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.     

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.     

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.     

Analysis of a <Emphasis Type="Italic">LEA</Emphasis> gene promoter via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the desiccation tolerant plant <Emphasis Type="Italic">Lindernia brevidens</Emphasis>

An Agrobacterium tumefaciens-based transformation procedure was developed for the desiccation tolerant species Lindernia brevidens. Leaf explants were infected with A. tumefaciens strain GV3101 harbouring a binary vector that carried the hygromycin resistance gene and an eGFP reporter gene under the control of a native dehydration responsive LEA promoter (Lb2745pro). PCR analysis of the selected hygromycin-resistant plants revealed that the transformation rates were high (14/14) and seeds were obtained from 13/14 of the transgenic lines. A combination of RNA gel blot and microscopic analyses demonstrated that eGFP expression was induced upon dehydration and ABA treatment. Comparison with existing procedures used to transform the well studied resurrection plant and close relative, Craterostigma plantagineum, revealed that the transformation process is both rapid and leads to the production of viable seed thus making L. brevidens a candidate species for functional genomics approaches to determine the genetic basis of desiccation tolerance.     

The novel gene<Emphasis Type="Italic"> CpEdi-9</Emphasis> from the resurrection plant<Emphasis Type="Italic"> C. plantagineum</Emphasis> encodes a hydrophilic protein and is expressed in mature seeds as well as in response to dehydration in leaf phloem tissues

The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants. CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.Abbreviations ABA Abscisic acid - ABRE ABA-responsive element - Edi Early dehydration induced - GUS Glucuronidase - LEA Late embryogenesis abundant - MU Methylumbelliferone This article is dedicated to Prof. Dr. Francesco Salamini on the occasion of his 65th birthday and his departure from the Max Planck Institute in Köln     

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     

Dynamics of Endogenous Phytohormones during Desiccation and Recovery of the Resurrection Plant Species Haberlea rhodopensis

Drought is one of the most significant threats to world agriculture and hampers the supply of food and energy. The mechanisms of drought responses can be studied using resurrection plants that are able to survive extreme dehydration. As plant hormones function in an intensive cross-talk, playing important regulatory roles in the perception and response to unfavorable environments, the dynamics of phytohormones was followed in the resurrection plant Haberlea rhodopensis Friv. during desiccation and subsequent recovery. Analysis of both leaves and roots revealed that jasmonic acid, along with and even earlier than abscisic acid, serves as a signal triggering the response of the resurrection plants to desiccation. The steady high levels of salicylic acid could be considered an integral part of the specific set of parameters that prime H. rhodopensis desiccation tolerance. The dynamic changes of cytokinins and auxins suggest that these hormones actively participate in the dehydration response and development of desiccation tolerance in the resurrection plants. Our data contribute to the elucidation of a global complex picture of the resurrection plant’s ability to withstand desiccation, which might be successfully utilized in crop improvement.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

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.     

Desiccation‐induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy,electrokinetic and optical measurements

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation‐induced alterations in surface topography of thylakoids isolated from well‐hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well‐hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well‐hydrated H. rhodopensis had a grainy surface, significantly different from the well‐structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross‐sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.     

Characterization of oxidative and antioxidative events during dehydration and rehydration of resurrection plant <Emphasis Type="Italic">Ramonda nathaliae</Emphasis>

In order to investigate changes of oxidative status in relation to the activity of the various protective mechanisms in resurrection plant Ramonda nathaliae, we have analysed time and relative water content (RWC) related changes in lipid peroxidation and ion leakage, hydrogen peroxide accumulation, changes of pigment content and antioxidative enzyme activity, together with expression of dehydrins. The results indicate that enhanced oxidative status during dehydration, not previously reported for resurrection plants, could play an active role in inducing the desiccation adaptive response in R. nathaliae. A critical phase is shown to exist during dehydration (in the range of RWC between 50 and 70%) during which a significant increase in hydrogen peroxide accumulation, lipid peroxidation and ion leakage, accompanied by a general decline in antioxidative enzyme activity, takes place. This phase is designated as a transition characterized by change in the type of stress response. The initial response, relying mainly on the enzymatic antioxidative system, is suspended but more effective, desiccation specific protective mechanisms, such as expression of dehydrins, are then switched on. The expression of dehydrins in R. nathaliae could be inducible as well as constitutive. In order to cope with the oxidative stress associated with rapid rewatering, R. nathaliae reactivated antioxidative enzymes. We propose that controlled elevation of reactive oxygen species, such as hydrogen peroxide, could be an important mechanism enabling resurrection plants to sense dehydration and to trigger an adaptive programme at an appropriate stage during the dehydration/rehydration cycle.