Xu et al. BMC Plant Biology (2019) 19:60 https://doi.org/10.1186/s12870-019-1662-9 RESEARCHARTICLE Open Access The expression of a tubby-like protein from Malus domestica (MdTLP7) enhances abiotic stress tolerance in Arabidopsis Jianing Xu†, Shanshan Xing†, Qinghua Sun, Chunyan Zhan, Xin Liu, Shizhong Zhang* and Xiaoyun Wang* Abstract Background: Tubby-like proteins (TLPs), characterized by a signature tubby domain, are widespread in plants and animals. To date, only plant TLPs involved in multifarious stress responses and male gametophyte development have been identified. However, studies on the molecular functions of plant TLPs are largely unknown. Results: In this investigation, the roles of a TLP from Malus domestica (MdTLP7) in response to abiotic stresses were characterized by expressing it in Arabidopsis. The expression of wild-type full-length MdTLP7 (FL) significantly increased the stress tolerance of Arabidopsis seedlings to osmotic, salt, cold and heat stress, while the expression of truncated MdTLP7 containing only the tubby domain (Tub) also showed some function. Located on a central α helix surrounded by 12 anti-parallel β strands in the tubby domain, the K190/R192 site may be involved in fixation to the plasma membrane, as shown by 3D homology modelling with animal TLPs. This site might play a crucial role in anti-stress functions since site-directed mutagenesis of MdTLP7 reduced stress tolerance. Subcellular localization showed that MdTLP7 was mainly localized in the plasma membrane in plant cells, suggesting that it might participate in the transduction of stress signals. Conclusions: The results of this study showed that MdTLP7 could improve abiotic stress tolerance not only in bacteria but also in plants. The K190/R192 residues in the tubby domain were not only the plasma membrane binding site of MdTLP7 but also played a key role in stress tolerance. These results may provide a basis for further exploring the mechanism of anti-stress functioning and downstream target genes of plant TLPs. Keywords: Tubby-like protein, MdTLP7, Functional site, Stress tolerance, Subcellular localization Background Several plant TLPs are upregulated under abiotic and The tubby-like proteins (TLPs) are widely distributed biotic stress [4, 10–13]. in the animal and plant kingdoms. There are 5 mem- Our previous study showed that a TLP gene from bers of the TLP family in mice, 4 in Homo sapiens,9 Malus domestica (MdTLP7) was upregulated in the tran- in apple and 11 in Arabidopsis [1–4]. In mammals, scriptional profile of apple under cold stress [14]. Heter- many cellular functions of TLPs are involved in ves- ologous expression of MdTLP7 significantly increased icular trafficking, the mediation of insulin signalling, the stress tolerance of E. coli cells against different abi- gene transcription, G-protein signalling, and riboso- otic stresses [15]. In contrast to animal TLPs, MdTLP7 mal RNA synthesis [5–9]. Although several TLPsin contains a highly conserved F-box domain at its plants have been identified, their roles are elusive. In N-terminus in addition to the characteristic tubby do- Arabidopsis, AtTLP3 and AtTLP9 are involved in main [15]. In eukaryotes, F-box proteins participate in ABA-dependent signalling during germination [3]. diverse cellular processes such as the response to stress, signal transduction, and the development of floral or- gans, primarily as a component of the Skp1-cullin-F-box * Correspondence: [email protected]; [email protected] (SCF) complex in protein ubiquitination [16–20]. †Jianing Xu and Shanshan Xing contributed equally to this work. College of Life Science, State Key Laboratory of Crop Biology, Shandong Subcellular localization of TLPs clarifies their func- Agricultural University, Shandong Taian 271018, People’s Republic of China tions. Many mammalian and plant TLPs are usually © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. BMC Plant Biology (2019) 19:60 Page 2 of 8 localized in the plasma membrane and nucleus [12, 21]. than 10 transgenic lines were identified by kanamycin As to the mechanism of TLP binding to the plasma mem- testing and PCR amplification (T1 generation, brane, a phosphatidylinositol-4,5-bisphosphate (PIP2) Additional file 1: Figure S1). Homozygous lines were binding site involving two conserved residues in the tubby identified by screening for non-segregation from each domain, lysine330 (K330) and arginine332 (R332), interacted independent transformant (T3 generation). The 2 homo- directlywiththemembranelipidoftheplasmamem- zygous FL transgenic lines (named FL-1, FL-2) and 2 brane, as demonstrated in two TLPs from mouse Tub transgenic lines (named Tub-1, Tub-2) with high (TUB and TULP1) [22]. These two positively charged expression were chosen for further analysis (Additional phosphate-coordinating residues (K330 and R332)are file 2: Figure S2). highly conserved not only in the animal TLPs but Under PEG and salt treatments, the growth of both also in quite a few plant TLPs. The corresponding plasma transgenic lines, FL and Tub, was better than that of membrane binding site in the tubby domain of AtTLP3 WT; little difference was found between the FL and (K187/R189)alsolocalizesAtTLP3 in the plasma mem- Tub lines (Fig. 1a and b). The results suggested that brane. Mutations of these conservative residues to other the expression of wild-type MdTLP7 (FL) or trun- residues could disrupt the localization of AtTLP3 to the cated MdTLP7 (Tub) enhanced tolerance to osmotic plasma membrane [23]. PIP2 affects cell signalling by and salt stresses. binding target proteins or enzymes [24]. When plants suf- To induce temperature stress, we placed seedlings in fer abiotic stress-inducing agents such as mannitol, NaCl hot (45 °C) and cold (− 20 °C) environments. After treat- and H2O2 treatments, AtTLP3 can trigger signals by ment at 45 °C for 12 h, obvious differences were ob- detaching from the plasma membrane and moving served among the transgenic lines (FL and Tub) and through the cytosol to the nucleus to regulate the expres- between the transgenic lines and WT Arabidopsis, and sion of genes [12, 23]. As bipartite transcription factors the growth of the Tub plants was better than that of the (TFs), mouse TLPs have DNA binding domains near their WT plants but worse than that of the FL plants. After 3 C-terminus and transcription modulation segments at days of recovery, all of the WT plants were dead their N-terminus [5]. TFs usually bind to cis-regulatory re- whereas 80% of the FL transgenic plants and 40% of the gions of a gene, which act as molecular switches control- Tub transgenic plants survived (Fig. 1c). Similar results ling various biological processes including abiotic and after cold treatment were observed among the WT, FL biotic stress responses [25, 26]. A chickpea TLP (CaTLP1) and Tub lines. The seedlings were pre-treated at 5°Cto was demonstrated to be a putative TF presumably − 5 °C for 10 h and then subjected to − 20 °C for 40 min. involved in multivariate stresses [11]. A rice TLP All of the WT plants were dead whereas 70% of the FL (OsTLP2) has been shown to bind to the promoter of plants and 30% of the Tub plants survived after 3 days of OsWRKY13 to regulate disease resistance [10]. These recovery (Fig. 1d). reports suggest that TLPs might participate in stress The results indicated that the expression of MdTLP7 signal transduction as TFs. in Arabidopsis could enhance tolerance to several abiotic To determine the functions of MdTLP7 in plants, stressors in Arabidopsis. Truncated MdTLP7 (Tub) still full-length and truncated MdTLP7 (without the F-box retained almost complete function compared to that of domain) were transformed into Arabidopsis in this wild-type MdTLP7 (FL) under both PEG and salt stress. study. The responses of transgenic plants to different Regarding temperature stress, truncated MdTLP7 (Tub) stresses were assessed. Using 3D structure modelling only had approximately half or less of the anti-stress and site-directed mutation experiments, we found a crit- function of wild-type MdTLP7 (FL). The results sug- ical site in the tubby domain of MdTLP7 for abiotic gested that the tubby domain of MdTLP7 has key roles stress tolerance. These results open up new horizons for and that the F-box domain may have synergistic effects studying the mechanism of action of stress tolerance in with the tubby domain in the stress response. plant TLPs and may promote the development of methods to improve plant stress resistance. MdTLP7 is localized in the plasma membrane A 3D homology model of the tubby domains of Results MdTLP7 was established using SWISS-MODEL that Expression of MdTLP7 enhanced abiotic stress tolerance is highly consistent with the mouse TLP (TULP3) in Arabidopsis structure. Both contain a central α helix and a closed To examine the function of MdTLP7 in the plant stress 12-stranded β barrel (Fig. 2a and b). Mouse TLPs are response, the full-length cDNAs of wild-type full-length tethered to the plasma membrane via a PIP2 binding MdTLP7 (FL) and truncated MdTLP7 with only the site (K330/R332) in the tubby domain [8]. The corre- tubby domain (Tub) were transformed into wild-type sponding amino acid residues in MdTLP7, K190/R192, Arabidopsis (WT) by the CaMV 35S promoter. More show high positional overlap with residues K330/R332 Xu et al.