Functional identification of apple MdMYB2 gene in phosphate-starvation response

https://doi.org/10.1016/j.jplph.2019.153089Get rights and content

Abstract

Inorganic phosphate (Pi) starvation severely affects the normal growth and development of plants. Here, a Pi-responsive gene, named MdMYB2 (MDP0000823458), was cloned and functionally identified in apple. Overexpression of MdMYB2 regulated the expression of Pi starvation-induced (PSI) genes and then promoted phosphate assimilation and utilization. The ectopic expression of MdMYB2 in Arabidopsis influenced plant growth and flowering, which was partially rescued by application of exogenous gibberellin (GA). These results indicated that MdMYB2 may be an essential regulator in phosphate utilization and GA-regulated plant growth and development.

Introduction

Phosphorus (P) is an essential macronutrient for plant growth and development, which can only be absorbed by plants in the form of inorganic phosphate (Pi). Because Pi is stable and insoluble, it is difficult for the roots of plants to acquire Pi from the soil (Holford, 1997; Schachtman, 1998). Hence, Pi is a restrictive factor that limits plant growth and development (Hinsinger, 2001). To counter this disadvantage, plants have developed numerous mechanisms to improve Pi assimilation and utilization (Marschner, 1995). During Pi starvation, significant changes in gene expression lead to genetic and morphological changes in the plant. PSI genes (AtPT1, AtPT2, AtPS2, AtPS3, AtIPS1, and AtRNS1) are regulated by transcription factors (TFs) during Pi starvation (Bariola et al., 1999; Baldwin et al., 2001; Shin et al., 2004; Franco-Zorrilla et al., 2007; Ramaiah et al., 2011). Additionally, plants may alter their root architecture, enhance anthocyanin pigments, and secrete phosphatases to overcome Pi starvation (Raghothama, 2000; Misson et al., 2005; Péret et al., 2011).

The activities of some plant-TFs vary in response to Pi starvation. These include MYB62, a R2R3-type MYB transcription factor; PHR1, a MYB transcription factor; bHLH32, a bHLH family transcription factor; WRKY75, a WRKY family transcription factor; and ZAT6, a C2H2-type zinc finger transcription factor in Arabidopsis thaliana L (Rubio et al., 2001; Chen et al., 2007; Devaiah et al., 2007a, b; Devaiah et al., 2009). Many of the changes observed during Pi starvation, such as changed root architecture and anthocyanin accumulation, are coordinated by several phytohormones, including auxin, ethylene, and cytokinin (Zorilla et al., 2004; Devaiah et al., 2009; Lei et al., 2011). Previous studies have shown that auxin cooperates with GA to regulate root architecture and anthocyanin accumulation (Fu and Harberd, 2003). Additionally, DELLA proteins, which constitute a core component of GA signaling, play crucial roles in regulating root architecture and increasing anthocyanin accumulation during Pi starvation (Jiang et al., 2007). Thus, there is a possible link between TFs and GA during Pi starvation.

The MYB family is one of the largest families of transcription factors in plants and is divided into three categories (3R-MYB, R2R3-MYB, and MYB-related group) according to the number of peptide repeats in the MYB domain. Of these TFs, R2R3-MYB TFs play crucial roles in dealing with Pi starvation (Jin and Martin, 1999). AtMYB2, an R2R3-type MYB transcription factor, regulates the plant response to Pi starvation by directly activating the expression of the miR399 gene (Dongwon et al., 2013). Overexpression of AtMYB2 in Arabidopsis increases Pi assimilation and utilization, PSI gene expression, and root hair density (Dongwon et al., 2013).

Apple, a widely consumed fruit, often faces serious Pi starvation in production. In this study, MdMYB2 (MDP0000823458) was identified as a Pi starvation-induced gene. We found that MdMYB2 may serve to connect Pi starvation and GA signaling. Our study reveals the role of MdMYB2 in the nutrient deficiency response, laying the theoretical foundation for the cultivation of apple varieties with greater resistance.

Section snippets

Plant materials and stress treatments

In this study, we used three wild-type plant materials, Malus domestica ‘Royal Gala’, ‘Columbia’ ecotype Arabidopsis thaliana and ‘Orin’ Cultivar apple calli. Gala seedlings were grown on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA), 0.4 mg/L naphthyl acetic acid (NAA), and 1.0 mg/L gibberellin (GA) (Liu et al., 2017a). Arabidopsis seeds were germinated and grown on MS medium. The apple calli were subcultured on MS medium supplemented with 3 mg/L 2,

MdMYB2 is a Pi-responsive gene

To identify the MYB2 gene in apple, we used the AtMYB2 gene as a query and identified 16 highly similar genes in apple. The phylogenetic tree analysis showed that MDP0000823458 was closely related to AtMYB2 (Fig. 1A), and was subsequently named MdMYB2.

The qRT-PCR analysis showed that MdMYB2 was expressed in all tissues of apple, with the highest expression level detected in leaves (Fig. 1B).

Since expression of AtMYB2 was Pi-starvation regulated (Dongwon et al., 2013), it was hypothesized that

Discussion

In this study, we identified MdMYB2 as a Pi-responsive gene in apple. During Pi starvation, MdMYB2 not only exhibited a sensitive phenotype, with accumulation of ROS, induced expression of PSI genes, and increased density of root hair, but also served as an essential gene to connect Pi starvation and GA biosynthesis.

We have experimentally demonstrated that expression of MdMYB2 was induced by Pi starvation in apple (Fig. 1). However, not all Pi stress-responsive transcription factors can be

Author contributions

Yu-Jin Hao, Xiao-Fei Wang, and Yu-Ying Yang conceived and designed the experiments; Xiao-Fei Wang and Yu-Jin Hao supervised the experiments; Yi-Ran Ren, Peng-Fei Zheng and Feng-Jia Qu performed most of the experiments; Yu-Jin Hao and Yu-Ying Yang conceived the project and wrote the article with contributions from all the authors.

Declaration of Competing Interest

All the authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by National Nature Science Foundation of China (31430074), Major Program of Shandong Provincial Natural Science Foundation (ZR2017ZC0328), Shandong Province Government (SDAIT-06-03), and Ministry of Agriculture of China (CARS-27).

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