Analysis Of The Mechanism Of Or Gene In Cauliflower And Arabidopsis

Orange (Or) gene was discovered in spontaneous, semidominant mutant of cauliflower which showed orange color of its curd, shoot pith, leaf base and shoot apical meristem due to the abnormal accumulation of high level of β-carotene. The homozygous mutant (or) causes no significant up-regulation of genes required for β-carotene biosynthesis or the upstream MEP pathways. It appears that the abnormal accumulation of high level of β-carotene is due to an enhanced ability of accumulation rather than increased capacity of biosynthesis. Further studies revealed that Or encodes a Cys-rich domain containing DnaJ-like protein. The or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the or allele. Or knock-out mutant didn’t show the high pigment accumulation pattern, indicating that the or mutant is not a loss-of-function mutation but a gain-of-function mutation. Plastid division is affected in the shoot apical meristem of or homozygous mutant, and only one or two large plastids were observed in a cell. Plastid development in leaves is normal. Genes involved in chromoplast differentiation are up-regulated in or mutant and or gene itself has a high expression in proplastid-rich or other noncolored plastid-rich tissues, suggesting that the function of OR is associated with a cellular process that promotes the formation of chromoplasts. Recently, OR was reported to interact with eRF1-2in the nucleus of Arabidopsis protoplast and was thought to be a plastid-nucleus bi-localized protein. or transgenic potato and Arabidopsis also showed increased carotenoids content in their tuber and inflorescence respectively, indicating the great potential of or gene in improving nutritional value of crops.To further understand the possible mechanism of OR, we generated or over-expression and Or dsRNA interference Arabidopsis mutants and investigated the phenotype, pigments content, transcriptome profile of the mutants. We also utilized de-etiolated wild-type (WT) and or mutant cauliflower seedlings as research objects. Pigment contents, plastid development, plastid-to-nucleus retrograde signaling pathways, transcription level and protein level of genes involved in the de-etiolation process were analyzed. Taken together, we preliminarily proposed the following conclusions:1) OR also functions in green tissues, such as Arabidopsis leaves and cauliflower cotyledons, although not that effective as in non-green tissues. In contrast to the great enhanced level of β-carotene content in mutant cauliflower curds,the increase of β-carotene content was not significant in green tissues and the contents of lutein and antheraxanthin were also increased, indicating that OR may play its role in a tissue-specific manner. There was no significant change in the content of chlorophylls, while anthocyanin content increased significantly in mutant cauliflower seedlings.2) Microarray analysis of the Arabidopsis mutants showed no remarkable change of the transcription pattern of the genes required for carotenoids biosynthesis. But the Q-PCR results exhibit increased expression level of PSY, LCYE, VDE in de-etiolated or mutant cauliflower cotyledons. The protein levels of PDS were also different between WT and mutant cauliflowers. These might account for the higher content of carotenoids in mutant cauliflower cotyledons. Due to the different tissues and different development stages investigated, the conclusion was not in contradiction with the previous studies.3) OR has no significant effect on the plastid development in de-etiolated cauliflower cotyledons, except for the slightly looser and thicker grana in6h de-etiolated mutant cauliflower cotyledons. This might related to the stress condition in the mutant.4) There were many cues implying that a stress condition existed in the mutant cauliflower seedlings, such as the increased content of antheraxanthin, anthocycanin and ABA, the enhanced expression of VDE, decreased level of Fv/Fm and increased level of NPQ. Microarray analysis also supported the point. The up-regulated genes in both flower buds and cauline leaves of Arabidopsis or mutants were simultaneously up-regulated or down-regulated in stress condition treated WT plants or in resistant-defective mutants, proving that OR may be involved in the regulation of plant physical activity responding to the environmental stimuli.5) The stress condition in homozygous cauliflower or mutant triggered the high expression of GUN1. It appears that or may function in the Redox/ROS pathways rather than tetrapyrroles metabolism pathway or the plastid gene expression pathway. The decreased level of total glutathione and the ratio of GSH/GSSG in or mutant cauliflower curd also supported the point. The more oxidized environment in mutant cauliflower seedlings may increase the activity of the enzymes required for carotenoids biosynthesis.6) or mutant is sensitive to light, especially the blue light. Its close co-expression relationship with CRY1and the high expression level of GUN1in or mutant imply that OR may participate in the integration network of light and plastid signals together with CRY1and GUN1.In this work, comprehensive analyses were carried out at various aspects in Arabidopsis and cauliflower mutants. The conclusions provide a probably orientation for the future work.