The DnaJ-like zinc finger domain protein ORANGE localizes to the nucleus in etiolated cotyledons of Arabidopsis thaliana.

Vitamin A deficiency (VAD) is a worldwide health problem. Overexpression of the DnaJ-like zinc finger domain protein ORANGE (OR) is a novel strategy for the biofortification of pro-vitamin A carotenoids in different staple crops to alleviate VAD. In plants, OR triggers the differentiation from non-pigmented plastids into carotenoid-accumulating plastids. There are different reports on the subcellular localization of this protein in either chloroplasts or the nucleus, both of which were supported by confocal observation and protein-protein interaction results. In this work, we studied the subcellular localization of OR in the cotyledons of germinating seedlings whose plastids were transitioning from non-pigmented proplastids into carotenoid-accumulating etioplasts in the dark, and then into chloroplasts upon illumination. Our Western blot analysis identified two bands of the Arabidopsis OR protein (AtOR) from the chloroplast fraction of the mature leaves (i.e., a 34-kDa form corresponding to the full-length peptide and a 30-kDa form suggesting the removal of the N-terminal chloroplast transit peptide). We found that the full-length AtOR was predominantly localized in the nucleus in etiolated cotyledons, although its abundance decreased upon illumination. Our bioinformatics analysis indicated a nuclear localization signal (NLS) after the N-terminal chloroplast transit peptide. When we substituted different N-terminal regions of AtOR with the green fluorescent protein, our confocal observations demonstrated that this NLS was sufficient to target AtOR to the nucleus. Our results demonstrate that AtOR is a dual-targeted protein that mainly localizes in the nucleus in etiolated cotyledons.

The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

The Artemisia annua L. ß-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic ß-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring ß-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation.

Coordinated regulation of gene expression for carotenoid metabolism in Chlamydomonas reinhardtii.

Carotenoids are important plant pigments for both light harvesting and photooxidation protection. Using the model system of the unicellular green alga Chlamydomonas reinhardtii, we characterized the regulation of gene expression for carotenoid metabolism by quantifying changes in the transcript abundance of dxs, dxr and ipi in the plastidic methylerythritol phosphate pathway and of ggps, psy, pds, lcyb and bchy, directly involved in carotenoid metabolism, under different photoperiod, light and metabolite treatments. The expression of these genes fluctuated with light/dark shifting. Light treatment also promoted the accumulation of transcripts of all these genes. Of the genes studied, dxs, ggps and lcyb displayed the typical circadian pattern by retaining a rhythmic fluctuation of transcript abundance under both constant light and constant dark entrainments. The expression of these genes could also be regulated by metabolic intermediates. For example, ggps was significantly suppressed by a geranylgeranyl pyrophosphate supplement and ipi was upregulated by isopentenyl pyrophosphate. Furthermore, CrOr, a C. reinhardtii homolog of the recently characterized Or gene that accounts for carotenoid accumulation, also showed co-expression with carotenoid biosynthetic genes such as pds and lcyb. Our data suggest a coordinated regulation on carotenoid metabolism in C. reinhardtii at the transcriptional level.