0.58 MPa. There were some visual symptoms in the solution grown plants characteristic of uptake of high molecular weight PEG. However the relative growth rates of these plants were equal to or greater than those of the soil grown plants. In view of the differences in plant water status of soil and PEG solution cultured plants it was concluded that the use of the latter system would not be entirely suitable for some studies of drought resistance in sorghum, as related to crop performance in the field.     

The effect of jasmonic acid on the accumulation of ABA,proline and spermidine and its influence on membrane injury under water deficit in two barley genotypes

Seedlings of two barley genotypes (‘Maresi’ and wild form of Hordeum spontaneum) were treated with jasmonic acid (JA 5 μM and 15 μM) for 24 h, and then subjected to water stress (PEG 6000 solution of − 1.5 MPa). JA caused an increase in the content of ABA but not in that of proline and spermidine in the two studied genotypes. The effect of the treatment did not depend on the applied JA concentration. The pre-stress treatment with JA changed plant response to water deficit with regard to membrane injury. Treatment with a lower JA concentration (5 μM) caused a substantial reduction of the stress-induced membrane damage in the both genotypes. A higher JA concentration (15 μM) caused the reduction of membrane injury only in H. spontaneum and was ineffective in ‘Maresi’. JA had no influence on the leaf water status in water-stressed plants. A possible role of JA in leaf ABA accumulation and alleviation of cell membrane injury under water deficit is discussed. The work was partly supported by the Polish Committee For Scientific Research, grant No 5 PO6A 036 18     

Relationship between osmotic stress-induced abscisic acid accumulation,biomass production and plant growth in drought-tolerant and -sensitive wheat cultivars

The effects of increasing osmotic stress induced by 100–400 mOsm (−0.976 MPa) polyethylene glycol (PEG 6000) were investigated in a drought-tolerant (Triticum aestivum L. cv. Mv Emese) and drought-sensitive (cv. GK élet) wheat cultivar at the three-leaf stage. During osmotic stress, the decline of the water potential (ψ _w) was more significant in the leaves, while the abscisic acid (ABA) levels of the roots increased earlier and remained higher in the sensitive than in the tolerant variety. There was an increasing gradient of ABA content toward the youngest leaves in the drought-sensitive GK élet, while more ABA accumulated in the fully developed, older leaves of the tolerant cultivar Mv Emese. In accordance with the rapid and significant accumulation of ABA, the stomatal conductance decreased earlier in the tolerant cultivar. The effect of water stress on the PSII photochemistry was pronounced only 1 week after the exposure to PEG, as indicated by the earlier decrease of the net CO₂ fixation, the effective quantum yield (Φ_PSII) and the photochemical quenching (q _P) in light-adapted samples of the tolerant variety in 400 mOsm PEG 6000. The stress treatment caused more significant reductions in these parameters toward the end of the experiment in the sensitive cultivar. In spite of small differences in the photosynthetic characteristics, the net biomass production was not significantly altered by this osmotic stress. The accumulation of ABA controlled the distribution of the biomass between the shoot and root systems under osmotic stress, and contributed to the development of stronger and deeper roots in the drought-sensitive cultivar GK élet. However, the root elongation did not correlate with the drought sensitivity of these cultivars on the basis of crop yield.     

ABA and proline accumulation in leaves and roots of wild (<Emphasis Type="Italic">Hordeum spontaneum</Emphasis>) and cultivated (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> ‘Maresi’) barley genotypes under water deficit conditions

The purpose of the study was to examine water stress-induced changes in the ABA and proline contents in roots and leaves of a potentially more resistant wild accession of Hordeum spontaneum and the modern cultivar Maresi (Hordeum vulgare). Leaves of H. spontaneum had higher contents of constitutive ABA and proline in comparison to those of ‘Maresi’. A moderate water deficit resulted only in root dehydration, which was higher in ‘Maresi’. Increases of water deficit in roots coincided with an increase of ABA content in roots, followed by that in leaves. The level of proline increased only in leaves and only in the case of H. spontaneum. Under conditions of severe water stress, the root dehydration levels were similar in the both genotypes, whereas leaf dehydration was higher in ‘Maresi’. H. spontaneum, as compared to ‘Maresi’ showed an earlier increase of ABA content in the roots and accumulated more ABA in the leaves. Free proline levels in the roots increased in both genotypes but H. spontaneum exhibited a 2-fold higher proline accumulation than ‘Maresi’. In H. spontaneum the accumulation of proline in the leaves occurred noticeably earlier and to a higher extent than in ‘Maresi’. A possible connection of these modifications with water stress resistance of the investigated genotypes is discussed in this paper.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

Relations between MAPK phosphorylation and ABA level in <Emphasis Type="Italic">Malus sieversii</Emphasis> under water stress

A correlation between protein kinase phosphorylation and ABA level was studied in Malus sieversii (Ledeb.) Roem. seedlings under water stress. The seedlings were treated with PEG 6000 for imitation of water stress, and the MAPK activity and ABA content in each treatment were then determined. We demonstrated that the increase in the activities of the total protein kinase (TPK) and mitogen-activated protein kinase (MAPK) after treatment with 20% PEG 6000 appeared to result in a high level of ABA. MAPK activity accounted for 76.8% of TPK activity. The activity peaks of TPK and MAPK preceded the highest level of ABA accumulation. It is interesting that the ABA level in roots and leaves of seedlings pretreated with 2 × 10⁻² mM exogenous ABA for 20 min following treatment by 20% PEG 6000 was much higher than that of seedlings treated with exogenous ABA only. We analyzed the influence of MAPK inhibitor ITU (5-iodotubercidin) on ABA accumulation in the seedlings of M. sieversii under water stress and showed that 1 μM ITU significantly decreased the ABA level induced by a water loss. However, the phosphoesterase inhibitor PAO (phenylarisine oxide) enhanced ABA accumulation, indicating that the phosphorylated MAPK was correlated to ABA synthesis. Together, these results suggest that MAPK phosphorylation played an important role in ABA accumulation under water stress, and MAPK might mediate the signal transduction of ABA synthesis.     

The Extent and Pattern of Osmotic Adjustment in White Clover (Trifolium repens L.) During the Development of Water Stress

White clover plants were subjected to water stress followingthe cessation of watering. As a water deficit developed, waterand osmotic potentials were measured in stolon tips, leavesfrom the stolon tip and leaves from the plant crown. Pressurepotentials were calculated. Pressure potential was maintainedin stolon tips even when water potential fell to around –2·0MPa. In contrast, pressure potential in leaves fell rapidlyas water stress developed. Total amino acid and potassium levels were largely unaffectedin both stolon tips and leaves. Water-soluble carbohydratesand proline accumulated during water stress. The increase inproline level in leaves did not follow the same pattern as thatin stolon tips, although toward the end of the water stressperiod the level had increased by a similar extent in both partsof the plant. Additionally, pressure potential and osmotic potentialappeared to be significantly related to proline content in stolontips. No such relationship was found for leaves. The role ofproline in osmotic adjustment is discussed. Trifolium repens L. cv. Olwen, white clover, water stress, osmotic adjustment, proline     

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

Contents of ethylene, osmoprotectants, levels and forms of polyamines (PAs) and activities of antioxidant enzymes in the leaves and roots were investigated for five wheat cultivar seedlings (differing in drought tolerance) exposed to osmotic stress (?1.5 MPa). Stress was induced by 2-day-long treatment of plants with polyethylene glycol 6000 (PEG) or NaCl added to hydroponic cultures. Nawra, Parabola and Manu cv. (drought tolerant) showed a marked increase in osmoprotectors (proline and soluble carbohydrates, mainly glucose, saccharose and maltose), free PAs (putrescine Put, spermidine Spd and spermine Spm) and Spd-conjugated levels, in both leaves and roots, after PEG-treatments. Radunia and Raweta (drought sensitive) exhibited smaller changes in the content of these substances. The analysis of enzymes involved in proline metabolism revealed the glutamate as a precursor of proline synthesis in PEG-induced stress conditions. The increase in the activity of antioxidative enzymes, especially catalase and peroxidases, was characteristic for tolerant wheat plants, but for sensitive ones, a decrease in superoxide dismutase and an increase in mainly glutathione reductase activities were observed. After NaCl-treatment smaller changes of all biochemical parameters were registered in comparison with PEG-induced stress. Exceptions were the higher values of ethylene content and a significant increase in saccharose, raffinose and maltose levels (only in stress sensitive plants). The proline synthesis pathway was stimulated from both glutamate and ornithine precursors. These results suggest that the accumulation of inorganic ions in NaCl-stressed plants may be involved in protective mechanisms as an additional osmoregultor. Thus, a weaker stressogenic effect as determined as water deficit by leaf relative water content and relative dry weight increase rate and differences in metabolite synthesis in comparison with PEG stress was observed. Proline seems to be the most important osmo-protector in osmotic stress initiated by both PEG and NaCl. The synthesis of sugars and PAs may be stimulated in a stronger stress conditions (PEG).     

Effect of abscisic acid on betacyanin leakage from plant tissues

Effect of abscisic acid on cell permeability in leaves ofIresine u allisi hort. and roots ofBeta vulgaris L. were examined. An increase of betacyanin leakage from leaf cells was shown by ABA at 10⁻⁴, 10⁻⁷ or 10⁻⁹ M concentrations in water solution at 25 °C. The efflux of batacyanin from tissues did not change during the joint action of ABA and PEG 1000. ABA could lower the betacyanin leakage fromIresine leaves and beet-root slices under severe osmotic stress, as was found by deplasmolysis. The results suggest that ABA elicits some alteration in density of tonoplast membranes under dehydration. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Role of Abscisic Acid in Water Stress-induced Antioxidant Defense in Leaves of Maize Seedlings

Roles of abscisic acid (ABA) in water stress-induced oxidative stress were investigated in leaves of maize ( Zea mays L.) seedlings exposed to water stress induced by polyethylene glycol (PEG 6000). Treatment with PEG at &#109 0.7 MPa for 12 and 24 h led to a reduction in leaf relative water content (RWC) by 7.8 and 14.1%, respectively. Duration of the osmotic treatments is considered as mild and moderate water stress. The mild water stress caused significant increases in the generation of superoxide radical ( O 2 &#109 ) and hydrogen peroxide (H 2 O 2 ), the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the contents of ascorbate (ASC), reduced glutathione (GSH). The moderate water stress failed to further enhance the capacity of antioxidant defense systems, as compared to the mild water stress. The contents of catalytic Fe, which is critical for H 2 O 2 -dependent hydroxyl radical ( &#148 OH) production, and the oxidized forms of ascorbate and glutathione pools, dehydroascorbate (DHA) and oxidized glutathione (GSSG), markedly increased, a significant oxidative damage to lipids and proteins took place under the moderate water stress. Pretreatment with ABA caused an obvious reduction in the content of catalytic Fe and significant increases in the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants, and then significantly reduced the contents of DHA and GSSG and the degrees of oxidative damage in leaves exposed to the moderate water stress. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA induced by water stress, reduced the enhancement in the capacity of antioxidant defense systems, and resulted in an increase in catalytic Fe, DHA and GSSG, and oxidative damage in the water-stressed leaves. These effects were completely prevented by addition of ABA, which raised the internal ABA content. Our data indicate that ABA plays an important role in water stress-induced antioxidant defense against oxidative stress.     

Physiological and morphological response to water deficit in seedlings of five provenances of Pinus canariensis: potential to detect variation in drought-tolerance

To assess the potential of short-term screenings for drought resistance at the seedling stage to detect ecotypic variation and predict field performance, we studied the responses to water deficit of seedlings of Pinus canariensis from five geographic origins under controlled conditions and compared these responses with the performance of provenances in a multi-site field trial. Leaf water potential, the osmotic component, leaf chlorophyll fluorescence and growth and biomass partitioning were measured as seedlings were subjected for 11 days to two levels of osmotic potential generated by polyethylene glycol (PEG 6000), −1 MPa (slowly imposed water deficit; S) and −1.5 MPa (fast imposed water deficit; F), and a control treatment (no PEG added to the nutrient solution; C). Leaf water potential declined to final mean values of −1.2, −2.7 and −4.7 MPa in the C, S and F treatments, respectively. The ratio of variable to maximum chlorophyll fluorescence declined to final mean values of 0.77, 0.66 and 0.40 in the C, S and F treatments, respectively, with no differences amongst provenances. All provenances showed an active osmotic adjustment (OA) in response to water deficit which varied depending on the drying rate. A slow imposition of water deficit favoured solute accumulation. Pooling all treatments, the index of OA ranged from 0.28 to 0.40, but rose considerably when only C and S treatments were considered (0.56 to 0.70). There was a positive and significant correlation between the overall index of OA (all treatments pooled) and the drought period in the site of origin, suggesting ecotypic variation in OA as a result of drought duration. Seedlings allocated more dry matter to roots than shoots when subjected to moderate and slowly imposed water deficit; only one provenance showed no increase in the root to shoot ratio at the end of the treatment period compared with control seedlings. Responses to controlled water deficits were only qualitatively related to performance (survival and growth) of provenances in several field sites, indicating the involvement of complex mechanisms to cope with drought under natural conditions. However, the provenance with the highest overall index of OA outgrew and outsurvived the other provenances in the most arid site, and the only provenance not modifying the root to shoot ratio in response to water deficit survived the least in all field sites. Acclimation of root to shoot ratio and net solute accumulation to water deficit could hence favour drought-tolerance beyond the seedling stage and be used as preliminary predictors of field performance.     

The role of organic and inorganic solutes in the osmotic adjustment of drought-stressed Jatropha curcas plants

This study aimed to assess the accumulation of organic and inorganic solutes and their relative contribution to osmotic adjustment in roots and leaves of Jatropha curcas subjected to different water deficit intensity. Plants were grown in vermiculite 50% (control), 40%, 30%, 20% and 10% expressed in gravimetric water content. The water potential, osmotic potential and turgor potential of leaves decreased progressively in parallel to CO₂ photosynthetic assimilation, transpiration and stomatal conductance, as the water deficit increased. However, the relative water content, succulence and water content in the leaves did not show differences between the control and stressed plants, indicating osmotic adjustment associated with an efficient mechanisms to prevent water loss by transpiration through stomatal closure. The K⁺ ions had greater quantitative participation in the osmotic adjustment in both leaves and roots followed by Na⁺ and Cl⁻, while the NO₃⁻ ion only showed minor involvement. Of the organic solutes studied, the total soluble sugars showed the highest relative contribution to the osmotic adjustment in both organs and its concentration positively increased with more severe water deficit. The free amino acids and glycinebetaine also effectively contributed to the osmotic potential reduction of both the root and leaves. The role of proline was quantitatively insignificant in terms of osmotic adjustment, in both the control and stressed roots and leaves. Our data reveal that roots and leaves of J. curcas young plants display osmotic adjustment in response to drought stress linked with mechanisms to prevent water loss by transpiration by means of the participation of inorganic and organic solutes and stomatal closure. Of all the solutes studied, soluble sugars uniquely display a prominent drought-induced synthesis and/or accumulation in both roots and leaves.     

Hydraulic and Chemical Responses of Citrus Seedlings to Drought and Osmotic Stress

In this work we investigated the function of abscisic acid (ABA) as a long-distance chemical signal communicating water shortage from the root to the shoot in citrus plants. Experiments indicated that stomatal conductance, transpiration rates, and leaf water potential decline progressively with drought. ABA content in roots, leaves, and xylem sap was also increased by the drought stress treatment three- to sevenfold. The addition of norflurazon, an inhibitor of ABA biosynthesis, significantly decreased the intensity of the responses and reduced ABA content in roots and xylem fluid, but not in leaves. Polyethylene glycol (PEG)-induced osmotic stress caused similar effects and, in general, was counteracted only by norflurazon at the lowest concentration (10%). Partial defoliation was able to diminish only leaf ABA content (22.5%) at the highest PEG concentration (30%), probably through a reduction of the active sites of biosynthesis. At least under moderate drought (3–6 days without irrigation), mechanisms other than leaf ABA concentration were required to explain stomatal closure in response to limited soil water supply. Measurements of xylem sap pH revealed a progressive alkalinization through the drought condition (6.4 vs. 7.1), that was not counteracted with the addition of norflurazon. Moreover, in vitro treatment of detached leaves with buffers iso-osmotically adjusted at pH 7.1 significantly decreased stomatal conductance (more than 30%) as much as 70% when supplemented with ABA. Taken together, our results suggest that increased pH generated in drought-stressed roots is transmitted by the xylem sap to the leaves, triggering reductions in shoot water loss. The parallel rise in ABA concentration may act synergistically with pH alkalinization in xylem sap, with an initial response generated from the roots and further promotion by the stressed leaves.