AtMYB77促进NO合成参与调控干旱胁迫下拟南芥侧根发育

doi:10.11983/CBB20207

植物学报 ›› 2021, Vol. 56 ›› Issue (4): 404-413.DOI: 10.11983/CBB20207

AtMYB77促进NO合成参与调控干旱胁迫下拟南芥侧根发育

车永梅^†, 孙艳君^†, 卢松冲, 侯丽霞, 范欣欣, 刘新^*()

青岛农业大学生命科学学院, 山东省高校植物生物技术重点实验室, 青岛 266109

收稿日期:2020-12-22 接受日期:2021-04-19 出版日期:2021-07-01 发布日期:2021-06-30
通讯作者: 刘新
作者简介:*E-mail: liuxin6080@126.com
第一联系人：
†共同第一作者
基金资助:
国家自然科学基金(31770275);国家自然科学基金(31701063)

AtMYB77 Involves in Lateral Root Development via Regulating Nitric Oxide Biosynthesis under Drought Stress in Arabidopsis thaliana

Yongmei Che^†, Yanjun Sun^†, Songchong Lu, Lixia Hou, Xinxin Fan, Xin Liu^*()

Key Lab of Plant Biotechnology in Universities of Shandong Province, Life Science College, Qingdao Agricultural University, Qingdao 266109, China

Received:2020-12-22 Accepted:2021-04-19 Online:2021-07-01 Published:2021-06-30
Contact: Xin Liu
About author:First author contact:
†These authors contributed equally to this paper

摘要/Abstract

摘要： 转录因子MYB77与信号分子一氧化碳(NO)是侧根发育的重要调节因子, 但MYB77和NO在干旱胁迫下侧根发生中的作用及机制尚不明确。该文以拟南芥(Arabidopsis thaliana)野生型、AtMYB77缺失突变体Atmyb77-1及过表达株系AtOE77-1和AtOE77-3为材料, 研究了MYB77和NO在干旱胁迫下侧根发生中的作用。结果表明, AtMYB77受干旱胁迫诱导, AtMYB77缺失导致干旱胁迫下侧根发育相关基因CYCA2;1和CDKA;1表达下调, 同时Atmyb77-1的侧根数目和长度显著低于野生型, AtMYB77过表达则作用相反, 表明AtMYB77参与干旱胁迫下侧根发育的调控过程。干旱胁迫下, 拟南芥根系NO含量显著升高, NO合成关键酶NO合酶(NOS)和硝酸还原酶(NR)活性及基因表达上调, Atmyb77-1中NO含量、NOS和NR活性及基因表达量显著低于野生型, 而AtOE77-1和AtOE77-3根系NO含量及合成酶活性和基因表达量显著高于野生型。外施NO供体硝普钠(SNP)能缓解AtMYB77缺失对CYCA2;1和CDKA;1表达及侧根生长的抑制, NO清除剂或合成抑制剂则削弱AtMYB77过表达对侧根生长的促进作用。上述结果表明, AtMYB77通过促进NO合成参与干旱诱导的拟南芥侧根生长过程, 研究结果为深入解析干旱诱导侧根生长的信号转导机制和培育耐旱植物奠定了理论基础。

关键词: AtMYB77, NO, 侧根发育, 干旱胁迫, 拟南芥

Abstract: Both transcription factor MYB77 and signal molecule nitric oxide (NO) are important regulators of lateral root development. However, our understanding about the role of MYB77 and NO in the regulation of lateral root formation in plants remains elusive. This study investigated the roles and interrelation of MYB77 and NO in regulating lateral root formation under drought stress by using wild type Arabidopsis, AtMYB77 deletion mutant Atmyb77-1 and overexpression lines AtOE77-1 and AtOE77-3. The results showed that the expression of AtMYB77 was induced by drought stress. When subjected to drought stress treatment, the Atmyb77-1 mutant showed down-regulation of CYCA2;1 and CDKA;1, two genes that are related with lateral root development. Meanwhile, the number and length of lateral roots in the Atmyb77-1 mutant were significantly lower than those in wild type, while AtOE77-1 and AtOE77-3 lines displayed more and longer lateral roots. These results indicated that AtMYB77 was involved in the regulation of lateral root development under drought stress. We also showed that drought stress could increase the NO content, as well as the nitric oxide synthase (NOS) and nitrate reductase (NR) enzymes activity and gene expression in roots of Arabidopsis. Such increase in NO content, NOS and NR activities as well as related gene transcript levels were attenuated by deletion of AtMYB77 but enhanced by AtMYB77 overexpression. Exogenous NO donor sodium nitroprusside (SNP) alleviated the inhibitive effects of AtMYB77 deletion on the expressions of CYCA2;1 and CDKA;1 as well as the lateral root formation, while NO sca-vengers or synthesis inhibitors attenuate the promoting effect of AtMYB77 overexpression on lateral root growth. Taken together, these results demonstrate that AtMYB77 participates in drought-induced lateral root growth by promoting NO synthesis.

Key words: AtMYB77, NO, lateral root development, drought stress, Arabidopsis thaliana

车永梅, 孙艳君, 卢松冲, 侯丽霞, 范欣欣, 刘新. AtMYB77促进NO合成参与调控干旱胁迫下拟南芥侧根发育. 植物学报, 2021, 56(4): 404-413.

Yongmei Che, Yanjun Sun, Songchong Lu, Lixia Hou, Xinxin Fan, Xin Liu. AtMYB77 Involves in Lateral Root Development via Regulating Nitric Oxide Biosynthesis under Drought Stress in Arabidopsis thaliana. Chinese Bulletin of Botany, 2021, 56(4): 404-413.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20207

https://www.chinbullbotany.com/CN/Y2021/V56/I4/404

图/表 7

表1 定量PCR引物序列

Table 1 The primers used for quantitative PCR analysis

Primer name	Primer sequence (5°-3°)
AtMYB77-FP	GGAGAAGGACGTAGAGGTGAG
AtMYB77-RP	GGTGTTATTACTCCACAATCCCTA
AtCDKA;1-FP	GAGGATACATGGCGTGGGGTA
AtCDKA;1-RP	GCGTTGATTCTTTTGGTCGGA
AtCYCA2;1-FP	GCCCCTGAAATCCACTACAAT
AtCYCA2;1-RP	AGAGACCTCCACAAGCCAATC
AtNia1-FP	AGGTCCACTAGGGCACATCG
AtNia1-RP	TTCGTCCTCTGGATCACTCAATAT
AtNia2-FP	TTCTTACAAACCTCCCGTTCCAG
AtNia2-RP	GATTTTCTTATCATCTCCTTGTAGT
AtNOS1-FP	GATTCTCCGGGATTTGTCGA
AtNOS1-RP	CCTCCATTACCACCAACTGCT

图1 干旱胁迫对拟南芥根部AtMYB77表达的影响 CK: 对照。不同小写字母表示不同处理间差异显著(P<0.05)。

Figure 1 The effect of drought stress on AtMYB77 expression in Arabidopsis roots CK: Control. Different lowercase letters indicate significant differences among different treatments at P<0.05.

图2 干旱对拟南芥Atmyb77-1突变体和AtMYB77过表达株系侧根生长和发育相关基因表达的影响 (A) 干旱胁迫下Atmyb77-1突变体根系生长表型(Bar=1 cm); (B) 干旱对拟南芥Atmyb77-1突变体和AtMYB77过表达株系侧根数目的影响; (C) 干旱对拟南芥Atmyb77-1突变体和AtMYB77过表达株系侧根长度的影响; (D) 干旱对拟南芥Atmyb77-1突变体和AtMYB77过表达株系根部AtCYCA2;1表达的影响; (E) 干旱对拟南芥Atmyb77-1突变体和AtMYB77过表达株系根部AtCDKA;1表达的影响。CK: 对照; WT: 野生型。不同小写字母表示不同株系的不同处理间差异显著(P<0.05)。

Figure 2 Effects of drought stress on lateral root growth and expression of lateral root development related genes in Arabidopsis Atmyb77-1 mutant and AtMYB77 overexpression lines (A) Root phenotypes of Atmyb77-1 mutant under drought stress (Bar=1 cm); (B) Effects of drought stress on lateral root number of Atmyb77-1 mutant and AtMYB77 overexpression lines; (C) Effects of drought stress on lateral root length of Atmyb77-1 mutant and AtMYB77 overexpression lines; (D) Effects of drought stress on relative expression level of AtCYCA2;1 in roots of Arabidopsis Atmyb77-1 mutant and AtMYB77 overexpression lines; (E) Effects of drought stress on relative expression level of AtCDKA;1 in roots of Arabidopsis Atmyb77-1 mutant and AtMYB77 overexpression lines. CK: Control; WT: Wild type. Different lowercase letters indicate significant differences among different treatments of different lines at P<0.05.

图3 干旱对拟南芥根部NO含量及NO合成酶活性和相关基因表达的影响 (A) 干旱胁迫下拟南芥根部NO荧光成像(Bars=100 μm); (B) 干旱对拟南芥根部NO含量的影响; (C) 干旱对拟南芥根部NOS活性的影响; (D) 干旱对拟南芥根部NR活性的影响; (E) 干旱对拟南芥根部AtNOS1表达量的影响; (F) 干旱对拟南芥根部AtNia1表达量的影响; (G) 干旱对拟南芥根部AtNia2表达量的影响。CK: 对照; WT: 野生型; NOS: 一氧化氮合酶; NR: 硝酸还原酶。不同小写字母表示不同株系的不同处理间差异显著(P<0.05)。

Figure 3 Effects of drought stress on NO content, activities and gene expression of NO synthesis enzymes in Arabidopsis roots (A) NO fluorescence imaging of Arabidopsis roots under drought stress (Bars=100 μm); (B) Effects of drought stress on NO content in Arabidopsis roots; (C) Effects of drought stress on NOS activity in Arabidopsis roots; (D) Effects of drought stress on NR activity in Arabidopsis roots; (E) Effects of drought stress on relative expression level of AtNOS1 in Arabidopsis roots; (F) Effects of drought stress on relative expression level of AtNia1 in Arabidopsis roots; (G) Effects of drought stress on relative expression level of AtNia2 in Arabidopsis roots. CK: Control; WT: Wild type; NOS: Nitric oxide synthase; NR: Nitrate reductase. Different lowercase letters indicate significant differences among different treatments of different lines at P<0.05.

图4 NO供体硝普钠(SNP)对干旱条件下拟南芥Atmyb77-1缺失突变体侧根生长和发育关键基因表达的影响 (A) SNP对干旱胁迫下Atmyb77-1突变体根系生长的影响(Bar=1 cm); (B) SNP对干旱条件下Atmyb77-1缺失突变体侧根数目的影响; (C) SNP对干旱条件下Atmyb77-1缺失突变体侧根长度的影响; (D) SNP对干旱条件下Atmyb77-1缺失突变体AtCYCA2;1表达的影响; (E) SNP对干旱条件下Atmyb77-1缺失突变体AtCDKA;1表达的影响。CK: 对照; WT: 野生型。不同小写字母表示不同株系的不同处理间差异显著(P<0.05)。

Figure 4 Effects of NO donor sodium nitroprusside (SNP) on lateral root growth and expression of lateral root development related genes in Arabidopsis Atmyb77-1 mutant under drought condition (A) The effect of SNP on root growth of Atmyb77-1 mutant under drought stress (Bar=1 cm); (B) The effect of SNP on lateral root number in Atmyb77-1 mutant under drought condition; (C) The effect of SNP on lateral root length in Atmyb77-1 mutant under drought condition; (D) The effect of SNP on relative expression level of AtCYCA2;1 in Atmyb77-1 mutant root under drought condition; (E) The effect of SNP on relative expression level of AtCDKA;1 in Atmyb77-1 mutant root under drought condition. CK: Control; WT: Wild type. Different lowercase letters indicate significant differences among different treatments of different lines at P<0.05.

图5 NO清除剂(c-PTIO)或合成抑制剂(L-NAME)对干旱条件下AtMYB77过表达株系侧根生长和发育关键基因表达的影响 (A) NO清除剂或合成抑制剂对干旱胁迫下AtMYB77过表达株系根系生长的影响(Bar=1 cm); (B) NO清除剂或合成抑制剂对干旱条件下AtMYB77过表达株系侧根数目的影响; (C) NO清除剂或合成抑制剂对干旱条件下AtMYB77过表达株系侧根长度的影响; (D) NO清除剂或合成抑制剂对干旱条件下AtMYB77过表达株系根部AtCYCA2;1表达量的影响; (E) NO清除剂或合成抑制剂对干旱条件下AtMYB77过表达株系根部AtCDKA;1表达量的影响。CK: 对照; WT: 野生型。不同小写字母表示不同株系的不同处理间差异显著(P<0.05)。

Figure 5 Effects of NO scavenger (c-PTIO) or biosynthesis inhibitor (L-NAME) on lateral root growth and expression of lateral root development related genes in AtMYB77 overexpression lines under drought condition (A) The effects of NO scavenger or biosynthesis inhibitor on root growth of AtMYB77 overexpression lines subjected to drought stress (Bar=1 cm); (B) The effects of NO scavenger or biosynthesis inhibitor on lateral root number of AtMYB77 overexpression lines under drought condition; (C) The effects of NO scavenger or biosynthesis inhibitor on lateral root length of AtMYB77 overexpression lines under drought condition; (D) The effects of NO scavenger or biosynthesis inhibitor on relative expression level of AtCYCA2;1 in AtMYB77 overexpression lines under drought condition; (E) The effects of NO scavenger or biosynthesis inhibitor on relative expression level of AtCDKA;1 in AtMYB77 overexpression lines under drought condition. CK: Control; WT: Wild type. Different lowercase letters indicate significant differences among different treatments of different lines at P<0.05.

图6 AtMYB77参与干旱胁迫下拟南芥侧根发育的工作模型实线部分为本文结果, 虚线部分为根据已有报道及推测可能存在的作用。ABA: 脱落酸; NR: 硝酸还原酶; NOS: 一氧化氮合酶

Figure 6 Working model of AtMYB77 function in regulating lateral roots development under drought stress in Arabidopsis The solid lines indicate the results of this study, and the dotted lines indicate the possible roles based on reports and speculation. ABA: Abscisic acid; NR; Nitrate reductase; NOS: Nitric oxide synthase

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AtMYB77促进NO合成参与调控干旱胁迫下拟南芥侧根发育

AtMYB77 Involves in Lateral Root Development via Regulating Nitric Oxide Biosynthesis under Drought Stress in Arabidopsis thaliana

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