Identification of LIGULELESS 2 Controlling the Lamina Joints and Screening of Its Interacting Proteins in Wheat

doi:10.7668/hbnxb.20196290

Abstract

Abstract:

The identification of the wheat lamina joint regulator LIGULELESS 2 (LG2) and analysis of its interacting proteins are of great significance for exploring the molecular mechanisms underlying lamina joint formation and leaf angle regulation, as well as for breeding new varieties with ideal plant architecture. First, two homologous genes of LG2, designated TaLG2 and TaLG2L, were identified in the wheat genome through phylogenetic analysis, each containing three homoeologous copies located in the A, B, and D subgenomes with more than 90% sequence similarity. Transcriptome-based expression profiling during lamina joint development and in different leaf tissues revealed that both TaLG2 and TaLG2L were specifically expressed in the lamina joint. Except for TaLG2-3A, the other five homoeologs exhibited specific expression at the early S1 stage of lamina joint development. The bait vector pGBKT7-TaLG2L-1A was constructed and used for yeast two-hybrid (Y2H) screening yielding 174 positive clones.Sequencing analysis identified 68 candidate interacting proteins of TaLG2L-1A. GO enrichment analysis showed that these proteins were mainly involved in ubiquitin binding, cation binding, and mRNA binding. Further rotation verification and pairwise Y2H assays demonstrated that TaLG2L-1A can interact with the auxin response factor TaARF17 and the blue light receptor cryptochrome TaCRY1, respectively. This study clarifies the tissue-specific expression patterns of wheat LG2 homologs, identifies and obtains key interacting proteins TaARF17 and TaCRY1 of TaLG2L-1A, and reveals the molecular mechanism by which LG2 may coordinate auxin and light signaling pathways to regulate lamina joint formation and leaf angle development.

Key words: Wheat, Lamina joint, LIGULELESS 2, Expression pattern, Yeast two-hybrid, Interaction proteins

CLC Number:

WANG Yimo, LIU Zitong, NIU Ziyu, GU Bingyan, LI Li, SONG Yilei, NIU Zelin, WANG Rongna. Identification of LIGULELESS 2 Controlling the Lamina Joints and Screening of Its Interacting Proteins in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 24-33. doi: 10.7668/hbnxb.20196290.

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Figures/Tables 13

Tab.1 Primer information

引物名称 Primer name	序列信息(5'—3') Sequence information(5'—3')	用途 Purpose
TaLG2-F	ATGGTGCAAGGTGAGGAGGC	TaLG2基因克隆
TaLG2-R	TCAAAATCCGGAGAACTGATTCTGTTGC
TaLG2L-F	ATGAGTAGAGTTCTTCCACTCATTGCC	TaLG2L基因克隆
TaLG2L-R	TCAGAGGCCGGAGAACTGG
BD-TaLG2L-1A-F	CATGGAGGCCGAATTCATGAGTAGAGTTCTTCCACTCATTGCC	pGBKT7-TaLG2L-1A载体构建
BD-TaLG2L-1A-R	GCAGGTCGACGGATCCTCCGAGGCCGGAGAACTG
AD-TaWD-F	GGAGGCCAGTGAATTCATGTCCAATTATCACGACGATCAC	pGADT7-TaWD载体构建
AD-TaWD-R	CGAGCTCGATGGATCCTCAAAGCATCGAGTCATGGT
AD-TaCRY1-F	GGAGGCCAGTGAATTCATGTCGGTCTCGTCCTCCTC	pGADT7-TaCRY1载体构建
AD-TaCRY1-R	CGAGCTCGATGGATCCCTAACCGATGAAATTTGGCTGCACG
AD-TaARF17-F	GGAGGCCAGTGAATTCATGAGGCTCTCGTCGCCG	pGADT7-TaARF17载体构建
AD-TaARF17-R	CGAGCTCGATGGATCCTCAGAACTCTACTTCGACCGAGCC

Fig.1 The phylogenetic tree and cloning of TaLG2L and TaLG2 A.Phylogenetic tree of LG2 proteins in different species;B.Clonal gel electrophoresis of TaLG2L and TaLG2.

Fig.2 Amino acid sequence alignment of ZmLG2,OsLG2,TaLG2-3A,TaLG2-3B,and TaLG2-3D

Fig.3 Amino acid sequence alignment of ZmLG2,OsLG2L,TaLG2L-1A,TaLG2L-1B,and TaLG2L-1D

Fig.4 Differential expression analysis of LG2 in different periods and different tissues In the transcriptome analysis,FPKM(Fragments per kilobase of transcript per million mapped reads)was used to measure gene expression levels,each sample includes three biological replicates.B and S represent the leaf blade and leaf sheath,respectively at the S3 stage(12 d after germination);LJ-S1,LJ-S2,and LJ-S3 represent the S1 stage(4 d after germination),S2 stage(8 d after germination),and S3 stage during lamina joint(LJ)development,respectively.Different lowercase letters indicate significant differences among treatments(P<0.05).

Fig.5 PCR detection of TaLG2L gene amplification product(A)and enzyme digestion verification(B)

Fig.6 pGBKT7-TaLG2L-1A decoy vector self-activation detection(A)and the 3AT titration results(B)

Fig.7 Conversion efficiency of TaLG2L-1A library screening

Fig.8 The results of positive cloning were verified by four missing plates(QDO/X)

Tab.2 Candidate interacting proteins identified through yeast two-hybrid screening

编号 Number	NCBI登录号 Accession	功能描述 Description
1	XM_044599280.1	类转录因子蛋白
2	XM_044592733.1	核糖体蛋白
3	XM_044583174.1	E3泛素蛋白连接酶
4	XM_044582607.1	转录共抑制因子
5	XM_044580925.1	含有Smr结构域的蛋白
6	XM_044568826.1	病原相关蛋白
7	XM_044563894.1	泛素相关蛋白
8	XM_044556763.1	激酶家族蛋白
9	XM_044539035.1	丝氨酸蛋白酶
10	XM_044532185.1	含锌指HIT结构域蛋白1
11	XM_044527595.1	ATP依赖性RNA解旋酶
12	XM_044509258.1	类同源框蛋白BEL1
13	XM_044475164.1	40S核糖体蛋白SA
14	XM_044601792.1	隐花色素-1
15	XM_044580216.1	生长素响应因子17
16	XM_044579331.1	过氧化氢酶
17	XM_044557948.1	含WD重复序列的类蛋白
18	XM_044509917.1	延伸因子
19	XM_044503465.1	锚蛋白重复家族蛋白
20	XM_044487387.1	脱落酸应激成熟蛋白

Fig.9 GO annotation analysis The ordinate represents-log₁₀(P-value),where a higher value indicates stronger enrichment significance.Yellow bars stand for cellular component(CC),and blue bars represent molecular function(MF).The main enriched terms in CC category include:1.Intracellular anatomical structure;2.Cytoplasm;3.Ribosome;4.Plastid stroma;5.Plastid;6.Thylakoid;7.Cytosolic ribosome;8.Thylakoid membrane;9.Organelle;10.Membrane-bounded organelle;11.Non-membrane-bounded organelle;12.Intracellular organelle;13.Intracellular membrane-bounded organelle;14.Intracellularnon-membrane-bounded organelle;15.Ribonucleoprotein complex.The main enriched terms in MF category include:16.Ubiquitin binding;17.Ubiquitin-like protein binding;18.Cation binding;19.mRNA binding;20.poly(A)RNA binding;21.Metal ion binding;22.Structural molecule activity;23.Transition metal ion binding;24.Ion binding.

Fig.10 Rotation verification From left to right,the first row shows the positive control and negative control;the second row displays BD-TaLG2L-1A+AD-TaWD (fragment),BD-TaLG2L-1A+AD-TaCRY1(fragment),and BD-TaLG2L-1A+AD-TaARF17(fragment),respectively.

Fig.11 Pairwise interaction validation between TaLG2L-1A and TaWD,TaCRY1,TaARF17

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