ABSTRACT
A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H2O2, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.
Subject(s)
Adaptation, Physiological/genetics , Arabidopsis/genetics , Fabaceae/genetics , Gene Expression Regulation, Plant , HSP70 Heat-Shock Proteins/genetics , Plant Proteins/genetics , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis/metabolism , Chlorophyll/biosynthesis , Cloning, Molecular , Cold Temperature , Droughts , Fabaceae/drug effects , Fabaceae/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Hot Temperature , Hydrogen Peroxide/pharmacology , Models, Molecular , Molecular Sequence Data , Oxidative Stress , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/metabolism , Plant Shoots/drug effects , Plant Shoots/genetics , Plant Shoots/metabolism , Plants, Genetically Modified , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Salinity , Sequence Alignment , Sodium Chloride/pharmacology , Stress, Physiological , Water/metabolismABSTRACT
This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 µg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 µg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set.
Subject(s)
Arabidopsis/growth & development , Carbon/metabolism , Diterpenes, Kaurane/biosynthesis , Gibberellins/metabolism , Plant Proteins/metabolism , Pollen/growth & development , Seeds/growth & development , Arabidopsis/anatomy & histology , Arabidopsis/drug effects , Arabidopsis/genetics , Gene Expression Regulation, Plant/drug effects , Germination , Gibberellins/pharmacology , Hypocotyl/ultrastructure , Phenotype , Plants, Genetically Modified , Pollen/drug effects , Seeds/drug effects , Stevia/metabolismABSTRACT
The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll.