Identification of regulatory elements involved in expression and induction by sucrose and UV-B light of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b

The promoter sequences required for expression of the Arabidopsis thaliana COX5b-2 gene, encoding an isoform of cytochrome c oxidase subunit 5b, were analyzed using plants transformed with deleted and mutagenized forms of the promoter fused to gus . A 1000-bp promoter fragment produces expression in root and shoot meristems, leaf and cotyledon tips, and anthers. Deletion analysis indicated the presence of positive and negative regulatory elements. A regulatory element located between −660 and −620 from the translation start site was identified as a G-box by mutagenic analysis. Mutation of the G-box, that is present within the coding region of the preceding gene in the genome, increases expression of COX5b-2 in cotyledon and leaf lamina and abolishes induction by ultraviolet-B (UV-B) light, which presumably acts through the removal of an inhibitory factor. Identified positive regulatory elements include a site II element (TGGGCC), a related element with the sequence TGGGTC and four initiator elements (YTCANTYY) that completely abolish expression when mutated in combination. Site II elements are also involved in the response to sucrose. The results imply that the COX5b-2 gene has retained expression characteristics presented by most respiratory chain component genes, but its expression mechanisms have diverged from those employed by COX5b-1 , the other gene encoding cytochrome c oxidase subunit 5b in Arabidopsis.     

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.     

Differential expression of the Arabidopsis cytochrome c genes Cytc-1 and Cytc-2. Evidence for the involvement of TCP-domain protein-binding elements in anther- and meristem-specific expression of the Cytc-1 gene

The promoters of the Arabidopsis (Arabidopsis thaliana) cytochrome c genes, Cytc-1 and Cytc-2, were analyzed using plants transformed with fusions to the beta-glucuronidase coding sequence. Histochemical staining of plants indicated that the Cytc-1 promoter directs preferential expression in root and shoot meristems and in anthers. In turn, plants transformed with the Cytc-2 promoter fusions showed preferential expression in vascular tissues of cotyledons, leaves, roots, and hypocotyls, and also in anthers. Quantitative measurements in extracts prepared from different organs suggested that expression of Cytc-1 is higher in flowers, while that of Cytc-2 is higher in leaves. The analysis of a set of deletions and site-directed mutants of the Cytc-1 promoter indicated that a segment located between -147 and -156 from the translation start site is required for expression and that site II elements (TGGGCC/T) located in this region, coupled with a downstream internal telomeric repeat (AAACCCTAA), are responsible for the expression pattern of this gene. Proteins present in cauliflower nuclear extracts, as well as a recombinant protein from the TCP-domain family, were able to specifically bind to the region required for expression. We propose that expression of the Cytc-1 gene is linked to cell proliferation through the elements described above. The fact that closely located site II motifs are present in similar locations in several genes encoding proteins involved in cytochrome c-dependent respiration suggests that these elements may be the target of factors that coordinate the expression of nuclear genes encoding components of this part of the mitochondrial respiratory chain.     

Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants.

The gln-gamma gene, which specifies the gamma subunit of glutamine synthetase in Phaseolus vulgaris L., has been isolated and the regulatory properties of its promoter region analyzed in transgenic Lotus corniculatus plants. A 2-kilobase fragment from the 5'-flanking region of gln-gamma conferred a strongly nodule-enhanced pattern of expression on the beta-glucuronidase reporter gene. Parallel studies on the promoter of another glutamine synthetase gene (gln-beta) showed that a 1.7-kilobase fragment directed 20-fold to 140-fold higher levels of beta-glucuronidase expression in roots than in shoots. Histochemical localization of beta-glucuronidase activity in nodules of the transgenic plants indicated that the chimeric gln-gamma gene was expressed specifically in the rhizobially infected cells; expression of the gln-beta construct was detected in both cortical and infected regions of young nodules, and became restricted to the vascular tissue as the nodule matured. We conclude that gln-beta and gln-gamma genes are differentially expressed both temporally and spatially in plant development and that the cis-acting regulatory elements responsible for conferring these contrasting expression patterns are located within a 2-kilobase region upstream of their coding sequences.     

Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution.

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated psi COX6b-1, psi COX6b-2 and psi COX6b-3, were determined. Pseudogene psi COX6b-1 bears all the hallmarks of a processed pseudogene and diverged from the parental gene after the divergence of man and cow. Alu repetitive elements were integrated into the structural sequences of the other two pseudogenes. Comparison with the human and bovine cDNA sequences encoding COX subunit VIb suggests that psi COX6b-2 and psi COX6b-3 were formed earlier in evolution than psi COX6b-1. Genomic Southern analysis indicated that a few more pseudogenes for COX subunit VIb are likely to be present in the human genome. Identical nt differences with respect to the human cDNA sequence in the pseudogenes provide some clues on the evolution of the ancestral gene coding for COX subunit VIb.     

Promoter analysis of iron-deficiency-inducible barley IDS3 gene in Arabidopsis and tobacco plants.

Under conditions of iron deficiency, graminaceous plants induce the expression of genes involved in the biosynthesis of mugineic acid family phytosiderophores. We previously identified the novel cis-acting elements IDE1 and IDE2 (iron-deficiency-responsive element 1 and 2) through promoter analysis of the barley (Hordeum vulgare L.) iron-deficiency-inducible IDS2 gene in tobacco (Nicotiana tabacum L.). To gain further insight into plant gene regulation under iron deficiency, we analyzed the barley iron-deficiency-inducible IDS3 gene, which encodes mugineic acid synthase. IDS3 promoter fragments were fused to the beta-glucuronidase (GUS) gene, and this construct was introduced into Arabidopsis thaliana L. and tobacco plants. In both Arabidopsis and tobacco, GUS activity driven by the IDS3 promoter showed strongly iron-deficiency-inducible and root-specific expression. Expression occurred mainly in the epidermis of Arabidopsis roots, whereas expression was dominant in the pericycle, endodermis, and cortex of tobacco roots, resembling the expression pattern conferred by IDE1 and IDE2. Deletion analysis revealed that a sequence within -305 nucleotides from the translation start site was sufficient for specific expression in both Arabidopsis and tobacco roots. Gain-of-function analysis revealed functional regions at -305/-169 and -168/-93, whose coexistence was required for the induction activity in Arabidopsis roots. Multiple IDE-like sequences were distributed in the IDS3 promoter and were especially abundant within the functional region at -305/-169. A sequence moderately homologous to that of IDE1 was also present within the -168/-93 region. These IDE-like sequences would be the first candidates for the functional iron-deficiency-responsive elements in the IDS3 promoter.