Identification, Mapping and Transcriptome Analysis of a New Leaf Color Mutant in Cucumber
- Manya Zhao ,
- Qiannan Sun ,
- Jingjing Xu ,
- Tianni Duan ,
- Jintao Cai ,
- Jing Zhou ,
- Tingting Fan ,
- Langtao Xiao ,
- Ruozhong Wang
- Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology Hunan Agricultural University, Changsha 410128, China
Received date: 2024-07-23
Accepted date: 2025-06-04
Online published: 2025-06-04
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.
Cite this article
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 . DOI: 10.11983/CBB24112
References
| [1] | Allan AC, Hellens RP, Laing WA (2008). MYB transcription factors that colour our fruit. Trends Plant Sci 13, 99-102. |
| [2] | Amaresh, Krishnan SG, Vinod KK, Chinnusamy V, Sevanthi ACRMV, Dhandapani R, Ellur RK, Bhowmick PK, Bollinedi H, Umadevi P, Senapati M, Verma RK, Nagarajan M, Dhawan G, Kumar P, Singh AK (2023). Phenotypic characterization of the novel seedling stage zebra leaf mutant, Pusa Zebra 18 in rice. Plant Physiol Rep 28, 500-512. |
| [3] | Awai K, Xu CC, Tamot B, Benning C (2006). A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc Natl Acad Sci USA 103, 10817-10822. |
| [4] | Boase MR, Brendolise C, Wang L, Ngo H, Espley RV, Hellens RP, Schwinn KE, Davies KM, Albert NW (2015). Failure to launch: the self-regulating Md-MYB10R6 gene from apple is active in flowers but not leaves of Petunia. Plant Cell Rep 34, 1817-1823. |
| [5] | Bogorad L (1962). Porphyrin synthesis. Methods Enzymol 5, 885-895. |
| [6] | Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EGWM, Hall RD, Bovy AG, Luo J, Martin C (2008). Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26, 1301-1308. |
| [7] | Chen H, Cheng ZJ, Ma XD, Wu H, Liu YL, Zhou KN, Chen YL, Ma WW, Bi JC, Zhang X, Guo XP, Wang JL, Lei CL, Wu FQ, Lin QB, Liu YQ, Liu LL, Jiang L (2013). A knockdown mutation of YELLOW-GREEN LEAF2blocks chlorophyll biosynthesis in rice. Plant Cell Rep 32, 1855-1867. |
| [8] | Chen JY, Zhao J, Liu X, Li C, Lin DZ, Dong YJ, Ye SH, Zhang XM (2010). Genetic analysis and molecular mapping of a new thermosensitive leaf-color mutant in Oryza sativa. Chin Bull Bot 45, 419-425. (in Chinese) |
| 陈佳颖, 赵剑, 刘晓, 李超, 林冬枝, 董彦君, 叶胜海, 张小明 (2010). 一个新水稻温敏感叶色突变体的遗传分析及其基因分子定位. 植物学报 45, 419-425. | |
| [9] | Cheng MZ, Meng FY, Mo FL, Chen XL, Zhang H, Wang AX (2022). Insights into the molecular basis of a yellow leaf color mutant (ym) in tomato (Solanum lycopersicum). Sci Hortic 293, 110743. |
| [10] | Dasgupta K, Thilmony R, Stover E, Oliveira ML, Thomson J (2017). Novel R2R3-MYB transcription factors from Prunus americana regulate differential patterns of anthocyanin accumulation in tobacco and citrus. GM Crops Food 8, 85-105. |
| [11] | Dei M (1985). Benzyladenine-induced stimulation of 5-aminolevulinic acid accumulation under various light intensities in levulinic acid-treated cotyledons of etiolated cucumber. Physiol Plant 64, 153-160. |
| [12] | Deng LC, Qin P, Liu Z, Wang GL, Chen WL, Tong JH, Xiao LT, Tu B, Sun YT, Yan W, He H, Tan J, Chen XW, Wang YP, Li SG, Ma BT (2017). Characterization and fine-mapping of a novel premature leaf senescence mutant yellow leaf and dwarf 1in rice. Plant Physiol Biochem 111, 50-58. |
| [13] | Ding Y, Yang W, Su CG, Ma HH, Pan Y, Zhang XG, Li JH (2019). Tandem 13-lipoxygenase genes in a cluster confers yellow-green leaf in cucumber. Int J Mol Sci 20, 3102. |
| [14] | Du WK, Hu FR, Yuan SX, Liu C (2020). The identification of key candidate genes mediating yellow seedling lethality in a Lilium regale mutant. Mol Biol Rep 47, 2487-2499. |
| [15] | Gao ML, Hu LL, Li YH, Weng YQ (2016). The chlorophyll-deficient golden leaf mutation in cucumber is due to a single nucleotide substitution in CsChlI for magnesium chelatase I subunit. Theor Appl Genet 129, 1961-1973. |
| [16] | Gao YF, Zhao DH, Zhang JQ, Chen JS, Li JL, Weng Z, Rong LP (2021). De novo transcriptome sequencing and anthocyanin metabolite analysis reveals leaf color of Acer pseudosieboldianum in autumn. BMC Genomics 22, 383. |
| [17] | Ghangal R, Rajkumar MS, Garg R, Jain M (2020). Genome-wide analysis of glutathione S-transferase gene family in chickpea suggests its role during seed development and abiotic stress. Mol Biol Rep 47, 2749-2761. |
| [18] | Guan HY, Xu XB, He CM, Liu CX, Liu Q, Dong R, Liu TS, Wang LM (2016). Fine mapping and candidate gene analysis of the leaf-color gene ygl-1 in maize. PLoS One 11, e0153962. |
| [19] | Han HW, Zhou Y, Liu HF, Chen XJ, Wang Q, Zhuang HM, Sun XX, Ling QH, Zhang HJ, Wang BK, Wang J, Tang YP, Wang H, Liu HY (2023). Transcriptomics and metabolomics analysis provides insight into leaf color and photosynthesis variation of the yellow-green leaf mutant of hami melon (Cucumis melo L.). Plants 12, 1623. |
| [20] | Hu LL, Zhang HQ, Xie C, Wang J, Zhang JY, Wang H, Weng YQ, Chen P, Li YH (2020). A mutation in CsHD encoding a histidine and aspartic acid domain-containing protein leads to yellow young leaf-1 (yyl-1) in cucumber (Cucumis sativus L.). Plant Sci 293, 110407. |
| [21] | Huang SN, Liu ZY, Li DY, Yao RP, Hou L, Li X, Feng H (2016). Physiological characterization and comparative transcriptome analysis of a slow-growing reduced-thylakoid mutant of Chinese cabbage (Brassica campestris ssp. pekinensis). Front Plant Sci 7, 3. |
| [22] | Huang WF, Zhang Y, Shen LQ, Fang Q, Liu Q, Gong CB, Zhang C, Zhou Y, Mao C, Zhu YL, Zhang JH, Chen HP, Zhang Y, Lin YJ, Bock R, Zhou F (2020). Accumulation of the RNA polymerase subunit RpoB depends on RNA editing by OsPPR16 and affects chloroplast development during early leaf development in rice. New Phytol 228, 1401-1416. |
| [23] | Jiang SH, Chen M, He NB, Chen XL, Wang N, Sun QG, Zhang TL, Xu HF, Fang HC, Wang YC, Zhang ZY, Wu SJ, Chen XS (2019). MdGSTF6, activated by MdMYB1, plays an essential role in anthocyanin accumulation in ap- ple. Hortic Res 6, 40. |
| [24] | Jiao FC, Zhao L, Wu XF, Song ZB, Li YP (2020). Metabolome and transcriptome analyses of the molecular mechanisms of flower color mutation in tobacco. BMC Genomics 21, 611. |
| [25] | Ladygin VG (2006). Spectral features and structure of chloroplasts under an early block of chlorophyll synthesis. Bio- physics 51, 635-644. |
| [26] | Li CH, Zhu LH, Yang HJ, Song RH, Gu WH (2019). Main agronomic characters and biochemical traits of xantha mutant of vegetable soybean. Mol Plant Breed 17, 3726-3734. (in Chinese) |
| 李超汉, 朱丽华, 杨红娟, 宋荣浩, 顾卫红 (2019). 菜用大豆黄化新突变体的主要农艺性状和生理特性. 分子植物育种 17, 3726-3734. | |
| [27] | Li XF, Zhang ZL (2016). Plant Physiology Laboratory Manual (5th edn). Beijing: Science Press. pp. 30-31. (in Chinese) |
| 李小方, 张志良 (2016). 植物生理学实验指导(第5版). 北京: 科学出版社. pp. 30-31. | |
| [28] | Lin N, Gao YM, Zhou QY, Ping XK, Li JN, Liu LZ, Yin JM (2022). Genetic mapping and physiological analysis of chlorophyll-deficient mutant in Brassica napus L. BMC Plant Biol 22, 244. |
| [29] | Lin SH, Kuo HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, Wang YY, Tsai CB, Gojon A, Tsay YF (2008). Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20, 2514-2528. |
| [30] | Liu J, Wang JY, Yao XY, Zhang Y, Li JQ, Wang XX, Xu ZJ, Chen WF (2015). Characterization and fine mapping of thermo-sensitive chlorophyll deficit mutant1 in rice (Oryza sativa L.). Breed Sci 65, 161-169. |
| [31] | Liu L, Sun TT, Liu XY, Guo Y, Huang X, Gao P, Wang XZ (2019). Genetic analysis and mapping of a striped rind gene (st3) in melon (Cucumis melo L.). Euphytica 215, 20. |
| [32] | Liu P, Li MJ (2016). Experiments in Plant Physiology (2nd edn). Beijing: Science Press. pp. 142-150. (in Chinese) |
| 刘萍, 李明军 (2016). 植物生理学实验(第2版). 北京: 科学出版社. pp. 142-150. | |
| [33] | Liu X, Huang QQ, Yang YR, Tang JY, Zhao YN, Zhang J (2021). Characterization and map-based cloning of the novel rice yellow leaf mutant yl3. J Plant Biol 64, 35-44. |
| [34] | Mao GZ, Wei HL, Hu W, Ma Q, Zhang M, Wang HT, Yu SX (2019). Fine mapping and molecular characterization of the virescent gene vsp in upland cotton (Gossypium hirsutum). Theor Appl Genet 132, 2069-2086. |
| [35] | Miao H, Zhang SP, Wang M, Wang Y, Weng YQ, Gu XF (2016). Fine mapping of virescent leaf gene v-1 in cucumber (Cucumis sativus L.). Int J Mol Sci 17, 1602. |
| [36] | Michelmore RW, Paran I, Kesseli RV (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88, 9828-9832. |
| [37] | Nagata N, Tanaka R, Satoh S, Tanaka A (2005). Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of prochlorococcus species. Plant Cell 17, 233-240. |
| [38] | Parks BM, Quail PH (1991). Phytochrome-deficient hy1 and hy2 long hypocotyl mutants of Arabidopsis are defective in phytochrome chromophore biosynthesis. Plant Cell 3, 1177-1186. |
| [39] | Pierce LK, Wehner TC (1990). Review of genes and linkage groups in cucumber. Hortscience 25, 605-615. |
| [40] | Qin R, Zeng DD, Liang R, Yang CC, Akhter D, Alamin M, Jin XL, Shi CH (2017). Rice gene SDL/RNRS1, encoding the small subunit of ribonucleotide reductase, is required for chlorophyll synthesis and plant growth development. Gene 627, 351-362. |
| [41] | Qiu J, Sun SQ, Luo SQ, Zhang JC, Xiao XZ, Zhang LQ, Wang F, Liu SZ (2014). Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum. Plant Cell Rep 33, 669-680. |
| [42] | Sakowska K, Alberti G, Genesio L, Peressotti A, Delle Vedove G, Gianelle D, Colombo R, Rodeghiero M, Panigada C, Juszczak R, Celesti M, Rossini M, Haworth M, Campbell BW, Mevy JP, Vescovo L, Cendrero-Mateo MP, Rascher U, Miglietta F (2018). Leaf and canopy photosynthesis of a chlorophyll deficient soybean mutant. Plant Cell Environ 41, 1427-1437. |
| [43] | Sandhu D, Atkinson T, Noll A, Johnson C, Espinosa K, Boelter J, Abel S, Dhatt BK, Barta T, Singsaas E, Sepsenwol S, Goggi AS, Palmer RG (2016). Soybean proteins GmTic110 and GmPsbP are crucial for chloroplast development and function. Plant Sci 252, 76-87. |
| [44] | Schmittgen TD, Livak KJ (2008). Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101-1108. |
| [45] | Shim KC, Kang YN, Song JH, Kim YJ, Kim JK, Kim C, Tai TH, Park I, Ahn SN (2023). A frameshift mutation in the Mg-chelatase I subunit gene OsCHLI is associated with a lethal chlorophyll-deficient, yellow seedling phenotype in rice. Plants 12, 2831. |
| [46] | Song MF, Wei QZ, Wang J, Fu WY, Qin XD, Lu XM, Cheng F, Yang K, Zhang L, Yu XQ, Li J, Chen JF, Lou QF (2018). Fine mapping of CsVYL, conferring virescent leaf through the regulation of chloroplast development in cucumber. Front Plant Sci 9, 432. |
| [47] | Stern DB, Hanson MR, Barkan A (2004). Genetics and genomics of chloroplast biogenesis: maize as a model system. Trends Plant Sci 9, 293-301. |
| [48] | Sun Y, Bai PP, Gu KJ, Yang SZ, Lin HY, Shi CG, Zhao YP (2022). Dynamic transcriptome and network-based analysis of yellow leaf mutant Ginkgo biloba. BMC Plant Biol 22, 465. |
| [49] | Tanaka R, Tanaka A (2011). Chlorophyll cycle regulates the construction and destruction of the light-harvesting complexes. Biochim Biophys Acta Bioenerg 1807, 968-976. |
| [50] | Wu ZM, Zhang X, He B, Diao LP, Sheng SL, Wang JL, Guo XP, Su N, Wang LF, Jiang L, Wang CM, Zhai HQ, Wan JM (2007). A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol 145, 29-40. |
| [51] | Xie Y, Tan HJ, Ma ZX, Huang JR (2016). DELLA proteins promote anthocyanin biosynthesis via sequestering MYBL2 and JAZ suppressors of the MYB/bHLH/WD40 complex in Arabidopsis thaliana. Mol Plant 9, 711-721. |
| [52] | Xiong LR, Du H, Zhang KY, Lv D, He HL, Pan JS, Cai R, Wang G (2021). A mutation in CsYL2.1 encoding a plastid isoform of triose phosphate isomerase leads to yellow leaf 2.1 (yl2.1) in cucumber (Cucumis sativus L.). Int J Mol Sci 22, 322. |
| [53] | Xu BH, Zhang CY, Gu Y, Cheng R, Huang DY, Liu X, Sun YD (2023). Physiological and transcriptomic analysis of a yellow leaf mutant in watermelon. Sci Rep 13, 9647. |
| [54] | Yin JJ, Zhu XB, Yuan C, Wang J, Li WT, Wang YP, He M, Cheng QS, Ye BQ, Chen WL, Linghu QY, Wang JC, Ma BT, Qin P, Li SG, Chen XW (2015). Characterization and fine mapping of a novel vegetative senescence lethal mutant locus in rice. J Genet Genomics 42, 511-514. |
| [55] | Zhang HT, Li JJ, Yoo JH, Yoo SC, Cho SH, Koh HJ, Seo HS, Paek NC (2006). Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol Biol 62, 325-337. |
| [56] | Zhang KJ, Li Y, Zhu WW, Wei YF, Njogu MK, Lou QF, Li J, Chen JF (2020b). Fine mapping and transcriptome analysis of virescent leaf gene v-2 in cucumber (Cucumis sativus L.). Front Plant Sci 11, 570817. |
| [57] | Zhang TT, Dong XY, Yuan X, Hong YY, Zhang LL, Zhang X, Chen SX (2022). Identification and characterization of CsSRP43, a major gene controlling leaf yellowing in cucumber. Hortic Res 9, uhac212. |
| [58] | Zhao CJ, Liu LJ, Safdar LB, Xie ML, Cheng XH, Liu YY, Xiang Y, Tong CB, Tu JX, Huang JY, Liu SY (2020a). Characterization and fine mapping of a yellow-virescent gene regulating chlorophyll biosynthesis and early stage chloroplast development in Brassica napus. G3(Bethesda) 10, 3201-3211. |
| [59] | Zhong XM, Sun SF, Li FH, Wang J, Shi ZS (2015). Photosynthesis of a yellow-green mutant line in maize. Photosynthetica 53, 499-505. |
| [60] | Zhou H, Lin-Wang K, Wang HL, Gu C, Dare AP, Espley RV, He HP, Allan AC, Han YP (2015). Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J 82, 105-121. |
| [61] | Zhu HY, Zhang MJ, Sun SR, Yang S, Li JX, Li H, Yang HH, Zhang KG, Hu JB, Liu DM, Yang LM (2019). A single nucleotide deletion in an ABC transporter gene leads to a dwarf phenotype in watermelon. Front Plant Sci 10, 1399. |
| [62] | Zhu XY, Guo S, Wang ZW, Du Q, Xing YD, Zhang TQ, Shen WQ, Sang XC, Ling YH, He GH (2016). Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice (Oryza sativa). BMC Plant Biol 16, 134. |