Histone Deacetylase Gene SlHDA3 Involves in ABA, Drought and Salt Response in Tomato

Histone deacetylation, one of vital modifying factors of post-translation modications, which is catalyzed by histone deacetylase. The genes of histone deacetylase(HDACs) play critical roles in various stress responses. However, detailed functions for most SlHDAC members in tomato still unknown. In this work, we found that a histone deacetylase, SlHDA3, involved in response to NaCl and drought abiotic stresses. The expression of SlHDA3 was also induced signicantly by NaCl, drought stress and endogenous hormone treatments. Silencing of SlHDA3 in tomato, the RNAi transgenic plants presented depressed tolerance to drought and salt stresses compared with WT tomato. The results of sensitivity analysis indicated that the length of hypocotyl and roots in RNAi plants were more inhibited by ABA and salt stress than that of WT at post-germination stage. Worse growth status were exhibited in SlHDA3 transgenic plants under salt and drought stress as are evaluated by a series of physiological parameters related to stress responses, such as decreased RWC, survival rate, ABA content, chlorophyll content and CAT activity, and increased MDA content and proline content. Besides, the expressions analysis of transgenic plants showed that the transcripts of genes which associated with responses to abiotic stress were down-regulated under salt-stressed conditions. To sum up, SlHDA3 acts as a stress-responsive gene, plays a role in the positive regulation of abiotic stress tolerance, and may be one of the new members in the engineering breeding of salt- and drought-tolerant tomato.


Introduction
Abiotic stresses, such as drought and high salinity, not only affects crop growth and development but also adversely affects the yield of crop. A series of compromising related to plant growth and development are continuously resulted from stresses, which may break plant homeostasis. Usually, tomato is mainly cultivated as nutritious edible fruit and vegetable crop, which can be commercially grown worldwide. Although tomato plants have a certain degree of tolerance to various abiotic stresses, crop losses are more severe under extreme weather conditions. It is urgent and necessary to obtain the stress-tolerance crop varieties and it is pro table to cultivate stress-tolerant crops that impressionable to abiotic stress for modern agriculture areas. It is well known that the stress response is regulated by both ABA-dependent and ABA-independent regulatory systems (Fujita, et al. 2010;Tran, et al. 2004). ABA plays an important part in abiotic stress adaptation by regulating stomatal closure and by stimulating numerous stress-related genes, and then enhancing the tolerance of plants to various stresses (Nakashima, et al. 2012).
Previous researches have shown that histone deacetylase act as indispensable components during the process plant growth and development, such as the construction of leaf morphology, the elongation of hypocotyl, the development of root, the regulation of owering time, the fruit ripening and so on ). HDACs genes have been identi ed and functionally studied in the model plant Arabidopsis. Detailed functional characterization of HD2 subfamilies genes have been reported involves in the regulation of responses to abiotic stresses including ABA, salt and drought (Chen and Wu 2010;Li-Ting, et al. 2010;; Song and C.-P. 2005; Sridha and Wu 2006). Members of HD2 subfamilies genes, AtHDT1, AtHDT2, AtHDT3 and AtHDT4, were related to ABA and salt stresses in Arabidopsis (Chinnusamy, et al. 2008;Yuan, et al. 2013;Zhaofen, et al. 2016).
Distinguish from HD2 subfamilies and SIR2 subfamilies, the members of RPD/HDA1 subfamilies are wide participation in diverse processes, including seed development and germination (G., et al. 2013;Georgieva, et al. 1991), root hair development , leaf morphogenesis ; Sco eld and Murray 2006), ower development (Gonzalez, et al. 2007;Tian, et al. 2003), light signaling and hypocotyls growth (Benhamed, et al. 2006;Kim, et al. 2013), ABA and salt stress response ), and plant cell cycle and development (Varotto, et al. 2003 (Tessadori, et al. 2009), DNA methylation (Aufsatz, et al. 2002), cold stress response and ethylene pathway ). These results suggested that histone deacetylation regulated by HDA6 is referred in plant growth and development and abiotic stress response in Arabidopsis. Our previous research showed that the expression level of SlHDA3 is signi cantly induced by various abiotic stresses such as NaCl, dehydration and high/low temperature, indicating that SlHDA3 may involves in abiotic stress tolerance (Guo, et al. 2017).
Herein, we reported the functional characterization of SlHDA3, we generated tomato RNAi plants of SlHDA3, and the transgenic lines showed reduced tolerance to ABA, drought, and salt. These phenotypes were further con rmed by analysis of physiological and biochemical features and related gene expression features.

Plant materials and growth conditions
All tomato seed of WT(wild type, Solanum lycopersicum Mill. cv. Ailsa Craig) and homozygous T3 SlHDA3-RNAi transgenic lines were surface sterilized and grown in soil under sodium lights(16h days, 25 C, and 8h nights, 18 C) and 80% humidity.
Hormone treatments 35-day-old WT tomato seedlings with similar growth status were chosen for hormone treatments. The experimental group tomato plants were sprayed with 100μM ABA, 50μM GA3, 50μM IAA and 50μM SA solution, while the control group tomato plants were sprayed with water (Fujita, et al. 2010). All seedlings were enclosed in plastic as soon as possible after spraying, the leaves were collected at 0, 1, 2, 4, 8, 12, 24 and 48h, and all the gathered samples were frozen as soon as possible in liquid nitrogen and stored at -80 °C until used for this study.

Sensitivity assays
The sensitivity assays were carry out for further research the sensitivity of SlHDA3 seedling growth to ABA and salt stress, the germinated seeds of WT and transgenic were selected and transferred to MS medium containing ABA (0, 4 and 8 μM) and NaCl (0, 100 and 150 mM) according to their consistent germination status. Pictures were taken and the length of root and hypocotyl was measured 7 days later.
Phenotype analyses and evaluation of salt stress tolerance and drought tolerance assay 35-day-old tomato plants that including WT and SlHDA3-RNAi lines were selected for salt stress tolerance and drought tolerance assay based on their uniformity. In the experiment of salt stress tolerance assay, all the plants were irrigated with 200 mL 400 mM NaCl solution every 48h, and the leaves from treated tomato plants at 24h were gathered for stress related-gene expression analysis. As for the experiments of drought tolerance assay, all the tomato plants were irrigated with water enough and with holding water until 21days. Total RNA extraction and quantitative RT-PCR analysis Total RNA was extracted using RNA trizol, the extract was digested by Dnase and after that total RNA was used to reverse-transcribed to rst-strand cDNA. The qRT-PCR reaction system and the analysis of qRT-PCR was performed as showed as our previous report (Guo, et al. 2017). NRT(no reverse transcription control) and NCT(no template control) were carried out for further analysis of each gene. For abiotic stress, the tomato SlEF1 gene was also performed as the internal standards (Nicot, et al. 2005).

SlHDA3 transcript was increased under hormone treatments
Previous research identi ed that HDACs were referred to plant responses to hormone treatments and involved in the processes of plant hormone-induced growth and development Ming 2015). To clarify the detailed function of SlHDA3 in hormone treatments during plant growth and development in tomato plants, we rst test whether SlHDA3 expression was affected by hormoneinduced such as ABA, GA 3 , IAA and SA. Figure 1 showed that the transcript of SlHDA3 were induced under ABA, GA 3 , IAA and SA. When suffered with exogenous ABA, the expression of SlHDA3 was obviously induced at 4h and 8h. While suffered with exogenous GA 3 , 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 ndings 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 signi cant difference in the statistical level. While signi cant 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 , were also obviously reduced in SlHDA3-RNAi plants.

Silencing of SlHDA3 signi cantly decreases drought tolerance
The results in our published previously indicated that the transcript of SlHDA3 was signi cantly increased under dehydration stress (Guo, et al. 2017), the effects of drought stress on WT and SlHDA3-RNAi tomato were conducted in soil. No signi cant 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). Signi cant 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 con rm 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 signi cantly 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 signi cant 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 ndings suggest that the sensitivity to NaCl was increased in SlHDA3-RNAi lines.
Silencing of SlHDA3 signi cantly 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 signi cant 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 signi cantly 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 signi cantly decreases the expression of stress-related genes under salt tolerance
To further comprehend the underlying mechanisms of the depressed tolerance to salt stress, 14 stressrelated genes including an endochitinase gene (Gawehns 2014), a potassium channel KAT3-like gene (Nakano, et (Fig.7), indicating that SlHDA3 is involved in regulating stress-related genes.

Discussion
Various stresses including drought and high salinity in the environment are major factors that affect the agricultural productivity. It is pro table to cultivate stress-tolerant crops that vulnerable to abiotic stress for modern agriculture areas. Functional study of histone deacetylase genes was more detailed, especially in the past few years. Histone deacetylase gene plays signi cant roles in plants development, bio-and abiotic stresses. Recently, it has been proved that some stress-responsive histone deacetylase genes can affect the stress tolerance of transgenic plants. The mutant and RNAi plants of HDA6 in Arabidopsis were involved in the salt-stress signaling pathways and the growth were inhibited 14 days after NaCl treatment, and the expression level also down-regulated when treated with ABA in HDA6-RNAi plants (Chen and Wu 2010). Besides, drought stress-up-regulated genes have been proved related to histone modi cation (To and Kim 2013). The more severe the drought, the higher expression levels in AtHDT3 was participated in the regulation of heating stress response and an increased sensitivity of seed germination to ABA and NaCl was displayed in the mutant of AtHDT3 in Arabidopsis (Buszewicz, et al. 2016;G., et al. 2013;). OsHDT701 is involved in mediating the seed germination in response to abiotic stresses ). The expression assay of SlHDA3 under various abiotic stresses showed that the transcripts of SlHDA3 were induced signi cantly in both salt and dehydration treatments (Guo, et al. 2017). In this study, we further a rmed that the expression level of SlHDA3 was markedly improved by ABA (Fig. 1), suggesting that SlHDA3 may play an indispensable role in various environmental stress corresponding. Experiment on post-germination seeds suggested that the length of SlHDA3 transgenic seedling hypocotyl and root was more inhibited by ABA than that of WT (Fig. 2), indicating that silencing of SlHDA3 improved seedling ABA sensitivity in tomato. The reduced expression of ABA biosynthesis-and signal transduction-related genes in SlHDA3 transgenic plants further substantiate SlHDA3 is involved in ABA biosynthesis/signal transduction through regulating the expression of related genes (Fig.3).
In the experiment of dehydration treatment, a lower survival ratio and worse growth phenotype were observed in SlHDA3 transgenic lines. The lower chlorophyll content, RWC, CAT activity and higher proline content, MDA content were shown in SlHDA3-RNAi plant in our work (Fig. 4). These physiological indices are consistent with the morphology change. When suffered with NaCl treatment, the post-germination seeds growth was restrained sharply and the elongation of SlHDA3-RNAi seedling hypocotyl and root was more inhibited by salt than that of WT. When watered with NaCl solution, SlHDA3 transgenic plants turned wilted and yellow earlier than that of WT. Chlorophyll content, RWC, survival ratio, CAT activity and ABA concentration were reduced while MDA content and proline content were enhanced than that in WT(Figs. 5 6). In conclusion, we speculated that SlHDA3 act as a positive regulator in answer to the osmotic stress caused by drought and salt.
The enhancement of tolerance to abiotic stresses are mainly due to sustaining and markedly induced transcripts of related abiotic stress-response genes (2007). The results in our study indicating that those six genes which we selected were signi cantly down-regulated in SlHDA3-RNAi transgenic plants. In this study, the expression of a series of biotic and abiotic stress-related genes were signi cantly down-  (Yu, et al. 2015), indicating that SlHDA3 not only play an important role the adaptation to abiotic in tomato, but also mediates signaling pathways of response to biotic and abiotic stress though modulating transcripts of related abiotic stress-response genes, including NaCl, drought and the pathogen, which further con rm our results.
In conclusion, the data indicated in our work not only elucidate the important role of SlHDA3 in salt stress and drought stress tolerance, but also provide a foundation for further research on the application of histone deacetylase genes in the signal transduction pathway of salt stress and drought stress. However, how to realize the regulation of drought stress and salt stress by SlHDA3 and the detailed regulation mechanism needs to be further studied.

Declarations Author Contributions
All the work was accomplished by JG. Expression pro les of SlHDA3 gene under hormone treatments. Gene expression was detected by qRT-PCR using total RNA from leaves of WT plants. The relative expression levels were normalized to 1 in control plants (0 h). Bars represent the mean of three biological replicates ± SE.  Values represent mean ± SD (n = 3). Asterisks above each column indicate a signi cant difference(p < 0.05) between WT and transgenic lines. Values represent mean ± SD (n = 3). Asterisks above each column indicate a signi cant difference(p < 0.05) between WT and transgenic lines.