Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: I. Stomatal and gas exchange responses to osmotic stress

Sixteen years growth in height and basal stem diameter of full-sib black spruce [ Picea mariana (Mill.) B. S. P.] progenies varied with soil moisture availability. The responses to water stress of two faster-growing progenies under drought were compared with two slower-growing progenies to determine the physiological basis of drought tolerance. Six-month-old seedlings were stressed using an osmoticum, polyethylene glycol-3350 (PEG). Seedlings were passed through a series of increasing concentration: 10, 18 and 25% PEG (w/v) each for 3 days to provide solution water potentials of -0.4, -1.0, and -2.0 MPa, respectively. The stress was then relieved by returning the seedlings to nutrient solution without PEG for 24 hours. Gas exchange and water relation parameters were similar in the 4 progenies prior to the imposition of the stress but varied during the stress and after stress relief. The two progenies which grew more vigorously on the driest site maintained significantly higher stomatal conductance, leaf transpiration rate, and net photosynthesis rate during mild (10% PEG) and moderate (18% PEG), but not severe (25% PEG) osmotic stress, and also recovered faster after release of the stress than the other two slower-growing progenies. Black spruce progenies did not differ in xylem water potential or water use efficiency. Progenies capable of faster growth under drought stress were thus characterized by a greater dehydration tolerance, rather than postponement, compared with slower-growing progenies.     

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.     

Abscisic acid induced electrolyte leakage in woody species with contrasting ecological requirements

To determine the relationship between drought, abscisic acid (ABA) levels, and membrane leakage, these aspects were studied in three woody species with contrasting ecological requirements. Physiological comparisons were made, during a 14-day drought-recovery cycle, on a drought resistant species, jack pine ( Pinus banksiana Lamb.), and two more drought susceptible species, black spruce ( Picea mariana (Mill) B.S.P.) and flooded gum ( Eucalyptus grandis W. Hill ex Maiden). As the xylem pressure potentials (Ψ), declined under drought, both foliar ABA and electrolyte leakage increased significantly. However, ABA increased prior to the increase in leakage, and there was a significant linear correlation between endogenous ABA and leakage in all 3 species. Addition of exogenous ABA to the bathing solutions also significantly increased leakage of both unstressed and (sorbitol) osmotically-stressed leaf segments in response to increasing (log) concentrations of (±) cis-trans ABA [0–10.0 μ M ]. Electrolyte leakage in black spruce and flooded gum was more sensitive to stress-induced ABA than in jack pine. The results suggest that drought increases foliar ABA which initiates membrane leakage, and that sensitivity of membranes to ABA accumulation varies, depending on the ecological requirements of a species.     

Hydraulic adjustment in jack pine and black spruce seedlings under controlled cycles of dehydration and rehydration

Drought adjustments were compared in black spruce ( Picea mariana [Mill] B.S.P), and jack pine ( Pinus banksiana [Lamb.]) by subjecting seedlings to five cycles of dehydration and rehydration. A computer-controlled root misting chamber system, supplied low (−1.5 MPa), moderate (−2.0 MPa), and severe (−2.5 MPa) dehydration, respectively, in cycles 1, 3 and 5. Although cell water relations failed to adjust to chronic dehydration, there was limited osmotic adjustment in black spruce (cycle 3), and water was re-allocated from the apoplast to the symplast in jack pine (cycles 1 and 3). Dehydration postponement was more important than dehydration tolerance. Jack pine was better able to postpone dehydration than black spruce. Specific conductivity, the hydraulic conductivity per unit stem cross-sectional area, was lower in jack pine and slower to decline during chronic dehydration. When specific conductivity was corrected for the greater leaf area in black spruce, the leaf-specific conductivity did not differ in the two species. There was no increase in needle leakage in jack pine and stomata in jack pine seedlings reopened fully after rehydration. Black spruce was more of a 'water spender', and less water stress (−2.0 MPa, cycle 3) was required to lower specific conductivity, compared to jack pine (−2.5 MPa, cycle 5). Leakage from needle membranes increased in black spruce, and stomata failed to reopen after rewatering (cycles 3 and 5). A greater needle area, smaller root system, and a higher specific conductivity lowered the water stress threshold for cavitation in black spruce, which is confined to moister sites in the boreal forest. Jack pine had a larger root system, smaller needle area and lower specific conductivity than black spruce. Because of these static features, jack pine is more drought tolerant and it is often found on sites that are too hot and dry for black spruce.     

The relative contribution of elastic and osmotic adjustments to turgor maintenance of woody species.

To determine how tissue water relations vary and contribute to turgor maintenance in species from contrasting ecological zones, seedlings of jack pine ( Pinus banksiana Lamb.), black spruce ( Picea mariana [Mill] B.S.P.) and flooded gum ( Eucalyptus grandis W. Hill ex Maiden) were subjected to an 8 day drought stress by water withholding with and without prior mild water stress conditioning. Jack pine, a deep-rooted species from dry, sandy boreal sites, lost turgor at the lowest relative water content (75–65%) and water potential, and had lowest maximum bulk elastic modulus (E_max of 5.2–5.8 MPa). Although this suggests a high inherent dehydration tolerance, jack pine did not further adjust its elasticity when repeatedly stressed. Black spruce, a shallow-rooted species from predominantly moist sites in the boreal region, lost turgor at intermediate relative water content (86–76%) and water potential, but could adjust its elasticity to maintain turgor in repeatedly stressed tissues. Flooded gum, a deep-rooted species from moist, warm temperate-subtropical regions, had a low inherent drought tolerance since it lost turgor at higher relative water content (88–84%) and water potential, but was capable of some adjustment when the stress was repeated. Elastic adjustment (<3.7 MPa) was more important for turgor maintenance than osmotic adjustment (<0.13 MPa), which was statistically nonsignificant. Maximum bulk modulus of elasticity, but not osmotic potentials at full turgor, was significantly correlated with the relative water content and water potential at zero turgor in droughted seedlings. These results highlight the importance of tissue shrinkage for dehydration tolerance. Both the inherent capacity for turgor maintenance of a species under drought and its ability to adjust to repeated drought should be considered in genetic selections for drought tolerance.     

Reciprocal grafting between clones with contrasting drought tolerance suggests a key role of abscisic acid in coffee acclimation to drought stress

The role of abscisic acid (ABA) in drought tolerance of Coffea canephora is unknown. To determine whether ABA is associated with drought tolerance and if the use of tolerant rootstocks could increase ABA and drought tolerance, we performed reciprocal grafting experiments between clones with contrasting tolerance to drought (clone 109, sensitive; and clone 120, tolerant). Plants were grown in large (120 L) pots in a greenhouse and subjected to drought stress by withholding irrigation. The non-grafted 120 plants and graft treatments with 120 as a rootstock showed a slower reduction of predawn leaf water potential (Ψ_pd) and a lower negative carbon isotopic composition ratio compared with the other grafting combinations in response to drought. The same 120 graft treatments also showed higher leaf ABA concentrations, lower levels of electrolyte leakage, and lower activities of ascorbate peroxidase and catalase under moderate (Ψ_pd?=???1.0 or ??1.5 MPa) and severe (Ψ_pd?=???3.0 MPa) drought. Root ABA concentrations were higher in plants with the 120 rootstocks regardless of watering regime. The 120 shoots could also contribute to drought tolerance because treatment with 120/109 rootstock/scion combination showed postponed dehydration, higher leaf ABA concentration, and lower leaf electrolyte leakage compared with the sensitive clone. We conclude that both the shoot and root systems of the tolerant clone can increase the concentrations of ABA in leaves in response to drought. This further suggests that ABA is associated with a delayed onset of severe water deficit and decreased oxidative damage in C. canephora.     

Different acclimatization mechanisms of two grass pea cultivars to osmotic stress in in vitro culture

Grass pea (Lathyrus sativus L.) is a legume crop known from its tolerance to various abiotic stresses, especially drought. In this study, we investigated: (1) the response of grass pea seedlings to osmotic stress generated in vitro by polyethylene glycol (PEG); (2) potential drought acclimatization mechanisms of two polish grass pea cultivars. Grass pea seeds of two cultivars were sown on media containing different PEG concentrations (0, 5.5, 11.0 mM) and cultivated for 14 days in controlled conditions. Plants’ dry matter increased under osmotic stress (regardless of PEG concentration). In turn, the highest dose of PEG caused a reduction in seedling growth in both cultivars. Furthermore, PEG caused the peroxidase activity increase in whole seedlings and catalase (CAT) activity in roots. However, differences between cultivars were noted in: CAT activity in shoots; while phenols and anthocyanin content as well as electrolyte leakage in shoots and roots. In turn, in both tested genotypes, accumulation of proline increased in shoots under osmotic stress. Obtained results indicate that the examined plants, although belonging to the same species, differ in acclimatization processes leading to elevated tolerance to osmotic stress.     

Drying rate and dehydrin synthesis associated with abscisic acid-induced dehydration tolerance in Spathoglottis plicata orchidaceae protocorms

Dehydration tolerance of in vitro orchid protocorms was investigated under controlled drying conditions and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on Murashige and Skoog (MS) medium containing 10% (v/v) coconut water, 2% (w/v) sucrose and 0.8% (w/v) agar, and were dehydrated in relative humidities (RH) ranging from 7% to 93% at 25 degrees C. The critical water content of dehydration tolerance was determined, using the electrolyte leakage method. Drying rate affected the critical water content. Slow drying under high RH conditions achieved the greatest tolerance to dehydration. ABA pretreatment decreased the drying rate of protocorms, and increased dehydration tolerance. Improved tolerance to dehydration after ABA treatment was correlated with the effect of ABA on drying rate of protocorms. When critical water content of protocorms dried under different RH was plotted as a function of actual drying rate, no significant difference in tolerance to dehydration was observed between ABA-treated and control protocorms. ABA pretreatment and dehydration of orchid protocorms induced the synthesis of dehydrin, especially under the slow drying conditions. ABA pretreatment also promoted dry matter accumulation such as carbohydrates and soluble proteins and increased the concentration of K(+) and Na(+) ions in protocorms. The ABA-induced decrease in drying rate was correlated with lower osmotic potential, the enhanced maturity of protocorms and the accumulation of dehydrin in protocorms during pretreatment.     

The effect of dehydration with or without abscisic acid pretreatment on buds regeneration from <Emphasis Type="Italic">Polypodium vulgare</Emphasis> L. rhizomes

Polypodium vulgare L., a widely distributed fern, is water-stress tolerant. Under controlled dehydration conditions (20% mannitol, 9 h) without and with abscisic acid (ABA) pretreatment (2 mg l⁻¹, 24 h), dehydration tolerance and regenerative, potential of common polypody rhizomes was investigated. We demonstrated the positive effect of ABA on changes in dehydrated rhizome metabolism. ABA pretreatment reduced electrolyte leakage from cells: it has also a role in regulating sucrose accumulation and thus, osmotic adjustment. Our findings confirm that P. vulgare rhizomes are well adapted to stress conditions through maintaining of ability to bud formation by dehydrated and rehydrated rhizomes.     

Ambiol,spermine, and aminoethoxyvinylglycine prevent water stress and protect membranes in Pinus strobus L under drought

The ability of antistress compounds to enhance the drought tolerance of conifer seedlings was tested by feeding plant growth regulators (PGRs) to 1-year-old white pine (Pinus strobus L.), which were then subjected to either a moderate (11 day) or a more severe (16 day) drought. The following PGRs were either fed directly into the xylem or applied as a root drench: the antioxidant Ambiol (2-methyl-4-[dimethylaminomethyl]- 5-hydroxybenzimidazole dihydrochloride), the polyamine, spermine, an anti-ethylene agent, aminoethoxyvinylglycine (AVG), and the inhibitor, abscisic acid (ABA). Leaf water potentials (ƒ_l) declined in untreated seedlings when they were exposed to drought. Preconditioning with PGRs postponed water deficits and prevented membrane leakage under drought. The specific physiological adjustments observed were found to vary, depending on the type of compound. Ambiol, AVG and spermine caused transpirant rates to decline under drought. Although the antitranspirant effects of Ambiol and spermine would explain the increase in water use efficiency under drought, spermine also enhanced photosynthesis. The same compounds promoted osmotic adjustment, which would help to maintain turgor under drought. This was shown by the decline in osmotic potential at full turgor, and at zero turgor, in Ambiol and spermine-treated seedlings. Seedlings treated with Ambiol and ABA could sustain a greater water loss before turgor declined to zero. The possibility that preconditioning may help to maintain leaf physiological functioning under drought by reducing water stress and stress-ethylene production is discussed.     

A relationship between tolerance to dehydration of rice cell lines and ability for ABA synthesis under stress.

Plant dehydration is commonly caused by some adverse environmental conditions such as salinity, drought and freezing. As the plant hormone abscisic acid (ABA) is involved in responses to water stress, we studied its putative relationship with the degree of tolerance to these abiotic stresses. For this purpose we used cell lines that had been established from mature embryos of rice (Oryza sativa L, cvs. Bahia and Bomba), and selected by their high (L-T) or low (L-S) levels of tolerance to each type of stress. Tolerance of rice calli to either osmotic, saline, or freezing stress was generally improved by a previous treatment with ABA. This ABA effect was evident in those callus lines with low tolerance (L-S), as their ability to recover from stress increased up to three fold. Independent of the cultivar used, there were no significant differences in the endogenous ABA contents between untreated L-T and L-S lines. However, upon stress, the increase in endogenous ABA was higher in L-T than in L-S lines. These results, together with those obtained by using Fluridone, an inhibitor of ABA synthesis, show that differences in the level of cell tolerance to osmotic, saline and freezing stress are related to their different capacity of ABA synthesis under stress conditions.     

Metabolic pathways regulated by abscisic acid,salicylic acid and γ‐aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera)

Abscisic acid (ABA), salicylic acid (SA) and γ‐aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 μM), GABA (0.5 mM) and SA (10 μM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5‐oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress‐defense secondary metabolism by GABA.     

Comparison of polyethylene glycol 3350 induced osmotic stress and soil drying for drought simulation in three woody species

 Drought simulation usually involves either soil drying or the use of an osmoticum, such as high molecular weight (>3000) polyethylene glycol (PEG). Although easy to apply, PEG absorption and toxicity remain a concern. This study compared the effects of soil drying and use of an osmoticum (PEG 3350). Osmotic stress and soil drought were applied to 5-month-old seedlings of jack pine (Pinus banksiana Lamb.) and black spruce [Picea mariana (Mill) B.S.P.] , which are both coniferous species from cold, boreal regions of North America, and flooded gum (Eucalyptus grandis W. Hill ex Maiden), a hardwood species growing in warmer, sub-tropical regions of Australia. Results showed that PEG 3350 was absorbed by roots, transported to shoots, and deposited on the leaves of both flooded gum and jack pine (but not black spruce). PEG lowered relative water content and damaged leaf tissues in both species, and also damaged stomata of flooded gum. Although 12 days of PEG-induced osmotic stress produced a decline in water potentials that was similiar to soil drying, it also caused significantly higher membrane injury and reduced net photosynthesis and stomatal conductance in leaves of all three species. Recovery of net photosynthesis and stomatal conductance in PEG-treated jack pine and black spruce was also slower after stress alleviation. Even a short exposure to PEG 3350 adversely affected seedlings compared to soil drought. These results confirmed that drought effects may vary, depending on the species and the method of stress induction. Received: 6 March 1996 / Accepted: 17 September 1996     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

Physiological and Biochemical Responses Reveal the Drought Tolerance Efficacy of the Halophyte Salicornia brachiata

The drought tolerance of Salicornia brachiata seedlings was assessed by monitoring growth, nutrient uptake, electrolyte leakage, lipid peroxidation, and biochemical responses under drought conditions simulated with 0, 10, 20, and 30 % polyethylene glycol (PEG 6000). After 7 days of drought induction, plants were harvested for measurement of various parameters. The biomass decreased and the plant height remained unchanged with PEG treatment. The total plant water content (TWC%) decreased by 11 % at the highest concentration of PEG (30 %). The electrolyte leakage and lipid peroxidation of shoots increased by 17 and 5 %, respectively, in 30 % PEG-treated plants. K⁺ and Ca²⁺ contents of shoots increased in a dose-dependent manner. However, in roots K⁺ content decreased and Ca²⁺ content remained unaffected by PEG treatment. Mg²⁺ content increased at high concentrations of PEG (20–30 %) in shoots and decreased at the highest concentration of PEG (30 %) in roots. Total free amino acids, proline, and polyphenol contents increased progressively with increase in severity of the drought stress. Total sugar content and reducing sugar content increased in 10 and 20 % PEG-treated plants and decreased in 30 % PEG-treated plants. Our results suggest that proline and other free amino acids, sugars, and polyphenols are the main compatible solutes in S. brachiata for maintenance of osmotic balance, protection of cellular macromolecules, detoxification of the cells, and scavenging of free radicals under drought stress. A greater accumulation of compatible solutes also facilitates the maintenance of nutrient uptake and adequate tissue water status and protection of membranes under drought conditions in S. brachiata. The results from the present study suggest that S. brachiata can be used for restoration of arid and semiarid lands of coastal ecosystems.     

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.     

Alleviation of Negative Effects of Water Stress in Two Contrasting Wheat Genotypes by Calcium and Abscisic Acid

The individual and interactive role of calcium and abscisic acid (ABA) in amelioration of water stress simulated by polyethylene glycol (PEG) 6000 was investigated in two contrasting wheat genotypes. PEG solution (osmotic potential –1.5 MPa) was applied to 10-d-old seedlings growing under controlled conditions and changes in photosynthetic rate, activities of ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase, water potential and stomatal conductance were observed in the presence of 0.1 mM ABA, 5 mM calcium chloride, 1 mM verapamil (Ca²⁺ channel blocker), and 1 mM fluridone (inhibitor of ABA biosynthesis). ABA and calcium chloride ameliorated the effects of water stress and the combination of the two was more effective. The two genotypes varied for their sensitivity to ABA and Ca²⁺ under stress. As was evident from application of their inhibitors, ABA caused more alleviation in C 306 (drought tolerant) while HD 2380 (drought susceptible) was more sensitive to Ca²⁺.