Abstract:
Among abiotic stresses, heat stress has the most devastating impact on plant growth. In the
present study, heat shock transcription factor HsfA1d was isolated from Arabidopsis
thaliana and cloned into an in-house constructed gateway compatible cloning vector
pUC57GW-CmRccdB through infusion cloning. Entry clone was confirmed through
restriction digestion and sequencing analysis. For subcellular localization, HsfA1d was
cloned in-frame with yellow florescent protein (YFP) in pGWB442 through LR clonase
reaction resulted in YFP-HsfA1d chimeric gene construct. Homodimerization of HsfA1d was
studied using Bimolecular Florescence complementation (BIFC) assay. For BIFC, HsfA1d
was cloned in-frame with sequences that codes for N and C termini of yellow florescent
protein, into 2 in-house constructed vectors pGSA002-nYFPn and pGSA002-nYFPc
respectively. After confirmation through restriction enzyme digestion, the newly constructed
vectors were transformed into Agrobacterium tumefaciens GV3101 for transient expression
in tobacco (Nicotiana benthamiana) through syringe-infiltration. Subcellular localization of
YFP-HsfA1d in DAPI-stained cells was evaluated using confocal microscopy. For plant
transformation, HsfA1d was cloned into 2 plant expression vectors pGWB402Ω and
pGWB442 through LR clonase reaction. After confirmation through RFLP analysis, the
vectors were transformed into Agrobacterium GV3101 for tobacco leaf disc infection.
Putative transgenic plants were selected and regenerated on appropriate selection media
using different growth regulators. After PCR and confocal-based confirmation, transgenic
plants were evaluated for their response to heat stress. HsfA1d, HSP70 and HSP90 exhibited
4.8, 2.8 and 2 folds increase respectively in their expression under heat stress compare to
room temperature. HsfA1d was found to significantly enhance the expression of downstream
gene HSP70 while no such effect on the expression of HSP90 was recorded. The plants
transformed with HsfA1d were found to retain more water and accumulate more proline
under heat stress. The transgenic plants exhibited efficient protective system, causing less
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electrolyte leakage and less chlorophyll damage under heat stress. It is concluded that
HsfA1d plays a vital role in thermotolerance enhancement and hence recommended for
increasing plant thermotolerance.
