Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Desiccation sensitivity and tolerance in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>: assessing limits and damage

The moss Physcomitrella patens is becoming the model of choice for functional genomic studies at the cellular level. Studies report that Physcomitrella survives moderate osmotic and salt stress, and that desiccation tolerance can be induced by exogenous ABA. Our goal was to quantify the extent of dehydration tolerance in wild type moss and to examine the nature of cellular damage caused by desiccation. We exposed Physcomitrella to humidities that generate water potentials from −4 (97% RH) to −273 MPa (13% RH) and monitored water loss until equilibrium. Water contents were measured on a dry matter basis to determine the extent of dehydration because fresh weights (FW) were found to be variable and, therefore, unreliable. We measured electrolyte leakage from rehydrating moss, assessed overall regrowth, and imaged cells to evaluate their response to drying and rehydration. Physcomitrella did not routinely survive water potentials <−13 MPa. Upon rehydration, moss dried to water contents >0.4 g g dm⁻¹ maintained levels of leakage similar to those of hydrated controls. Moss dried to lower water contents leaked extensively, suggesting that plasma membranes were damaged. Moss protonemal cells were shrunken and their walls twisted, even at −13 MPa. Moss cells rehydrated after drying to −273 MPa failed to re-expand completely, again indicating membrane damage. ABA treatment elicited tolerance of desiccation to at least −273 MPa and limited membrane damage. Results of this work will form the basis for ongoing studies on the functional genomics of desiccation tolerance at the cellular level.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis

Recalcitrant seed axes were reported to survive to lower water contents under fast-drying conditions. The present study was to examine the hypothesis that drying rate and dehydration duration could interact to determine desiccation tolerance through different physico-chemical mechanisms. The effect of drying rate on desiccation tolerance of Theobroma cacao seed axes at 16 degrees C was examined. Rapid-drying at low relative humidity (RH) and slow-drying at high RH were more harmful to cocoa axes, because electrolyte leakage began to increase and axis viability began to decrease at high water contents. Maximum desiccation tolerance was observed with intermediate drying rates at RH between 88% and 91%, indicating the existence of an optimal drying rate or optimal desiccation duration. This maximum level of desiccation tolerance for cocoa axes (corresponding to a critical water potential of -9 MPa) was also detected using the equilibration method, in which axes were dehydrated over a series of salt solutions or glycerol solutions until the equilibrium. These data confirmed that the physiological basis of the optimal drying rate is related to both mechanical stress during desiccation and the length of desiccation duration during which deleterious reactions may occur. The optimal drying rate represents a situation where combined damages from mechanical and metabolic stresses become minimal.     

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475-483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G6-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.     

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.     

Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.

Three-week-old protonemata of Funaria hygrometrica Hedw. cultivated in Petri dishes tolerate slow drying (24 h to complete dryness) but not rapid drying (1h to complete dryness). Slowly dried mosses show, on a dry-weight basis, a sixfold increase in abscisic-acid (ABA) contents during the drying process. Rehydrated, slowly dried protonemata have the ability to tolerate subsequent rapid drying. When ABA is added to three-week-old protonemata at a concentration of 10 M for 16 h, tolerance to rapid drying is induced. These data indicate that the induction of drought tolerance in Funaria hygrometrica is mediated by ABA. Mosses treated with ABA loose their water as fast as controls do; therefore, ABA does not act via reduced water loss. However, induction of synthesis of new proteins by ABA may form an important part of the drought tolerance because 10 M cycloheximide inhibits the ABA-mediated tolerance to rapid drying.Abbreviations ABA abscisic acid - CHI cycloheximide - DW dry weight - FW fresh weight - RWL relative water loss This work was supported by grants from the Deutsche Forschungs-gemeinschaft and by a NATO fellowship awarded to R.M. Ros Espin.     

Increased activities of superoxide dismutase and catalase are not the mechanism of desiccation tolerance induced by hardening in the moss Atrichum androgynum

Abstract

The effects of treatments that increase desiccation tolerance were tested on the activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) in the moss Atrichum androgynum subjected to a drying/wetting cycle. Hardening by both abscisic acid (ABA) pretreatment and partial dehydration significantly increased the rate of recovery of photosynthesis during rehydration following desiccation. Hardening treatments had little effect on SOD activity. In non-hardened plants, SOD activity increased three-fold during desiccation for 32 h at 52% rh, but hardened material tended to display smaller increases in activity. During rehydration, SOD activities rapidly declined to their initial values in all treatments. Hardening by partial dehydration, but not ABA, reduced CAT activity. After desiccation for 32 h, material from all treatments displayed about half the initial CAT activity, and activity did not change during subsequent rehydration. Results show that, while the induction of SOD appears to play a role in desiccation tolerance, a similar induction occurred in both hardened and non-hardened mosses. Induction of greater activities of enzymes that scavenge reactive oxygen species is not responsible for the added tolerance induced by hardening treatments.     

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.     

Effects of abscisic acid on growth and dehydration tolerance of <Emphasis Type="Italic">Cynanchum komarovii</Emphasis> seedlings

Cynanchum komarovii is well adapted to hot and dry adverse environments. To determine if exogenous abscisic acid (ABA) affects the growth and dehydration tolerance of this wild plant, ABA was added into the hydroponic solution at a final concentration of 10 μM for 14 days. Root growth is less inhibited than shoot growth under well-watered condition by ABA treatment. ABA reduced the drying rate of seedlings, indicating the acquisition of increased dehydration tolerance. Increased dehydration tolerance is associated with osmotic adjustment and with accumulation of soluble sugars and dehydrins. Results from immunological detection showed that a band of dehydrins with molecular mass of 28.3 kDa appeared in ABA-treated fresh seedlings, which coincided with that in nontreated seedling dessicated for 4 h in 75% relative humidity. The increased ratio of higher to lower amide I bands in Fourier transform infrared spectroscopy analysis indicated that the proportion of unordered structures in proteins was higher after ABA treatment. We concluded that exogenous ABA improved dehydration tolerance of C. komarovii seedlings.     

Different plant provenance same seed tolerance to abiotic stress: implications for <Emphasis Type="Italic">ex situ</Emphasis> germplasm conservation of a widely distributed coastal dune grass (<Emphasis Type="Italic">Uniola paniculata</Emphasis> L.)

Preservation of genetic diversity within germplasm repositories represents an important tool for plant conservation. However, seeds must tolerate extreme levels of post-harvest desiccation and cold to realize benefits of ex situ storage. Factors including local climate and habitat impact expression of desiccation and freezing tolerance especially for widely distributed species. Our aim here was to understand the influence of a latitudinal gradient on seed desiccation and cryo-freezing tolerance. We sampled mature U. paniculata seeds from two geographically and genetically distinct populations then examined seed-water relations and germination following desiccation via equilibrium drying assays (0.5 to 91% RH; ?797 to ?12.9 MPa). Germination ability after drying and subsequent cryo-freezing treatments (?196?°C, 1 to 1440 min) was also evaluated. Seeds of both populations displayed similar reverse sigmoid moisture sorption isotherms characteristic of desiccation tolerant tissues. Furthermore, initial seed water potential (?63 and ?90 MPa) was considerably lower than the lethal limit (?20 MPa) identified for desiccation sensitive tissues. Final germination (range 58–93%) and temporal patterns differed significantly between populations following desiccation and cryo-freezing stress, but these germination responses were similar to initial germination. A higher proportion of non-germinated, yet viable seeds remained for the northern compared to southern population. Location does influence germination response, but differential germination is related to seed dormancy rather than desiccation or cryo-freezing sensitivity. Ex situ conservation of U. paniculata is therefore feasible across the latitudinal gradient studied here.     

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.     

Stress-associated factors increase after desiccation of germinated seeds of <Emphasis Type="Italic">Tabebuia impetiginosa</Emphasis> Mart.

Improved re-establishment of desiccation tolerance was studied in germinated seeds of Tabebuia impetiginosa Mart. by exposing to a polyethylene glycol solution prior to desiccation. The effects of different osmotic potentials and drying rates were studied. In addition, temporary temperature stress and exogenous abscisic acid were applied to evaluate their effect on desiccation tolerance of the protruded radicle. An osmotic potential of −1.7 MPa at 5°C followed by slow drying was most effective in the re-establishment of desiccation tolerance in protruded radicles with a length up to 3 mm. An osmotic potential of −1.4 or −2.0 MPa was less effective. Fast drying completely prevented the re-induction of desiccation tolerance. Cold shock or heat shock prior to osmotic treatment as well as abscisic acid added to the osmotic solution improved desiccation tolerance of protruded radicles. Surprisingly, survival of the germinated seed did not depend on re-establishment of desiccation tolerance in the protruded radicle. Even after the protruded radicle became necrotic and died, the production of adventitious roots from the hypocotyls allowed for survival and the development of high quality seedlings. Thus, T. impetiginosa appeared to be well adapted to the seasonally dry biome in which the species thrives via mechanisms that offer protection against desiccation in the young seedling stage.     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

Dry artificial seeds and desiccation tolerance induction in microspore-derived embryos of broccoli

Desiccation tolerance of broccoli microspore-derived embryos was induced by exogenous application of abscisic acid (ABA). Embryos, which were desiccated to about 10% water content, were estimated for viability after rehydration. Survival was dependent on the ABA concentration and the development stage of embryo, but not on the length of exposure period to ABA or genotype. Cotyledonary stage embryos acquired the highest desiccation tolerance when treated with 1×10^-4M ABA. Under this condition, on average 27–48% of the desiccated embryos could convert into plants. Embryos treated with 1×10^-6M ABA or no ABA or earlier development-staged embryos, such as globular and heart stages, lost viability after desiccation. A one day exposure to ABA had the similar effect on the induction of desiccation tolerance as a 7-day treatment. The dried embryos maintained their ability of plant conversion after three months of storage under room conditions. The plants derived from the desiccated embryos were not different in the morphology or ploidy level from those from non-desiccated ones.Abbreviations ABA abscisic acid - RH relative humidity