Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation

Increased endogenous plant cytokinin (CK) content through transformation with an adenine isopentyl transferase (ipt) gene has been associated with improved plant drought tolerance. The objective of this study is to determine metabolic changes associated with elevated CK production in ipt transgenic creeping bentgrass (Agrostis stolonifera L.) with improved drought tolerance. Null transformants (NTs) and plants transformed with ipt controlled by a stress- or senescence-activated promoter (SAG12-ipt) were exposed to well-watered conditions or drought stress by withholding irrigation in an environmental growth chamber. Physiological analysis confirmed that the SAG12-ipt line (S41) had improved drought tolerance compared with the NT plants. Specific metabolite changes over the course of drought stress and differential accumulation of metabolites in SAG12-ipt plants compared with NT plants at the same level of leaf relative water content (47% RWC) were identified using gas chromatography-mass spectroscopy. The metabolite profiling analysis detected 45 metabolites differentially accumulated in response to ipt expression or drought stress, which included amino acids, carbohydrates, organic acids, and organic alcohols. The enhanced drought tolerance of SAG12-ipt plants was associated with the maintenance of accumulation of several metabolites, particularly amino acids (proline, γ-aminobutyric acid, alanine, and glycine) carbohydrates (sucrose, fructose, maltose, and ribose), and organic acids that are mainly involved in the citric acid cycle. The accumulation of these metabolites could contribute to improved drought tolerance due to their roles in the stress response pathways such as stress signalling, osmotic adjustment, and respiration for energy production.     

Quercitol and osmotic adaptation of field-grown Eucalyptus under seasonal drought stress

This study investigated the role of quercitol in osmotic adjustment in field-grown Eucalyptus astringens Maiden subject to seasonal drought stress over the course of 1 year. The trees grew in a native woodland and a farm plantation in the semi-arid wheatbelt region of south Western Australia. Plantation trees allocated relatively more biomass to leaves than woodland trees, but they suffered greater drought stress over summer, as indicated by lower water potentials, CO₂ assimilation rates and stomatal conductances. In contrast, woodland trees had relatively fewer leaves and suffered less drought stress. Plantation trees under drought stress engaged in osmotic adjustment, but woodland trees did not. Quercitol made a significant contribution to osmotic adjustment in drought-stressed trees (25% of total solutes), and substantially more quercitol was measured in the leaves of plantation trees (5% dry matter) than in the leaves of woodland trees (2% dry matter). We found no evidence that quercitol was used as a carbon storage compound while starch reserves were depleted under drought stress. Differences in stomatal conductance, biomass allocation and quercitol production clearly indicate that E. astringens is both morphologically and physiologically 'plastic' in response to growth environment, and that osmotic adjustment is only one part of a complex strategy employed by this species to tolerate drought.     

Changes in gas exchange versus leaf solutes as a means to cope with summer drought in Eucalyptus marginata

Two of the ways in which plants cope with water deficits are stomatal closure and “osmotic adjustment”. We sought to assess the contributions of these processes to maintenance of leaf hydration in field-grown, 7-year-old Eucalyptus marginata. Plants were exposed to their normal summer drought (controls) or supplied with additional water (irrigated). Irrigation increased photosynthesis by 30% in E. marginata. These increases in photosynthesis were related to an 80% increase in g _s. However, there was no difference in substomatal CO₂ concentrations between treatments, or in chloroplast CO₂ concentrations, as indicated by carbon isotope composition of leaf soluble sugars. This suggests that impaired mesophyll metabolism may partially explain slower rates of photosynthesis in plants exposed to their normal summer drought. There was no difference in concentrations of solutes or osmotic potential between non-irrigated and irrigated individuals, perhaps because relative water content was the same in non-irrigated and irrigated plants due to stomatal sensitivity to water deficits. Irrespective of the absence of osmotic adjustment, analysis of leaf solutes gave a clear indication of the major groups of compounds responsible for maintaining cell osmotic potential. Soluble sugars were three times as abundant as amino acids. Proline, a putatively osmotically active amino acid, contributed less than 1% of total solutes. These patterns of solutes in E. marginata are consistent with a growing body of literature arguing a greater role for carbohydrates and cyclitols and lesser role for amino acids in maintaining osmotic potential. Our data suggest the primary mechanism by which E. marginata coped with drought was partial stomatal closure; however, we cannot discount the possibility of osmotic adjustment under more severe water deficits.     

Associations between plant production and some physiological components of drought resistance in wheat

Abstract. Drought resistance in terms of plant production under conditions of drought stress was previously defined for several spring wheat ( Triticum aestivum L.) varieties. Four varieties, differing in their drought resistance by this definition, were compared in their physiological responses to water stress, as induced by polyethylene glycol 6000 in the growth medium.
Drought resistance was associated with osmotic adjustment, total root mass production under stress, maintenance of some stomatal permeability under stress, and maintenance of turgor at a given level of drought stress, by either osmotic adjustment or elevated plant water potential.
Drought resistance was not associated, in this experiment, with plant top growth under stress or non-stress conditions, maximum leaf area per plant, plant transpiration, and total root mass production under non-stress conditions.     

Effects of water stress on internal water relations of apple leaves

The capacity of apple ( Malus pumila Mill. cv. James Grieve and Golden Delicious) pot- and orchard-grown trees to adjust osmotically in response to drought was investigated. Stressed leaves exhibited alterations in the moisture release curves when compared to well hydrated control leaves. Results suggest that osmotic adjustment occurred in both field- and pot-grown trees. Water potential for zero turgor was lowered by 0.5 MPa in leaves of potted trees and by 1.1 MPa in leaves of field-grown trees as a result of stress treatments. A decrease in the osmotic potential was responsible for that adjustment allowing the leaf to maintain turgor at lower water potentials and relative water contents. The extent of adjustment was similar for both potted and orchard trees despite the difference in the rate of stress imposition and its intensity. Changes in the concentration of sugars apparently contributed to this adjustment.     

Leaf age effects on solute accumulation in water-stressed grapevines

The effects of leaf age on water relations, organic solute, and total ion accumulation were studied in mature and immature leaves of two-year-old grapevines (Vitis vinifera L., cv. Savatiano) grown under water stress conditions. Osmotic potential at full turgor decreased significantly in leaves of stressed plants, irrespective of leaf age, indicating the occurrence of an active osmotic adjustment. The apoplastic water fraction (A) increased during leaf ontogeny in both control and stressed plants. However, the values of A were lower in stressed plants. Starch concentration decreased significantly in both mature and immature leaves during the drought cycle, while the relative proportion of monosaccharides and sucrose was markedly different in immature leaves compared to mature. The accumulation of total inorganic ions, induced by drought, was also age dependent, increasing significantly with leaf age, while there were no significant differences in total amino acids content. Inorganic ions and carbohydrates seem to be the major component of osmotic adjustment in mature and immature grapevine leaves, respectively.     

Osmotic adjustment increases water uptake, remobilization of assimilates and maintains photosynthesis in chickpea under drought

Eight chickpea advanced breeding lines (ABLs) and their parents were evaluated for osmotic adjustment (OA), leaf carbohydrates and gas exchange under dryland field . These (ABLs) were derived from crosses between CTS 60543 x Kaniva and Tyson x Kaniva. Mean leaf water potential (LWP) fell down from -1.00 MPa at pre-stress level to about -2.25 MPa during terminal stress. Relative water content (RWC) showed periodic changes with alternate decrease or increase at certain interval, which also influenced the values of OA (low or high) in number of genotypes e.g. Kaniva, CTS 60543, Tyson and M 75. Significant variation in OA ranging 0.45 to 0.88 MPa was observed at high level of stress at -2.5 MPa. However, none of the genotypes showed stability of OA over the period of stress. Leaf starch declined even at mild stress (LWP, -1.6 MPa) resulting in an increase in hexose sugars and activation state of sucrose-phosphate synthase (SPS) that led to accumulation of sucrose. Both photosynthesis (Pmax) and transpiration decreased concurrently in two chickpea lines M 129 and Tyson with increasing water stress. However, rate of decline in the photosynthesis slowed down even drought was further intensified. The observed periodic changes in OA, RWC and photosynthesis appeared to be associated with drought-induced changes in SPS and carbohydrates which modify water uptake of the leaves.     

UPLC‐HRMS‐based untargeted metabolic profiling reveals changes in chickpea (Cicer arietinum) metabolome following long‐term drought stress

Genetic improvement for drought tolerance in chickpea requires a solid understanding of biochemical processes involved with different physiological mechanisms. The objective of this study is to demonstrate genetic variations in altered metabolic levels in chickpea varieties (tolerant and sensitive) grown under contrasting water regimes through ultrahigh‐performance liquid chromatography/high‐resolution mass spectrometry‐based untargeted metabolomic profiling. Chickpea plants were exposed to drought stress at the 3‐leaf stage for 25 days, and the leaves were harvested at 14 and 25 days after the imposition of drought stress. Stress produced significant reduction in chlorophyll content, F_v/F_m, relative water content, and shoot and root dry weight. Twenty known metabolites were identified as most important by 2 different methods including significant analysis of metabolites and partial least squares discriminant analysis. The most pronounced increase in accumulation due to drought stress was demonstrated for allantoin, l ‐proline, l ‐arginine, l ‐histidine, l ‐isoleucine, and tryptophan. Metabolites that showed a decreased level of accumulation under drought conditions were choline, phenylalanine, gamma‐aminobutyric acid, alanine, phenylalanine, tyrosine, glucosamine, guanine, and aspartic acid. Aminoacyl‐tRNA and plant secondary metabolite biosynthesis and amino acid metabolism or synthesis pathways were involved in producing genetic variation under drought conditions. Metabolic changes in light of drought conditions highlighted pools of metabolites that affect the metabolic and physiological adjustment in chickpea that reduced drought impacts.     

Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach

In peach (Prunus persica [L.] Batsch.), sorbitol and sucrose are the two main forms of photosynthetic and translocated carbon and may have different functions depending on the organ of utilization and its developmental stage. The role and interaction of sorbitol and sucrose metabolism was studied in mature leaves (source) and shoot tips (sinks) of ‘Nemaguard’ peach under drought stress. Plants were irrigated daily at rates of 100, 67, and 33% of evapotranspiration (ET). The relative elongation rate (RER) of growing shoots was measured daily. In mature leaves, water potential (Ψ_w), osmotic potential (Ψ_s), sorbitol‐6‐phosphate dehydrogenase (S6PDH, EC 1.1.1.200), and sucrose‐phosphate synthase (SPS, EC 2.4.1.14) activities were measured weekly. Measurements of Ψ_s, sorbitol dehydrogenase (SDH, 1.1.1.14), sucrose synthase (SS, EC 2.4.1.13), acid invertase (AI, EC 3.2.1.26), and neutral invertase (NI, EC 3.2.1.27) activities were taken weekly in shoot tips. Drought stress reduced RER and Ψ_w of plants in proportion to water supply. Osmotic adjustment was detected by the second week of treatment in mature leaves and by the third week in shoot tips. Both SDH and S6PDH activities were reduced by drought stress within 4 days of treatment and positively correlated with overall Ψ_w levels. However, only SDH activity was correlated with Ψ_s. Among the sucrose enzymes, only SS was affected by drought, being reduced after 3 weeks. Sorbitol accumulation in both mature leaves and shoot tips of stressed plants was observed starting from the second week of treatment and reached up to 80% of total solutes involved in osmotic adjustment. Sucrose content was up to 8‐fold lower than sorbitol content and accumulated only occasionally. We conclude that a loss of SDH activity in sinks leads to osmotic adjustment via sorbitol accumulation in peach. We propose an adaptive role of sorbitol metabolism versus a maintenance role of sucrose metabolism in peach under drought stress.     

Facing drought in a Mediterranean post-fire community: tissue water relations in species with different life traits

Bulk shoot water potential, the osmotic component and the bulk modulus of elasticity were measured throughout one growing season in four species co-occurring in a post-fire Mediterranean community in southern Italy: Pinus halepensis, Phillyrea latifolia, Cistus salvifolius and Rosmarinus officinalis. A severe drought occurred throughout the measurement period. Large seasonal fluctuations have been observed for both predawn and afternoon water potential in all species. Although minimum values down to –4 MPa have been measured, plant water potential always recovered to less negative values after drought. Daily amplitude of water potential decreased with increasing plant water stress in all species. In Cistus and Rosmarinus less ability for short-term control of plant water status has been assessed. Osmotic potential at full turgor did not display clear seasonal patterns, with no consistent ranking of species by their osmotic values. In most cases, no osmotic adjustment (lowering of osmotic potentials) and no change in tissue elastic properties were observed in response to increasing summer drought and intensity of water stress.     

Osmotic Adjustment in Cotton (Gossypium hirsutum L.) Leaves and Roots in Response to Water Stress

The relative magnitude of adjustment in osmotic potential (ψ_s) of water-stressed cotton (Gossypium hirsutum L.) leaves and roots was studied using plants raised in pots of sand and grown in a growth chamber. One and three water-stress preconditioning cycles were imposed by withholding water, and the subsequent adjustment in solute potential upon relief of the stress and complete rehydration was monitored with thermocouple psychrometers. Both leaves and roots exhibited a substantial adjustment in ψ_s in response to water stress with the former exhibiting the larger absolute adjustment. The osmotic adjustment of leaves was 0.41 megapascal compared to 0.19 megapascal in the roots. The roots, however, exhibited much larger percentage osmotic adjustments of 46 and 63% in the one and three stress cycles, respectively, compared to 22 and 40% in the leaves in similar stress cycles. The osmotically adjusted condition of leaves and roots decreased after relief of the single cycle stress to about half the initial value within 3 days, and to the well-watered control level within 6 days. In contrast, increasing the number of water-stress preconditioning cycles resulted in significant percentage osmotic adjustment still being present after 6 days in roots but not in the leaves. The decrease in ψ_s of leaves persisted longer in field-grown cotton plants compared to plants of the same age grown in the growth chamber. The advantage of decreased ψ_s in leaves and roots of water-stressed cotton plants was associated with the maintenance of turgor during periods of decreasing water potentials.     

Influence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants

Accumulation of various osmolytes was examined in plants of sugar beet cv. Janus grown under two soil water treatments: control (60% of the field water capacity; FWC) and drought (30–35% FWC). The water shortage started on the 61st day after emergence (DAE), at the stage of the beginning of tap-roots development and was imposed for 35 days. Osmotic potential of sugar beet plant organs, particularly tap-roots, was decreased significantly as a consequence of a long-term drought. Water shortage reduced univalent (K⁺, Na⁺) cations concentrations in the petioles and divalent (Ca²⁺, Mg²⁺) ions level in the mature and old leaves. Cation concentrations in the tap-roots were not affected by water shortage. The ratio of univalent to divalent cations was significantly increased in young leaves and petioles as a consequence of drought. Long-term water deficit caused a significant reduction of inorganic phosphorus (P_i) concentration in young and old leaves. Under the water stress condition, the concentration of proline was increased in all individual plant organs, except proline concentration in the youngest leaves. Drought treatment caused a significant increase of glycine betaine content in shoot without any change in tap-roots. Glucose concentrations were significantly increased only in tap-roots as the effect of drought. In response to water shortage the accumulation of sucrose was observed in all the examined leaves and tap-roots. Overall, a long-term drought activated an effective mechanism for osmotic adjustment both in the shoot and in the root tissues which may be critical to survival rather than to maintain plant growth but sugar beet organs accumulate different solutes as a response to water cessation.     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: II. Osmotic adjustment and changes of soluble carbohydrates and amino acids under osmotic stress

The possibility was considered that osmotic adjustment, the ability to accumulate solutes in response to water stress, may contribute to growth rate differences among closely-related genotypes of trees. Progeny variation in osmotic adjustment and turgor regulation was investigated by comparing changes in osmotic and pressure potentials, soluble carbohydrates, and amino acids in osmotically stressed seedlings in 4 full-sib progenies of black spruce [ Picea mariana (Mill.) B. S. P.] that differed in growth rate under drought. Osmotic stress was induced by a stepwise increase in the concentration of polyethylene glycol (PEG)-3350 from 10 (w/v) to 18 and 25%, which provided osmotic potentials in solution culture of -0.4, -1.0 and -2.0 MPa each for 3 days. All 4 progenies maintained a positive cell turgor even at 25% PEG, due to a significant decline in osmotic potential. Although total amino acids, principally proline, increased, ca 60% of the decrease in osmotic potential was attributable to soluble carbohydrates and glucose was the major osmoregulating solute. There was little progeny variation in any of measured parameters in unstressed seedlings. Compared to two slower-growing progenies, the two progenies capable of more vigorous growth under drought in the field accumulated more soluble carbohydrates (mainly glucose and fructose), developed lower osmotic potential and maintained higher turgor pressure when osmotically-stressed in solution culture. The ability to adjust osmotically and maintain turgor under drought stress could thus be a useful criterion for the early selection of faster-growing, drought-tolerant genotypes.     

The role of solute accumulation, osmotic adjustment and changes in cell wall elasticity in drought tolerance in Ziziphus mauritiana (Lamk.)

Ber (Ziziphus mauritiana Lamk.) is a major fruit tree crop of the north-west Indian arid zone. In a study of the physiological basis of drought tolerance in this species, two glasshouse experiments were conducted in which trees were droughted during single stress-cycles. In the first experiment, during a 13 d drying cycle, pre-dawn leaf water (leaf) and osmotic () potentials in droughted trees declined from -0.5 and -1.4 MPa to -1.7 and -2.2 MPa, respectively, for a decrease in relative water content () of 14%. During drought stress, changes in sugar metabolism were associated with significant increases in concentrations of hexose sugars (3.8-fold), cyclitol (scyllo-inositol; 1.5-fold), and proline (35-fold; expressed per unit dry weight), suggesting that altered solute partitioning may be an important factor in drought tolerance of Ziziphus. On rewatering pre-dawn leaf and recovered fully, but remained depressed by 0.4 MPa relative to control values, indicating that solute concentration per unit water content had changed during the drought cycle.Evidence for osmotic adjustment was provided from a second study during which a gradual drought was imposed. Pressure-volume analysis revealed a 0.7 MPa reduction in osmotic potential at full turgor, with leaf at turgor loss depressed by 1 MPa in drought-stressed leaves. Coupled with osmotic adjustment, during gradual drought, was a 65% increase in bulk tissue elastic modulus (wall rigidity) which resulted in turgor loss at the same in both stressed and unstressed leaves. The possible ecological significance of maintenance of turgor potential and cell volume at low water potentials for drought tolerance in Ziziphus is discussed.Keywords: Ziziphus mauritiana, drought, solute accumulation, osmotic adjustment, proline.      

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.     

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.     

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     

Hydraulic regulation strategies for whole-plant water balance of two maize inbred lines differing in drought resistance under short-term osmotic stress

The conservation of water in agriculture requires an understanding of the mechanisms of plant–water relations. This study aimed to reveal hydraulic regulation strategies of maize (Zea mays L.) for maintaining the plant water balance during drought. The water relations of two maize inbred lines (Tian4 and 478) that differ in their resistance to drought in the field were investigated under well-watered conditions and osmotic stress induced with 10 % PEG 6000. The leaf transpiration rate and leaf water potential of 478 varied diurnally, but remained constant in Tian4, which is more drought resistant. Tian4 plants showed morphological, anatomical and physiological advantages that protected them from foliar water loss. The strategies of leaf hydraulics to regulate leaf water balance during the day and during short-term osmotic stress also differed between Tian4 and 478. The leaf hydraulic conductivity of Tian4 and 478 increased temporarily, but their root hydraulic conductivities were reduced under osmotic stress. However, the root hydraulic conductivity of Tian4 subsequently recovered. Lower and rapidly reduced leaf transpiration and the ability of root hydraulics to recover from short-term osmotic stress can help explain the strategies for plant water balance of drought-tolerant maize.     

Responses of pomegranate cultivars to severe water stress and recovery: changes on antioxidant enzyme activities,gene expression patterns and water stress responsive metabolites

The biochemical and molecular responses of five commercially well-known pomegranate cultivars to severe water stress were studied. The cultivars were subjected to 14-day water stress by withholding irrigation, followed by re-watering for 7 days. Results showed clear differences in metabolites contents and activities of antioxidant enzymes among various pomegranate cultivars during severe water stress and recovery. According to our results, increased accumulation of proline in pomegranate was not related to osmotic adjustment during severe water stress. Except for ‘Ghojagh’, leaves grown under severe water stress conditions showed symptoms of oxidative stress such as reduced chlorophyll concentration. The improved performance of ‘Ghojagh’ under drought stress may be associated with an efficient osmotic adjustment. The up- or down regulated expression of cytosolic glutathione reductase (cytosolic GR) and glutathione peroxidase were observed under drought conditions. Moreover, the suppressed expression of cytosolic GR was also noted. Comparatively, ‘Rabab’ exhibited higher antioxidant capacity and an efficient ROS-scavenging mechanism under drought stress. Lower levels of membrane lipid peroxidation in ‘Ghojagh’ and ‘Rabab’ under drought stress and the marked reduction of malondialdehyde concentration after re-watering represents that these cultivars have a good tolerance to drought stress. As a first step towards the study of the biochemical and molecular responses of pomegranate plants to water stress, this research provides new information into the mechanisms of drought tolerance in the plants.