Identification, Mapping and Transcriptome Analysis of a New Leaf Color Mutant in Cucumber

doi:10.11983/CBB24112

Abstract

Abstract: INTRODUCTION Cucumber (Cucumis sativus) is one of the foremost vegetable crops globally. Photosynthesis intricately influences the fruit yield of cucumber, and leaf color determines the photosynthetic efficiency to a large extent. Therefore, Leaf color mutants serve as ideal materials for scrutinizing diverse physiological processes, including photomorphogenesis, chloroplast development, chlorophyll metabolism, and photosynthetic mechanisms. Currently, the molecular mechanisms underlying the yellowing lethal phenotype remain unclear. RATIONALE In this study, a stable cucumber yellowing lethal mutant, ycl(yellow cotyledon lethal), was isolated from the near-isogenic line XYYH-2-1-1. The phenotype, leaf microstructure and chloroplast ultrastructure, as well as physiological and biochemical analyses, were conducted on the mutant ycl and the wild-type XYYH-3-1 to explore the physiological mechanisms underlying the yellowing lethal phenotype. Preliminary localisation of yellowing lethal mutation genes was performed by whole genome resequencing using BSA. The integration of transcriptome sequencing allowed us to analyze the expression of genes related to yellowing death and the main pathways. This approach laid a solid foundation for further investigation into the molecular mechanisms responsible for the lethal phenotype associated with yclyellowing. RESULTS The ycl mutant exhibited yellow cotyledons, which ultimately withered and perished within approximately two weeks. Notably, its growth-inhibiting phenotype appeared to be light-independent. Compared to the wild type, ycl accumulated extremely low Chl a and Chl b contents, which was consistent with the blockade in the magnesium ion chelation process within the chlorophyll biosynthesis pathway. Microscopic and ultrastructural analyses revealed disordered ycl leaf structure and inhibited chloroplast development. Additionally, the ycl mutant displayed significantly increased antioxidant enzyme activities and malondialdehyde contents, suggesting elevated oxidative stress levels and robust antioxidant capacities. The substantial decrease in net photosynthetic rate and rise in intercellular CO₂ concentration in ycl were hypothesized to stem from reduced stomatal conductance, diminished chlorophyll content, and impaired chloroplast development in the mutant. Transcriptomic analyses suggested that key pathways including photosynthesis, flavonoid biosynthesis, chlorophyll metabolism, and reactive oxygen species metabolism were affected in ycl. The ycl mutant gene was preliminarily mapped to a region between 1.48 to 1.9 Mb on chromosome 3 through BSA-seq analysis, encompassing 41 candidate genes. CONCLUSION The study investigated the physiological mechanisms underlying the yellowing lethal phenotype of the yclmutant, preliminarily mapped the mutant gene to chromosome 3, and identified differentially expressed genes (DEGs) and key pathways associated with the lethal phenotype. These findings provide valuable insights into the molecular mechanisms of chloroplast development in cucumber.
Phenotypic changes of WT and the ycl mutant at the cotyledon stage under natural light conditions, and preliminary mapping of the mutant gene.

Key words: cucumber, yellowing lethal mutant ycl, physiological characteristics, gene mapping, transcriptome analysis

Manya Zhao, Qiannan Sun, Jingjing Xu, Tianni Duan, Jintao Cai, Jing Zhou, Tingting Fan, Langtao Xiao, Ruozhong Wang. Identification, Mapping and Transcriptome Analysis of a New Leaf Color Mutant in Cucumber[J]. Chinese Bulletin of Botany, 2025, 60(4): 515-532.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24112

https://www.chinbullbotany.com/EN/Y2025/V60/I4/515

Figures/Tables 12

Table 1 Primers used for qRT-PCR

Primer name	Forward primer (5′→3′)	Reverse primer (5'→3')
Cs-Actin	GTTACGCCCTCCCTCATGCCATTC	TCCCGTTCGGCAGTGGTGGT
CsaV3_3G002180	GTCGTCCTGCCATTCGATCA	AGCACCAAGTTCACTCCAACT
CsaV3_5G025230	AATTCTTCCGACCCGAACCC	AGTAGCCTTCTGCGGACCTA
CsaV3_1G030370	CAGTGGCTGGATACGTCCTC	GTGAGCTCCCGCCATAAAGT
CsaV3_7G000620	TGGAGCATCTCCGAAAGTGG	GGCAAGGAATTGTGATGCCA
CsaV3_5G028880	TAGAACCCAGGCTCCCTCAA	CCGTGTTTTCACAAGCTTCTCT
CsaV3_3G041340	AACATGTTACTGGTGGGGGC	CACATTGAAATCATTGGGTACCTG
CsaV3_6G037230	CCCACTCAAGCGATGTG CTA	CCATTGACCTCAGCATTGCG
CsaV3_5G006200	GACCCAGTTCAAGCTAGCCA	ACTGAGACAACAAGCGCGTA
CsaV3_7G008610	GGCTCCAAGGGCCAATACAT	GTAGGCCTTGGACAGGCATT

Figure 1 Comparison of phenotypic changes between ycl and XYYH-3-1 at the cotyledon stage of cucumber (A) Phenotypic changes of wild type (WT) and ycl in 4, 5, 6, 7, 8 and 14 days (natural light); (B) Phenotypic differences of WT and ycl between the two groups at 7 d of age (dark treatment for 7 d); (C) Phenotypic differences of WT and ycl between the two groups at 8 d of age (light culture for 1 d). Bars=1 cm

Table 2 Agronomic characters of ycl and XYYH-3-1 at cotyledon stage (7 d) of cucumber

Material	Plant height (cm)	Root length (cm)	Stem diameter (cm)	Leaf area (cm²)
WT	4.86±0.32	15.56±0.94	0.22±0.03	9.04±1.66
ycl	2.64±0.56**	5.5±0.9**	0.18±0.02*	1.95±0.26**

Figure 2 Content of chlorophyll and its biosynthetic intermediate metabolites in ycl and XYYH-3-1 (A) Relative content of chlorophyll biosynthetic intermediate metabolites; (B) Photosynthetic pigment content. WT: Wild type; 5-ALA: 5-aminolevulinic acid; PBG: Porphobilinogen; Urogen III: Uroporphyrinogen III; Coprogen III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX; Mg-Proto IX: Mg-protoporphyrin IX. * denote significant differences (t-test, P<0.05); ** denote extremely significant differences (t-test, P<0.01); ns denote no significant difference.

Table 3 Photosynthetic pigment content in leaves of ycl and XYYH-3-1

Material	Chl a (mg·g⁻¹)	Chl b (mg·g⁻¹)	Caro (mg·g⁻¹)	Total Chl (mg·g⁻¹)	Chl a/b	Total Chl/Caro
WT	0.6483±0.0162	0.2157±0.0063	0.1304±0.0018	0.8641±0.0225	3.0057	6.6272
ycl	0.0008±0**	0.0004±0**	0.0265±0.0007**	0.0012±0**	1.9303	0.0443

Figure 3 Microstructure and chloroplast ultrastructure of ycl and XYYH-3-1 leaves (A), (B) Wild type cotyledon microstructures; (C), (D) Mutant cotyledon microstructures; (E), (F) Wild type chloroplast ultrastructures; (G), (H) ycl chloroplast ultrastructures. (F) is an enlarged version of the chloroplast in the red circle in (E). (H) is an enlarged version of the chloroplast in the red circle in (G). Sg: Starch grain; TH: Thylakoid; GL: Grana lamella; O: Osmiophilic body

Table 4 Photosynthesis parameters of the ycl and XYYH-3-1

Material	P_n(μmol·m^-2·s^-1)	G_s(mmol·m^-2·s^-1)	C_i (μmol·mol^-1)	T_r (mol·m^-2·s^-1)
WT	15.85±1.39	0.37±0.08	331±12	4.53±0.62
ycl	-2.49±0.15**	0.14±0.02**	458±7**	2.17±0.31**

Figure 4 Antioxidant enzyme activities and malondialdehyde contents of ycl and XYYH-3-1 (A) Superoxide dismutase (SOD) activity; (B) Peroxidase (POD) activity; (C) Malondialdehyde (MDA) contents. WT: Wild type. ** denote extremely significant differences (t-test, P<0.01).

Figure 5 Preliminary localisation and screening of mutant genes (A)-(C) Distribution of single nucleotide polymorphisms ((A) Population 18; (B) XY cotyledon population; (C) XY true leaf population); (D) Relative expression of CsaV3_3G001980 (** denote extremely significant differences, t-test, P<0.01); (E)-(G) SNP index plot ((E) Population 18; (F) XY cotyledon population; (G) XY true leaf population). WT: Wild type; SNP: Single nucleotide polymorphism

ycl突变位点初定位区间内基因 Figure 5 The genes within the preliminarily mapped region of ycl mutant site

Genes within the interval	Annotation
CsaV3_3G001960	Cytochrome P450 family ent-kaurenoic acid oxidase
CsaV3_3G001970	Cytochrome P450 family ent-kaurenoic acid oxidase
CsaV3_3G001980	Protein NRT1/PTR FAMILY 7.3-like
CsaV3_3G001990	Hsp90 co-chaperone Cdc37, N-terminally processed like
CsaV3_3G002000	Ribose-phosphate pyrophosphokinase
CsaV3_3G002010	Type I inositol polyphosphate 5-phosphatase, puta
CsaV3_3G002020	Phenylalanine-tRNA ligase alpha subunit like
CsaV3_3G002030	Two-component response regulator-like aprr2
CsaV3_3G002040	S-adenosyl-L-methionine-dependent methyltransferases superfamily protein
CsaV3_3G002050	Vacuolar protein sorting-associated protein 55 homolog
CsaV3_3G002060	Haloacid dehalogenase-like hydrolase
CsaV3_3G002070	Haloacid dehalogenase-like hydrolase domain-containing protein
CsaV3_3G002080	Regulator of nonsense transcripts 1 homolog
CsaV3_3G002090	Lon protease homolog 2, peroxisomal
CsaV3_3G002100	Electron carrier/iron ion-binding protein
CsaV3_3G002110	BOI-related E3 ubiquitin-protein ligase 1
CsaV3_3G002120	Protein of unknown function (DUF581)
CsaV3_3G002130	Poly(U)-specific endoribonuclease
CsaV3_3G002140	Methyltransferase-like protein 13
CsaV3_3G002150	Beta-amylase
CsaV3_3G002160	Protein kinase, putative
CsaV3_3G002170	Novel plant snare, putative
CsaV3_3G002180	Phosphatidylinositol 4-phosphate 5-kinase 1
CsaV3_3G002190	Coiled-coil domain-containing protein 97
CsaV3_3G002200	Calcineurin B-like protein
CsaV3_3G002210	Unknown protein
CsaV3_3G002220	Protein kinase
CsaV3_3G002230	Unknown protein
CsaV3_3G002240	Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein
CsaV3_3G002250	Peptidyl-prolyl cis-trans isomerase-like
CsaV3_3G002260	DVA-1 polyprotein
CsaV3_3G002270	tRNA/rRNA methyltransferase (SpoU) family protein
CsaV3_3G002280	Protein lingerer like
CsaV3_3G002290	Chlorophyll a/b binding family protein
CsaV3_3G002300	ATP-dependent Clp protease proteolytic subunit
CsaV3_3G002310	Unknown protein
CsaV3_3G002320	Histone-lysine N-methyltransferase, H3 lysine-9 specific SUVH6-like
CsaV3_3G002330	Kelch repeat-containing protein
CsaV3_3G002340	GATA transcription factor 26-like
CsaV3_3G002350	Vesicle-associated protein 2-1
CsaV3_3G002360	50S ribosomal protein L19, chloroplastic

ycl突变位点初定位区间内基因 Figure 5 The genes within the preliminarily mapped region of ycl mutant site

Genes within the interval	Annotation
CsaV3_3G001960	Cytochrome P450 family ent-kaurenoic acid oxidase
CsaV3_3G001970	Cytochrome P450 family ent-kaurenoic acid oxidase
CsaV3_3G001980	Protein NRT1/PTR FAMILY 7.3-like
CsaV3_3G001990	Hsp90 co-chaperone Cdc37, N-terminally processed like
CsaV3_3G002000	Ribose-phosphate pyrophosphokinase
CsaV3_3G002010	Type I inositol polyphosphate 5-phosphatase, puta
CsaV3_3G002020	Phenylalanine-tRNA ligase alpha subunit like
CsaV3_3G002030	Two-component response regulator-like aprr2
CsaV3_3G002040	S-adenosyl-L-methionine-dependent methyltransferases superfamily protein
CsaV3_3G002050	Vacuolar protein sorting-associated protein 55 homolog
CsaV3_3G002060	Haloacid dehalogenase-like hydrolase
CsaV3_3G002070	Haloacid dehalogenase-like hydrolase domain-containing protein
CsaV3_3G002080	Regulator of nonsense transcripts 1 homolog
CsaV3_3G002090	Lon protease homolog 2, peroxisomal
CsaV3_3G002100	Electron carrier/iron ion-binding protein
CsaV3_3G002110	BOI-related E3 ubiquitin-protein ligase 1
CsaV3_3G002120	Protein of unknown function (DUF581)
CsaV3_3G002130	Poly(U)-specific endoribonuclease
CsaV3_3G002140	Methyltransferase-like protein 13
CsaV3_3G002150	Beta-amylase
CsaV3_3G002160	Protein kinase, putative
CsaV3_3G002170	Novel plant snare, putative
CsaV3_3G002180	Phosphatidylinositol 4-phosphate 5-kinase 1
CsaV3_3G002190	Coiled-coil domain-containing protein 97
CsaV3_3G002200	Calcineurin B-like protein
CsaV3_3G002210	Unknown protein
CsaV3_3G002220	Protein kinase
CsaV3_3G002230	Unknown protein
CsaV3_3G002240	Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein
CsaV3_3G002250	Peptidyl-prolyl cis-trans isomerase-like
CsaV3_3G002260	DVA-1 polyprotein
CsaV3_3G002270	tRNA/rRNA methyltransferase (SpoU) family protein
CsaV3_3G002280	Protein lingerer like
CsaV3_3G002290	Chlorophyll a/b binding family protein
CsaV3_3G002300	ATP-dependent Clp protease proteolytic subunit
CsaV3_3G002310	Unknown protein
CsaV3_3G002320	Histone-lysine N-methyltransferase, H3 lysine-9 specific SUVH6-like
CsaV3_3G002330	Kelch repeat-containing protein
CsaV3_3G002340	GATA transcription factor 26-like
CsaV3_3G002350	Vesicle-associated protein 2-1
CsaV3_3G002360	50S ribosomal protein L19, chloroplastic

Figure 6 RNA-Seq analysis of differential expression between ycl and XYYH-3-1 (A) Differentially expressed genes volcano map; (B) Scatter plot of differentially expressed genes KEGG pathway enrichment; (C) GO term secondary classification statistics; (D) The qRT-PCR verification of transcriptome results. BP: Biological process; MF: Molecular function; CC: Cellular component; WT: Wild type; DEG: Differential expressed gene; FPKM: Fragments per kilobase per million mapped reads; FC: Fold Change. ** denote extremely significant differences (t-test, P<0.01).

Figure 7 Differentially expressed genes (DEGs) associated with the ycl phenotype (A) DEGs involved in photosynthesis; (B) DEGs involved in flavonoid biosynthesis; (C) DEGs involved in anthocyanin transport; (D) DEGs encoding transcription factors. WT: Wild type; LHCP: Chlorophyll a/b binding protein; PC: Plastocyanin; PEPC: Phosphoenolpyruvate carboxylase; Chlase1: Chlorophyllase 1; SAT: Stemmadenine O-acetyltransferase; CCoAMT: Trans- caffeoyl-CoA 3-O-methyltransferase; GST: Glutathione-S-transferase; MATE: Multidrug and toxic compound extrusion

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