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CmNAC73 Mediates the Formation of Green Color in Chrysanthemum Flowers by Directly Activating the Expression of Chlorophyll Biosynthesis Genes HEMA1 and CRD1.

ABSTRACT: Chrysanthemum is one of the most beautiful and popular flowers in the world, and the flower color is an important ornamental trait of chrysanthemum. Compared with other flower colors, green flowers are relatively rare. The formation of green flower color is attributed to the accumulation of chlorophyll; however, the regulatory mechanism of chlorophyll metabolism in chrysanthemum with green flowers remains largely unknown. In this study, we performed Illumina RNA sequencing on three chrysanthemum materials, Chrysanthemum vestitum and Chrysanthemum morifolium cultivars 'Chunxiao' and 'Green anna', which produce white, light green and dark green flowers, respectively. Based on the results of comparative transcriptome analysis, a gene encoding a novel NAC family transcription factor, CmNAC73, was found to be highly correlated to chlorophyll accumulation in the outer whorl of ray florets in chrysanthemum. The results of transient overexpression in chrysanthemum leaves showed that CmNAC73 acts as a positive regulator of chlorophyll biosynthesis. Furthermore, transactivation and yeast one-hybrid assays indicated that CmNAC73 directly binds to the promoters of chlorophyll synthesis-related genes HEMA1 and CRD1. Thus, this study uncovers the transcriptional regulation of chlorophyll synthesis-related genes HEMA1 and CRD1 by CmNAC73 and provides new insights into the development of green flower color in chrysanthemum and chlorophyll metabolism in plants.

SUBMITTER: Luo J

PROVIDER: S-EPMC8151904 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.

Project description:Background'Regal Splendour' (Hosta variety) is famous for its multi-color leaves, which are useful resources for exploring chloroplast development and color changes. The expressions of chlorophyll biosynthesis-related genes (HrHEMA, HrPOR and HrCAO) in Hosta have been demonstrated to be associated with leaf color. Herein, we isolated, sequenced, and analyzed HrHEMA, HrPOR and HrCAO genes. Subcellular localization was also performed to determine the location of the corresponding enzymes. After plasmid construction, virus-induced gene silencing (VIGS) was carried out to reduce the expressions of those genes. In addition, HrHEMA-, HrPOR- and HrCAO-overexpressing tobacco plants were made to verify the genes function. Changes of transgenic tobacco were recorded under 2000 lx, 6000 lx and 10,000 lx light intensity. Additionally, the contents of enzyme 5-aminolevulinic acid (5-ALA), porphobilinogen (PBG), chlorophyll a and b (Chla and Chlb), carotenoid (Cxc), superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (Pro) and catalase (CAT) under different light intensities were evaluated.ResultsThe silencing of HrHEMA, HrPOR and HrCAO genes can induce leaf yellowing and chloroplast structure changes in Hosta. Specifically, leaves of Hosta with HrCAO silencing were the most affected, while those with HrPOR silencing were the least affected. Moreover, all three genes in tobacco were highly expressed, whereas no expression was detected in wild-type (WT). However, the sensitivities of the three genes to different light intensities were different. The highest expression level of HrHEMA and HrPOR was detected under 10,000 lx of illumination, while HrCAO showed the highest expression level under 6000 lx. Lastly, the 5-ALA, Chla, Cxc, SOD, POD, MDA, Pro and CAT contents in different transgenic tobaccos changed significantly under different light intensities.ConclusionThe overexpression of these three genes in tobacco enhanced photosynthesis by accumulating chlorophyll content, but the influential level varied under different light intensities. Furthermore, HrHEMA-, HrPOR- and HrCAO- overexpressing in tobacco can enhance the antioxidant capacity of plants to cope with stress under higher light intensity. However, under lower light intensity, the antioxidant capacity was declined in HrHEMA-, HrPOR- and HrCAO- overexpressing tobaccos.

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CmNAC73 Mediates the Formation of Green Color in Chrysanthemum Flowers by Directly Activating the Expression of Chlorophyll Biosynthesis Genes HEMA1 and CRD1.

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