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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (11): 2731-2741.doi: 10.3724/SP.J.1006.2024.44024

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Soybean GmRSM1 promotes apical hook disappearance by regulating PIN gene expression

FU Jia-Qi1,2(), LI Shi-Kuan1,2, TAN Meng-Hui1,2, LUO Fang3, ZHANG Chuan-Ling3, LIU Ling-Yue3, LU Qian1,*(), GU Yong-Zhe1,2,*()   

  1. 1College of Life Sciences and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3Arong Banner Agricultural Development Center, Arong Banner 162750, Inner Mongolia, China
  • Received:2024-02-05 Accepted:2024-06-20 Online:2024-11-12 Published:2024-07-10
  • Contact: *E-mail: luqian@hrbnu.edu.cn;E-mail: guyongzhe@caas.cn
  • Supported by:
    National Natural Science Foundation of China(32372190)

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Abstract:

The apical hook is a transient structure found in etiolated dicot seedlings, and while the current model for the apical hook in Arabidopsis thaliana is relatively well-established, there is limited research on soybean. In this study, the pBAR- GmRSM1 overexpression vector was constructed, and three homozygous lines each of Arabidopsis thaliana (OE-GmRSM1#64, #69, and #70) and soybean (OE-GmRSM1#103, #78, and #95) were generated through genetic transformation and positive plant selection. The expression levels of the transgenic lines were significantly higher than the wild type. Both Arabidopsis thaliana and soybean cultures were grown in darkness, and the apical hooks in the overexpressed transformants disappeared or exhibited faster disappearance than the wild type. Scanning electron microscopy observations of cell length in the apical hook of Arabidopsis thaliana wild-type and transformants revealed that the inner hook cells were shorter than the outer hook cells during the hook maintenance stage, while the cell lengths were same on both sides in the transformant plants. The inner and outer sides of the soybean apical hook were separated, and the expression of the PIN gene in the wild type and three overexpressed transformants of soybean was analyzed. The results demonstrated that the expression levels of the PIN1e, PIN3d, and PIN6a genes were significantly higher in the apical hook after cotyledon unfolding, and during the hook maintenance stage, these three genes exhibited significantly higher expression levels in the transformants than the wild type, but there was no significant difference in expression levels after unfolding. Transient expression in tobacco confirmed than the GmRSM1 protein were localized in the nucleus and cell membrane. Thus, the apical hook formation is attributed to differential cell elongation between the inner and outer sides of the hook, leading to hypocotyl curvature. GmRSM1 regulates the expression of auxin transporters PIN1e, PIN3d, and PIN6a, thereby shortening the duration of hook maintenance and unfolding. These genes are positively regulated by GmRSM1 during the hook stage, and the study confirmed the regulatory effect of this gene on auxin transport. Overall, this study verified the role of GmRSM1 in the disappearance of the apical hook phenotype and further elucidated the gene pathway involved in apical curvature, laying a foundation for future research.

Key words: apical hook, soybean, Arabidopsis, PIN family, auxin

Table 1

Websites used for bioinformatics prediction and analysis"

网站或软件名
Website or software		 网址
Uniform resource locator		 用途
Purpose
ExPASy ProtParam http://us.expasy.org/tools 氨基酸组成及理化性质预测
Prediction of amino acid composition and physicochemical properties
Cell-PLoc 2.0 http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 蛋白亚细胞定位预测
Predict protein subcellular localization
SPOMA https://npsa.lyon.inserm.fr/cgi-bin/
secpred_sopma.pl		 蛋白二级结构预测
Protein secondary structure prediction
TMHMM-2.0 https://services.healthtech.dtu.dk/
services/TMHMM-2.0/		 蛋白跨膜分析
Protein transmembrane analysis
Phytozome 13 https://phytozome-next.jgi.doe.gov/ 获得GmRSM1的启动子序列、CDS序列、编码序列和蛋白序列, 并进行基因结构分析
The promoter sequence, CDS sequence, coding sequence and protein sequence of GmRSM1 were obtained, and the gene structure was analyzed
PlantCARE https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ 启动子区顺式作用元件分析
Analysis of cis-acting elements in the promoter region

Fig. 3

Phenotypic differences of apical hooks between GmRSM1 overexpressing lines and wild-type in soybean A: qRT-PCR was used to determine the relative expression level of positive lines overexpressing soybean. ** in the figure represents a significant difference compared with wild-type WT (P < 0.01). B: soybean phenotype, From top to bottom, the apical hooks of yellowed seedlings of wild-type soybean, transformants OE-GmRSM1#103, #78, and #95 were phenotyped by culture time. C: schematic diagram of the apical hook angle measurement of soybeans. D: line chart of tip hook angle changes detected and measured in chronological order."

Table S1

Primers used in this experiment and their functions"

引物名称
Primer name		 上游引物
Forward primer (5°-3°)		 上游引物
Reverse primer (5°-3°)		 引物用途
Primer function
pBAR-GmRSM1 CTCGGTACCCGGGGATCCATGGCCT-CAAGCTCAGCTT GATGAAGGTTCTAAGCCTCCACTGA-TCTAGAGTCCGCAAATCACCAGTCT 基因克隆和构建过表达载体
Gene cloning and overexpression vector construction
qPCR-GmRSM1 GTAAGACTGTGGAGGAAGTGAA AGCATTTCTGTAATTGGGCAAG 荧光定量Real-time PCR
qPCR-GmActin GGTGGTTCTATCTTGGCATC CTTTCGCTTCAATAACCCTA 内参引物Reference primer
qPCR-AtUBQ1 TTCCTTGATGATGCTTGCTC TTGACAGCTCTTGGGTGAAG 内参引物Reference primer
qPCR-PIN1a GCAACCGAGGATCATAGCAT AGAGAACGCCTTTGAGTCCA 荧光定量Real-time PCR
qPCR-PIN1b TGTTGATTGCTTTGCCCATA TGTCTGATGCTCAACAAGCC 荧光定量Real-time PCR
qPCR-PIN1c ATGCAAAGCTTGGTTGAGGT GATCCTGGGGTTCTTCTTCC 荧光定量Real-time PCR
qPCR-PIN1d GCATAAAAAGTGGGACCGAA ATGACAACCTGTGCCATTCA 荧光定量Real-time PCR
qPCR-PIN1e GGGATGCTAATTGCTCTTCCTA GGTAGTTTGATCCACACTGCAA 荧光定量Real-time PCR
qPCR-PIN3a CCCCAACACTTACTCCAGTCTC ATAGCTATACGCAAGAGGGTGC 荧光定量Real-time PCR
qPCR-PIN3b AGAATTCGCAGACACAGCCT GTTGGCTTTGTTCCCACTGT 荧光定量Real-time PCR
qPCR-PIN3c GTGACGGTAGCTTCTCCTCG GAATTCTGGCTCTGGCTCTG 荧光定量Real-time PCR
qPCR-PIN3d ACACTTGCAAAATGGGGAAG GCCCAACTTGTTGAGTCCAT 荧光定量Real-time PCR
qPCR-PIN6a TGCAACTCGTGGTTCTTCAG CGTCGAATTTCGCTATGGAT 荧光定量Real-time PCR
qPCR-PIN6b AGTTCTTGACATGCCCTGCT TTCCCACAAGCTTTTCCAAC 荧光定量Real-time PCR

Fig. 1

Bioinformatics analysis of GmRSM1 A: evolutionary tree analysis. The red circle of Glyma.14G074500.1CDS is the protein sequence corresponding to the coding sequence of GmRSM1, and the blue circle is marked with the protein sequence of ATRL1-ATRL6 in Arabidopsis thaliana, and the AmRAD protein sequence in snapdragon. B: protein sequence conservation alignment. The numbers in the figure represent the number of amino acids in each peptide, the asterisk indicates 100% conservation of the entire sequence, and the arranged triangles represent residues specific to RAD-like. C: gene structure analysis; D: prediction of tertiary structure of proteins; E: transmembrane structure prediction."

Table 2

Analysis of promoter elements 2000 bp upstream of GmRSM1"

元件
Motif		 数量
Number		 功能
Function
CAAT-box 47 启动子和增强子区域中常见的顺式作用元件
Common cis-acting element in promoter and enhancer regions
TATA-box 38 转录起始点−30左右的核心启动子元件 Core promoter element around −30 of transcription start
G-box 7 参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness
ABRE 6 参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness
MYC 5 MYC结合位点元件 MYC binding site element
ABRE3a 3 ABA (脱落酸)应答元件ABA (abscisic acid) transponder
ABRE4 3 ABA (脱落酸)应答元件ABA (abscisic acid) transponder
AT~TATA-box 3 核心启动子元件 Core promoter element
Box 4 3 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness
ACE 2 参与光响应的顺式作用元件 Cis-acting element involved in light responsiveness
ERE 2 乙烯诱导顺式作用元件 Ethylene-induced cis-acting element
TCCC-motif 2 光响应元件的一部分 Part of a light responsive element
TCT-motif 2 光响应元件的一部分 Part of a light responsive element
ATCT-motif 1 参与光响应的保守DNA模块的一部分
Part of a conserved DNA module involved in light responsiveness
Box II 1 光响应元件的一部分 Part of a light responsive element
Box III 1 蛋白结合位点 Protein binding site
CCAAT-box 1 MYB Hv1结合位点 MYB Hv1 binding site
GATA-motif 1 光响应元件的一部分 Part of a light responsive element
GT1-motif 1 光响应元件 Light responsive element
I-box 1 光响应元件的一部分 Part of a light responsive element
MBS 1 MYB结合位点参与干旱诱导 MYB binding site involved in drought-inducibility
MBSI 1 MYB结合位点参与类黄酮生物合成基因调控
MYB binding site involved in flavonoid biosynthetic genes regulation
MYB 2 MYB结合位点元件 MYB binding site element
MYB recognition site 1 MYB识别位点元件 MYB recognizes site-based elements
MYB-like sequence 1 MYB-like序列 MYB-like sequence
STRE 1 应激响应的顺式作用元件 Cis-acting element of stress response
TATC-box 1 参与赤霉素反应的顺式作用元件 Cis-acting element involved in gibberellin-responsiveness
TCA-element 1 参与水杨酸反应性的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness

Fig. 2

Phenotypic differences between overexpressing GmRSM1 lines and wild-type apical hooks in Arabidopsis thaliana A: qRT-PCR was used to determine the relative expression level of positive lines overexpressing Arabidopsis; ** in the figure represents a significant difference compared with wild-type WT (P < 0.01). B: Arabidopsis phenotype: from left to right, the apical hook of yellowed seedlings of wild-type, transformants OE-GmRSM1#64, #69, and #70. C: electromicroscope scans of Arabidopsis thaliana overexpression line #69 and wild-type apical hooks."

Fig. 4

Relative expression of PIN genes inside and outside the apical hook in the maintenance and unfolding stages"

Fig. 5

Subcellular localization of GmRSM1 protein Photographs were taken in four fields of view: green fluorescence, red fluorescence, bright-field, and superimposed field. The scale bar is 10 μm."

Fig. 6

Phenotypic differences between soybean GmRSM1 overexpressing lines and wild-type apical hooks under deep sowing conditions"

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