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.     

Desiccation tolerance in Physcomitrella patens: Rate of dehydration and the involvement of endogenous abscisic acid (ABA)

The moss Physcomitrella patens , a model system for basal land plants, tolerates several abiotic stresses, including dehydration. We previously reported that Physcomitrella patens survives equilibrium dehydration to ?13 MPa in a closed system at 91% RH. Tolerance of desiccation to water potentials below ?100 MPa was only achieved by pretreatment with exogenous abscisic acid (ABA). We report here that gametophores, but not protonemata, can survive desiccation below ?100 MPa after a gradual drying regime in an open system, without exogenous ABA. In contrast, faster equilibrium drying at 90% RH for 3–5 days did not induce desiccation tolerance in either tissue. Endogenous ABA accumulated in protonemata and gametophores under both drying regimes, so did not correlate directly with desiccation tolerance. Gametophores of a Ppabi3a/b/c triple knock out transgenic line also survived the gradual dehydration regime, despite impaired ABA signaling. Our results suggest that the initial drying rate, and not the amount of endogenous ABA, may be critical in the acquisition of desiccation tolerance. Results from this work will provide insight into ongoing studies to uncover the role of ABA in the dehydration response and the underlying mechanisms of desiccation tolerance in this bryophyte.     

Abscisic acid and osmotic stress or slow drying independently induce desiccation tolerance in mutant seeds of Arabidopsis thaliana

Action of endogenous abscisic acid (ABA) is absent in the ABA-deficient and -insensitive double mutant ( aba-1abi3–1 ) seeds of Arabidopsis thaliana . Thus, responses to osmotic stress and dehydration can be studied without interference of endogenous ABA. Seeds of this double mutunt are viable hut desiceation-intolerant. However, desiccation tolerance can he induced by either (1) slow dehydration of immature seeds; (2) treatment of immature seeds with osmotica or; (31 due to the leakiness of the ABA-insensitivty mutation, by application of exogenous ABA. Consequently it is concluded that either ABA or osmotic- or dehydration-stress and related gene expression meets the minimal requirements for acquisition of desiccation tolerance in seeds of Arabidopsis thalianna .     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Drought tolerance, abscisic acid and electrolyte leakage in fast-and slow-growing black spruce (Picea mariana) progenies

To investigate the role that drought tolerance plays in growth, abscisic acid (ABA) accumulation and electrolyte leakage during water stress were compared in fast- and slow-growing black spruce ( Picea mariana [Mill.] B. S. P.) progenies. Changes in the ABA content of the needles were quantified using an indirect enzyme-linked immuno-sorbent assay validated by gas chromatography electron capture detection. Needle electrolyte leakage was estimated using a conductivity bridge. Seedlings were stressed using (1) osmotic stress, induced by a stepwise increase in concentrations of polyethylene glycol 3 350 (PEG) for ABA study and (2) air drying for electrolyte leakage study. Progenies did not differ in ABA levels under unstressed conditions, but progeny differences were observed under osmotic stress. Needle ABA content increased up to 500% under osmotic stress. Slow-growing black spruce progenies (25 and 46) accumulated more ABA under moderate (18% PEG), but not severe (25% PEG), osmotic stress. The slow-growing progenies also leaked more electrolytes under moderate to severe water stress and lost 50% electrolytes at a higher xylem tension, suggesting they suffered more injury and were less dehydration tolerant. Our previously-published results showed that slow-growing progenies lost their photosynthesis and stomatal conductance more quickly during osmotic stress and recovered more slowly after rehydration. Therefore, tolerance of dehydration leading to a maintenance of physiological integrity during drought stress could explain the fast growth rates of more vigorous black spruce progenies.     

Expression of an mRNA with sequence similarity to pea dehydrin (Psdhn 1) in guard cells of Vicia faba in response to exogenous abscisic acid

The function and location of guard cells uniquely subject them to stress. First, stomatal movements require large fluctuations in the concentration of potassium salts. Second, guard cell inner walls are the first surfaces exposed to evaporation and apoplastic solutes may accumulate there as a result. We have therefore investigated whether guard cells exhibit atypical expression of dehydrin genes because dehydrins accumulate in vegetative tissues in response to water stress. We have also assayed for osmotin mRNA, which is up-regulated in leaves in response to various stresses. mRNA probes for several representative genes were used with RNA extracts from control and water-stressed Vicia faba leaflets. Correlatively, these probes were used with RNA extracts from "isolated' guard cells that had been incubated with combinations of abscisic acid, mannitol and Ca²⁺. (Isolated guard cells are epidermal strips sonicated to destroy cells other than guard cells.) Hybridization with the probe prepared for a dehydrin from Pisum sativum (Psdhn 1) was detected in leaf extracts only if the leaf had been stressed. Similarly, after 1- and 6-h incubations with abscisic acid, isolated guard cells contained an mRNA that hybridized with the probe for Psdhn 1. Appearance of this abscisic acid-dependent mRNA required neither mannitol nor exogenous Ca²⁺. Regardless of the conditions or tissue, no hybridization was detected with the probe against osmotin, but our interpretation of this result is qualified. The simplest conclusion is that atypical expression of dehydrin is not the mechanism by which guard cells cope with their peculiar function and location.     

Contribution of osmotic adjustment to the dehydration tolerance of water-stressed pigeon pea (Cajanus cajan (L.) millsp.) leaves

Abstract Water-stressed pigeonpea leaves have high levels of osmotic adjustment at low leaf water potentials. The possible contribution of this adjustment of dehydration tolerance of leaves was examined in plants grown in a controlled environment. Osmotic adjustment was varied by withholding water from plants growing in differing amounts of soil, which resulted in different rates of decline of leaf water potential. The level of osmotic adjustment was inversely related to leaf water potential in all treatments. In addition, at any particular water potential, plants that had experienced a rapid development of stress exhibited less osmotic adjustment than plants that experienced a slower development of stress. Leaves with different levels of osmotic adjustment died at water potentials between –3.4 and –6.3 MPa, but all leaves died at a similar relative water content (32%). Consequently, leaves died when relative water content reached a lethal value, rather than when a lethal leaf water potential was reached. Osmotic adjustment delayed the time and lowered the leaf water potential when the lethal relative water content occurred, because it helped maintain higher relative water contents at low leaf water potentials. The consequences of osmotic adjustment for leaf survival in water-stressed pigeonpea are discussed.     

Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants

The hypothesis that physiologically activeconcentrations of salicylic acid (SA) and itsderivatives can confer stress tolerance in plants wasevaluated using bean (Phaseolus vulgaris L.) andtomato (Lycopersicon esculentum L.). Plantsgrown from seeds imbibed in aqueous solutions (0.1--0.5 mM) of salicylic acid or acetyl salicylic acid(ASA) displayed enhanced tolerance to heat, chillingand drought stresses. Seedlings acquired similarstress tolerance when SA or ASA treatments wereapplied as soil drenches. The fact that seedimbibition with SA or ASA confers stress tolerance inplants is more consistent with a signaling role ofthese molecules, leading to the expression oftolerance rather than a direct effect. Induction ofmultiple stress tolerance in plants by exogenousapplication of SA and its derivatives may have asignificant practical application in agriculture,horticulture and forestry.     

The effect of abscisic acid on the growth and storage of germinating rape (Brassica napus L.) seed dried following selection on the basis of a newly-emerged radicle

Abscisic acid at 1 × 10^–4M concentration controlled the progress of the emerging radicle from germinating rape seeds and apparently restricted it to the dehydration tolerant phase. ABA treatment during germination followed by washing reduced the deleterious effects of drying the seeds following selection based on a newly-emerged radicle. Furthermore, the longevity of these low-moisture-content germinating seeds at a range of temperatures was improved by the ABA treatment. The viability of the treated seeds stored at –20°C was maintained for up to 100 days.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Genetic variation in photosynthetic performance and tolerance to osmotic stress (desiccation,freezing, hyposalinity) in the rocky littoral foundation species Fucus vesiculosus (Fucales,Phaeophyceae)

Genetic diversity may play an analogous role to species diversity, as it can contribute to ecosystem function and stability, and provision of ecosystem services. In the Baltic Sea, perennial algal beds are often comprised of only Fucus vesiculosus and the amount of genetic variation in fitness‐related traits (i.e., the ability of the alga to photosynthesize or withstand stress) will thus determine the alga's local persistence in a changing environment. To study genetic variation in the crucial traits behind persistence we grew replicate vegetative branches that came from the same genotype in common gardens. We quantified osmotic stress tolerance and recovery responses by exposing branches to desiccation, freezing, and hyposalinity regimens. Our results show that genetic variation among genotypes was apparent for some photosynthetic parameters (maximal electron transport rate, saturation irradiance for electron transport, nonphotochemical quenching) and growth. Algae tolerated freezing (1,440 min at ?2.5°C) and hyposalinity (1,560 min at 2.5) well, but did not recover from desiccation (70 min at 12°C, causing ~94% water loss). Furthermore, we found very little if any evidence on genetic variation in tolerance to these stressors. Our results suggest that low salinity and cold winters in the northern marginal populations selected for hyposalinity and freezing tolerant genotypes, possibly eroding genetic variation in tolerance, but that tolerance to harsh desiccation has been lost, likely due to relaxed selection. The overall availability of genetic variation in fitness related traits might be supportive for F. vesiculosus during adaptation to gradual changes of its environment.     

Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings

Changes in abscisic acid (ABA) contents in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl2, the ABA content rapidly increased in the leaves and roots of Cd-tolerant cultivar (cv. Tainung 67, TNG67) but not in the Cd-sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 caused by Cd was less than that of TNG67. Exogenous application of ABA reduced transpiration rate, decreased Cd content, and enhanced Cd tolerance of TN1 seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone, reduced ABA accumulation, increased transpiration rate and Cd content, and decreased Cd tolerance of TNG67 seedlings. Fluridone effect on Cd toxicity of TNG67 seedlings was reversed by the application of ABA. The roles of endogenous ABA in Cd tolerance of rice seedlings are discussed and suggested.     

Growth, Water Content, and Proline Accumulation in Drought-Stressed Callus of Date Palm

This study was conducted to examine the response of date palm (Phoenix dactylifera L., cvs. Barhee and Hillali) calli to water stress. Callus derived from shoot tip explants was inoculated in liquid Murashige and Skoog medium containing 10 mg dm^–3-naphthaleneacetic acid, 1.5 mg dm^–3 2-isopentenyladenine, and 0 to 30 % (m/v) polyethylene glycol (PEG 8000) to examine the effect of water stress. After 2 weeks, callus growth, water content, and proline accumulation were assessed. Increasing water stress caused a progressive reduction in growth as expressed in callus fresh mass, relative growth rate, and index of tolerance. Both genotypes tested followed this general trend, however, cv. Barhee was more tolerant to drought stress than cv. Hillali. Increasing PEG concentration was also associated with a progressive reduction in water content and increased content of endogenous free proline.     

Differential proteomics of dehydration and rehydration in bryophytes: evidence towards a common desiccation tolerance mechanism

All bryophytes evolved desiccation tolerance (DT) mechanisms during the invasion of terrestrial habitats by early land plants. Are these DT mechanisms still present in bryophytes that colonize aquatic habitats? The aquatic bryophyte Fontinalis antipyretica Hedw. was subjected to two drying regimes and alterations in protein profiles and sucrose accumulation during dehydration and rehydration were investigated. Results show that during fast dehydration, there is very little variation in protein profiles, and upon rehydration proteins are leaked. On the other hand, slow dehydration induces changes in both dehydration and rehydration protein profiles, being similar to the protein profiles displayed by the terrestrial bryophytes Physcomitrella patens (Hedw.) Bruch and Schimp. and, to what is comparable with Syntrichia ruralis (Hedw.) F. Weber and D. Mohr. During dehydration there was a reduction in proteins associated with photosynthesis and the cytoskeleton, and an associated accumulation of proteins involved in sugar metabolism and plant defence mechanisms. Upon rehydration, protein accumulation patterns return to control values for both photosynthesis and cytoskeleton whereas proteins associated with sugar metabolism and defence proteins remain high. The current results suggest that bryophytes from different ecological adaptations may share common DT mechanisms.     

Changes in abscisic acid levels in embryo axes of Norway maple and sycamore seeds during maturation and dehydration

Changes in the abscisic acid (ABA) levels in embryo axes of seeds, belonging to the orthodox (Norway maple — Acer platanoides L.) and recalcitrant (sycamore — Acer pseudoplatanus L.) categories, were investigated throughout maturation using an ELISA (enzyme-linked immunosorbent assay) test. Concentration of ABA in embryo axes substantially differed depending on species and sampling date. ABA was always higher in Norway maple except at the end of seed maturation when ABA content was similar in both species. During maturation ABA decreased in both species but the decline was more marked in Norway maple than in sycamore (11 vs. 3 fold). These species also differed in the pattern of ABA changes, which in sycamore embryo axes was very regular, while in Norway maple a sharp decrease was recorded after acquisition by the seeds of tolerance to desiccation. Dehydration of embryo axes of Norway maple caused a further significant decrease of ABA level. In contrast, in dehydrated sycamore embryo axes ABA content did not decrease, but slightly increased. The role of ABA in desiccation tolerance and dormancy of Norway maple and sycamore seeds is discussed.     

Ecological and evolutionary consequences of desiccation tolerance in tropical fern gametophytes

Ferns have radiated into the same diverse environments as spermatophytes, and have done so with an independent gametophyte that is not protected by the parent plant. The degree and extent of desiccation tolerance (DT) in the gametophytes of tropical fern species was assessed to understand mechanisms that have allowed ferns to radiate into a diversity of habitats. Species from several functional groups were subjected to a series of desiccation events, including varying degrees of intensity and multiple desiccation cycles. Measurements of chlorophyll fluorescence were used to assess recovery ability and compared with species ecology and gametophyte morphology. It is shown that vegetative DT (rare in vascular plants) is widely exhibited in fern gametophytes and the degree of tolerance is linked to species habitat preference. It is proposed that gametophyte morphology influences water-holding capacity, a novel mechanism that may help to explain how ferns have radiated into drought-prone habitats. Fern gametophytes have often been portrayed as extreme mesophytes with little tolerance for desiccation. The discovery of DT in gametophytes holds potential for improving our understanding of both the controls on fern species distribution and their evolution. It also advances a new system with which to study the evolution of DT in vascular plants.     

Hardening by partial dehydration and ABA increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon

* BACKGROUND AND AIMS: The ability of partial dehydration and abscisic acid pretreatments to increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon was tested. * METHODS: Net photosynthesis and respiration were measured using infrared gas analysis during a drying and rehydration cycle. At the same time, the efficiency of photosystem two was measured using chlorophyll fluorescence, and the concentrations of chlorophyll a were spectrophotometrically assayed. Heat production was also measured during a shorter drying and rehydration cycle using differential dark microcalorimetry. * KEY RESULTS: Pretreating lichens by dehydrating them to a relative water content of approx. 0.65 for 3 d, followed by storing thalli hydrated for 1 d in the light, significantly improved their ability to recover net photosynthesis during rehydration after desiccation for 15 but not 30 d. Abscisic acid pretreatment could substitute for partial dehydration. The improved rates of photosynthesis during the rehydration of pretreated material were not accompanied by preservation of photosystem two activity or chlorophyll a concentrations compared with untreated lichens. Partial dehydration and ABA pretreatments appeared to have little direct effect on the desiccation tolerance of the mycobiont, because the bursts of respiration and heat production that occurred during rehydration were similar in control and pretreated lichens. * CONCLUSIONS: Results indicate that the photobiont of P. polydactylon possesses inducible tolerance mechanisms that reduce desiccation-induced damage to carbon fixation, and will therefore improve the supply of carbohydrates to the whole thallus following stress. In this lichen, ABA is involved in signal transduction pathways that increase tolerance of the photobiont.     

Genotypic variation in coleus in the ability to accumulate abscisic acid in response to water deficit

Terry, P. H., Krizek, D. T. and Mirecki, R. M. 1988. Genotypic variation in coleus in the ability to accumulate abscisic acid in response to water deficit. - Physiol. Plant. 72: 441–449.
Abscisic acid (ABA) concentration and plant and soil water potentials were determined in leaves of three cultivars of Coleus blumei Benth. cvs. Marty, Buckley Supreme and PI354190, chosen for their differences in sensitivity to drought, SO₂ and/or chilling stress. Plants were subjected to 'gradual' soil moisture stress (SMS) for 0–6 days, during which time the soil dried out slowly and to more 'rapid' SMS for 0–9 days where the soil dried out at a faster rate. Plants were propagated from clonal stock in the greenhouse and transferred to the growth chamber, where they were maintained for 1 week prior to beginning water stress treatment. ABA concentration was determined as the methyl ester using a gas chromatography - electron capture detector method. Samples for ABA determinations were taken from the third pair of leaves from the apex at the same time each day (1430 h). Measurements of stomatal conductance (C₅) and leaf water potential (φ_l) were made on the fourth pair of leaves from the apex, using the same plants as those sampled for ABA. During the more rapid stress portion of the study soil matric potentials (φ_m) were monitored on a daily basis. Despite large cultivar differences in ABA concentration at 0–6 days, by 7–9 days these differences had largely disappeared. Except for drought-insensitive cv. Marty, there was generally little correlation between ABA levels and measurements of plant and soil water status at 7–9 days.