The Tomato 14-3-3 Protein TFT4 Modulates H+ Efflux,Basipetal Auxin Transport,and the PKS5-J3 Pathway in the Root Growth Response to Alkaline Stress

Alkaline stress is a common environmental stress, in particular in salinized soils. Plant roots respond to a variety of soil stresses by regulating their growth, but the nature of the regulatory pathways engaged in the alkaline stress response (ASR) is not yet understood. Previous studies show that PIN-FORMED2, an auxin (indole-3-acetic acid IAA]) efflux transporter, PKS5, a protein kinase, and DNAJ HOMOLOG3 (J3), a chaperone, play key roles in root H⁺ secretion by regulating plasma membrane (PM) H⁺-ATPases directly or by targeting 14-3-3 proteins. Here, we investigated the expression of all 14-3-3 gene family members (TOMATO 14-3-3 PROTEIN1 TFT1]–TFT12) in tomato (Solanum lycopersicum) under ASR, showing the involvement of four of them, TFT1, TFT4, TFT6, and TFT7. When these genes were separately introduced into Arabidopsis (Arabidopsis thaliana) and overexpressed, only the growth of TFT4 overexpressors was significantly enhanced when compared with the wild type under stress. H⁺ efflux and the activity of PM H⁺-ATPase were significantly enhanced in the root tips of TFT4 overexpressors. Microarray analysis and pharmacological examination of the overexpressor and mutant plants revealed that overexpression of TFT4 maintains primary root elongation by modulating PM H⁺-ATPase-mediated H⁺ efflux and basipetal IAA transport in root tips under alkaline stress. TFT4 further plays important roles in the PKS5-J3 signaling pathway. Our study demonstrates that TFT4 acts as a regulator in the integration of H⁺ efflux, basipetal IAA transport, and the PKS5-J3 pathway in the ASR of roots and coordinates root apex responses to alkaline stress for the maintenance of primary root elongation.Alkaline soils occur commonly in terrestrial ecology, in particular in areas affected by salinity, thus contributing to one of the most widespread environmental challenges that limit agricultural productivity globally (Kawanabe and Zhu, 1991; Ge et al., 2010; Xu et al., 2012a). Worldwide, it is estimated that up to 831 × 10⁶ ha of land is saline, and more than half of this area is alkalinized. High-pH stress limits the survival of most plants under these conditions and can be a more significant factor in reducing plant growth than the stress resulting from salinity (Guo et al., 2010). Improved understanding of the basic mechanisms of plant responses to alkaline stress is urgently needed and will aid biotechnological efforts focused on breeding suitable crops for fodder and human food on these unproductive lands.Primary root elongation regulated by a sensory zone in the root tip plays a pivotal role in the plastic acclimation response to fluctuating soil environments (Baluška et al., 2010). The root functions simultaneously as an organ for the uptake and transport of water and nutrients and as the primary site for the perception of soil stresses. Thus, roots must be the obvious first focus in any examination of the adaptive and acclimation mechanisms underpinning the alkaline stress response. However, currently, only limited information is available on this particular form of stress (Degenhardt et al., 2000; Zhu, 2001; Yang et al., 2008).Acidification of the aqueous fraction of the cell wall apoplast by H⁺ excretion via the plasma membrane (PM) H⁺-ATPase is a critical component of the growth-promoting effect and a key factor determining the elongation of the primary root (Moloney et al., 1981; Palmgren, 2001). Optimal primary root elongation requires the fine regulation of H⁺-ATPase-mediated H⁺ efflux, particularly at the root tip (Staal et al., 2011; Haruta and Sussman, 2012). Under alkaline stress, in Arabidopsis (Arabidopsis thaliana), PROTEIN KINASE5 (PKS5) and the chaperone DNAJ HOMOLOG3 (J3) play important roles in H⁺ efflux by regulating the interaction between PM H⁺-ATPase and 14-3-3 proteins (Fuglsang et al., 2007; Yang et al., 2010). Furthermore, PIN-FORMED2 (PIN2), an auxin (indole-3-acetic acid IAA]) efflux transporter, is required for the acclimation of roots to alkaline stress through the modulation of H⁺ secretion in the root tip, maintaining primary root elongation (Xu et al., 2012a). However, these mechanisms, and other physiologically relevant processes that may fine-tune root-apical responses to alkaline stress, have not been investigated in depth.The 14-3-3 proteins are highly conserved, and nearly ubiquitous, phosphoserine-binding proteins that regulate the activities of a wide array of targets via direct protein-protein interactions (Moore and Perez, 1967; Comparot et al., 2003). In higher plants, 14-3-3 proteins are encoded by a multigene family and play important roles in regulating plant development and stress responses (Mayfield et al., 2012). Although 14-3-3 proteins in plants possess a highly conserved target-binding domain, several studies indicate that various 14-3-3 isoforms may regulate different targets or act in distinct locations under variable abiotic stresses (Sehnke et al., 2002; Xu et al., 2012b). At least 12 genes predicted to encode 14-3-3 proteins (TOMATO 14-3-3 PROTEIN1 TFT1]–TFT12) have been identified in tomato (Solanum lycopersicum; Roberts, 2003; Xu and Shi, 2006). However, little is known about the detailed actions of tomato 14-3-3 proteins in response to alkaline stress in relation to H⁺ secretion, auxin modulation, or specific signaling pathways. Thus, in this study, we investigated the roles of tomato 14-3-3 proteins, incorporated into Arabidopsis, in root acclimation to alkaline stress and the involvement of PKS5 and J3 in modulating H⁺ secretion and basipetal (shoot-ward) IAA transport for maintaining primary root elongation.