Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli

Trehalose considerably increased the tolerance of Escherichia coli to air drying, whether added as an excipient prior to drying or accumulated as a compatible solute in response to osmotic stress. The protective effect of exogenously added trehalose was concentration dependent, up to a threshold value of 350 mM. However, trehalose alone cannot explain the intrinsically greater desiccation tolerance of stationary compared to exponential phase E. coli cells, although their tolerance was also enhanced by exogenous or endogenously accumulated trehalose. In contrast, glycine betaine whether added as an excipient or accumulated intracellularly had no influence on desiccation tolerance. These data demonstrate that the protection provided by compatible solutes to cells subjected to desiccation differs from that during osmotic stress, due to the much greater reduction in available cell water. The protective effects of trehalose during desiccation appear to be due to its stabilising influence on membrane structure, its chemically inert nature and the propensity of trehalose solutions to form glasses upon drying, properties which are not shared by glycine betaine.     

Responses to desiccation stress in bryophytes and an important role of dithiothreitol-insensitive non-photochemical quenching against photoinhibition in dehydrated states

The effects of air drying and hypertonic treatments in the dark on seven bryophytes, which had grown under different water environments, were studied. All the desiccation-tolerant species tested lost most of their PSII photochemical activity when photosynthetic electron transport was inhibited by air drying, while, in all the sensitive species, the PSII photochemical activity remained at a high level even when photosynthesis was totally inhibited. The PSI reaction center remained active under drying conditions in both sensitive and tolerant species, but the activity became non-detectable in the light only in tolerant species due to deactivation of the cyclic electron flow around PSI and of the back reaction in PSI. Light-induced non-photochemical quenching (NPQ) was found to be induced not only by the xanthophyll cycle but also by a DeltapH-induced, dithiothreitol-insensitive mechanism in both the desiccation-tolerant and -intolerant bryophytes. Both mechanisms are thought to have an important role in protecting desiccation-tolerant species from photoinhibition under drying conditions. Fluorescence emission spectra at 77K showed that dehydration-induced quenching of PSII fluorescence was observed only in tolerant species and was due to neither state 1-state 2 transition nor detachment of light-harvesting chlorophyll protein complexes from PSII core complexes.The presence of dehydration-induced quenching of PSI fluorescence was also suggested.     

Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.     

Acquisition of desiccation tolerance in soybeans

The entry into a desiccation-tolerant state is a major developmental component of seed maturation. Development of desiccation tolerance of embryonic axes of soybean [Glycine max (L.) Merrill cv. Chippewa 64] was studied by measuring changes in electrolyte leakage. germination and relative growth rate after axes were rapidly air-dried to various water contents. Axes acquired the full capacity for germination at 34 days after flowering (DAF). and reached physiological maturity (maximum dry weight) at 48 DAF. When dried to water content h = 0. 08 (g water g⁻¹ dry weight). few axes germinated before 42 DAF. but more than 90% germinated after 48 DAF. However, electrolyte leakage of rehydrated axes showed a linear decline from 30 to 55 DAF. For developing axes there was a critical water content or desiccation threshold. which could be estimated by using the electrolyte leakage method. The threshold of desiccation tolerance decreased gradually from h = 1. 10 to 0. 18 as axes matured from 28 to 55 DAF. The development of desiccation tolerance continued after physiological maturity at 48 DAF. We conclude that the acquisition of desiccation tolerance of soybean axes is a gradual event, rather than an abrupt transition.     

Salt tolerance requires cortical microtubule reorganization in Arabidopsis

Although the results of some studies indicate that salt stress affects the organization of microtubules, it remains an open question whether microtubules play an active role in the plant's ability to withstand salt stress. In the present study, we showed that salt stress-induced wild-type Arabidopsis seedling roots display right-handed skewed growth and depolymerization of the cortical microtubules. The results of a long-term observational study showed that cortical microtubules depolymerized then reorganized themselves under salt stress. Stabilization of microtubules with paclitaxel resulted in more seedling death under salt stress, while disruption of microtubules with oryzalin or propyzamide rescued seedlings from death. Seedlings in which the cortical microtubules were reorganized did not succumb to salt stress. These results suggest that both depolymerization and reorganization of the cortical microtubules are important for the plant's ability to withstand salt stress. Depolymerizing microtubules by drugs rescues seedlings from death under salt stress. This rescue effect was abolished by removing calcium from the medium or treatment with a calcium channel inhibitor. Depolymerization of the microtubules is followed by an increase in the free cytoplasmic calcium concentration. The addition of calcium to the growth medium increased the number of seedlings in which recovery of the cortical microtubules occurred, whereas the removal of calcium decreased the number of seedlings in which recovery occurred. Therefore, depolymerization of the cortical microtubules raises intracellular calcium concentrations, while reorganization of the cortical microtubules and seedling survival may be mediated by calcium influx in salt stress.     

HPLC法测定卷柏及其炮制品中穗花杉双黄酮的含量

采用HPLC方法测定卷柏及炮制品中穗花杉双黄酮的含量。方法:Shim-pack VP-ODS柱(4.6 mm×250mm,5μm);流动相:甲醇-0.1%磷酸水(65∶35);流速:0.8 mL/min;检测波长:337 nm;柱温:25℃。实验结果表明,卷柏生品中穗花杉双黄酮的含量为0.84%,焦卷柏为1.07%,卷柏炭为0.53%。不同的炮制方法对穗花杉双黄酮的含量产生不同的影响。     

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.     

Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.     

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.     

An ABC transporter gene of Arabidopsis thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion

Cuticle, including wax and cutin, is the barrier covering plant aerial organs and protecting the inner tissues. The Arabidopsis thaliana ATP-binding cassette (ABC) transporter CER5 (AtWBC12) has been identified as a wax exporter. In agreement with the latest report of another wax exporter, AtWBC11, here we show that atwbc11 mutants displayed organ fusions and stunted growth, and became vulnerable to chlorophyll leaching and toluidine blue staining. Chemical analysis showed that wax and cutin monomers were both reduced in the atwbc11 mutant. AtWBC11 was widely expressed in aerial organs. Interestingly, we found that the expression was light dependent, and the phytohormone ABA up-regulated AtWBC11 expression. We also found that while the AtWBC11 promoter had a broad pattern of activity, the expression was converted to epidermis specific when the reporter gene was fused to AtWBC11 cDNA. Furthermore, RNA blot analysis supported epidermis-specific expression of AtWBC11. Our results support that AtWBC11 is involved in cuticle development.     

Biochemical bases for a widespread tolerance of cyanobacteria to the phosphonate herbicide glyphosate

Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.     

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

Trehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria.     

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, showsunique responses to dehydration. These responses are: (i) lossof PSII activity in parallel with the loss of photosynthesis;(ii) loss of PSI activity; and (iii) dissipation of light energyabsorbed by pigment–protein complexes. In this study,the deactivation of PSII is shown to be important in avoidingphotoinhibition when the Calvin–Benson cycle is repressedby dehydration. Furthermore, our evidence suggests that dissipationof light energy absorbed by PSII blocks photoinhibition understrong light in dehydrated states.     

Antisense expression of 3-oxoacyl-ACP reductase affects whole plant productivity and causes collateral changes in activity of fatty acid synthase components

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.     

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts

Tissues of three species of in vitro grown liverworts, Riccia fluitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 μM ABA. ABA preculture initiated total desiccation tolerance in R. fluitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABA-treated R. fluitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reflect different adaptations of these three species to conditions in situ.     

The discovery,scope, and puzzle of desiccation tolerance in plants

The modern scientific study of desiccation tolerance began in 1702 when Anthony von Leeuwenhoek discovered that rotifers could survive without water for months. By 1860, the controversy over whether organisms could dry up without dying had reached such a pitch that a special French commission was convened to adjudicate the dispute. In 2000, we know that a few groups of animals and a wide variety of plants can tolerate desiccation in the active, adult stages of their life cycles. Among plants, this includes many lichens and bryophytes, a few ferns, and a very few flowering plants, but no gymnosperms nor trees. Some desiccation-tolerant species can survive without water for over ten years, recover from desiccation to unmeasurably low water potentials, and, when plants are desiccated, endure temperature extremes from ?272 to 100 °C. Desiccation-tolerant plants occur on all continents but mainly in xeric habitats or microhabitats where the cover of desiccation-sensitive species is low. Two main puzzles arise from these patterns: What are the mechanisms by which plants tolerate desiccation? and Why are desiccation-tolerant plants not more ecologically widespread? Recent molecular and biochemical studies suggest that there are multiple mechanisms of tolerance, many of which involve protection from oxidants and from the loss of configuration of macromolecules during dehydration. Hypotheses to explain the restricted ecological range of desiccation-tolerance plants include inability to maintain a cumulative positive carbon balance during repeated cycles of wetting and drying and inherent trade offs between desiccation tolerance and growth rate.

     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate