ABSTRACT
The APETALA2/Ethylene-Responsive Factor (AP2/ERF) is one of the largest gene families encoding several plant specific transcription factors. It plays significant roles in growth and development process, biotic and abiotic stresses, and responses to hormones. AP2 is a homeotic gene governing floral meristem specification, floral organ determination and floral homeotic gene expression in Arabidopsis. The basic structure of AP2 gene was unchanged during evolution in diploid species. The present study was undertaken to find whether AP2 has undergone any change in structure or expression pattern during evolution of allopolyploid Brassica juncea. We cloned AP2 orthologs and c-DNAs from B. juncea and B. nigra. B. juncea was found to carry three AtAP2 orthologs. Comparison of BjAP2 genes with AP2 orthologs from progenitor species, B. rapa and B. nigra showed that two of the BjAP2 genes were derived from B. rapa and one from B. nigra. BjAP2 genes have retained its characteristic AP2 domain and miR172 complementary sequences. mRNAs originated from three AP2 orthologs were detected in all the tissues examined, namely, leaf, flower buds and seedling, indicating absence of sub-functionalization of AP2 during polyploid evolution. However, one of the B. rapa copies gave alternatively spliced AP2 transcript which lacked the second exon. Consequently, the splice variant could not be translated into functional AP2 protein. Considering that miR172 suppresses translation of AP2 transcripts, the alternatively spliced transcript could still play important regulatory role by limiting the availability of miR172 molecules to bind to functional AP2 transcripts. qRT-PCR analysis of BjAP2 expression in different accessions of B. juncea with contrasting seed size indicated that BjAP2 is not a major determinant of seed size in mustard
ABSTRACT
Centromeres are epigenetically specified by the centromeric histone H3 protein (CENH3). The timing and level of expression of CENH3 is tightly regulated to match the demands of the host cell. So far in plants, only CENH3 promoter of Arabidopsis thaliana (L.) Heynh. has been characterized. However, whether CENH3 promoters retain their characteristic mode of regulation in other species remains to be established. In the present study, activity of AtCENH3 promoter was investigated using reporter gene assay in Brassica juncea (L.) Czern. A 1156 bp promoter fragment of AtCENH3 gene (At1g01370) including the first 111 nucleotides of the coding sequence was amplified and cloned into the pORE-R2 binary vector to ensure translation fusion with the uidA coding sequences. The Agrobacterium tumefaciens strain GV3101 harbouring the recombinant construct was used to transform B. juncea cv. RLM198 hypocotyl explants. Histochemical assay of T0 and T1 transgenics showed GUS expression in shoot apical meristem, leaf, sepal, flower pedicel and root tip. Intense GUS expression was observed in meristematic tissues, particularly at shoot and root apices. However, mature leaves, flowers, pollen and ovules exhibited very low or no GUS expression. Our results showed that AtCENH3 promoter regulates cognate gene expression in Brassica juncea as it does in A. thaliana, and hence a suitable candidate for developing haploid inducer line in B. juncea.