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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (11): 2764-2774.doi: 10.3724/SP.J.1006.2024.42008

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Phenotype identification and gene mapping of lesion mimic and early senescence mutant lmes6 in rice

HU Yao-Jie1(), LIU Ya-Ping1, ZHENG Jun-Yan1, HAN Ting1, MA Bo-Jun1, YE Ya-Feng2, LIU Bin-Mei2,*(), CHEN Xi-Feng1,*()   

  1. 1College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
    2Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei 230031, Anhui, China
  • Received:2024-02-05 Accepted:2024-06-20 Online:2024-11-12 Published:2024-07-12
  • Contact: *E-mail: liubm@ipp.ac.cn; E-mail: xfchen@zjnu.cn
  • Supported by:
    National Natural Science Foundation of China(32071987);National Natural Science Foundation of China(32101697);National Natural Science Foundation of China(32272096);Natural Science Foundation of Zhejiang Province(LQ22C130005);Natural Science Foundation of Zhejiang Province(LZ23C130004);Natural Science Foundation of Zhejiang Province(LZ24C130016)

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Abstract:

The occurrence of lesion mimic and early senescence is affected by multiple factors, including hormones, metabolism, and ambient signals. However, the molecular mechanism underlying this process remains incompletely understood. In this study, we identified a lesion mimic and early senescence mutant, named lmes6 (lesion mimic and early senescence 6), through screening of a heavy ion beam radiation mutagenesis library. Comparative analysis with the wild-type control revealed that the leaves of the lmes6 mutant exhibited lesion mimic, chlorosis, and a significant reduction in chlorophyll content from the tillering stage. Additionally, the mutant displayed a notable decrease in panicle length, grain number per panicle, grain length, and yield per plant. Tissue staining showed that the mutant exhibited programmed cell death and excessive accumulation of reactive oxygen species in the leaves. Furthermore, the mutant displayed enhanced resistance to bacterial blight and bacterial leaf streak. Genetic analysis showed that the mutant phenotype was governed by a single recessive nuclear gene. Through map-based cloning techniques, the gene was precisely mapped to a 53 kb region on rice chromosome 7. Candidate gene prediction and PCR sequencing analysis identified a gene, LOC_Os07g46460, which encodes Fd-GOGAT1 (ferredoxin-dependent glutamate synthase 1) in the lmes6 mutant. A single base substitution in this gene resulted in the conversion of L-phenylalanine (F) to leucine (L), which is one of new multiple alleles of this gene. Interestingly, compared to the three allelic mutations of this gene reported, lc7, abc1, and spl32, the lmes6 mutant exhibited less impact on growth and yield of rice. Phylogenetic analysis and alignment of the homologous protein sequences of Fd-GOGAT1 revealed its high conservation among monocots.

Key words: lesion mimic, early senescence, lmes6, fine mapping, sequence analysis

Table 1

PCR primer sequences of molecular markers used in gene mapping"

分子标记
Molecular marker		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
R7M30 ATGTCGCCTACGAGTTTTC TTCATGTGACCATTTGTGC
RM22017 GCTACCATGCTAGTCGTAATGC GCTAGTGTAAACTTTGGCATCG
RM22091 CCGTGAGGGCCATGTACAGACG TGCCACGTCAGCACTCTTTCTTCC
RM22144 GCAGAAGCAGGAGCCCAATATCC CTGTCTGTCCAGCACCAACACC
RM22171 TAGTACCGCCATTACCATTCATCC GACGGTGGGACTCCTAATTACAGC
Indel 1 CCACCTTCAATCCCTGCC AGTGAAGACCGCCCAGATC
Indel 2 GCACAGACACAACCTTTAGGA GTGTCAGGTTTTGGCTCGG
Indel 3 CTTACAATGCCCACAAAGGA TTGCTGGTAAAGAATGCTAATC
Indel 4 CGTTTGATCGTCCGTCTTAT AATTGCCATGACTCCTCCTC
Indel 5 CGCAGAGAGTTCAAGGAAGC AGACTGTTGCTAACCATCATGG

Table 2

Primer sequences for PCR amplification of rice LMES6 gene"

引物名称
Primer name		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
LMES6-1 TTCCCGCATCCACCACCA CATCTAGGGCTTGTATTGGTAC
LMES6-2 GATCTGAGATTCTTGGGCAATT TCACGCCCACGCCATACAG
LMES6-3 CAATGGCTTCACAAGGCAAGG TCACCGTGGGCATCTTCC
LMES6-4 CCCTGCCATCCCGATACTTC CCCTTCACCAGGCTTTGC
LMES6-5 TGAGCCTGCAACCTCCAT GGGCCATTACTGTGGACATCTT
LMES6-6 TTTCCTGGTGTTCCTGGTG GTGGACCGTGGTGTTATCGT

Fig. 1

Mutational phenotype and disease resistance of the rice mutant lmes6 A: plants phenotype at tillering stage; B: plants phenotype at mature stage; C: flag-leaf at mature stage; D: leaves phenotype of WT and lmes6 before staining; E-G: leaves strained by TB, DAB, and NBT; H: rice leaves after inoculation with Xoc and Xoo strains; I: chlorophyll and carotenoids content determination of leaves; J: statistics on the proportion of leaf lesions to total leaf area after inoculation with Xoc strain; K: statistics on the lesion length of leaf lesions after inoculation with Xoo strains; WT: wild-type control Wuyungjing 7; lmes6: lesion mimic and early senescence mutant in rice; ** indicates a highly significant difference (P < 0.01) by the t test between WT and lmes6; error bars in I-K indicate SD. Bar: 5 cm (A); 15 cm (B); 2 cm (C); 1 cm (D-H)."

Fig. 2

Comparison of agronomic traits between rice lmes6 mutant and its wild-type control A-L: statistical measurement of plant height (A), flag-leaf length (B), flag-leaf width (C), effective tiller number (D), panicle length (E), grain number per panicle (F), setting ratio (G), grain length (H), grain width (I), grain thickness (J), 1000-grain weight (K), and yield per plant (L); M-O: photograph of ten grains in length (M), width (N), and thickness (O) between WT and lmes6 mutant; P: panicle length. * and ** indicate significant (P < 0.05) and highly significant difference (P < 0.01) by the t-test between the wild-type (WT) control and the rice lmes6 mutant, respectively; error bars in A-L indicate SD. Bar: 1 cm (M-O); 5 cm (P)."

Fig. 3

Fine mapping and candidate gene sequencing of the target gene of the lmes6 mutant A: genetic map of the lmes6 gene by fine mapping; n refers to the number of F2 mutants used for mapping; the number under each marker represents the number of recombination detected by the corresponding marker. B: all annotation genes in the localization interval; C: the sequencing results of candidate gene LOC_Os07g46460 in the mutation sites."

Fig. 4

Phylogenetic tree of Fd-GOGAT1 homologous proteins in plants"

Fig. 5

Sequence alignment of rice Fd-GOGAT1 and its homologous proteins Red arrows or parentheses indicate sites where the sequence has changed; lc7, abc1, and lmes6 gene were different variational allele of the spl32 gene. In the amidotransferase domain of Fd-GOGAT1, 19 amino acids were lost in spl32 mutant, and asparagine (N) was replaced by serine (S) in lc7 mutant. In the FMN-binding domain, lmes6 mutants undergo phenylalanine (F) to leucine (L) substitution, and abc1 mutants undergo lysine (K) to glutamate (E) substitution."

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