LHD3 Encoding a J-Domain Protein Controls Heading Date in Rice

doi:10.1016/j.rsci.2023.03.015

Abstract

Abstract:

Heading date is one of the most important agronomic traits of rice, which critically affects rice ecogeographical adaptation, yield and quality. In this study, a late heading date 3 (lhd3) mutant was screened from the⁶⁰Co-γ irradiation mutant library. The lhd3 delayed heading date in rice under both short day and long day conditions. Map-based cloning combined with Mutmap strategy was adopted to isolate the causal LHD3 gene. The LHD3 gene encodes a DNA_J domain protein, which was ubiquitously expressed in various plant organs, and dominant expressed in stems and leaves. Subcellular localization analysis showed that LHD3 was localized to nucleus, indicating that LHD3 may interact with other elements to regulate the expression of flowering genes. The transcriptions of the heading activators Ehd1, Hd3a and RFT1 significantly decreased in the lhd3 mutant, suggesting that LHD3 may control the heading date through the Ehd1-Hd3a/RFT1 photoperiodic flowering pathway. The variation and haplotype analyses of the genomic region of LHD3 showed that there were 7 haplotypes in the LHD3 region from 4 702 accessions. The haplotypes of LHD3 can be divided into two classes: class a and class b, and the heading dates of these two classes were significantly different. Further study showed that two single nucleotide polymorphisms (SNPs), SNP10 (G2100C) in Hap II and SNP3 (C861T) in Hap VII, may be the functional sites causing early and late heading in accessions. Nucleotide diversity analysis showed LHD3 had been selected in the indica population, rather than in the japonica population. Therefore, the present study sheds light on the regulation of LHD3 on heading date in rice and suggests that LHD3 is a novel promising new target for rice molecular design and breeding improvement.

Key words: rice, LHD3, heading stage, J-domain protein, photoperiod flowering pathway

Liu Qiao, Qiu Linlin, Hua Yangguang, Li Jing, Pang Bo, Zhai Yufeng, Wang Dekai. LHD3 Encoding a J-Domain Protein Controls Heading Date in Rice[J]. Rice Science, 2023, 30(5): 437-448.

Figures/Tables 4

Fig. 1. Phenotypes and agronomic traits of lhd3. A, Phenotypes of wild type (WT) and mutant (lhd3) rice at the heading stage. Scale bar, 10 cm. B?I, Heading dates (B), grain lengths (C and E), grain widths (D and F), 1000-grain weights (G), plant heights (H) and panicle lengths (I) of WT and lhd3. Scale bars, 1 mm in C and D. NSD, Natural short-day; NLD, Natural long-day. Values are Mean ± SD (n = 10 in B, E, F, H and I; n = 3 in G). **, P < 0.01 compared with WT by the Student’s t-test.

Fig. 2. Cloning and expression analysis of LHD3. A, Molecular mapping of LHD3. The numbers below the lines indicate the number of recombinants. B, Distribution of SNP-index between bulked pools lhd3 and Zhonghua 11 on rice chromosome 3 (Chr. 3). Blue dot, Variant; Orange line, p99; Green line, p95; Red line, SNP-index. C, Schematic diagrams of LHD3 and mutated gene. The 1-bp deletion at 1 242 bp of LHD3 leads to premature termination of the encoded amino acid of LOC_Os03g28310. D, Phenotypes of wild type (WT), mutant lhd3 and its complementary lines. #1 and #2 represent positive transgenic lines from the complementation test. Scale bar, 20 cm. E, Expression of LHD3 in different organs by qRT-PCR analysis. LS, Leaf sheath; YP, Young panicle. Samples were harvested at natural long-day condition. The rice Ubiquitin-5 gene was used as an internal control. Values are Mean ± SD of three independent experiments and three biological replicates. F, Diurnal expression of LHD3 under controlled short-day (SD) and long-day (LD) conditions. The rice Ubiquitin-5 gene was used as an internal control. Values are Mean ± SD of three independent experiments and two biological replicates. G, Subcellular localization of LHD3 in Nicotiana benthamiana mesophyll cells. H2B-mCherry is a nucleus marker. Scale bars, 20 μm.

Fig. 3. Rhythmic expression pattern of flowering genes in wild type (WT) and mutant lhd3. A, C, E and G showed short-day conditions. B, D, F and H showed long-day conditions. The open and filled bars at the bottom represent the light and dark periods, respectively. The rice Ubiquitin-5 (UBQ5) gene was used as an internal control. Values are Mean ± SD of three independent experiments and three biological replicates.

Fig. 4. Haplotype and nucleotide diversity analysis of LHD3. A, Natural variation in LHD3 genomic region revealed 7 haplotypes in 4 702 rice accessions (Oryza sativa ssp. indica, O. sativa ssp. japonica, O. sativa ssp. aus, and intermediate). B, Haplotype analysis of LHD3. The neighbor-joining phylogenetic tree was constructed using MEGA 11 software with 1 000 bootstrap replicates (left). C, Haplotype network analysis of LHD3 in 4 702 rice accessions. Allele frequencies are proportional to the size of circles. D, Box diagram of heading date in seven haplotypes. Different lowercase letters represent significant difference at 5% level according to the Duncan’s multiple range. E, Nucleotide diversity of LHD3 in wild, japonica and indica rice. Red box marked the position of LHD3. F, Nucleotide diversity, the ratios of πwild to πindica and πwild to πjaponica surrounding LHD3.

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