C-repeat binding factor (CBF) dependent cold stress signaling cascade is well studied in the model plant arabidopsis but is relatively lesser studied in the crop plants. In the present study， two novel isoforms of an upstream regulator of CBF， Inducer of CBF expression (ICE)， BjICE46 (1314 bp， accession number HQ446510) and BjICE53 (1494 bp， accession number HQ857208) were isolated from Brassica juncea seedlings. Genomic clones of both the isoforms (accession numbers HQ433510 and JX571043) showed three introns， out of which one intron was spanning the bHLH (basic helix-loop-helix) domain. Interestingly， the constitutive expression of BjICE53 was 21 fold higher than BjICE46. Real time quantitat... More
C-repeat binding factor (CBF) dependent cold stress signaling cascade is well studied in the model plant arabidopsis but is relatively lesser studied in the crop plants. In the present study， two novel isoforms of an upstream regulator of CBF， Inducer of CBF expression (ICE)， BjICE46 (1314 bp， accession number HQ446510) and BjICE53 (1494 bp， accession number HQ857208) were isolated from Brassica juncea seedlings. Genomic clones of both the isoforms (accession numbers HQ433510 and JX571043) showed three introns， out of which one intron was spanning the bHLH (basic helix-loop-helix) domain. Interestingly， the constitutive expression of BjICE53 was 21 fold higher than BjICE46. Real time quantitative expression (RT-qPCR) showed BjICE53 to be cold induced but non-responsive to phytohormones. Interestingly， BjICE46 was salinity stress induced and showed upregulation with methyl jasmonate (MeJa) and abscisic acid (ABA). This was supported by the presence of ABA， MeJa and defense related cis- acting regulatory elements in the promoter region of BjICE46. The downstream transcription factor BjCBF (645 bp) was also isolated. The promoter region of BjCBF showed three E-boxes， the binding site for ICE. BjCBF was expressed and purified from E. coli and binding of purified BjCBF with the DRE/CRT elements (present in the promoter of cold responsive genes) was EMSA confirmed. Overall， this study shows that ICE-CBF pathway is conserved in Brassica juncea along with the differential regulation of the ICE isoforms indicating cross-talk between cold and defense signaling.
