Expression Analysis of the Cucumber Mg-Dechelatase Gene CsMDC and Functional Verification of Its Role in Regulating Chlorlorophyll Degradation

doi:10.16420/j.issn.0513-353x.2024-0160

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (6): 1463-1476.doi: 10.16420/j.issn.0513-353x.2024-0160

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Expression Analysis of the Cucumber Mg-Dechelatase Gene CsMDC and Functional Verification of Its Role in Regulating Chlorlorophyll Degradation

LI Meiqing, LUO Sifei, JIA Yaohao, WANG Weigui, and SUN Jin^*()

College of Horticulture，Nanjing Agricultural University，Nanjing 210095，China

Received:2025-03-19 Revised:2025-05-20 Online:2025-06-20 Published:2025-06-20
Contact: and SUN Jin

Abstract

Abstract:

In this study，cucumber cultivar‘Zhongnong 12’（Cucumis sativus L.）was used to analyze the expression of CsMDC in cucumber and further verify its role in the process of chlorophyll degradation. The analysis and prediction of the physicochemical properties of the CsMDC protein revealed that it has the highest similarity to melon（Cucumis melo L.）from the same family and genus，followed by plants from the same family but different genera such as wax gourd（Benincasa hispida Cogn.），pumpkin（Cucurbita moschata Duch.），and squash（Cucurbita maxima Duch.）. Its similarity to plants from other families is relatively low，indicating that MDC has distinct species-specific characteristics. qRT-PCR analysis of the expression patterns of CsMDC under different exogenous growth regulator treatments and abiotic stresses showed that the gene has the highest relative expression in senescent tissues and exhibits different response characteristics to various conditions. Functional analysis through the construction of CsMDC-overexpressing tobacco plants demonstrated that the overexpression of CsMDC resulted in significantly lower chlorophyll content，dry-to-fresh weight ratio，and net photosynthetic rate compared to wild-type plants，while the reactive oxygen species content was significantly higher. qRT-PCR results showed that the expression of photosystem protein genes in the CsMDC-overexpressing tobacco plants significantly decreased after dark stress，while the expression of genes related to chlorophyll degradation significantly increased. These findings suggest that CsMDC in cucumber is closely related to chlorophyll degradation，induced by different hormones and environmental stresses，and positively regulates chlorophyll degradation and plant senescence.

Key words: cucumber, Mg-dechelatase, CsMDC, expression pattern, chlorophyll degradation, senescence, abiotic stress

LI Meiqing, LUO Sifei, JIA Yaohao, WANG Weigui, and SUN Jin. Expression Analysis of the Cucumber Mg-Dechelatase Gene CsMDC and Functional Verification of Its Role in Regulating Chlorlorophyll Degradation[J]. Acta Horticulturae Sinica, 2025, 52(6): 1463-1476.

Figures/Tables 11

Table 1 The primers of CsMDC gene of PCR or qRT-PCR

用途Application	引物名称Primer name	引物序列（5′-3′）Primer sequence
CsMDC全长PCR PCR of CsMDC full length	CsMDC-F	TTAAACCGATCACAAACC
CsMDC全长PCR PCR of CsMDC full length	CsMDC-R	AGGGCAGTCAATAAAATG
CsMDC实时荧光定量PCR qRT-PCR of CsMDC	qPCR-CsMDC-F	CGCAAAGAACTCCCTGTGGT
CsMDC实时荧光定量PCR qRT-PCR of CsMDC	qPCR-CsMDC-R	AATGCAAGTAATTTGCTGATAGTGT
CsAction实时荧光定量PCR qRT-PCR of CsActin	CsActin-F	CAGGAATCCACGAAACTACT
CsAction实时荧光定量PCR qRT-PCR of CsActin	CsActin-R	AGACCCTCCAATCCAAACAC

Table 2 qPCR primers for the Chl. degredation genes of tobacco

基因Gene	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
NtRbcL	GTCCCCTGTTGGGATGTACTATT	TGTGAGTTCACGTTCTCATCATC
NtRbcS	TCATTGGATTCGACAACGTG	CACAACCCCTAAAGACAAGACA
NtLhcb1	GCTGCTACAATGGCTCTTT	TGGCGACAGTCTTTCTCA
NtLhcb2	TCCGAGCAAACTCCATCT	CAGTGTCCCATCCGTAA
NtLhcb4	GAGATGGGCTATGTTGGC	TGGAGAATGGGAGTGGTT
NtNYC	TAAACAACGCTGGGACAA	TAAACAACGCTGGGACAA
NtRCCR	CTGTGGAGAATCGGCTTGG	ACCTGGGAAGAGGAGTGGC
NtActin	CATTGGCGCTGAGAGATTCC	GCAGCTTCCATTCCGATCA

Fig. 1 Cucumber CsMDC cloning result

Fig. 2 Evolutionary relationships of MDC amino acid sequences of 19 species The decimal above the horizontal line represents the length of the evolutionary branch，and the integer near the node is the bootstrap value

Fig. 3 qRT-PCR analysis of CSMDC in cucumber leaves under different exogenous plant growth regulator treatments and abiotic stresses Control group：foliar spray with distilled water；Exogenous ABA，GA3，SA，and MeJA treatments：foliar spray with 100 μmoL · L⁻¹ ABA，GA3，SA，and MeJA，respectively；Drought treatment：20%（w/v）polyethylene glycol（PEG-6000）；High temperature treatment：42 ℃；Low temperature treatment：4 ℃；Darkness treatment：Light intensity 0 μmoL · m-2 · s-1. Relative expression was calculated using the 2-∆∆CTmethod（Livak et al.，2001）. Independent samples t-test was applied for significance analysis after confirming normality of the data through normality tests. * P < 0.05，** P < 0.01，*** P < 0.001

Fig. 4 Tissue-specific expression of CsMDC in cucumber Relative expression was calculated using the 2-∆∆CT method. Normality tests confirmed that the data met the normality assumption. Statistical significance was analyzed by one-way ANOVA followed by Duncan’s multiple range test and different lowercase letters indicate significant differences，P < 0.05，the same below

Fig. 5 The PCR analysis（A），qRT-PCR analysis（B），phenotype images of tobacco before and after dark treatment（C），and dry weight to fresh weight ratio（D）of cucumber CsMDC transgenic tobacco

Fig. 6 Chlorophyll content，net photosynthetic rate，maximum photochemical efficiency of leaves，and quantum yield of non-regulated energy dissipationin cucumber CsMDC transgenic tobacco plants before and after dark treatment

Fig. 7 H2O2 content，O2- content，and O2- production rate in cucumber CsMDC transgenic tobacco before and after dark treatment

Fig. 8 Relative expression of chlorophyll degradation-related genes in cucumber CsMDC transgenic tobacco before and after dark treatment Wild type tobacco lacks the CsMDC gene，resulting in no detectable signal in this figure

Fig. 9 Relative expression of photosystem-related genes in cucumber CsMDC transgenic tobacco before and after dark treatment Lhcb1、Lhcb2、Lhcb4、RbcL and RbcS：Photosystem protein genes；GLN1：Glutamine synthetase gene，which encodes a protein that indirectly supports photosynthesis through nitrogen assimilation and photorespiration regulation

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Expression Analysis of the Cucumber Mg-Dechelatase Gene CsMDC and Functional Verification of Its Role in Regulating Chlorlorophyll Degradation

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