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.     

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.     

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.     

A simple method for cryopreservation of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> callus

Two-years-old Ginkgo biloba cell culture initiated from cotyledonary explants was cryopreserved by a simple desiccation method. Preliminary incubation of callus clumps on MS preculture medium supplemented with 100 g l⁻¹ sucrose and 2 mg l⁻¹ ABA for 7 and 14 days resulted in accumulation of endogenous soluble sugars and was essential for cell culture post-cryopreservation survival. The optimal time for the preculture on sucrose-and-ABA containing medium was found to be 14 days. The sufficient desiccation duration was determined as 150 min. FCM profiles of calli maintained for 2 years remained stable and were not affected by cryopreservation.     

Responses of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> to high irradiance

The effect of high irradiance (HI) during desiccation and subsequent rehydration of the homoiochlorophyllous desiccation-tolerant shade plant Haberlea rhodopensis was investigated. Plants were irradiated with a high quantum fluence rate (HI; 350 μmol m⁻² s⁻¹ compared to ca. 30 μmol m⁻² s⁻¹ at the natural rock habitat below trees) and subjected either to fast desiccation (tufts dehydrated with naturally occurring thin soil layers) or slow desiccation (tufts planted in pots in peat-soil dehydrated by withholding irrigation). Leaf water content was 5 % of the control after 4 d of fast and 19 d of slow desiccation. Haberlea was very sensitive to HI under all conditions. After 19 d at HI, even in well-watered plants there was a strong reduction of rates of net photosynthesis and transpiration, contents of chlorophyll (Chl) and carotenoids, as well as photosystem 2 activity (detected by the Chl fluorescence ratio R_Fd). Simultaneously, the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis of polyphenolic compounds. Desiccation of plants in HI induced irreversible changes in the photosynthetic apparatus and leaves did not recover after rehydration regardless of fast or slow desiccation. Only young leaves survived desiccation.     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

Physiological consequences of desiccation in the aquatic bryophyte <Emphasis Type="Italic">Fontinalis antipyretica</Emphasis>

The moss Fontinalis antipyretica, an aquatic bryophyte previously described as desiccation-intolerant, is known to survive intermittent desiccation events in Mediterranean rivers. To better understand the mechanisms of desiccation tolerance in this species and to reconcile the apparently conflicting evidence between desiccation tolerance classifications and field observations, gross photosynthesis and chlorophyll a fluorescence were measured in field-desiccated bryophyte tips and in bryophyte tips subjected in the laboratory to slow, fast, and very fast drying followed by either a short (30 min) or prolonged (5 days) recovery. Our results show, for the first time, that the metabolic response of F. antipyretica to desiccation, both under field and laboratory conditions, is consistent with a desiccation-tolerance pattern; however, drying must proceed slowly for the bryophyte to regain its pre-desiccation state following rehydration. In addition, the extent of dehydration was found to influence metabolism whereas the drying rate determined the degree of recovery. Photosystem II (PSII) regulation and structural maintenance may be part of the induced desiccation tolerance mechanism allowing this moss to recover from slow drying. The decrease in the photochemical quenching coefficient (qP) immediately following rehydration may serve to alleviate the effects of excess energy on photosystem I (PSI), while low-level non-photochemical quenching (NPQ) would allow an energy shift enabling recovery subsequent to extended periods of desiccation. The findings were confirmed in field-desiccated samples, whose behavior was similar to that of samples slowly dried in the laboratory.     

Development of desiccation tolerance and vitrification by preculture treatment in suspension-cultured cells of the liverwort Marchantia polymorpha

Some cultured plant cells are able to acquire tolerance to various stresses when they are cultured under suitably controlled conditions. Induction of a high level of desiccation tolerance in suspension-cultured cells of the liverwort Marchantia polymorpha was examined for studying the mechanisms of desiccation tolerance and vitrification at the cellular level. Desiccation tolerance level of cells was very low and the survival rate was less than 10% after exposure to drying below 0.1 g H₂O g⁻¹ dry weight (DW). Preculture treatment in 0.5 M sucrose medium was the most effective method for inducing a high level of desiccation tolerance in cells and the survival rate was 87% even after being desiccated to below 0.1 g H₂O g⁻¹ DW. Preculture treatment caused alteration of cell structures and accumulation of a large amount of sucrose and newly synthesized proteins in cells. Abundant sucrose and preculture-induced proteins were necessary for full development of desiccation tolerance in the cells. When water content decreased to below 0.1 g H₂O g⁻¹ DW, desiccation-tolerant cells that had been precultured were vitrified above 0°C and maintained stable viability. We have succeeded in the induction of desiccation tolerance that allows formation of intracellular glass with cell viability at ambient temperatures by controlling culture conditions, and our results suggest that suspension-cultured cells of M. polymorpha are useful for studying cellular mechanisms for the development of desiccation tolerance and the stabilization of vitrified cells.     

Plant desiccation and protein synthesis: an in vitro system from dry and hydrated mosses using endogenous and synthetic messenger ribonucleic Acid

The conditions and requirements for an in vitro protein synthesizing system from the moss Tortula ruralis are outlined. Using this system the effects of desiccation, achieved quickly or slowly, were studied. Slowly dried moss retained fewer polyribosomes on desiccation but more active ribosomes than rapidly dried moss. Even in the completely desiccated moss the polyribosomes and/or free ribosomes present have retained their synthetic capacities. On rehydration, the slowly dried moss resumed protein synthesis more quickly than moss previously desiccated rapidly. Moss ribosomes are cycloheximide sensitive and chloramphenicol insensitive and thus the major protein synthesis occurs within the cytoplasm on rehydration. Extracted polyribosomes per se can withstand desiccation to a significant extent, suggesting that protection by the cytoplasm might not be necessary. The aquatic moss Hygrohypnum luridum can retain polyribosomal and ribosomal activity during desiccation, but this decreases greatly on rehydration.     

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.     

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     

ABA treatment increases both the desiccation tolerance of photosynthesis,and nonphotochemical quenching in the moss Atrichum undulatum

Pulse amplitude modulation fluorescence was used to investigate whether abscisic acid (ABA) pretreatment increases the desiccation tolerance of photosynthesis in the moss Atrichum undulatum. In unstressed plants, ABA pretreatment decreased the F _V/F _m ratio, largely as a result of an increase in F _o. This indicated a reduction in energy transfer between LHCII and PSII, possibly hardening the moss to subsequent stress by reducing the production of the reactive oxygen species near PSII. During desiccation, F ₀, F _m, F _v/F _m, PSII, and NPQ and F ₀ quenching declined in ABA-treated and nontreated mosses. However, during rehydration, F ₀, F _m, F _v/F _m, and PSII recovered faster in ABA-treated plants, suggesting that ABA improved the tolerance of photosystem II to desiccation. NPQ increased upon rehydration in mosses from both treatments, but much more rapidly in ABA-treated plants; during the first hour of rehydration, NPQ was two-fold greater in plants treated with ABA. F ₀quenching followed a similar pattern, indicating that ABA treatment stimulated zeaxanthin-based quenching. The implications of these results for the mechanisms of ABA-induced desiccation tolerance in A. undulatum are discussed.     

The Conservation of Polyribosomes in the Moss Tortula ruralis during Total Desiccation

Total desiccation of the moss Tortula ruralis was achieved byplacing it in a dry atmosphere for 90 min. Reintroduction ofthe moss to water resulted in the recovery of its normal morphologicalform within 15–30 s. The sedimentation profile on a sucrosegradient of the ribosomal content of the totally dry moss showsthe presence of distinct polyribosomal peaks. The levels ofthese polyribosomes rise upon rehydration of the moss. The differencebetween the tolerance to water deficit by this moss and by higherplants is outlined.     

Influence of freezable/non-freezable water and sucrose on the viability of <Emphasis Type="Italic">Theobroma cacao</Emphasis> somatic embryos following desiccation and freezing

Encapsulated cocoa (Theobroma cacao L.) somatic embryos subjected to 0.08–1.25 M sucrose treatments were analyzed for embryo soluble sugar content, non-freezable water content, moisture level after desiccation and viability after desiccation and freezing. Results indicated that the higher the sucrose concentration in the treatment medium, the greater was the extent of sucrose accumulation in the embryos. Sucrose treatment greatly assisted embryo post-desiccation recovery since only 40% of the control embryos survived desiccation, whereas a survival rate of 60–95% was recorded for embryos exposed to 0.5–1.25 M sucrose. The non-freezable water content of the embryos was estimated at between 0.26 and 0.61 g H₂O g⁻¹dw depending on the sucrose treatment, and no obvious relationship could be found between the endogenous sucrose level and the amount of non-freezable water in the embryos. Cocoa somatic embryos could withstand the loss of a fraction of their non-freezable water without losing viability following desiccation. Nevertheless, the complete removal of potentially freezable water was not sufficient for most embryos to survive freezing.     

Rate of dehydration,state of subcellular organisation and nature of cryoprotection are critical factors contributing to the variable success of cryopreservation: studies on recalcitrant zygotic embryos of <Emphasis Type="Italic">Haemanthus montanus</Emphasis>

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g⁻¹ dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g⁻¹ showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g⁻¹, embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g⁻¹, lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g⁻¹—in which the degree of vacuolation remained moderate—were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.     

Desiccation Tolerance and Global Change: Implications for Tropical Bryophytes in Lowland Forests

Global change puts an increasing pressure on tropical forests and their inherent diversity by the risk of longer droughts and drier microclimatic conditions within the forest. How organisms will respond is uncertain, especially for organisms highly depending on their microclimatic environment such as bryophytes. An adequate tolerance to desiccation is important to face these changes, however, little is known for tropical bryophytes. We investigated for the first time the desiccation tolerance of epiphytic bryophytes from contrasting microsites at the tropical lowland forest in French Guiana. Using chlorophyll‐fluorescence (F_v/F_m) as an indicator of recovery, we tested: (1) desiccation tolerance for short (3 d) and long (9 d) desiccation events; (2) different desiccation intensities; and (3) recovery by rehydration with water vapor. Species from the canopy were well adapted to desiccation events. Thirteen of 18 species maintained more than 75 percent of their photosynthetic capacity after recovery at the strongest desiccation treatment of 9 d at 43 percent relative humidity (RH). In contrast, species from the understory were sensitive and withstood desiccation only at humid conditions of 75 percent RH and higher. The photosystem of the studied bryophytes was reactivated efficiently in equilibration with water vapor only—a yet neglected phenomenon in bryology. A novel introduced desiccation tolerance index allows global comparison of desiccation tolerances and highlights the sensitivity of understory species. Our results suggest that decreasing humidity caused by climate change and forest degradation could be a concerning threat for understory species.     

<Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> mutant with high inhibitor tolerance obtained by low-energy ion implantation

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l⁻¹ of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g⁻¹. When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l⁻¹ of ABE with a yield of 0.34 g g⁻¹, including 2.2 g l⁻¹ acetone, 6.8 g l⁻¹ butanol, and 0.5 g l⁻¹ ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.     

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to −20°C) by ³¹P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (−400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.     

Insecticide-tolerant and plant growth promoting <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (<Emphasis Type="Italic">vigna</Emphasis>) improves the growth and yield of greengram [<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek] in insecticide-stressed soils

This study was designed to identify rhizobial strains specific to greengram expressing higher tolerance against insecticides, fipronil and pyriproxyfen, and synthesizing plant growth regulators even amid insecticide-stress. Of the 50 bradyrhizobial isolates, the Bradyrhizobium sp. strain MRM6 showed tolerance up to 1,600 μg mL⁻¹ against each of fipronil and pyriproxyfen. The tolerant Bradyrhizobium sp. (vigna) produced plant growth promoting substances in substantial amounts, both in the presence and absence of insecticides. The strain MRM6 was further used to investigate its impact on greengram grown in soils treated with 200 (the recommended dose), 400 and 600 μg kg⁻¹ soil of fipronil and 1,300 (the recommended dose), 2,600 and 3,900 μg kg⁻¹ soil of pyriproxyfen. Fipronil at 600 μg kg⁻¹ soils and pyriproxyfen at 3,900 μg kg⁻¹ soils had greatest toxic effects and decreased plant biomass, symbiotic efficiency, nutrient uptake and seed yield of greengram plants. The Bradyrhizobium sp. (vigna) inoculant when used with fipronil and pyriproxyfen significantly increased the measured parameters compared to the plants grown in soils treated solely with the same concentration of each insecticide. This study inferred that the Bradyrhizobium sp. (vigna) strain MRM6 may be exploited as bio-inoculant to increase the productivity of greengram exposed to insecticide-stressed soils.