Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

Although well known as an aging phytohormone for the acceleration of fruit ripening, organ senescence, and abscission, ethylene also regulates many aspects of vegetative growth and development (Abeles et al., 1992; Vandenbussche et al., 2012). In general, ethylene tends to function as an inhibitor of vegetative growth but also, promotes reproductive growth, accelerating the lifecycle, especially under environmental stress. Over a century ago, Neljubow (1901) first discovered that ethylene inhibits hypocotyl elongation of pea (Pisum sativum) seedlings, enhances horizontal growth, and leads to radial swelling in the dark, which is known as the triple response. Based on this discovery, a series of mutants was identified in Arabidopsis (Arabidopsis thaliana), and the classical signaling transduction model was established by genetic analysis. In the absence of ethylene, negative regulator Constitutive Triple Response1 (CTR1) tightly interacts with receptor complex (Clark et al., 1998), phosphorylates the central factor Ethylene-Insensitive2 (EIN2), and prevents its translocation into nucleus, thus blocking ethylene signaling transduction. In the presence of ethylene, receptors and CTR1 are inactivated, leading to the dephosphorylation and cleavage of EIN2. The C terminus of EIN2 is then translocated into the nucleus to activate downstream transcription factors EIN3/Ethylene-Insensitive3-Like (EIL)s followed by expression of target genes (Ju et al., 2012; Qiao et al., 2012; Wen et al., 2012). In this pathway, the ethylene receptors EIN2 and EIN3 are regulated by ubiquitination and proteasome-mediated degradation (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004; Binder et al., 2007; Chen et al., 2007; Kevany et al., 2007; Qiao et al., 2009; An et al., 2010).

As for the role of ethylene in the regulation of vegetative growth, one of the most prominent pieces of evidence is the variant seedling morphology of signaling mutant plants. In Arabidopsis, mutant plants with enhanced ethylene response, such as ethylene response1-6 (etr1-6) and ctr1-1, often show retarded growth, leading to dwarfed seedlings and smaller leaves compared with the wild type (Kieber et al., 1993; Hua and Meyerowitz, 1998). On the contrary, etr1-1, ein2-1, ein3-1, and other ethylene-insensitive or low-sensitivity mutants often show accelerated seedling growth (Chang et al., 1993; Chao et al., 1997; Alonso et al., 1999). Unlike ctr1-1, ein2-1, or ein3-1, mutants of individual ethylene receptors show only weak effects on seedling growth because of the functional redundancy of different receptors (Hua and Meyerowitz, 1998; Hall and Bleecker, 2003). However, each receptor seems to have its own features of protein structure and expression pattern and therefore, may play unique roles in addition to the general role of ethylene perception (Shakeel et al., 2013). In Arabidopsis, mutation and transgenic analysis illustrate that subfamily I receptors play more significant roles in ethylene-regulated growth responses than subfamily II receptors do, probably because of the more efficient activation of CTR1 by subfamily I receptors (Hua and Meyerowitz, 1998; Hall and Bleecker, 2003; Wang et al., 2003; Qu et al., 2007). Additionally, the kinase activity of ETR1 may play a role in the activation of CTR1 (Hall et al., 2012).

Moreover, subfamily I receptors have stronger associations with CTR1 than subfamily II receptors (Clark et al., 1998; Cancel and Larsen, 2002). A similar preference is observed for the associations between ethylene receptors and CTRs in tomato (Solanum lycopersicum; Lin et al., 2008a, 2009b). In addition, ctr1 loss-of-function mutants continue to exhibit a residual ethylene response (Larsen and Chang, 2001; Larsen and Cancel, 2003), and the quadruple ethylene receptor loss-of-function mutant etr1 etr2 ein4 ethylene response sensor2 (ers2) and the etr1 ers1 double mutant display a more severe phenotype than the ctr1 loss-of-function mutants (Hua and Meyerowitz, 1998; Hall and Bleecker, 2003). Therefore, other than the classical CTR1-dependent pathway, other pathways might exist, possibly through unique receptor-interacting factors (Ju et al., 2012). One possible pathway is the two-component signaling pathway involving the phosphorelay transduction (Hass et al., 2004; Mason et al., 2005; Scharein et al., 2008; Scharein and Groth, 2011), but direct evidence is still lacking. Gel filtration analysis of ethylene receptor complexes in Arabidopsis further indicates that isoform-specific interacting proteins may exist for different receptors to mediate ethylene signaling (Chen et al., 2010). Indeed, Arabidopsis RTE1 was identified as an ETR1-specific interacting protein to mediate CTR1-independent ethylene response (Resnick et al., 2006, 2008; Zhou et al., 2007; Dong et al., 2010; Qiu et al., 2012). Expression of the N-terminal fragment of ETR1 (1-349) in ctr1-1 partially suppresses its constitutive ethylene-response phenotype, and this effect is dependent on the Ethylene Sensitivity1 (RTE1; Qiu et al., 2012). Another example is that Arabidopsis Tetatricopeptide Repeat Protein1 and tomato Tetratricopeptide Repeat Protein1 were identified as interacting factors with subfamily I ethylene receptors to modulate ethylene response and plant development (Lin et al., 2008b, 2009a). We recently showed that tobacco Histidine Kinase1 (NTHK1) Ethylene Receptor-interacting Protein2 (NEIP2), an ankyrin domain protein, interacts with subfamily II receptors to regulate plant response to abiotic stresses in tobacco (Nicotiana tabacum; Cao et al., 2015).

Previously, we studied roles of tobacco subfamily II ethylene receptor NTHK1 in plant growth and stress response. The transcripts of NTHK1 were inducible under salt stress and wounding (Zhang et al., 1999, 2001). Overexpression of NTHK1 in both tobacco and Arabidopsis exhibited reduced ethylene sensitivity, accelerated growth, and increased salt sensitivity of transgenic plants (Xie et al., 2002; Cao et al., 2006, 2007). The kinase domain and Ser/Thr kinase activity of NTHK1 were differentially required for salt response and seedling growth (Zhou et al., 2006; Chen et al., 2009). Surprisingly, NTHK1 seems to play more prominent roles in these responses than the subfamily I member NtETR1 (Chen et al., 2009). Characterized downstream factors involved in NTHK1-regulated stress response and plant growth processes include two transcription factors AtNAC2 and AtMYB59 (He et al., 2005; Mu et al., 2009), an NIMA-related kinase NEK6 (Zhang et al., 2011), and an ankyrin domain protein NEIP2 (Cao et al., 2015). To further elucidate the mechanism underlying the function of NTHK1, we initiated a yeast (Saccharomyces cerevisiae) two-hybrid assay to screen components that interact with NTHK1. Finally, a translationally controlled tumor protein (TCTP) was identified and further investigated. NtTCTP protein accumulation is induced by ethylene treatment. Overexpression of NtTCTP promotes seedling growth, mainly through the control of cell proliferation. Genetic analysis reveals that NtTCTP is required for NTHK1-mediated ethylene response and seedling growth.

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