Abstract
BackgroundThe bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Still, nothing is known about the expression of OSBZ8 at protein level in vegetative tissue of salt sensitive and salt tolerant rice plants. In our previous study, Electrophoretic Mobility Shift Assay (EMSA) of [32P]ABRE-DNA and nuclear extracts prepared from the lamina of Pokkali rice plants has detected the presence of an ABRE-binding factor. Northern analysis has also detected salinity stress induced accumulation of transcripts for bZIP class of factor. Therefore, OSBZ8 was considered to play an important role in the regulation of transcription in the vegetative tissue of rice. The aim of this study is to find out whether OSBZ8 has any role in regulating the NaCl-stress induced gene expression in vegetative tissue and whether the expression of OSBZ8 factor directly correlates with the stress tolerance of different varieties of indica type rice.ResultsNorthern analysis of total RNA from roots and lamina of salt-sensitive M-I-48 and salt-tolerant Nonabokra, when probed with the N-terminal unique region of OSBZ8 (OSBZ8p, without the highly conserved basic region), a transcript of 1.3 kb hybridized and its level was much higher in tolerant cultivar. EMSA with Em1a, the strongest ABA Responsive Element till reported from the upstream of EmBP1, and the nuclear extracts from laminar tissue of untreated and salt-treated seedlings of three salt sensitive, one moderately sensitive and two salt tolerant indica rice cultivars showed specific binding of nuclear factor to ABRE element. Intensity of binding was low and inducible in salt sensitive rice cultivars while high and constitutive in salt tolerant cultivars. EMSA with 300 bp 5'upstream region of Rab16A gene, a well known salt stress and ABA-inducible gene of rice, showed formation of two complexes, again very weak in salt sensitive and strong in salt tolerant rice cultivar.ConclusionThe bZIP factor OSBZ8 was found to be present in the ABRE-DNA: protein complex as shown by the supershift of the complex by the purified antiserum raised against OSBZ8p. Treatment of the seedlings with NaCl was found to enhance the complex formation, suggesting the regulation of OSBZ8 gene at both transcriptional and post-translational steps. Comparative EMSA with different varieties of rice suggests a positive correlation with the expression pattern of OSBZ8 and salt tolerance in rice cultivars.
Highlights
The basic leucine zipper (bZIP) class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate abscisic acid (ABA)-mediated transcription in the suspension cultured cells of japonica rice
Northern blot analysis with GC19, OSBZ8p and Rab16A full length probe Ten-day-old Pokkali plants were treated with NaCl or ABA or NaCl along with cycloheximide and total RNA was extracted from the roots and lamina
The size of the upper transcript matches well with the reported size of TRAB1 [25] and that of the lower one (1.3 kb) matches well with the reported size of OSBZ8 [24]. Since both TRAB1 and OSBZ8 are bZIP class of factors, presence of well-conserved sequence encoding basic DNA-binding domain in the GC19 probe was the cause of hybridization with two different transcripts
Summary
The bZIP class Abscisic acid Responsive Element (ABRE)-binding factor, OSBZ8 (38.5 kD) has been considered to regulate ABA-mediated transcription in the suspension cultured cells of japonica rice. Extensive effort to monitor and clone salinity stress induced genes, subtractive hybridization followed by EST, resulted in cloning and identification of 1400 cDNAs from Pokkali rice plants [6]. Several such abiotic stress inducible genes, inducible in vegetative tissues by exogenous application of the plant hormone abscisic acid (ABA) have been cloned and characterized from different plant species; e.g. Em from wheat [7], Osem, Rab16A-D, SalT from rice [8,9,10], LEA, Dehydrin from cotton and barley [11,12], Rab from maize [13], etc. Several abiotic stress inducible genes are not responsive to exogenous ABA treatment, suggesting the existence of both ABA-dependent and ABA-independent pathways [4,5]
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