Isolation and functional characterization of High affinity potassium transporter (HKT) gene in yeast and expression profiling in tobacco (Nicotiana tabacum)

Abstract

Alternative splicing is widely observed in animals and plants. Intron retention in transcripts and presence of 5ʹ and 3ʹ splice sites within these introns mediate alternate splicing. This research work was intended to characterize the high affinity potassium transporter (HKT) from barley in model system (tobacco and arabidopsis) and functional complementation in yeast (Saccharomyces cervisceae) heterologous system. Relative expression analysis detected different HvHKT2;1 isoforms in barley (Hordeum vulgare) under both normal and high saline conditions. Besides barley, stable integration of insertions corresponding to two introns, were also observed at the 3ʹ-end in kallar grass (Leptochloa fusca). Transcript profiling of HvHKT2;1 in barley showed differential splicing events, which were regulated by NaCl concentration in soil. Accumulation of intron retaining transcripts was observed with abundance of short intron retaining HKT isoforms. Conventional PCR detected different splice variants which were cloned and sequenced. Intron retaining full length cDNA (HvHKT2;1-i) was transformed in tobacco (Nicotiana tabacum) and arabidopsis. Different HvHKT2;1 isoforms were detected in model plant systems with abundance of short intron retaining transcripts. Furthermore, HKT transcript analysis in both model plant systems showed similar results as observed in barley under native promoter conditions. To functionally characterize HvHKT2;1-i, a wild type and potassium uptake deficient mutant yeast strain (trk1, trk2) was used. Both, the wild type and trk1, trk2 yeast expressing HvHKT2;1-i, showed growth activities on YPD (Yeast Peptone Dextrose) solid medium. Growth sensitivities of both wild type and the trk1, trk2 yeast were observed on YPD solid medium containing hygromycin under similar conditions. Addition of milimolar (mM) concentrations of KCl and NaCl in hygromycin supplemented YPD solid resulted in growth recovery of trk1, trk2 yeast expressing HvHKT2;1-i suggesting the presence of functional transcripts in the pool of intron retaining transcripts. It is also clear from yeast functional analysis that HvHKT2;1-i cDNA with introns still enabled the trk1, trk2 yeast to complement K+/Na+ phenotype xiii while maintaining the high affinity K+ transport function. Expression analysis from barley, putative transgenic Arabidopsis, transgenic tobacco and the trk1, trk2 yeast mutant expressing HvHKT2;1-i, showed alternate splicing, intron retention and differential splicing events. Changes in HKT transcript patterns in response to varying K+ and Na+ concentrations, in both heterologous and plant system, suggest a role of these ions in regulation of HKT expression under salt stress. Nicotiana benthamiana plants were used for transient expression of HvHKT2;1-i. Confocal studies detected GFP signals on plasma membrane suggesting the presence of functional HvHKT2;1 fusion protein and hence, the evidence of properly spliced transcripts.