Citrate synthase (CS) and NADP-dependent isocitrate dehydrogenase (NADP-ICDH) have been considered as candidate enzymes to provide carbon skeletons for nitrogen assimilation, i.e., production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The CS and NADP-ICDH cDNAs were encoded for polypeptides of 402 and 480 amino acids with an estimated molecular weight of 53.01 and 45 kDa and an isoelectric point of 9.08 and 5.98, respectively. Phylogenetic analysis of these proteins in wheat across kingdoms confirmed the close relationship with Aegilops tauschii and Hordeum vulgare. Further, their amino acid sequences were demonstrated to have some conserved motifs such as Mg2+ or Mn2 binding site, catalytic sites, NADP binding sites, and active sites. In-silico-identified genomic sequences for the three homeologues A, B, and Dof CS and NADP-ICDH were found to be located on long arm of chromosomes 5 and 3, and sequence analysis also revealed that the three homeologues consisted of 13 and 15 exons, respectively. The total expression analysis indicated that both genes are ubiquitously expressed in shoot and root tissues under chronic as well as transient nitrogen stress. However, they are differentially and contrastingly expressed but almost in a coordinated manner in both the tissues. Under chronic as well as transient stress, both the genes in shoot tissue showed downregulation, lowest at 6 h of transient stress. However, in the root tissue, trend was found opposite except with exceptions. Moreover, all the three homeologues of both the genes were transcribed differentially, and the ratio of the individual homeologues transcripts to total homeologues transcripts also varied with the tissue, i.e., shoots or roots, as well as with nitrogen stress treatments. Thus, cDNA as well as genomic sequence information, apparent expression at different time point of nitrogen stress, and coordination between these enzymes would be ultimately linked to nitrate assimilation and nitrogen use efficiency in wheat.
Keywords: CS; Gene expression; Homeologues; NADP-ICDH; Nitrogen stress; Wheat.