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 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.     

Desiccation Tolerance Studied in the Resurrection Plant Craterostigma plantagineum

This review will focus on the acquisition of desiccation tolerancein the resurrection plant Craterostigma plantagineum. Molecularaspects of desiccation tolerance in this plant will be comparedwith the response of non-tolerant plants to dehydration. Uniquefeatures of C. plantagineum are described like the CDT-1 (Craterostigmadesiccation tolerance gene-1) gene and the carbohydrate metabolism.Abundant proteins which are associated with the desiccationtolerance phenomenon are the late embryogenesis abundant (=LEA)proteins. These proteins are very hydrophilic and occur in severalother species which have acquired desiccation tolerance.     

Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.     

Drought- and desiccation-induced modulation of gene expression in plants

Desiccation is the extreme form of dehydration. Tolerance of desiccation is acquired by seeds and in resurrection plants, a small group of angiosperms. Desiccation tolerance is the result of a complex cascade of molecular events, which can be divided into signal perception, signal transduction, gene activation and biochemical alterations leading to acquisition of tolerance. Many of these molecular processes are also observed during the dehydration of non-tolerant plants. Here we try to give an overview of the gene expression programmes that are triggered by dehydration, with particular reference to protective molecules and the regulation of their expression. Potential transgenic approaches to manipulating stress tolerance are discussed.     

Resurrection Plants and the Secrets of Eternal Leaf

Most higher plants possess a phase in their life cycle in whichtissues can survive desiccation. However, this is restrictedto specialized tissues such as seeds and pollen. Resurrectionplants are remarkable in that they can tolerate almost completewater loss in their vegetative tissues. The desiccated plantcan remain alive in the dried state for several years. However,upon watering the plants rehydrate and are fully functionalwithin 48 h. Underpinning this amazing ability is the capacityto accumulate large amounts of sucrose in the tissues. Thissugar has the property of stabilizing enzymes and cellular structuresin the absence of water. The sources of carbon that fuel sucrosesynthesis are not known, but temporary carbohydrate stores andphotosynthesis are the most likely candidates. On rewatering,the sucrose is metabolized rapidly as the tissues rehydrate.Increased expression of a number of genes in response to droughtstress have been noted. A number of these are associated withmetabolic pathways linked with primary carbohydrate metabolism.However, some genes related to LEA (Late Embryogenic Abundant)proteins have been isolated which suggests they too may playa role in maintaining tissue integrity during desiccation. Howthese mechanisms are integrated to enable resurrection plantsto survive desiccation is discussed. Copyright 2000 Annals ofBotany Company ABA, Craterostigma, desiccation tolerance, poikilohydric, resurrection.     

Inactivation of two homologues of proteins presumed to be involved in the desiccation tolerance of plants sensitizes Deinococcus radiodurans R1 to desiccation

Mutational inactivation of the genes designated DR1172 and DRB0118 in Deinococcus radiodurans R1 greatly sensitizes this species to desiccation, but not to ionizing radiation. These genes encode proteins that share features with the desiccation-induced LEA76 proteins of many plants and the PCC13-62 protein of Craterostigma plantagineum, suggesting that D. radiodurans may serve as a useful model for the study of desiccation tolerance in higher organisms.     

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.     

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     

Resurrection Plants: The Puzzle of Surviving Extreme Vegetative Desiccation

Tolerance to near complete desiccation of vegetative organs is a widespread capability in bryophytes and is also shared by a small group of vascular plants known as resurrection plants. To date more than 300 species, belonging to pteridophytes and angiosperms, have been identified that possess this kind of desiccation-tolerance. The vegetative desiccation-tolerance of resurrection plants is an inductive process displayed only under environmental stress with or without the involvement of abscisic acid as molecular signal. The different problems associated with desiccation encountered by resurrection plants render the employment of many interacting mechanisms necessary. Preservation of cell order and correct structure of membranes and macromolecules is underpinned by the synthesis of large amounts of sugars, amino acids, and small polypeptides such as late embryogenesis abundant (LEA) proteins and dehydrins. Some of these compatible solutes, such as sucrose and LEA proteins, are also involved in cytoplasm vitrification, which occurs during the last phase of desiccation. Mechanical damage due to vacuole shrinkage in dehydrating cells is avoided by cell wall folding or by replacing the water in vacuoles with nonaqueous substances. Oxidative stress, due to enhanced production of reactive oxygen species (ROS) especially by chloroplasts, is minimized through two different strategies. The homoiochlorophyllous resurrection plants, which conserve chloroplasts with chlorophylls and thylakoids upon drying, fold leaf blades and synthesize anthocyanins, as both sunscreens and free radical scavengers, and additionally increase the activity of antioxidant systems in cells. In contrast, the chloroplasts in poikilochlorophyllous species degrade chlorophylls and thylakoid membranes yielding desiccoplasts that are devoid of any internal structures. These adaptive mechanisms preserve cells from damage by desiccation and allow them to resume vital functions once rehydrated. Even if based mainly on cell protection during drying, the vegetative desiccation-tolerance of resurrection plants also relies on systems of cell recovery and repair upon rehydration. However, most of these systems are prepared during cell dehydration.