SUMMARY OF PRESENT RESEARCH:
ISOLATION AND CHARACTERIZATION OF GENES FROM THE RESURRECTION PLANT Xerophyta viscosa Baker THAT ARE FUNCTIONALLY IMPORTANT IN CONFERRING TOLERANCE TO OSMOTIC STRESS 

COLLABORATORS ON THIS PROJECT: 
Professor Jennifer A. Thomson and Assoc. Prof. Jill M.Farrant, Department of Molecular and Cell Biology, University of Cape Town.

Water has become a major limiting factor in world Agriculture. In general, most crop plants are highly sensitive to even a mild dehydration stress. There are however, a few genera of plants unique to Southern Africa called "resurrection plants" which can tolerate extreme water loss or desiccation. The unique ability of these plants to withstand severe waterloss (greater than 90%) makes them an ideal system to study desiccation stress tolerance. Xerophyta viscosa, a representative of the monocotyledonous resurrection plants, was used to isolate genes that are associated with desiccation stress tolerance. The strategies of "Complementation by Functional Sufficiency" and "Differential screening" were used to isolate genes that are important in osmotic stress tolerance. 

A cDNA library, based on mRNA isolated during various dehydration states was constructed in Lambda Phage Zap II vector (Stratagene). The advantage of using the above vector is that it allows for the rescue of phagemids containing the cDNA inserts by helper phage-mediated in vivo excission. The rescued phagemids were then used to infect a specific strain of E. coli (Srl : Tn10 mutant) which was grown under severe osmotic stress. With IPTG present in the medium, bacterial cells containing cDNAs that conferred osmo-tolerance, emerged as colonies on LB/AGAR plates. Nine colonies containing cDNAs which conferred functional sufficiency to the osmotically-stressed bacterial cells were identified. Two of the cDNAs have been characterised extensively viz. ALDRXV4, an aldose reductase homologue and XVCOR, a stress regulated gene. The characterisation of ALDRXV4 in the resurrection plant revealed that it accumulates abundantly during dehydration stress and that it potentially catalyses the conversion of glucose to sorbitol. Sorbitol is well documented to be an important osmoprotectant which accumulates during severe osmotic and NaCl stresses. Our studies on transgenic Arabidopsis thaliana and Digitaria sanguinalis plants expressing ALDRXV4 revealed significant tolerance to severe osmotic and NaCl stresses, respectively. We are currently transforming maize with the above gene. XVCOR has also been found to be a novel stress-responsive gene that is induced by a variety of stresses including heat, osmotic, NaCl and low tempperature. Transgenic Arabisopsis thaliana and Nicotina tabacum plants expressing XVCOR have been generated and are ready for analysis. 

The second strategy involved the differential screening of 192 randomly selected cDNA clones from the above cDNA library. Of the 192 cDNAs screened, 30 were found to be upregulated during dehydration while 20 were downregulated during dehydration. Some of the upregulated cDNAs have been sequenced and characterised. The identities of these proteins included: XVDH, a dehydrin; XVLEA, a lea-like homologue; XVGS, galactinol synthase; XVCBP, EF-hand calcium-binding; XVHSP90, a heat shock 90 protein; and XVPER, a peroxiredoxin. All six of the cDNAs have been found to be expressed during dehydration stress in the resurrection plant and they have also been implicated in other stresses, particularly those that have an osmotic stress component to it. Currently, the model plant A. thaliana is being used to generate knockouts of the above genes so as to determine the function of the above genes by reverse genetics. Transgenic A. thaliana and D. sanguinalis plants expressing the above genes, are being generated to determine the effects of the respective gene products on osmotic stress tolerance.