Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration

doi:10.16420/j.issn.0513-353x.2023-0441

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (5): 939-955.doi: 10.16420/j.issn.0513-353x.2023-0441

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration

DENG Shufang¹, LIU Qian¹, LIU Ling¹, CHEN Ou¹, WANG Wenjun¹^,², ZENG Kaifang¹^,², DENG Lili¹^,²^,^*()

¹ College of Food Science，Southwest University，Chongqing 400715，China
² Research Center of Food Storage & Logistics，Southwest University，Chongqing 400715，China

Received:2024-01-26 Revised:2024-03-11 Online:2024-05-25 Published:2024-05-29

Abstract

Abstract:

25 ℃ storage accelerated the coloration of mandarin fruits，while 32 ℃ storage inhibited their coloring. Using mandarin peel stored at these two temperatures as material，the key transcription factor CcHY5 was discovered that has a significant impact on chlorophyll metabolism. The structural characteristics of CcHY5 and conducted in-depth research on the gene function of this transcription factor were analyzed using the transient overexpression system mediated by Agrobacterium EHA105 on tobacco leaves and mandarin fruits，respectively. The results showed that the total length of CcHY5 was 507 bp，coded 168 amino acids，and had high homology with the AtHY5 of the model crop Arabidopsis. Its promoter region contains various response elements such as light responsive elements，abscisic acid responsive elements，and auxin responsive elements；subcellular localization in the nucleus with transcriptional activation activity. Tobacco leaves showed significant yellowing after transient overexpression of CcHY5，and the yellowing rate was faster at 25 ℃ than at 32 ℃. After transient overexpression of CcHY5 in the peel of mandarin fruit，a significant coloration occurred near the injection well，while the negative control remained green. CcHY5 was significantly up-regulated in the peel at different temperatures compared to the negative control，but the expression level was significantly reduced at 32 ℃ compared to 25 ℃. The content of chlorophyll and its metabolites（chlorophyll a，chlorophyll b，pheophorbide a，pheophorbide b and pheophytin a）in tobacco leaves and mandarin fruits peel significantly decreased，however，there was no significant difference in the content of carotenoids and their metabolites in the peel of mandarin fruits compared to the negative control. After transient overexpression，the expression of chlorophyll cycle genes CcCHL1，CcCHL2，and CcCHL3 in mandarin peel was inhibited to varying degrees，while the expression of chlorophyll degradation metabolism genes CcNYC and CcRCCR was significantly up-regulated. In summary，at an appropriate temperature（25 ℃），CcHY5 may accelerate the coloration process of mandarin peel by promoting chlorophyll degradation.

Key words: mandarin fruit, HY5 transcription factor, chlorophyll, function, coloration, storage temperature

DENG Shufang, LIU Qian, LIU Ling, CHEN Ou, WANG Wenjun, ZENG Kaifang, DENG Lili. Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration[J]. Acta Horticulturae Sinica, 2024, 51(5): 939-955.

Figures/Tables 15

Table 1 Primers used in qRT-PCR

基因名称 Gene name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
Actin	TGGATTCTGGTGATGGTGTG	TGGATTCTGGTGATGGTGTG
CcHY5	AAGGCGTACTTGAATGAGTT	AGGTAACTTTCAACCAGCAA
CcNYC	GGCACGGTTTTCCTTTACAGATG	TGTTGTAGTTCTGACGCTTTCTG
CcRCCR	CAAACGTTGCAGGTCTTCGG	CAAACGTTGCAGGTCTTCGG
CcCHL1	CCCGTCTCCGTTGAAGCTAA	GCAGTGCCGTGGAAAAACAA
CcCHL2	CGTTCTCGCGCGGAAAATAA	TTTGGAGGAGGTGGTGAGGA
CcCHL3	CCTTGTGCTCCGAAAGGAGT	CCTTGTGCTCCGAAAGGAGT

Fig. 1 Effect of different storage temperatures on the expression of CcHY5 in mandarin fruit peel A：FPKM values from RNAseq data，different lowercase letters indicate significant difference at 0.05 level；B：Gene quantification by RT-qPCR. * α = 0.05，** α = 0.01.

Fig. 2 Cloning of CcHY5

Fig. 3 Phylogenetic tree analysis of citrus CcHY5 and Arabidopsis AtbZIP

Table 2 Analysis of cis-elements in CcHY5 promoter region

顺式作用元件 cis-Element	序列 Sequence	预测功能 Predictive function	数量 Quantity
ABRE	ACGTG	脱落酸响应元件 Response element for abscisic acid	2
	CACGTG	脱落酸响应元件 Response element for abscisic acid	1
Box 4	ATTAAT	光响应元件 Response element for light	4
G-box	CACGTG	光响应元件 Response element for light	2
	TACGTG	光响应元件 Response element for light	1
ACE	CTAACGTATT	光响应元件 Response element for light	1
CAT-box	GCCACT	分生组织表达元件 Expression element of meristem tissue	2
AuxRR-core	GGTCCAT	生长素响应元件 Response element for auxin	1
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件 Response element for methyl jasmonate	1
TGACG-motif	TGACG	茉莉酸甲酯响应元件 Response element for methyl jasmonate	1
ARE	AAACCA	厌氧诱导响应元件 Anaerobic induced response element	3
CCAAT-box	CAACGG	MYB结合位点 MYB binding site	1

Fig. 4 Subcellular localization of CcHY5 in tobacco leaf cells

Fig. 5 Analysis of transcriptional activation activity of CcHY5 in yeast The pGBKT7-CcHY5 transformed yeast cells were cultured in single-deficient and triple-deficient medium with pGBKT7-53 + pGADT7-T as positive control and pGBKT7 as negative control. And transcriptional activation activity was analyzed based on yeast growth status and color change with addition of X-α-Gal.

Fig. 6 Changes in appearance of tobacco leaves after transient overexpression of CcHY5 for four days

Fig. 7 Changes in pigment content of tobacco leaves after transient overexpression of CcHY5（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

Fig. 8 Effect of different storage temperatures on the expression of CcHY5 in mandarin fruit peel after transient overexpression Significant difference analysis used Duncan’s test，different lowercase letters indicate significant difference between different treatments at the same time，P < 0.05.

Fig. 9 Changes in appearance and color index of citrus peel after transient overexpression of CcHY5（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

Fig. 10 Effects of transient overexpression of CcHY5 on the content of carotenoid and its metabolites in the peel of mandarin fruit（25 ℃） Student’s t-test，* α = 0.05.

Fig. 11 Effects of transient overexpression of CcHY5 on the content of chlorophyll and its metabolites in the peel of mandarin fruit（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

Fig. 12 Effects of transient overexpression of CcHY5 on the relative expression of CcHY5 and chlorophyll metabolism pathway related genes in mandarin fruit peel（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

Fig. 13 Pearson correlation analysis of chlorophyll and its metabolites and chlorophyll metabolism genes in mandarin fruit peel after transient overexpression of CcHY5 *P < 0.05，** P < 0.01.

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Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration

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