Protection mechanisms in the resurrection plant Xerophyta viscosa: cloning, expression, characterisation and role of XvINO1, a gene coding for a myo-inositol 1-phosphate synthase

Department of Molecular and Cellular Biology, A University of Cape Town, 7701, Rondebosch, Cape Town, South Africa; Institute of Plant Biology, Molecular Plant Physiology, B University of urich, Switzerland; C Present address, Université Paris 7, 75005, Paris, France
Functional Plant Biology (Impact Factor: 2.57). 01/2008; 35:26--39. DOI: 10.1071/FP07142

ABSTRACT We have used reverse transcription-PCR coupled with 5 -and 3 -RACE to isolate a full length INO1 cDNA (1692 bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (∼300 mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6 h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.

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    ABSTRACT: Desiccation tolerance (DT) is defined as the equilibration of protoplasmic water potential with that of the surrounding air (generally dry) without loss of viability upon rehydration. Vegetative DT is widespread among mosses and lichens, but is relatively rare in vascular plants (0.15%). Recent studies of selected resurrection species indicate that while resurrection plants might have evolved unique adaptive proteins, enzymes, and antioxidants, the molecular genetic basis of DT lies in the orchestration of transcriptional and posttranscriptional regulatory programs that operate during drying and rehydration. DT requires signaling pathways and regulatory mechanisms, aspects of which resemble developmental programs present in orthodox seeds, which result in the accumulation of oligosaccharides, stress adaptive proteins, antioxidants, reactive oxygen scavenging enzymes, as well as alterations in the composition and structure of membrane lipids. Functional genomics studies using transcriptome, proteome, and metabolome analyses are just beginning to unravel the system complexity required to orchestrate the metabolic symphony that is DT. The status of current gene discovery efforts is summarized along with major transcriptome technologies available currently to conduct desiccation sensitive versus tolerant species comparisons. These strategies, integrated with large-scale proteomic and metabolomic investigations currently in progress, promise to revolutionize our understanding of the mechanistic basis of desiccation tolerance in resurrection plants.
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    ABSTRACT: Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.
    BMC Molecular Biology 01/2010; 11:6. · 2.80 Impact Factor
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    ABSTRACT: In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.
    Plant Growth Regulation 01/2010; 62(3):203-211. · 1.67 Impact Factor

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