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.

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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

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