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

doi:10.11983/CBB20207

Abstract

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

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[J]. Chinese Bulletin of Botany, 2021, 56(4): 404-413.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB20207

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

Figures/Tables 7

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

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.

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.

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.

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.

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.

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

References 30

[1]	车永梅, 孙艳君, 卢松冲, 赵方贵, 侯丽霞, 刘新 (2018). AtWRKY40参与拟南芥干旱胁迫响应过程. 植物生理学报 54, 456-464.
[2]	刘国华, 刘菁, 侯丽霞, 唐静, 刘新 (2009). NO可能作为Ca²⁺的下游信号介导乙烯诱导的蚕豆气孔关闭. 分子细胞生物学报 42, 145-155.
[3]	张玲玲, 吴丹, 赵子捷, 赵立群 (2017). 植物一氧化氮信号分子的研究进展. 植物学报 52, 337-345. DOI
[4]	张雨, 赵明洁, 张蔚 (2020). 植物次生细胞壁生物合成的转录调控网络. 植物学报 55, 351-368. DOI
[5]	An JP, Wang XF, Zhang XW, Xu HF, Bi SQ, You CX, Hao YJ (2020). An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J 18, 337-353. DOI URL
[6]	Bashir W, Anwar S, Zhao Q, Hussain I, Xie FT (2019). Interactive effect of drought and cadmium stress on soybean root morphology and gene expression. Ecotoxicol Environ Saf 175, 90-101. DOI URL
[7]	Cao XC, Zhu CQ, Zhong C, Zhang JH, Wu LH, Jin QY, Ma QX (2019). Nitric oxide synthase-mediated early nitric oxide burst alleviates water stress-induced oxidative damage in ammonium-supplied rice roots. BMC Plant Biol 19, 108. DOI URL
[8]	Chakhchar A, Chaguer N, Ferradous A, Filali-Maltouf A, El Modafar C (2018). Root system response in Argania spinosa plants under drought stress and recovery. Plant Signal Behav 13, e1489669. DOI URL
[9]	Correa-Aragunde N, Graziano M, Chevalier C, Lamattina L (2006). Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato. J Exp Bot 57, 581-588. PMID
[10]	Correa-Aragunde N, Graziano M, Lamattina L (2004). Nitric oxide plays a central role in determining lateral root development in tomato. Planta 218, 900-905. PMID
[11]	Dash M, Yordanov YS, Georgieva T, Tschaplinski TJ, Yordanova E, Busov V (2017). Poplar PtabZIP1-like enhances lateral root formation and biomass growth under drought stress. Plant J 89, 692-705. DOI URL
[12]	Fang Q, Jiang TZ, Xu LX, Liu H, Mao H, Wang XQ, Jiao B, Duan YJ, Wang Q, Dong QN, Yang L, Tian GZ, Zhang C, Zhou YF, Liu XP, Wang HY, Fan D, Wang BJ, Luo KM (2017). A salt-stress-regulator from the poplar R2R3 MYB family integrates the regulation of lateral root emergence and ABA signaling to mediate salt stress tolerance in Arabidopsis. Plant Physiol Biochem 114, 100-110. DOI URL
[13]	Fukaki H, Okushima Y, Tasaka M (2007). Auxin-mediated lateral root formation in higher plants. Int Rev Cytol 256, 111-137.
[14]	Gibbs DJ, Voß U, Harding SA, Fannon J, Moody LA, Yamada E, Swarup K, Nibau C, Bassel GW, Choudhary A, Lavenus J, Bradshaw SJ, Stekel DJ, Bennett MJ, Coates JC (2014). AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. New Phytol 203, 1194-1207. DOI URL
[15]	Gu M, Zhang J, Li HH, Meng DQ, Li R, Dai XL, Wang SC, Liu W, Qu HY, Xu GH (2017). Maintenance of phosphate homeostasis and root development are coordinately regulated by MYB1, an R2R3-type MYB transcription factor in rice. J Exp Bot 68, 3603-3615. DOI URL
[16]	Hu ZR, Wang R, Zheng M, Liu XB, Meng F, Wu HL, Yao YY, Xin MM, Peng HR, Ni ZF, Sun QX (2018). TaWRKY51 promotes lateral root formation through negative regulation of ethylene biosynthesis in wheat (Triticum aes-tivum L.). Plant J 96, 372-388. DOI URL
[17]	Lee HK, Cho SK, Son O, Xu ZY, Hwang I, Kim WT (2009). Drought stress-induced Rma1H1, a RING membrane- anchor E3 ubiquitin ligase homolog, regulates aquaporin levels via ubiquitination in transgenic Arabidopsis plants. Plant Cell 21, 622-641. DOI URL
[18]	Nie J, Wen C, Xi L, Lv SH, Zhao QC, Kou YP, Ma N, Zhao LJ, Zhou XF (2018). The AP2/ERF transcription factor CmERF053 of chrysanthemum positively regulates shoot branching, lateral root, and drought tolerance. Plant Cell Rep 37, 1049-1060. DOI URL
[19]	Romano JM, Dubos C, Prouse MB, Wilkins O, Hong H, Poole M, Kang KY, Li EY, Douglas CJ, Western TL, Mansfield SD, Campbell MM (2012). AtMYB61, an R2R3-MYB transcription factor, functions as a pleiotropic regulator via a small gene network. New Phytol 195, 774-786. DOI PMID
[20]	Sahay S, Khan E, Gupta M (2019). Nitric oxide and abscisic acid protects against PEG-induced drought stress differentially in Brassica genotypes by combining the role of stress modulators, markers and antioxidants. Nitric Oxide 89, 81-92. DOI URL
[21]	Santisree P, Bhatnagar-Mathur P, Sharma KK (2015). NO to drought-multifunctional role of nitric oxide in plant drought: do we have all the answers? Plant Sci 239, 44-55. DOI URL
[22]	Seo PJ, Park CM (2009). Auxin homeostasis during lateral root development under drought condition. Plant Signal Behav 4, 1002-1004. DOI URL
[23]	Shin R, Burch AY, Huppert KA, Tiwari SB, Murphy AS, Guilfoyle TJ, Schachtman DP (2007). The Arabidopsis transcription factor MYB77 modulates auxin signal transduction. Plant Cell 19, 2440-2453. DOI URL
[24]	Wang PC, Du YY, Hou YJ, Zhao Y, Hsu CC, Yuan FJ, Zhu XH, Tao WA, Song CP, Zhu JK (2015). Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1. Proc Natl Acad Sci USA 112, 613-618. DOI URL
[25]	Willems E, Leyns L, Vandesompele J (2008). Standardization of realtime PCR gene expression data from independent biological replicates. Anal Biochem 379, 127-129. DOI PMID
[26]	Xie YJ, Mao Y, Lai DW, Zhang W, Zheng TQ, Shen WB (2013). Roles of NIA/NR/NOA1-dependent nitric oxide production and HY1 expression in the modulation of Arabidopsis salt tolerance. J Exp Bot 64, 3045-3060. DOI URL
[27]	Xing L, Zhao Y, Gao JH, Xiang CB, Zhu JK (2016). The ABA receptor PYL9 together with PYL8 plays an important role in regulating lateral root growth. Sci Rep 6, 27177. DOI PMID
[28]	Zhao Y, Xing L, Wang XG, Hou YJ, Gao JH, Wang PC, Duan CG, Zhu XH, Zhu JK (2014). The ABA receptor PYL8 promotes lateral root growth by enhancing MYB77- dependent transcription of auxin-responsive gene. Sci Signal 7, ra53.
[29]	Zhou GY, Zhou XH, Nie YY, Bai SH, Zhou LY, Shao JJ, Cheng WS, Wang JW, Hu FQ, Fu YL (2018). Drought- induced changes in root biomass largely result from altered root morphological traits: evidence from a synthesis of global field trials. Plant Cell Environ 41, 2589-2599. DOI URL
[30]	Che YM, Sun YJ, Lu SC, Hou LX, Fan XX, Liu X (2021). AtMYB77 involves in lateral root development via regulating nitric oxide biosynthesis under drought stress in Arabidopsis thaliana. Chin Bull Bot 56, 404-413.