Cloning of the CsCRD1 in Tea Plants and Its Impact on Chlorophyll Biosynthesis

doi:10.13560/j.cnki.biotech.bull.1985.2025-1048

Abstract

Abstract:

Objective Mg-protoporphyrin Ⅸ monomethyl ester cyclase (MgPEC) is a key enzyme in chlorophyll biosynthesis pathway. Exploring the effect of the CsCRD1 gene that encodes this enzyme, on chlorophyll biosynthesis will help elucidate the molecular mechanisms of leaf color formation in tea plants, providing a scientific theoretical basis for the selection and breeding of tea plant varieties with leaf color mutations. Method The CsCRD1 gene was cloned and bioinformatics, phylogenetic, expression pattern, and subcellular localization analysis were performed. Antisense oligonucleotide silencing technology was used to explore the function of CsCRD1. Result The CDS length of CsCRD1 is 1 215 bp, encoding 404 aa, with a molecular weight of 46.87 kD and the pI of 8.56. CsCRD1 is located on chromosome 10, and subcellular localization results showed that the CsCRD1 protein is located in the chloroplasts of tobacco mesophyll cells. The CsCRD1 protein contains a highly conserved Rubrerythrin domain, and it is most closely related to the tomato CRD1. The conserved motif types and sequence are consistent with other plant CRD1 proteins, indicating strong evolutionary conservation. Promoter element analysis revealed that it contains light-responsive, hormone-responsive, and stress-responsive elements. Tissue-specific expression analysis showed that CsCRD1 is most highly expressed in the young and mature leaves of tea plants, and least in the flowers. In tea plants with different leaf colors, the relative expression of CsCRD1 in green leaves is significantly higher than in albino leaves, and the expression of this gene increases during the re-greening process of the albino leaves. Correlation analysis showed that the expression level of CsCRD1 is positively correlated with chlorophyll content in tea leaves. Results from antisense oligonucleotide silencing experiments showed that silencing CsCRD1 significantly reduces the content of chlorophyll a, b, and total chlorophyll, and downregulates the expressions of chlorophyll biosynthesis-related genes CsCHLM, CsDVR, and CsPORA. Conclusion CsCRD1 is localized in the chloroplasts and highly expressed in green leaves. Silencing CsCRD1 expression reduces chlorophyll biosynthesis, thus confirming its critical role in chlorophyll metabolism and leaf color development.

Key words: tea plant, CsCRD1, gene cloning, bioinformatics analysis, expression analysis, subcellular localization, chlorophyll biosynthesis, silencing of antisense oligonucleotide

ZHANG Xin, CHEN Cheng, HUO Yuan-Bo, HUANG Ren-Li, ZHANG Li, ZHANG Kai, TONG Hua-rong, YUAN Lian-Yu. Cloning of the CsCRD1 in Tea Plants and Its Impact on Chlorophyll Biosynthesis[J]. Biotechnology Bulletin, 2026, 42(5): 280-291.

Figures/Tables 9

Fig. 1 Cloning and characterization analysis of CsCRD1 gene in tea plantA: Agarose gel electrophoresis image of the CsCRD1 gene. B: Chromosomal localization of CsCRD1 gene. C: Gene structural analysis of CsCRD1

Fig. 2 Analysis of phylogenetic tree of CRD1 proteins and its sequence characteristics in plantA: Phylogenetic tree of CRD1 proteins in plant. The CRD1 homologous proteins used to construct the phylogenetic tree come from tea plant (Camellia sinensis, CsCRD1), Arabidopsis (Arabidopsis thaliana, AtCRD1), tomato (Solanum lycopersicum, Solyc10T002193), corn (Zea mays, Zm00001d040463), rice (Oryza sativa, LOC_Os02g46700), cotton (Gossypium darwinii, Godar.D01G235000), mustard (Brassica juncea, Carub.0005s2368), arugula (Eruca vesicaria, Eruve.0383s0052), poplar (Populus trichocarpa, Potri.006G027300), grape (Vitis vinifera, VIT_208s0040g00390), cucumber (Cucumis sativus, Cucsa.059080), sweet orange (Citrus sinensis, orange1.1g014877m), pecan (Carya illinoinensis Pawnee, CiPaw.09G162600), clementine (Citrus clementina, Ciclev10011842m), and Miscanthus (Miscanthus, Misin06G112500). B: Conserved motifs of CRD1 proteins in plant. C: The schematic representation for conserved structural domains of CRD1 proteins in plant. D: Amino acid multiple sequence alignment of CRD1 proteins in plant. Blue-black: Amino acid identity is 100%. Pink: Amino acid identity is 75%. Blue: amino acid identity is 50%. ▬ indicates predicted the typical conserved domain Rubreythin of CRD1 protein in plants

Fig. 3 Analysis of cis-acting elements in the promoters of gene CsCRD1 in tea plantA: Distribution of cis-acting elements in the promoter. B: Quantity of cis-acting elements in the promoter

Fig. 4 Subcellular localization of CsCRD1 protein in tea plantGFP: Green fluorescent signal. Chlorophyll fluorescence: chloroplast autofluorescence. Bright: Bright field. Merged: Signal merge

Fig. 5 Analysis of tissue-specific expression of CsCRD1 gene in tea plantA: Relative expression of the CsCRD1 gene in different tissues of the tea plant. B: Relative expression of the CsCRD1 gene in different leaf development stages of leaves from ‘Fudingdabaicha’ (FDDB) and ‘Huangjinye’ (HJY). Different lowercase letters indicate significant differences at P<0.05 level (One-way ANOVA with Tukey’s post hoc test). The same below

Fig. 6 Expression analysis of the CsCRD1 gene during the leaf color change process in tea plantsA: Relative expression of the CsCRD1 gene. B: Chlorophyll a content. C: Chlorophyll b content. D: Total chlorophyll content. E: Correlation analysis between total chlorophyll content and the relative expression of the CsCRD1 gene

Fig. 7 Interacting network analysis of CsCRD1 protein in tea plant

Fig. 8 Analysis of the impact of CsCRD1 gene silencing on chlorophyll biosynthesis in tea plantsA: Schematic diagram of CsCRD1 gene AsODN silencing. B: Relative expression of the CsCRD1 gene after AsODNs-CsCRD1 silencing. C‒E: Changes in chlorophyll content after CsCRD1 gene silencing. F‒H: Relative expressions of chlorophyll biosynthesis-related genes after CsCRD1 gene silencing. *, ** and *** indicate the significances of P<0.05, P<0.01, and P<0.001, respectively (one-way ANOVA with Tukey’s post hoc test)

Fig. 9 Effects of CsCRD1 gene on chlorophyll biosynthesis in tea plants

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