Tomato plants ectopically expressing Arabidopsis GRF9 show enhanced resistance to phosphate deficiency and improved fruit production in the field
Introduction
Phosphorus is an essential macronutrient for plant growth and development, and serves various basic biological functions in the plant life cycle (Raghothama, 1999; Cordell et al., 2009). However, the availability of inorganic P in soils is very low, as it is easily bound by cations such as Fe3+ and Al3+, or converted to organic matter via soil-microbial activity, thereby becoming immobile and difficult to utilize for plants (Raghothama, 1999; Tiessen, 2008). The P concentration in the soil, typically 10 μM or less, results in P deficiency for plant growth and compromises crop productivity on ∼30–40% of arable lands worldwide (Runge-Metzger, 1995). Plants have evolved a series of adaptive strategies to overcome limited P availability in soils (Vance et al., 2003; Hoffland et al., 2006). In response to P deficiency, plants increase P uptake by altering root architecture (Ticconi et al., 2004; Osmont et al., 2007; Guo et al., 2011), by altering the expression of P-related genes (Bustos et al., 2010; Muneer and Jeong, 2015) and by changing their metabolic and developmental processes (Raghothama and Karthikeyan, 2005; Liang et al., 2013).
14-3-3 proteins, a large family of phosphoserine-binding proteins found in virtually every eukaryotic organism and tissue, play important roles in regulating plant development and stress responses in higher plants (Moore and Perez, 1967; Roberts et al., 2002; Comparot et al., 2003; Mayfield et al., 2007). Some recent studies suggest that plant 14-3-3 proteins might play a role in response to low P-deficiency by interacting with some phosphorus-deficiency response factors, such as protein kinases and phosphatases (Cao et al., 2007; Baldwin et al., 2008). In addition, 14-3-3 proteins are thought to be involved in directly regulating the plasma-membrane (PM) H+-ATPase, which affects root growth and may enhance P absorption in phosphorus-deficient soils (Palmgren, 2001; Shen et al., 2006; He et al., 2015). In Arabidopsis and tomato, thirteen and twelve 14-3-3 protein isoforms have been found, respectively (Rosenquist et al., 2000; Roberts, 2003; Xu and Shi, 2006). GRF9 is one of the 14-3-3 gene family members identified in Arabidopsis plants (Rooney and Ferl, 1995; Roberts, 2003; Mayfield et al., 2007; Xu and Shi, 2007). Arabidopsis GRF9 is involved in plant root responses to water stress by participating in shoot carbon allocation, which leads to improved root growth under water stress (Comparot et al., 2003; Mayfield et al., 2012; He et al., 2015). Although AtGRF9 has been identified to be involved in the response to P-deficiency stress, the clear function of this gene in response to P deficiency is still unknown (Cao et al., 2007).
Tomato (Solanum lycopersicum L.), aside from its enormous importance as a globally utilized fruit crop, is considered to be a model vegetable plant for the investigation of nutrition deficiencies and other abiotic stresses (Ivanov et al., 2012; Paolacci et al., 2014). To evaluate whether GRF9 can perform conserved functions across species in vegetable crops, we generated transgenic tomato plants overexpressing GRF9 derived from Arabidopsis (AtGRF9). Hydroponic experiments were carried out to elucidate the coordinated regulation of root architecture and proton exudation under differential P availability. Subsequent field experimentation was conducted to analyze tomato plant phenotypes and fruit productivity associated with P uptake. The results show that the constitutive expression of AtGRF9 in tomato increases the degree of tolerance to P deficiency in controlled hydroponic systems and promotes fruit production in the field.
Section snippets
Plant materials, growth conditions, and stress treatment
The tomato (Solanum lycopersicum L.) var. ‘Zhongshu NO.4’ was used to obtain transgenic lines. The full-length coding sequence of the AtGRF9 gene was obtained by the polymerase chain reaction (PCR) using the primers 5′-CACCATGGGTTCTGGAAAAGAGCGTG-3′ and 5′-ATTTGATTTACCCCGAGTAAAGG-3′. The 474-bp PCR product was cloned into the pMD18-T simple vector (TaKaRa, Tokyo, Japan) and digested using BamHI/SacI (New English Biolabs, inc., America) double digestion. The DNA was cloned into pBI121 (Jefferson
Overexpression of heterologous Arabidopsis GRF9 in transgenic tomato plants
To evaluate the effect of overexpression of GRF9 in tomato plants, an Arabidopsis GRF9 was transferred into the tomato genome using Agrobacterium tumefaciens-mediated transformation. After selection on kanamycin-containing medium, eleven independent kanamycin-resistant transformants (T0) were identified by quantitative real-time PCR. From each T0 plant, seeds (T1) were harvested, T2 plants were selected from kanamycin-resistant T1 transformants, all T2 generation plants used for experiment were
Discussion
Low P availability is a major abiotic stress, affecting up to 40% of the world’s arable land (Vance et al., 2003), and severely restricting crop productivity globally. A primary strategy of plants to acclimate to low-P stress is to enhance the root surface area and, thus, enhance P-uptake capacity (Yao et al., 2014; Sun et al., 2016). The molecular mechanisms of root development and P-uptake regulation during P deficiency are poorly understood. The AtGRF9 gene has been implicated in the
Acknowledgements
This work was supported by the National Key R&D Program of China (2017YFD0200200/2017YFD0200208), the National Natural Science Foundation of China (41171234), and the University of Melbourne.
We thank Dr. Nan Gao (Nanjing Industry University) for providing the transgenic tomato seeds. We also thank Professor Weifeng Xu for previous guidance of this work and Dr Si’en Li for his assistance in field sampling in Wuwei, Gansu, China.
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