Expression Patterns of Tomato SlIAMT1 and SlIAMT2 and Their Functions During Hypocotyl and Root Development

doi:10.16420/j.issn.0513-353x.2020-1063

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (12): 2385-2402.doi: 10.16420/j.issn.0513-353x.2020-1063

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Expression Patterns of Tomato SlIAMT1 and SlIAMT2 and Their Functions During Hypocotyl and Root Development

ZHANG Jingya, LEI Lei, SHANG Qingmao, XIE Lulu, DONG Chunjuan()

Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture and Rural Affairs,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Received:2021-04-19 Revised:2021-07-21 Published:2022-01-04
Contact: DONG Chunjuan E-mail:dongchunjuan@caas.cn

Abstract

Abstract:

Two indole-3-acetic acid methyltransferase（IAMT）genes,SlIAMT1 and SlIAMT2,were cloned from tomato cultivar‘Ailsa Craig’. The full-length of SlIAMT1 and SlIAMT2 genes were 2 510 and 4 620 bp,respectively,and both of them encode 390 amino acids. The sequence alignment suggested that both SlIAMT1 and SlIAMT2 contained all of the substrate binding sites and catalytic sites for IAMT,and showed more than 70% sequence identity with AtIAMT1 from Arabidopsis. In silico analysis suggested that the promoter sequences of SlIAMT1 and SlIAMT2 contained several typical cis-acting elements,including auxin-,abscisic acid-,salicylic acid- and light-responsive elements by using PlantCARE databases. Real-time PCR assays showed that SlIAMT1 was highly expressed in the germinating seeds,roots of 5-d-old seedlings,young leaves and green fruits,while SlIAMT2 was extensively expressed in the flower buds. In tomato seedlings,the expression of SlIAMT1 was remarkably induced by high light intensity（150 μmol · m^-2 · s^-1）,especially in the hypocotyls. However,the inducible level of SlIAMT2 was much lower. The induction of SlIAMT1 and SlIAMT2 was low by low temperature（15 ℃）treatment. IAA also exerted inducible effects on SlIAMT1 and SlIAMT2 expression. ABA induced the expression of SlIAMT1 in root and hypocotyl,but repressed its expression in cotyledons,while for SlIAMT2,its expression was repressed by ABA in root. SA repressed the expression of SlIAMT1 in roots and cotyledons,but slightly induced SlIAMT2 expression in cotyledons. The function of SlIAMT1 gene was further investigated by Agrobacterium-mediated genetic transformation into Arabidopsis. The iamt1 mutant showed longer hypocotyl and primary roots than wild type,and these phenotypes of mutant were functionally complemented by overexpression of SlIAMT1. Overexpression of SlIAMT1 could also reduce the sensitivity to IAA in lateral roots. These results indicated that SlIAMT genes respond to light and hormone signals,and regulate the IAA homeostasis via methylation and thereby regulate the hypocotyl and root development.

Key words: Solanum lycopersicum, IAMT, expression analysis, root, hypocotyl

CLC Number:

S641.2

ZHANG Jingya, LEI Lei, SHANG Qingmao, XIE Lulu, DONG Chunjuan. Expression Patterns of Tomato SlIAMT1 and SlIAMT2 and Their Functions During Hypocotyl and Root Development[J]. Acta Horticulturae Sinica, 2021, 48(12): 2385-2402.

Figures/Tables 14

Table 1 Primers used in this study

用途Use	基因Gene	引物序列（5′-3′）Sequence
基因克隆 Gene clone	SlIAMT1	F：CACTAGCAAAACAATAGAGACA;R：TCTAATACATGAGAGACATATAGACTT
基因克隆 Gene clone	SlIAMT2	F：TCTACTTTTATGGTTAGATTCTG;R：TATCCCAAATGTTGCTTAA
qRT-PCR	SlIAMT1	F：CTCAAGCTCAGGGGCAACAT;R：ATCTCCGGCGAGTTTAGCTG
	SlIAMT2	F：CTCAAGCCCAGGGACAACAT;R：CGAAGGGGATGTCGTCGTTAT
	SlActin41	F：CTTCCAGCAGATGTGGATTGC;R：GCATCTCTGGTCCAGTAGGAAA
	AtIAMT1	F：CGGTCTACTCTTCGGCACTC;R：GGTGCGTACACCGGGATATT
	AtActin2	F：ACACTGTGCCAATCTACGAGGGTT;R：ACAATTTCCCGCTCTGCTGTTGTG
载体构建 Transgenic vector construction	35S:SlIAMT1	F：CGGTACCCGGGGATCCATGGCACCTTTAGGAGACAA; R：CGACTCTAGAGGATCCCTACACAAGAGAAAGTGAAGCAACA

Fig. 1 Effects of exogenous MeIAA application on growth of hypocotyls and roots in tomato seedlings under light and dark conditions Different lowercases indicate significant differences between groups under same culture condition（P < 0.05）.

Table 2 Sequence characteristics of SlIAMT1 and SlIAMT2 genes in tomato

基因 Gene	序列号 Accession No.	基因全长/bp Gene length	外显子数 Number of exons	内含子数 Number of introns	ORF/ bp	蛋白长度/aa Protein length	分子量/ kD MW	等电点 pI	亚细胞定位 Subcellular localization
SlIAMT1	Solyc07G064990	2 510	4	3	1 173	390	42.92	5.58	细胞质Cytoplasm
SlIAMT2	Solyc12G014500	4 620	4	3	1 173	390	43.28	5.90	细胞质Cytoplasm

Fig. 2 Homology alignment of SlIAMT1 and SlIAMT2 with Arabidopsis AtIAMT1 protein sequences ▼：SAM/SAH-binding site;*：Interaction site with carboxyl moiety of IAA;†：Interaction site with aromatic moiety of IAA.

Fig. 3 Phylogenetic tree of SlIAMT proteins and IAMTs from other plant species

Table 3 Some important cis-acting regulatory elements in the promoters of SlIAMT1 and SlIAMT2

调控元件 Cis-element	核心序列 Core sequence	元件功能描述 Functional description of cis-element	位置Positions^*
调控元件 Cis-element	核心序列 Core sequence	元件功能描述 Functional description of cis-element	SlIAMT1	SlIAMT2
ABRE	CACGTG	ABA响应元件 cis-Acting element involved in the abscisic acid responsiveness	-1 547	-552、 -1 129、 -1 921
TCA-element	CCATCTTTTT	水杨酸响应元件 cis-Acting element involved in salicylic acid responsiveness	-1 513	/
TGA-element	AACGAC	生长素响应元件 Auxin-responsive element	/	-791
TGACG-motif	TGACG	茉莉酸甲酯响应元件 cis-Acting regulatory element involved in the methyl jasmonate responsiveness	/	-367
MBS	CAACTG	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility	/	-712
ACE	CTAACGTATT	光响应元件cis-Acting element for light responsive	-1 754	/
G-box	TACGTG	光响应元件cis-Acting element for light responsive	-1 546,-1 981	/
GT1-motif	GGTTAA	光响应元件Light responsive element	-263,-170, -1 500,-1 810	/
AE-box	AGAAACAA	光响应元件的一部分 Part of a module for light response	-1 867	/
AT1-motif	AATTATTTTTTATT	光响应元件的一部分 Part of a light responsive module	-1 711、-698	/
I-box	TGATAATGT	光响应元件的一部分 Part of a light responsive element	-1 331	/
MRE	AACCTAA	参与光响应的MYB结合位点 MYB binding site for light responsive	/	-1 614、 -1 079

Fig. 4 Expression levels of SlIAMT1 and SlIAMT2 in different tissues of tomato The different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 5 Expression levels of SlIAMT1 and SlIAMT2 in root,hypocotyl,and cotyledon of tomato seedlings in response to high light intensity（150 μmol · m-2 · s -1 Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 6 Expression levels of SlIAMT1 and SlIAMT2 in root,hypocotyl,and cotyledon of tomato seedlings in response to low temperature（15 ℃/15 ℃） Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 7 Expression levels of SlIAMT1 and SlIAMT2 in root,hypocotyl,and cotyledonof tomato seedlings in response to IAA Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 8 Expression levels of SlIAMT1 and SlIAMT2 in root,hypocotyl,and cotyledon of tomato seedlings in response to ABA Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 9 Expression levels of SlIAMT1 and SlIAMT2 in root,hypocotyl,and cotyledon of tomato seedlings in response to SA Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 10 Effects of SlIAMT1 overexpression on hypocotyl and root growth in Arabidopsis iamt1 mutant Different lowercases indicate significant differences between groups（P < 0.05）.

Fig. 11 Effects of overexpression of SlIAMT1 on the response of Arabidopsis iamt1 mutant seedlings to IAA The concentration of IAA is the logarithmic value. **,*** indicate significant differences between groups at 0.01 and 0.001 level,respectively.

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Expression Patterns of Tomato SlIAMT1 and SlIAMT2 and Their Functions During Hypocotyl and Root Development

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