SlHDA3 transcript was increased under hormone treatments
Previous research identified that HDACs were referred to plant responses to hormone treatments and involved in the processes of plant hormone-induced growth and development (Liu, et al. 2014; Luo, et al. 2012; Ming 2015). To clarify the detailed function of SlHDA3 in hormone treatments during plant growth and development in tomato plants, we first test whether SlHDA3 expression was affected by hormone-induced such as ABA, GA3, IAA and SA. Figure 1 showed that the transcript of SlHDA3 were induced under ABA, GA3, IAA and SA. When suffered with exogenous ABA, the expression of SlHDA3 was obviously induced at 4h and 8h. While suffered with exogenous GA3, IAA and SA, the transcripts accumulated of SlHDA3 were up-regulated continuously and peaked at 8h, 4h and 12h respectively, then declined to the lowest level at 48h.
The sensitivity to ABA was increased in SlHDA3 RNAi seedlings
The results in our previously work showed that SlHDA3 is highly homologous to the histone deacetylase gene AtHDA19 in Arabidopsis (Guo, et al. 2017). The 3D structures of SlHDA3 and AtHDA6(encoded by HDA6 gene in Arabidopsis) were generated by utilizing the SWISS-MODEL tool (http://www.swissmodel.expasy. org; Fig. S1) and the 3D structures models of SlHDA3 and AtHDA6 were very similar, indicating that SlHDA3 in tomato may play analogous roles to that of AtHDA6 in Arabidopsis.
Based on the similarity to the AtHDA19, we speculated that SlHDA3 may be involved in the regulation of ABA signaling pathway in tomato. To measure the sensitivity of SlHDA3-RNAi plants, the experiment of ABA treatment(0, 4 and 8μM) was performed. The results suggested that the length of roots and hypocotyl in SlHDA3-RNAi was relatively small deviations with WT in the absence of ABA(0μM), while there were obviously shorter than that in the control in the presence of ABA(4 and 8μM)(Fig.2). These findings demonstrate that suppress the expression of SlHDA3 in tomato may be results in increased sensitivity to ABA.
The expression of ABA biosynthesis- and signal transduction-related genes were down-regulated
Increased transcript accumulation in SlHDA3-RNAi when suffered with ABA treatment prompted us to test whether the expression level of ABA biosynthesis- and signal transduction-related genes were affected in SlHDA3-RNAi plants. The expression of SlPYL1-SlPYL8, eight ABA-dependent receptor genes (Danquah, et al. 2014), were detected both in WT and SlHDA3-RNAi plants. As it is showed in Figure 3, no obvious differences was observed in transgenic lines and WT tomato at 0h. However, various degrees of down-regulation was presented in SlHDA3-RNAi plants compared with WT at 4h. SlNCED1 and SlNCED2，two ABA biosynthesis-related genes (Ji, et al. 2014), also were measured in WT and SlHDA3-RNAi plants. The transcription of SlNCED1 and SlNCED2 was in SlHDA3-RNAi plants slightly higher than that in WT at 0h but no significant difference in the statistical level. While significant difference was exhibited in WT and SlHDA3-RNAi plants and the expression level was down-regulated in SlHDA3-RNAi plants at 4h. Besides, SlABF2 and SlABF4, two ABA-responsive element binding factor (ABF) genes (Chen, et al. 2016), were also obviously reduced in SlHDA3-RNAi plants.
Silencing of SlHDA3 significantly decreases drought tolerance
The results in our published previously indicated that the transcript of SlHDA3 was significantly increased under dehydration stress (Guo, et al. 2017), the effects of drought stress on WT and SlHDA3-RNAi tomato were conducted in soil. No significant difference in morphological phenotype was observed between WT and SlHDA3-RNAi plants (0d, Fig.4a). The leaves of SlHDA3-RNAi lines started turning to yellow and rolling, while the WT were less withered after 14 days of drought tolerance (Fig. 4b). Significant difference was exhibited on the 21th day after drought treatment, most leaves in RNAi plants were yellow and wilting (or even dead), whereas the WT plants began changing into yellow and rolling (Fig.4c). Based on drought tolerance differences between WT and SlHDA3-RNAi plants, the survival rates were tested. A lower survival rate of SlHDA3 transgenic plants than that of WT plants was observed 21 days after drought tolerance (Fig. 4d). Besides, the leaves of WT and transgenic line at 0, 14 and 21days were gathered to measure contents of total chlorophyll and RWC for further confirm this stress tolerance phenotype.The degradation of total chlorophyll in SlHDA3 transgenic plants leaves was faster than that in WT at both 14 and 21 days after drought treatment (Fig. 4e). As shown in Fig. 4f, the decreased of RWC in SlHDA3 transgenic plants was more faster than that in WT plants (Fig. 4f). Meanwhile, parameters of other physiological indicator were further tested including CAT activity, proline content and MDA content. CAT activity in WT leaves was higher than that in transgenic lines during the post-drought treatment. However, proline content and MDA content were significantly higher in transgenic plants than that in WT under drought treatment (Fig. 4g-i). The results indicate that SlHDA3 is involved in tomato drought resistance.
The sensitivity to NaCl was increased in SlHDA3-RNAi seedlings
The transcript of SlHDA3 was obviously increased under salt stress especially in leaves (Guo, et al. 2017) prompting us to analyses whether the sensitivity to NaCl was effected in SlHDA3-RNAi seedlings. The measurement of sensitivity experiment was designed for further detecting whether SlHDA3 seedlings had differences with WT seedlings under NaCl stress. The length of hypocotyl and roots in SlHDA3-RNAi lines and WT seedings has no significant difference in the absence of NaCl(0μM)(Fig.5a,d-e). However, the hypocotyl and roots length of SlHDA3-RNAi lines was distinctly shorter than that of WT in the incubation medium (100 and 150 μM NaCl) (Fig. 5b-e). These findings suggest that the sensitivity to NaCl was increased in SlHDA3-RNAi lines.
Silencing of SlHDA3 significantly decreases salt tolerance
Based on the results of induced expression of SlHDA3 under salt stress in our published previously and the increased sensitivity to NaCl in SlHDA3-RNAi seedlings, related research was performed to study whether silencing of SlHDA3 effected salt tolerance. Before NaCl stress(0d), the growth status of SlHDA3-RNAi plants was similarity to WT(Fig.6a). The lower leaves of SlHDA3-RNAi plants turning to wilt and chlorosis, while no obvious change in WT plants after 7 days of salt stress(Fig.6b). Severe chlorotic leaves and collapsed shoot tissue was observed in SlHDA3-RNAi plants on the 14th day after salt stress. Whereas, the leaves in WT plant exhibit wilting and degradation of chlorophyll (Fig.6c). Related physiological and biochemical indicators were measured to further illustrate the potential physiological mechanism cause of the reduced salt stress tolerance in SlHDA3-RNAi plants. A significant decrease survival rate of SlHDA3-RNAi plants than that of WT plants (Fig.6d). No obvious change were found in chlorophyll contents, RWC, CAT activity, proline content, ABA concentration and MDA content between WT and SlHDA3-RNAi plants under normal conditions (Fig.6e-j). 7 days and 14 days after NaCl treatment, the decrease of The chlorophyll contents, RWC and CAT activity in SlHDA3-RNAi plants were faster than that in WT plants (Fig.6e-g). On the contrary, the proline content, ABA concentration and MDA content in SlHDA3-RNAi plants were significantly higher than that in WT plants (Fig.6h-j). These results above suggest that the transcript of SlHDA3 confer to salt stress in SlHDA3-RNAi plants.
Silencing of SlHDA3 significantly decreases the expression of stress-related genes under salt tolerance
To further comprehend the underlying mechanisms of the depressed tolerance to salt stress, 14 stress-related genes including an endochitinase gene (Gawehns 2014), a potassium channel KAT3-like gene (Nakano, et al. 2013), a peroxidase gene, a key proline (Pro) synthetase gene SlP5CS (Kishor, et al. 1995), two ascorbate peroxidase (APX) genes SlAPX1 and SlAPX2 (Najami, et al. 2008), two pathogenesis-related (PR) genes SlPR1 and SlPR5 (Lim, et al. 2010), a MAP kinase kinase kinase gene SlMAPKKK11 (Wu, et al. 2014), two MYB TF genes SlMYB46 and SlMYB106 (Zhao, et al. 2014), a key ascorbic acid (AsA) synthetase gene SlGME2 (Chanjuan, et al. 2011), a pathogenesis-related protein gene SlSTH-2 (Liu, et al. 2016), and Trihelix TF gene SlGT26 (Yu, et al. 2015), were chosen and detected for further compared the expression between WT and SlHDA3-RNAi plants under salt stress for 48h. Varying degrees down-regulated of these stress-related genes were presented in SlHDA3-RNAi plants under stressed conditions(Fig.7), indicating that SlHDA3 is involved in regulating stress-related genes.