Identification of further <Emphasis Type="Italic">Craterostigma plantagineum cdt</Emphasis> mutants affected in abscisic acid mediated desiccation tolerance

The resurrection plant (Craterostigma plantagineum) is desiccation tolerant. However, callus derived from this plant, when propagated in vitro, requires exogenously applied abscisic acid (ABA) in order to survive desiccation. Treatment of callus tissue with ABA induces most of the genes that are induced by dehydration in the whole plant. This property has been exploited for the isolation of mutants that show dominant phenotypes resulting from the ectopic expression of endogenous genes induced by the insertion of a foreign promoter. Here we describe new T-DNA tagged Craterostigma desiccation-tolerant (cdt) mutants with different molecular and physiological characteristics, suggesting that different pathways of desiccation tolerance are affected. One of the mutants, cdt-2, constitutively expresses known osmoprotective Lea genes in callus and leaf tissue. Further analysis of this mutant revealed that the tagged locus is similar to a previously characterised gene, CDT-1, which codes for a signalling molecule that confers desiccation tolerance. The nature of the T-DNA insertion provides insight into the mechanism by which the CDT-1/2 gene family functions in ABA signal transduction.     

Molecular characterization of two alanine-rich Lea genes abundantly expressed in the resurrection plant C. plantagineum in response to osmotic stress and ABA

Expression of many genes is induced during dehydration in vegetative tissues of the desiccation tolerant resurrection plantCraterostigma plantagineum. The most abundant group of desiccation-related gene products belong to the LEA (= Late Embryogenesis Abundant) proteins. Here we describe structures and expression patterns of members of group 3 and group 4Lea genes fromC. plantagineum. The most intriguing observation is the strong conservation of repeat motifs inLea genes found across divers plant species includingC. plantagineum and non-desiccation tolerant plants. This conservation of structural elements leads to speculations about evolution of desiccation tolerance in the resurrection plant.     

The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach

Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation‐tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation‐sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x‐DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x‐DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation‐sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer‐ to non‐bilayer‐forming lipids, thus contributing to protein and membrane stabilization.     

Irrigation and Seed Quality Development in Rapid-cycling Brassica: Soluble Carbohydrates and Heat-stable Proteins

Changes in soluble carbohydrates and heat-stable proteins havebeen examined in relation to the acquisition of desiccationtolerance and/or potential seed longevity during seed developmentin rapid-cycling brassica [Brassica campestris (rapa)L.]. Ratesof seed development were moderated by different irrigation regimes.At the early stages, glucose, fructose and sucrose predominated.The raffinose series oligosaccharides accumulated during seedmaturation, and occurred earliest in seeds from plants irrigatedonly until 16 days after pollination. Stachyose content correlatedpositively, and monosaccharide content correlated negatively,with the ability of seeds to tolerate rapid desiccation andwith their potential longevity (the constantK_iof the seed viabilityequation). Similarly, the ratio of oligosaccharide[ratio]totalsugars provided strong positive correlations with ability totolerate desiccation and with potential longevity. Most of theheat-stable proteins selected for study accumulated comparativelylate, i.e. during maturation drying. The imposition of waterstress induced earlier accumulation of heat-stable proteins.The ability to tolerate desiccation was correlated with thecontent of selected heat-stable proteins, but potential longevityprovided stronger correlations. The content of a 58 kDa heat-stableprotein provided the strongest positive correlation with potentiallongevity. A simple multiple regression model of the relationsbetween potential longevity and both the oligosaccharide[ratio]totalsugar ratio and the 58 kDa heat-stable protein content was developedfor all three plant irrigation regimes to show the combinedeffect of certain sugars and proteins on seed quality. The modelsuggests that these sugars and proteins are equally likely tobe required for seed quality development, and that initiallythe sugars tend to accumulate at a greater rate than the proteins,but that during maturation drying the heat-stable proteins accumulateat the greater rate.Copyright 1998 Annals of Botany Company Brassica campestris (rapa) L., rapid-cycling brassica, potential longevity, seed development, desiccation tolerance, soluble sugars, oligosaccharides, dehydrins, heat-stable proteins.     

Abscisic Acid Content and Effects During Dehydration of Detached Leaves of Desiccation Tolerant Plants

Borya nitida is an angiospcrm whose detached leaves developcomplete tolerance to dehydration when they are equilibratedto air of 96% r.h. This treatment causes leaves to yellow aschlorophyll is destroyed, and abscisic acid contents increaseseveral-fold. Exogenous ABA (at 0.038–0.38 mol m^–3)promoted desiccation tolerance (a) in leaves undergoing toleranceinduction at 96% r.h., (b) only slightly during rapid dryingat rates which are normally injurious, and (c) considerablyin turgid tissue treated with ABA 48 h before rapid drying. ABA content also increased with intense water stress in Myrothamnusflabellifolia, a desiccation tolerant angiosperm which, unlikeBorya, retains most of its chlorophyll when dehydrated. Preliminaryincubation in ABA of detached leaves of this ‘resurrectionplant’ also promoted survival during rapid drying. Theability of ABA to substitute for the normal induction periodsuggests that this hormone participates in the development ofdesiccation tolerance. Key words: Abscisic acid, ABA, Drought tolerance, Resurrection plant     

The signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues

Desiccation-tolerance in vegetative tissues of angiosperms hasa polyphyletic origin and could be due to 1) appropriation ofthe seed-specific program of gene expression that protects orthodoxseeds against desiccation, and/or 2) a sustainable version ofthe abiotic stress response. We tested these hypotheses by comparingmolecular and physiological data from the development of orthodoxseeds, the response of desiccation-sensitive plants to abioticstress, and the response of desiccation-tolerant plants to extremewater loss. Analysis of publicly-available gene expression dataof 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitiveArabidopsis thaliana identified 13 LEAs and 4 anti-oxidantsexclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin)are not expressed in vegetative tissues in A. thaliana, buthave orthologues that are specifically activated in desiccatingleaves of Xerophyta humilis. A comparison of antioxidant enzymeactivity in two desiccation-sensitive species of Eragrostiswith the desiccation-tolerant E. nindensis showed equivalentresponses upon initial dehydration, but activity was retainedat low water content in E. nindensis only. We propose that theseantioxidants are housekeeping enzymes and that they are protectedfrom damage in the desiccation-tolerant species. Sucrose isconsidered an important protectant against desiccation in orthodoxseeds, and we show that sucrose accumulates in drying leavesof E. nindensis, but not in the desiccation-sensitive Eragrostisspecies. The activation of "seed-specific" desiccation protectionmechanisms (sucrose accumulation and expression of LEA6 and1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerantplants points towards acquisition of desiccation tolerance fromseeds.     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author     

Arabinose-rich polymers as an evolutionary strategy to plasticize resurrection plant cell walls against desiccation

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as ‘pectic plasticizers’. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of ‘plasticising’ the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.     

Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst

In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of β-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway of the resurrection plant are not active in tobacco or Arabidopsis. Received: 26 April 1997 / Accepted: 16 July 1997     

Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum

Abscisic acid-treated callus of the resurrection plant Craterostigma plantagineum tolerates extreme desiccation. Nuclear proteins from tolerant callus bind specific sequence elements in the promoter region of the ABA and desiccation-inducible CDeT27–45 gene. One specific region of the promoter, which is protected from DNAse I treatment by DNA-binding activities, is different from previously reported ABA response elements. Four complexes of nuclear proteins and this DNA region are detected by electrophoretic mobility shift assay: two of these complexes (I and II) are readily detectable in untreated samples and are increased by ABA treatment while two other complexes (III and IV) accumulate only following ABA treatment and are prevented from accumulating by protein synthesis inhibitors. When a fragment containing the novel binding site is deleted from the wild-type promoter the ABA responsiveness of the promoter is removed; however, gain of function experiments using synthetic promoters in a protoplast transient assay suggest that besides the binding site other promoter elements are required. A second region of the promoter, containing the sequence element ACGT which is found in abscisic acid response elements, is also bound by nuclear proteins. The level of this second binding activity is similar in both untreated and ABA-treated cells and promoter/reporter gene constructs which contain only the four ACGT elements of the CDeT27–45 promoter are not ABA responsive in a C. plantagineum transient assay system     

Molecular anhydrobiology: identifying molecules implicated in invertebrate anhydrobiosis

Studies in anhydrobiotic plants have defined many genes whichare upregulated during desiccation, but comparable studies ininvertebrates are at an early stage. To develop a better understandingof invertebrate anhydrobiosis, we have begun to characterisedehydration-inducible genes and their proteins in anhydrobioticnematodes and bdelloid rotifers; this review emphasises recentfindings with a hydrophilic nematode protein. Initial work withthe fungivorous nematode Aphelenchus avenae led to the identificationof two genes, both of which were markedly induced on slow drying(90–98% relative humidity, 24 hr) and also by osmoticstress, but not by heat or cold or oxidative stresses. The firstof these genes encodes a novel protein we have named anhydrin;it is a small, basic polypeptide, with no counterparts in sequencedatabases, which is predicted to be natively unstructured andhighly hydrophilic. The second is a member of the Group 3 LEAprotein family; this and other families of LEA proteins arewidely described in plants, where they are most commonly associatedwith the acquisition of desiccation tolerance in maturing seeds.Like anhydrin, the nematode LEA protein, Aav-LEA-1, is highlyhydrophilic and a recombinant form has been shown to be unstructuredin solution. In vitro functional studies suggest that Aav-LEA-1is able to stabilise other proteins against desiccation-inducedaggregation, which is in keeping with a role of LEA proteinsin anhydrobiosis. In vivo, however, Aav-LEA-1 is apparentlyprocessed into smaller forms during desiccation. A processingactivity was found in protein extracts of dehydrated, but nothydrated, nematodes; these shorter polypeptides are also activeanti-aggregants and we hypothesise that processing LEA proteinserves to increase the number of active molecules availableto the dehydrating animal. Other LEA-like proteins are beingidentified in nematodes and it seems likely therefore that theywill play a major role in the molecular anhydrobiology of invertebrates,as they are thought to do in plants.     

Recalcitrant Seed Storage Physiology in Three Aquatic Grasses (Zizania palustris, Spartina anglica and Porteresia coarctata)

The moisture content/probit viability relationship for storedseeds of Zizania palustris L. and Spartina anglica C. E. Hubbardwas linear and independent of the rate of embryo drying. Theseresults provide firm evidence of recalcitrant storage physiologyin these taxa. Preliminary tests strongly suggest that freshseeds of Porteresia coarctata (Roxb.) Tateoka are also intolerantof desiccation In Z. palustris apparent differences in desiccation tolerancebetween individuals can be partly explained by wide variationin individual embryo moisture contents during desiccation. Long-termstorage experiments in solutions of polyethylene glycol 6000(PEG) suggest that the actual variation in desiccation toleranceis confined to a narrow range of embryo water potentials inthe range –2 to –3 MPa. Despite the presence of prolonged dormancy in seeds of Z. palustrisand S. anglica there is no evidence of a significant effectof dormancy or storage period (up to the point of visible germination)on the limits of desiccation tolerance Aquatic grasses, seeds, storage, desiccation intolerance     

Coping mechanisms for crop plants in drought-prone environments

Background: Drought is a major limitation to plant productivity. Variousoptions are available for increasing water availability andsustaining growth of crop plants in drought-prone environments. Scope: After a general introduction to the problems of water availability,this review focuses on a critical evaluation of recent progressin unravelling mechanisms for modifying plant growth responsesto drought. Conclusions: Investigations of key regulatory mechanisms integrating plantgrowth responses to water deficits at the whole-organism, cellularand genomic levels continue to provide novel and exiting researchfindings. For example, recent reports contradict the widespreadconception that root-derived abscisic acid is necessarily involvedin signalling for stomatal and shoot-growth responses to soilwater deficits. The findings bring into question the theoreticalbasis for alternate-side root-irrigation techniques. Similarly,recent reports indicate that increased ABA production or increasedaquaporin expression did not lead to improved drought resistance.Other reports have concerned key genes and proteins involvedin regulation of flowering (FT), vegetative growth (DELLA),leaf senescence (IPT) and desiccation tolerance (LEA). Introgressionof such genes, with suitable promoters, can greatly impact onwhole-plant responses to drought. Further developments couldfacilitate the introduction by breeders of new crop varietieswith growth physiologies tailored to improved field performanceunder drought. Parallel efforts to encourage the introductionof supplementary irrigation with water made available by improvedconservation measures and by sea- or brackish-water desalination,will probably provide comprehensive solutions to coping withdrought-prone environments.     

MS-desi, a desiccation-related protein in the floral nectar of the evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization

Plant desiccation-related proteins (DRPs) were first identified as pcC13-62 from the resurrection plant Craterostigma plantagineum and it has been suggested they are involved in plant desiccation tolerance. We identified and characterized a plant DRP, which we called MS-desi, in the floral nectar of a subtropical bean species, Mucuna sempervirens (MS). MS-desi is a major nectar protein (nectarin) of the bean plant and expresses exclusively in the stylopodium, where the nectary is located. The full-length MS-desi gene encodes for a protein of 306 amino acids with a molecular mass of 33,248 Da, and possesses a ferritin-like domain and a signal peptide of 30 amino acids. Structural and phylogenetic analysis demonstrated MS-desi has high similarity to members of the plant DRPs, including pcC 13-62 protein. MS-desi has a similar hydropathy profile to that of pcC13-62 with a grand average of hydropathy index of 0.130 for MS-desi and 0.106 for pcC13-62 protein, which is very different from those of dehydrins and late embryogenesis abundant proteins. The protein’s secondary structures, both predicted from the amino acid sequence and directly analysed by far UV circular dichroism, showed that MS-desi is mainly composed of alpha helices and is relatively temperature dependent. The structure change is reversible within a wide range of temperatures. Purified MS-desi and raw MS floral nectar showed dose-dependent citrate synthase inhibition activity, but insensitivity to lactate dehydrogenase, suggesting that, unlike dehydrins, it does not act as a chaperone. The overall results constitute, to our knowledge, the first study on a desiccation-related protein in plant floral nectar.