Identification and Functional Analysis of the Rice Vein Development Gene OsLVD1

doi:10.16819/j.1001-7216.2026.251016

Abstract

Abstract:

【Objective】Photosynthesis is a major determinant of dry matter accumulation in rice, with chlorophyll content and vein density being critical for photosynthetic efficiency. Here, a rice gene regulating chlorophyll content and vein density, OsLVD1, was identified and functionally analyzed.【Method】OsLVD1 was identified based on homology to maize vein development genes, and its protein features, phylogeny, conserved motifs, and subcellular localization were analyzed bioinformatically. An lvd1 mutant was generated using CRISPR/Cas9, with ZH11 as the wild type. Chlorophyll content, vein density, soluble sugar content, and major agronomic traits were examined. The expression of genes related to chlorophyll metabolism and vein development was analyzed by qRT-PCR.【Result】OsLVD1 encodes a nuclear-localized WRKY transcription factor, and the lvd1 mutation caused a frameshift leading to premature termination. Compared with ZH11, the lvd1 mutant showed reductions of 25.90%, 30.75%, and 27.05% in chlorophyll a, chlorophyll b, and total chlorophyll content, respectively, accompanied by increases in minor vein density and bulliform cell number. Plant height was reduced and growth duration was slightly delayed, whereas soluble sugar content, seed-setting rate, and thousand-grain weight were unaffected. qRT-PCR showed that the expression levels of chlorophyll biosynthesis genes OsHEMA1 and OsCHLI were downregulated, while those of the chlorophyll degradation gene OsNYC1 and the vein development gene OsPIN1b were upregulated in the lvd1 mutant. OsLVD1 was predominantly expressed in leaves, with the highest expression at the jointing stage.【Conclusion】Loss of OsLVD1 resulted in a “low chlorophyll-high vein density” phenotype, which may compensate for reduced photosynthesis by enhancing vein-mediated transport and water retention, thereby maintaining yield stability. This study demonstrates a novel role of OsLVD1 in coordinating leaf pigment metabolism and vascular development in rice.

Key words: Oryza sativa, OsLVD1, chlorophyll content, leaf vein density, WRKY transcription factor, photosynthesis

摘要：

【目的】光合作用决定水稻干物质积累，叶绿素含量和叶脉密度与光合作用密切相关。本研究在水稻中鉴定调控叶绿素含量和叶脉密度的基因OsLVD1，并对其功能进行解析。【方法】通过玉米叶脉发育基因同源比对获得OsLVD1，并利用生物信息学分析其蛋白特性、系统发育关系、信号肽、跨膜结构域、保守结构域及亚细胞定位。通过CRISPR获得lvd1编辑株，以野生型ZH11为对照，测定叶绿素含量、叶脉密度、可溶性糖含量及株高、分蘖数、每穗粒数等主要农艺性状，并通过qRT-PCR分析相关基因的表达水平。【结果】生物信息学分析表明，OsLVD1编码WRKY家族转录因子，定位于细胞核。lvd1因移码突变导致蛋白提前终止。与ZH11相比，lvd1叶绿素a、叶绿素b和总叶绿素分别下降25.90%、30.75%和27.05%，剑叶小脉密度和泡状细胞数目增加。lvd1株高降低且生育期略延迟，但叶片可溶性糖、结实率、千粒重等均无显著差异。qRT-PCR分析表明，lvd1中叶绿素合成相关基因OsHEMA1和OsCHLI的表达显著下调，而叶绿素降解基因OsNYC1及叶脉发育相关基因OsPIN1b表达上调。组织表达分析显示，OsLVD1在叶片中高表达，且在拔节期达到峰值。【结论】OsLVD1功能缺失导致叶绿素含量降低、叶脉密度增加，可能通过增加叶脉密度和改变泡状细胞发育补偿光合产物运输与水分保持，从而维持产量稳定。本研究揭示OsLVD1在叶片色素代谢与维管系统发育中的新功能，为光合结构优化提供理论基础。

关键词: 水稻, OsLVD1, 叶绿素含量, 叶脉密度, WRKY转录因子, 光合作用

QIU Yijie, QI Feiyang, DU Hewei, HUANG Min, LI Manfei. Identification and Functional Analysis of the Rice Vein Development Gene OsLVD1[J]. Chinese Journal OF Rice Science, 2026, 40(4): 447-459.

邱一洁, 齐飞扬, 杜何为, 黄敏, 李曼菲. 水稻叶脉基因OsLVD1的鉴定及功能分析[J]. 中国水稻科学, 2026, 40(4): 447-459.

Figures/Tables 9

Table 1. Online platforms used in the research

序号 No.	软件名称 Software name	网址 Website
1	国家水稻数据中心National Rice Data Center	https://www.ricedata.cn/
2	NCBI	https://www.ncbi.nlm.nih.gov/
3	SMART	http://smart.embl.de/
4	ProtParam	https://web.expasy.org/protparam/
5	SOPMA	https://npsa-prabi.ibcp.fr/
6	SWISS-MODEL	https://swissmodel.expasy.org/
7	EvolView	http://evolgenius.info/
8	SignalP4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/
9	TMHMM	http://www.cbs.dtu.dk/services/TMHMM/
10	Predict Protein	https://www.predictprotein.org
11	Plant-mPLoc	http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/
12	CELLO	http://cello.life.nctu.edu.tw/

Fig. 1. Bioinformatic analysis of the OsLVD1 protein A, Domain analysis of LVD1 protein, including multiple low-complexity regions (purple squares), one conserved segment (green box), and a WRKY conserved domain (green pentagon); B, Hydrophobicity analysis of LVD1 protein, which is mainly hydrophilic; C, Secondary structure prediction of LVD1 protein, with high proportions of α-helix (blue) and random coil (purple); D, Predicted tertiary structure model of OsLVD1 protein; E, Phylogenetic analysis of WRKY1 proteins from different species; F, Multiple sequence alignment of WRKY1 domain-containing proteins from different species; G, Chromosomal localization analysis.

Table 2. Cis-acting elements of promoters of rice OsLVD1 genes

元件类别 Element category	元件名称 Element name	核心序列 Core sequence	数量 Number	功能注释 Function annotation
光响应元件 Light-responsive elements	GT1-motif	GGTTAAT/GCTTAA	2	光响应Light responsiveness
	G-Box	CACGTC/CACGAC	13	光响应Light responsiveness
	BOX4	ATTAAT	1	光响应Light responsiveness
	TCT-motif	TCTTAC	2	光响应Light responsiveness
	Spl	GGCCGG	1	光响应Light responsiveness
	AE-BOX	AGAAACTT	2	光响应Light responsiveness
	ATC-motif	AGTAATCT	1	光响应Light responsiveness
激素响应元件 Hormone-responsive elements	ABRE	ACGTG/CACGTG	13	脱落酸响应Abscisic acid responsiveness
	TGACG-motif	TGACG	2	茉莉酸响应MeJA responsiveness
	CGTCA-motif	CGTCA	2	茉莉酸响应MeJA responsiveness
	TGA-element	AACGAC	1	生长素响应Auxin responsiveness
	P-box	CCTTTTG	1	赤霉素响应Gibberellin responsiveness
	TATC-box	TATCCCA	1	赤霉素响应Gibberellin responsiveness
胁迫响应元件 Stress-responsive elements	LTR	CCGAAA	1	低温响应Low-temperature responsiveness
胁迫响应元件 Stress-responsive elements	MBS	CAACTG	1	干旱诱导Drought inducibility
生长发育相关元件 Growth and development-related elements	CAT-box	GCCACT	2	分生组织发育相关 Meristem expression and development

Fig. 2. Effects of OsLVD1 mutation on the expression of related genes and its tissue-specific expression pattern A, Relative expression levels of chlorophyll synthesis genes (OsHEMA1, OsCHLI), chlorophyll degradation genes (OsNYC1, OsPAO), and vein development gene (OsPIN1b) in leaves of ZH11 and lvd1 mutant at jointing stage by qRT-PCR, with ZH11 as the control for normalization. B, Relative expression of OsLVD1 in roots, stems, and leaves of ZH11 at different growth stages analyzed by qRT-PCR. Data are presented as mean ± SD (n = 3). Significant differences were determined by one-way ANOVA and LSD multiple comparison test (P < 0.05).

Fig. 3. Phenotypic characterization of ZH11 and lvd1 A, Overall morphology of ZH11 (left) and lvd1 (right) plants at jointing stage; B, Flag leaf morphology of ZH11 (left) and lvd1 (right) plants; C, Plant height of ZH11 and lvd1 at jointing stage (mean ± SD, n = 30). ns, not significant (P < 0.05), analyzed by Student’s t-test; D, Flag leaf width of ZH11 and lvd1 at jointing stage (mean ± SD, n = 30). ***, Significant (P < 0.05) by Student’s t-test; E, Genomic DNA PCR. Lanes 1-2, ZH11; Lanes 3-5: lvd1; F, Tertiary structure of lvd1 protein; G, CRISPR editing sites and mutant sequence alignment of OsLVD1. Yellow highlight indicates a 1 bp insertion in lvd1 compared with wild type; PAM sequence is marked in red.

Table 3. Statistical table of growth periods of ZH11 and lvd1

材料 Material	出芽期 Germination stage	幼苗期 Seedling stage	分蘖期 Tillering stage	拔节期 Jointing stage	孕穗期 Booting stage	抽穗期 Heading stage	扬花期 Anthesis stage	灌浆期 Grain filling stage	成熟期 Maturity stage
ZH11	3.67±0.99	10.47±1.33	40.00±1.53	61.77±1.99	70.70±1.15	84.93±1.84	99.37±1.47	119.63±1.92	155.47±1.93
lvd1	4.03±1.03	11.00±1.41	40.50±1.31	62.03±1.85	73.03±1.07	88.63±1.56	102.20±1.69	122.03±1.99	161.03±1.75
P值 P value	0.856	0.14	0.21	0.60	6.65×10⁻⁹	5.47×10⁻⁸	4.74×10⁻⁷	3.63×10⁻⁵	1.13×10⁻¹¹

Fig. 4. Comparison of physiological and anatomical features of flag leaves between ZH11 and lvd1 A, Chlorophyll content measurement (chla: chlorophyll a, chlb: chlorophyll b, chls: total chlorophyll); B−E, Paraffin sections of flag leaves. B, D, midribs of ZH11 and lvd1; C, E, Bulliform cells of ZH11 and lvd1 (marked by black triangles), with small veins indicated by black boxes. Scale bar; 400 μm. F, Bulliform cell number in ZH11 and lvd1; G, Soluble sugar content measurement in ZH11 and lvd1. Data are presented as mean ± SD (n≥3). Statistical analysis was performed by one-way ANOVA using GraphPad Prism, ns indicates no significant difference; **, ***, and **** indicate P < 0.01, P < 0.001, and P < 0.0001, respectively.

Table 4. Leaf vein density of flag leaves of ZH11 and lvd1 mm−2

材料 Material	大脉密度 Major vein density	小脉密度 Minor vein density
ZH11	3.6 ± 0.5	21.0 ± 0.6
lvd1	4.2 ± 0.4	22.9 ± 0.7
P值P value	0.16	8.00×10⁻³

Fig. 5. Statistical analysis of agronomic traits of ZH11 and lvd1 in field conditions A, Panicle morphology of ZH11 (left) and lvd1 (right); B, Grain length comparison of ZH11 (top) and lvd1 (bottom); C, Grain width comparison of ZH11 (top) and lvd1 (bottom); D-K, Plant height, tiller number, panicle length, grain length, grain width, seed-setting rate, grains per panicle, and 1000-grain weight of ZH11 and lvd1. Data are presented as mean ± SD (n = 30). Differences between ZH11 and lvd1 were analyzed by one-way ANOVA using GraphPad Prism; * indicates significant difference at P < 0.05.

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