Cloning and Functional Analysis of Rice Yellow Green Leaf Regulatory Gene YGL18

doi:10.11983/CBB22018

Abstract

Abstract:

Leaf color mutants are often accompanied by changes in chlorophyll content and abnormal chloroplast structure, and serve as essential materials for studying the functions of chloroplast development and photosynthesis-related genes. In this study, we obtained a yellow-green leaf mutant named yellow-green leaf 18 (ygl18) from Oryza sativa subsp. indica cv. ‘HZ’ with ethyl methanesulfonate (EMS). Compared with the wild type, the leaves of ygl18 turned yellow at three-leaf stage and the degree of yellowing increased as it grew, accompanied by decreasing photosynthetic rate and chlorophyll content. The seed-setting rate, 1 000-grain weight, and effective panicle number were significantly lower than those of the wild type. We observed disordered chloroplast structure, loose stromal lamellas, and stalled development in the mutant using transmission electron microscopy. Genetic analysis indicated that the mutant feature (or phenotype) of ygl18 is controlled by a pair of recessive nuclear alleles, which were located in a 115.2 kb region between markers InDel2 and InDel3 on the long arm of chromosome 3. We found mutations in the 5′UTR of LOC_Os03g48040 encoding FdC2 (ferredoxin C2). The gene’s function on controlling the mutant phenotype was verified using CRISPR transgenic experiments. Our results revealed a genetic basis for leaf color regulatory network and provide new clues for breeding photosynthetically efficient rice varieties in the future.

Key words: Oryza sativa (rice), leaf color variation, genetic analysis, map-based cloning, ferredoxin

Kairu Yang, Qiwei Jia, Jiayi Jin, Hanfei Ye, Sheng Wang, Qianyu Chen, Yian Guan, Chenyang Pan, Dedong Xin, Yuan Fang, Yuexing Wang, Yuchun Rao. Cloning and Functional Analysis of Rice Yellow Green Leaf Regulatory Gene YGL18[J]. Chinese Bulletin of Botany, 2022, 57(3): 276-287.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB22018

https://www.chinbullbotany.com/EN/Y2022/V57/I3/276

Figures/Tables 12

Table 1 Primer sequences for fine mapping

Primer name	Forward primer (5′-3′)	Reverse primer (5′-3′)
B3-14	AGGATTTGGGCTGTCAATGC	AGCCTACCACAAGACATATAATCG
B3-15	TCCAGTCCACTAAAAGTTTTC	ATTATGCTATAGGCTTTGAGC
M1	TGGAAATGGGTGGAATACCT	CCTCTGTGAGGACCATCTCC
M2	GAATTATTTTTCCTTGGTCATTGTG	GTACCAACGAGGGTCCAAAG
M3	GGAGGCAGGCTACACCAGTA	GCACAATTCTTAAAGAGATGAACC
M4	TTTGATTGGGCTCAAAGCAC	GCCAGGATTTGTTGGATTAGG
M5	AAAAACTCCCAGCGGATAGA	GCTAGAACTGGCAAAATGCTG
Indel1	TTAGGCAAATGAAACCAAGG	CCCAACCACCTAAGTAAACGA
Indel2	TCCTTTGATGACTGGTCGGC	ATGGGAAGACGGGGGAGTAA
Indel3	GTTTGGGAGAGACATGGGGG	GTTTGGCCCATTCCCTGAAG
Indel4	ATGATTTGGAGCCAGCAGTT	GGAAGTGGAATTGCAGATGG

Table 2 Primer sequences for qRT-PCR

Primer name	Forward primer (5′-3′)	Reverse primer (5′-3′)
CAO1-qrt	GCTGCTCTACCGGATGTCTC	ACAACCGATACCGGATACCA
DVR-qrt	TTCACCTACAGCATCGTCCG	AACAGCATCTCCCCTTGCTC
YGL1-qrt	TGTTGGTGGGTCCTTGCTTT	ACTGAAGCCCCAGAGCTCTA
PORA-qrt	CATGCTCGACGACCTCAAGA	TCCTGCATCGTCAGCATGTT
PORB-qrt	CATCATCGTCGGCTCCATCA	TCCTGCATCGTCAGCATGTT
ChlH-qrt	GGGAACTTGGCGTTTCATTA	TCAAATGTCTTGCGTTGCTC
ChlD-qrt	TCCTTTAGCTCACGGCCTTA	GTCCAACATCACCGCTCTTT
NOL-qrt	TCACAAGCCTTACGACCCAC	GCTCCCTCTCCATCATCTGC
NYC1-qrt	TAGTTGGCTCGGGGGAGTAA	AAGGACTGATTCAGGGCTGC
OsFdC2-qrt	AGCTGTGCGGTTCGGATAAA	AACAGGTCCTCGTGCGAAAT
rpoA-qrt	CCATTCCCACAAGCAAAAAT	TCTTACCGCCTTCCGTAGAA
rpoB-qrt	TGGTACATATCCCTTATCTCAA	CTCCAGGACCCAAACAACTC
rbcL-qrt	CTTGGCAGCATTCCGAGTAA	ACAACGGGCTCGATGTGATA
rbcS-qrt	GCTTGGAGTTCAGCAAGGTC	AACGAAGGCATCAGGGTATG
PsaA-qrt	TTAGAAATCCGCCAATCCA	TGCTAGGCTCTACAACCATT
PsaD-qrt	CCGCTCCAAGTACAAGATCA	AAGAGCAGCCTGACAGATGA
PsbA-qrt	ACCCTCATTAGCAGATTCGT	GATTGTATTCCAGGCAGAGC
PsbD-qrt	AAGACAGATTCCGAGGGTGG	TGATTCGCTAGGGATTAAAGAG

Table 3 Primer sequences for CRISPR/Cas9

Primer name	Forward primer (5′-3′)	Reverse primer (5′-3′)
CRISPR/Cas9	CTCCCTCTCTCGATCGTCAG	GCTACTGCTGCTTGAGACGG

Figure 1 Phenotypic of wild-type HZ and mutant ygl18 of rice (A) Phenotype of plant at tillering stage (bar=6 cm); (B) Phenotype of plant at maturity stage (bar=10 cm)

Figure 2 Agronomic characters of wild-type HZ and mutant ygl18 of rice (A) Panicle length; (B) Effective number of panicle; (C) Secondary branch number; (D) Tiller number; (E) Seed-setting rate; (F) 1000-grain weight. * and ** indicate significant differences at 0.05 and 0.01 levels, respectively.

Figure 3 Photosynthetic pigment contents and net photosynthetic rate in leaves of wild-type HZ and mutant ygl18 of rice (A) Photosynthetic pigment content of leaves at tillering stage; (B) Photosynthetic pigment content of leaves at heading stage; (C) Net photosynthetic rate of leaves. * and ** indicate significant differences at 0.05 and 0.01 levels, respectively.

Figure 4 Ultrastructural observation of mesophyll cells in wild-type HZ and mutant ygl18 of rice (A) HZ mesophyll cells (bar=2 μm); (B) HZ mesophyll cells (bar=1 μm); (C) ygl18 mesophyll cells (bar=2 μm); (D) ygl18 mesophyll cells (bar=1 μm). G: Grana lamella; Sg: Starch granule; O: Ophilic granule

Figure 5 Expression levels of chlorophyll synthesis, chloroplast development and photosynthesis related genes in wild-type HZ and mutant ygl18 of rice at tillering stage (A) Expression of genes related to chlorophyll synthesis; (B) Expression of genes related to chloroplast development and photosynthesis

Table 4 Genetic analysis of F2 population of rice mutant

Cross	No. of F₂ individuals			χ²_0.05<3.841
Cross	Normal	yellow-green leaf	Total	χ²_0.05<3.841
ygl18 × NIP	836	331	1167	0.833

Figure 6 Fine mapping of YGL18 in rice (A) YGL18 was located between B3-14 and B3-15 on long arm of rice chromosome 3; (B) Then it was narrowed to a region between M3 and M4 using 148 F2 mutants; (C) Then it was limited to an area of about 115.2 kb between InDel2 and InDel3 with a total of 15 open reading frames (ORFs), using 504 F2 mutants; (D) YGL18 gene structure modeling (green boxes indicate exons, white boxes indicate UTRs, and black lines between boxes indicate introns); (E) Peak sequencing result of YGL18 gene between wild type and mutant

Figure 7 CRISPR/Cas9 knockout verification of YGL18 in rice (A) The position of two separate knockout targets (black boxes represent exons, gray boxes represent UTRs, black lines represent introns, and Target 1 and Target 2 are two separate targets); (B) Sequencing verification of gene knockout strains; (C) Phenotype of wild type (WT), mutant ygl18, KO1 and KO2 lines (bar=2 cm). PAM: Protospacer adjacent motif

Figure 8 Expression pattern of gene YGL18 in wild-type HZ and mutant ygl18 of rice (A) Tissue specific expression of YGL18; (B) The expression level of YGL18 in different development stage

References 29

[1]	陈昆松, 李方, 徐昌杰, 张上隆, 傅承新 (2004). 改良CTAB法用于多年生植物组织基因组DNA的大量提取. 遗传 26, 529-531.
[2]	杜芳芳, 马骏杰, 杨泽伟, 赵雪莲, 刘东宇, 杨秀芹 (2021). 5'UTR在基因表达调控中的研究进展. 中国畜牧杂志 57(8), 60-67.
[3]	赫磊 (2020). 铁氧还蛋白调控水稻光合电子传递的分子机理研究. 博士论文. 北京: 中国农业科学院. pp. 1-101.
[4]	林添资, 孙立亭, 景德道, 钱华飞, 余波, 曾生元, 李闯, 龚红兵 (2018). 一个水稻黄绿叶突变体ygl14(t)的鉴定及基因定位. 核农学报 32, 216-226. DOI
[5]	任永梅, 王嘉宇, 王琬璐, 王棋, 姜鑫, 刘振宇, 朱琳 (2019). 水稻白条纹叶突变体wsl1的生理特性分析及基因定位. 沈阳农业大学学报 50, 87-92.
[6]	韦敏益, 吴子帅, 刘立龙, 李允振, 农春晓, 覃宝祥, 李容柏 (2016). 一个水稻长芒基因AWN-2的定位与克隆. 基因组学与应用生物学 35, 949-956.
[7]	徐娜, 徐江民, 蒋玲欢, 饶玉春 (2017). 水稻叶片早衰成因及分子机理研究进展. 植物学报 52, 102-112. DOI
[8]	许子怡, 程行, 沈奇, 赵亚男, 汤佳玉, 刘喜 (2021). 水稻黄绿叶突变体ygl3的鉴定与基因功能分析. 中国农业科学 54, 3149-3157.
[9]	杨颜榕, 黄纤纤, 赵亚男, 汤佳玉, 刘喜 (2020). 水稻叶色基因克隆与分子机制研究进展. 植物遗传资源学报 21, 794- 803.
[10]	张芳燕, 罗翔, 董娜, 张鹏 (2011). 5'UTR与基因表达的关系. 科技经济市场 (3), 13-16.
[11]	张力科, 高用明 (2009). 水稻叶色突变体及其基因定位和克隆的研究进展. 作物杂志 (2), 12-16.
[12]	周纯, 焦然, 胡萍, 林晗, 胡娟, 徐娜, 吴先美, 饶玉春, 王跃星 (2019). 水稻早衰突变体LS-es1的基因定位及候选基因分析. 植物学报 54, 606-619. DOI
[13]	周亭亭, 饶玉春, 任德勇 (2018). 水稻卷叶细胞学与分子机制研究进展. 植物学报 53, 848-855. DOI
[14]	Akhter D (2019). 水稻叶色基因OsBML和OsPL的基因定位和功能分析. 博士论文. 杭州: 浙江大学. pp. 52-54.
[15]	Chiu FY, Chen YR, Tu SL (2010). Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore. J Biol Chem 285, 5056-5065. DOI URL
[16]	Dong H, Fei GL, Wu CY, Wu FQ, Sun YY, Chen MJ, Ren YL, Zhou KN, Cheng ZJ, Wang JL, Jiang L, Zhang X, Guo XP, Lei CL, Su N, Wang HY, Wan JM (2013). A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants. Plant Physiol 162, 1867-1880. DOI PMID
[17]	Gou P, Hanke GT, Kimata-Ariga Y, Standley DM, Kubo A, Taniguchi I, Nakamura H, Hase T (2006). Higher order structure contributes to specific differences in redox potential and electron transfer efficiency of root and leaf ferredoxins. Biochemistry 45, 14389-14396. DOI URL
[18]	Jung KH, Hur J, Ryu CH, Choi Y, Chung YY, Miyao A, Hirochika H, An G (2003). Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system. Plant Cell Physiol 44, 463-472. DOI URL
[19]	Li CM, Hu Y, Huang R, Ma XZ, Wang Y, Liao TT, Zhong P, Xiao FL, Sun CH, Xu ZJ, Deng XJ, Wang PR (2015). Mutation of FdC2 gene encoding a ferredoxin-like protein with C-terminal extension causes yellow-green leaf phenotype in rice. Plant Sci 238, 127-134. DOI URL
[20]	Lichtenthaler HK (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148, 350-382.
[21]	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. DOI URL
[22]	Wang PY, Li CM, Wang Y, Huang R, Sun CH, Xu ZJ, Zhu JQ, Gao XL, Deng XJ, Wang PR (2014). Identification of a geranylgeranyl reductase gene for chlorophyll synthesis in rice. Springerplus 3, 201. DOI URL
[23]	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. DOI URL
[24]	Yoo SC, Cho SH, Sugimoto H, Li JJ, Kusumi K, Koh HJ, Iba K, Paek NC (2009). Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development. Plant Physiol 150, 388-401.
[25]	Zeng DC, Liu TL, Ma XL, Wang B, Zheng ZY, Zhang YL, Xie XR, Yang BW, Zhao Z, Zhu QL, Liu YG (2020a). Quantitative regulation of Waxy expression by CRISPR/ Cas9-based promoter and 5'UTR-intron editing improves grain quality in rice. Plant Biotechnol J 18, 2385-2387. DOI URL
[26]	Zeng ZQ, Lin TZ, Zhao JY, Zheng TH, Xu LF, Wang YH, Liu LL, Jiang L, Chen SH, Wan JM (2020b). OsHemA gene, encoding glutamyl-tRNA reductase (GluTR) is essential for chlorophyll biosynthesis in rice (Oryza sativa). J Integr Agricul 19, 612-623. DOI URL
[27]	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. DOI URL
[28]	Zhao J, Qiu ZN, Ruan BP, Kang SJ, Lei H, Zhang S, Dong GJ, Jiang H, Zeng DL, Zhang GH, Gao ZY, Ren DY, Hu XM, Chen G, Guo LB, Qian Q, Zhu L (2015). Functional inactivation of putative photosynthetic electron acceptor ferredoxin C2 (FdC2) induces delayed heading date and decreased photosynthetic rate in rice. PLoS One 10, e0143-361.
[29]	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. DOI URL