Protection mechanisms in the resurrection plant Xerophyta viscosa (Baker): both sucrose and raffinose family oligosaccharides (RFOs) accumulate in leaves in response to water deficit

Changes in water-soluble carbohydrates were examined in the leaves of the resurrection plant Xerophyta viscosa under conditions of water deficit. Sucrose and raffinose family oligosaccharides (RFOs), particularly raffinose, increased under these conditions, with the highest concentrations evident at 5% relative water content [RWC; 23.5 mg g−1 dry weight (DW) and 17.7 mg g−1 DW, respectively]. Importantly, these effects were reversible, with concentrations returning to levels comparable with that of the full turgor state 7 d after water deficit conditions were alleviated, providing evidence that both sucrose and RFOs may play a protective role in desiccated leaf tissue of X. viscosa. Further, because the sucrose-to-raffinose mass ratio of 1.3:1 observed in the dehydrated state was very low, compared with published data for other resurrection plants (always >5), it is suggested that, in X. viscosa leaves, RFOs serve the dual purpose of stress protection and carbon storage. XvGolS, a gene encoding a galactinol synthase enzyme responsible for the first catalytic step in RFO biosynthesis, was cloned and functionally expressed. In leaf tissue exposed to water deficit, XvGolS transcript levels were shown to increase at 19% RWC. GolS activity in planta could not be correlated with RFO accumulation, but a negative correlation was observed between RFO accumulation and myo-inositol depletion, during water deficit stress. This correlation was reversed after rehydration, suggesting that during water deficit myo-inositol is channelled into RFO synthesis, but during the rehydration process it is channelled to metabolic pathways related to the repair of desiccation-induced damag

Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea

Chickpea transformation is an important component for the genetic improvement of this crop, achieved through modern biotechnological approaches. However, recalcitrant tissue cultures and occasional chimerism, encountered during transformation, hinder the efficient generation of transgenic chickpeas. Two key parameters, namely micro-injury and light emitting diode (LED)-based lighting were used to increase transformation efficiency. Early PCR confirmation of positive in vitro transgenic shoots, together with efficient grafting and an extended acclimatization procedure contributed to the rapid generation of transgenic plants. High intensity LED light facilitate chickpea plants to complete their life cycle within 9 weeks thus enabling up to two generations of stable transgenic chickpea lines within 8 months. The method was validated with several genes from different sources, either as single or multi-gene cassettes. Stable transgenic chickpea lines containing GUS (uidA), stress tolerance (AtBAG4 and TlBAG), as well as Fe-biofortification (OsNAS2 and CaNAS2) genes have successfully been produced

Some physiological and molecular insights into the mechanisms of desiccation tolerance in the resurrection plant Xerophyta viscosa Baker

Few organisms are able to survive desiccation to the air-dried state. Nevertheless, there are a small number of species from every major class of plants (with the exception of gymnosperms) that exhibit this characteristic (Bewley and Oliver, 1992; Oliver and Bewley, 1997). Many of these “resurrection” plants (Gaff, 1971) are being studied in an attempt to understand the mechanisms which enable vegetative tissues to withstand desiccation, with the ultimate aim of identifying characteristics which may be used to produce crops with improved tolerance to osmotic stress

Novel determinants of salinity tolerance

A novel method for the isolation of plant cDNA clones that provide functional sufficiency for salinity tolerance in E. coli cells was developed. A cDNA library containing genes expressed in salt-adapted tobacco cells was constructed in the AZAPII vector. E. coli cells were infected with the rescued pBluescript phagemid library, and the infected bacterial cells were selected on agar plates containing LB medium supplemented with a non-permissive concentration of NaC1, and 10 mM IPTG. After repeated selections, 10 colonies were chosen. These colonies contained plasmids with inserts from 0.8 to 2.0 kb in size. The inserts were isolated and introduced into several strains of E. coll. Cells transformed with plasmids containing the selected inserts exhibited tolerance to 1.17 M NaCI in LB medium in the presence of IPTG. Southern blot analysis using the respective cDNA inserts confirmed the presence of the DNA sequences in the tobacco genome. Northern blot analysis demonstrated greatly increased expression of the corresponding genes in salt-adapted tobacco cells. Expression of any of these cDNA sequences in E. coli was sufficient to provide enhanced tolerance to salinity. Nucleotide sequence analysis and a subsequent search for homology with other genes in the GenBank database resulted in the identification of a homolog of a MIP protein similar to aquaporins, a novel elongation factor eFla, a CCAAT-box binding protein, and several novel genes not present in the gene bank. Functional sufficiency of plant cDNA clones in a prokaryotic model system provides a very powerful tool for the isolation of functionally important novel genes

Molecular cloning, bacterial overexpression and characterization of L-myo-inositol 1-phosphate synthase from a monocotyle-donous resurrection plant, xerophyta viscosa baker

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4 ; MIPS), an evolutionarily conserved enzyme-protein, catalyses the first and rate limiting step of inositol biosynthesis. Inositol and its derivatives play important roles in biological kingdom like growth regulation, membrane biogenesis, signal transduction and also acts as an osmolyte or osmoprotectant in abiotic stress tolerance. Here we report the cloning, sequencing and the characterization of the INO1 gene from Xerophyta viscosa(XINO1), a monocotyledonous resurrection plant. Nucleotide sequences of XINO1 show striking homology (70-99%) with a number of INO1 genes from plant sources particularly with the monocots. The gene is functionally identified through genetic complementation using a yeast inositol auxotrophic strain FY250. The gene is expressed in E. coli BL21, recombinant protein purified to homogeneity, biochemically characterized and compared with Oryza INO1 (RINO1) gene product. The XINO1 gene product is catalytically active in a broader range of lower temperature (between 10-40°C) than the RINO1 gene-product. This is the first report of MIPS gene from any resurrection plant

A novel stress-inducible antioxidant enzyme identified from the resurrection plant Xerophyta viscosa Baker.

A cDNA corresponding to 1-Cys peroxiredoxin, an evolutionarily conserved thiol-specific antioxidant enzyme, was isolated from Xerophyta viscosa Baker, a resurrection plant indigenous to Southern Africa and belonging to the family Velloziaceae. The cDNA, designated XvPer1, contains an open reading frame that encodes a polypeptide of 219 residues with a predicted molecular weight of 24.2 kDa. The XvPer1 polypeptide shows significant sequence identity (approx. 70%) to other recently identified plant 1-Cys peroxiredoxins and relatively high levels of sequence similarity (approx. 40%) to non-plant 1-Cys peroxiredoxins. The XvPer1 cDNA contains a putative polyadenylation site. As for all 1-Cys peroxiredoxins identified to date, the amino acid sequence proposed to constitute the active site of the enzyme, PVCTTE, is highly conserved in XvPer1. It also contains a putative bipartite nuclear localization signal. Southern blot analysis revealed that there is a single copy of XvPer1 in the X. viscosa genome. All angiosperm 1-Cys peroxiredoxins described to date are seed-specific and absent in vegetative tissues even under stress conditions; therefore, XvPer1 is unique in that it is expressed in the vegetative tissues of X. viscosa. The XvPer1 transcript was absent in fully hydrated X. viscosa tissue but levels increased in tissues subjected to abiotic stresses such as dehydration, heat (42 °C), high light intensity (1,500 µmol photons m–2 s–1) and when treated with abscisic acid (100 µM ABA) and sodium chloride (100 mM NaCl). Western blot analyses correlated with the patterns of expression of XvPer1 transcripts under different stress conditions. Immunofluorescence analyses revealed that XvPer1 is localized in the nucleus of dehydrated X. viscosa leaf cells. These results suggest that XvPer1 is a stress-inducible gene, which may function to protect nucleic acids within the nucleus against oxidative injury