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miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco Lei Chen a, Jun Meng b, Yushi Luan a, * a b
School of Life Sciences and Biotechnology, Dalian University of Technology, Dalian, 116024, China School of Computer Science and Technology, Dalian University of Technology, Dalian, 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2018 Accepted 27 November 2018 Available online xxx
MicroRNAs (miRNAs) play important roles in regulating plant responses to various environmental stresses. In our study, overexpression of miR1916 in tomato (OE-1) reduced its tolerance to drought. The miR1916-silenced transgenic plants (ST-1 and Anti-7) significantly increased resistance to drought stress. The transgenic tobacco plants also have a similar result in displaying the tolerance of drought. Physiological analysis revealed that miR1916 affected the osmoregulation and reactive oxygen species (ROS) accumulation. In addition, transcript levels of the miR1916 target genes, histone deacetylases (HDAC) and strictosidine synthase (STR), decreased in miR1916-overexpressing transgenic tobacco plants. Our results suggested that miR1916 is a passive regulator in the plant resistance to drought stress and has potentially impacting on abiotic stress responses in Solanaceae. © 2018 Elsevier Inc. All rights reserved.
Keywords: mir1916 Drought stress Tomato Tobacco
1. Introduction Drought is a major abiotic stress factor that affects crops growth and productivity [1]. To cope up with this adverse challenge from environment, the plants have evolved multiple mechanisms to activate stress responses at the molecular, biochemical, and physiological levels [2]. Previous studies have demonstrated that high relative water content and chlorophyll content are two typical mechanisms of resistance to drought [3e5]. Moreover, osmolyte accumulation is also a part of these adaptive mechanisms. Soluble sugars and proline can act as osmoprotectants for plants to counteract the effects drought stress by protecting the cell structures and scavenging the reactive oxygen species [6e8]. An abundance of stress-responsive genes are known to involve the responses to abiotic stress by modulating proline and soluble sugars accumulation, such as hydroxyproline-rich glycoproteins (HRGPs) and UDP-glycosyltransferases (UGTs) genes [9,10]. MicroRNAs (miRNAs) are 18e25 nucleotides small non-coding RNA molecules and negatively regulate the expression of their target genes [11]. Plant miRNAs have been shown to play a crucial
Abbreviations: HDAC, histone deacetylases; HRGP, Hydroxyproline-rich glycoproteins; miR1916, microRNA1916; ROS, reactive oxygen species; S. lycopersicum, Solanum lycopersicum; STR, strictosidine synthase; UGT, UDP-glycosyltransferases. * Corresponding author. Tel.: þ86 411 84706365; fax: 86 411 84706365. E-mail address: [email protected] (Y. Luan).
role in the regulation of switches the response to abiotic stresses [12,13]. For instance, transgenic plants that overexpress miR396a are more tolerance to abiotic stresses by negatively regulating GRFs transcription in tobacco [14]. Similarly, overexpression of miR393a in creeping bentgrass results in reduced the expressions of AsAFB2 and AsTIR1 to improve multiple stresses [15]. Unlike the other miRNAs showing positive modulation of abiotic stresses tolerance, miR417 are overexpressed in Arabidopsis and have been demonstrated to retard seed germination under salt stress [16]. Currently, miR1916 is a nonconserved miRNAs that has been discovered in tomato, tobacco, potato, eggplant, pepper, petunia, prickly pear cactus and bean [17e21]. And according to the previous relevant study, the expression of miR1916 is detected in leaf tissue only in the sample of drought stress, but not in the control sample by miRNA microarray [22]. In tomato, miR1916 in leaves are increased under cold stress treatments [23]. Similarly, treatment with ethylene induces expression of miR1916 in tomato pedicels [24]. Our previous study also found that miR1916 acts as a negative regulator in the defense responses of tomato against Phytophthora infestans and Botrytis cinerea infection [25]. Although some miR1916 expressions have been studied, the biological function of miR1916 in plants response to abiotic remains unknown. In this study, we investigated whether change in the expression of miR1916 in tomato and tobacco affect tolerance against drought stress. To explore the function of miR1916, we examined these
https://doi.org/10.1016/j.bbrc.2018.11.165 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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transgenic plants that altered water, chlorophyll, proline and soluble sugars content, and reactive oxygen species accumulation. We also revealed two target genes of miR1916, HDAC and STR, whose expression is inhibited in the transgenic plants. This finding implies that miR1916 is an underlying molecular in regulatory network of Solanaceae responds to drought stress. 2. Materials and methods
was determined using the Shan et al. method [29]. The sugar content was measured with the Li et al. method [30]. 2.7. ROS quantification After 14 d of treatment with drought stress, the tobacco leaves were harvested. These leaves were treated with nitro blue tetrazolium (NBT) and diamino benzidine (DAB) staining solution at 28 C in the dark as previously described [31].
2.1. Plant material and growth conditions 2.8. Prediction the targets Tomato, including one wild-type (Solanum lycopersicum cv. Zaofen No.2), one transgenic plant that overexpressed sly-miR1916 (OE-1) and two silenced transgenic plants (ST-3 and Anti-7) were grown in a greenhouse at 23e27 C/18e22 C under 16 h photoperiod. Tobacco (Nicotiana tabacum cv. NC89) was used for heterologous transformation.
The miR1916 putative targets were predicted in tobacco using psRNATarget software (version 2017) (http://plantgrn.noble.org/ psRNATarget/) and the library (Nicotiana tabacum(tobacco), unigene, SGN unigene). The expectation and top target genes adjusted to 5.0 and 250, respectively.
2.2. Tobacco transformation and identification
2.9. Statistical analysis
The Agrobacterrium tumefaciens strain GV3101 harboring the vectors (pBI121-MIR1916, pBI121-STTM1916 and pBI121-AntimiR1916) were used for transformation. Positive transgenic plants were obtained on 1/2 MS medium containing 50 mg l1 kanamycin and identified by PCR.
The results were analyzed using Gradpad Prism 5.0 software. The data are expressed as means ± SD. All experiments were repeated three times. The statistical significances were considered at the P < 0.05 level revealed by Duncan's multiple-range test. 3. Results
2.3. Drought stress tolerance assays of tomato 3.1. miR1916 negatively regulates drought tolerance in tomato For drought tolerance tests, 4-week-old wild-type, overexpression of miR1916 and miR1916-silenced plants were withheld from watering for 14 d. The phenotypes and the leaf survival rate were recorded. The wild-type and transgenic tomato seedlings were grown on 1/2 MS medium for 7 d, and then transferred into 1/ 2 MS medium with 300 mM mannitol, and the root length was calculated after 14 d. 2.4. RNA extraction and qRT-PCR Total RNA was prepared from the leaves of tobacco seedlings using the RNAiso Plus (Takara Dalian, China). For miRNA analysis, 5 mg of total RNA was reverse-transcribed into cDNA using the MirX miRNA First-Strand Synthesis Kit (Takara Dalian, China) according to the supplier's protocol. For target genes analysis, the first strand cDNA was synthesized from 300 ng RNA with the PrimeScript™ RT Master Mix (Perfect Real Time) (Takara Dalian, China). The relative expression levels were analyzed using the deltaedelta Ct method [26]. The data were collected in an ABI7500 thermal cycler with Real-time PCR System using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Dalian, China). The primers used in this study are listed in Table S1. 2.5. Transgenic tobacco drought stress treatments The wild-type and transgenic tobacco plants were grown on 1/2 MS medium, and then transferred them into sterilized soil in a greenhouse during root formation (16 h light/8 h dark, 25 ± 2/ 20 ± 2 C). After 2 weeks, they were treated without water for 14 d. The phenotypes and the leaf survival rate were recorded. 2.6. Determination of relative water, chlorophyll, proline and soluble sugars content The relative water content (RWC) of the leaf was determined according to previously described method [27]. Chlorophyll content was measured as previously described [28]. The proline content
The three independent transgenic tomato plants (OE 1, ST-3 and Anti-7), which are shown to change the expression of miR1916 and to display affected plant tolerance to P. infestans and B.cinerea infection in our previous studies [25]. The expression of miR1916 was detected in leaf tissue after drought stress [22]. To investigate whether miR1916 plays a role in the drought-stress tolerance, we selected these transgenic plants for use in this study. After 14 d of growth in the absence of irrigation, OE 1 displayed more wilt symptoms and lower leaf survival rate than wild-type plants (Fig. 1A and B). However, higher levels of leaf survival rates were observed in the ST-3 and Anti-7 transgenic plants compared to wild-type plants after drought stress (Fig. 1A and B). Concomitantly, wild-type and transgenic tomato seedlings were cultured in 1/2 MS agar medium with 300 mM mannitol. After 14 d treatment, the root growth was analyzed for evaluate the drought stress. The roots length of OE-1 transgenic plants was shorter than wild-type, while the ST-3 and Anti-7 transgenic plants was observed a significant longer (Fig. 1C and D). These results revealed that by regulating the expression of miR1916 in tomato, the plant tolerance and resistance to drought stress could be altered. 3.2. Overexpression and silencing of miR1916 affects the tolerance of tobacco to drought stresses To elucidate miR1916 functions in Solanaceae, transgenic tobacco plants overexpressing or silencing miR1916 were generated. The expression of miR1916 in all three transgenic plants miR1916OE (OE-1, OE-4 and OE-5) that overexpressed sly-miR1916 were upregulated at least by 15-fold compared to wild-type plants (Fig. 2A). As expected, the expression of miR1916 was significantly reduced in transgenic plants expressing STTM1916 (ST-1, ST-2 and ST-3) and Anti-miR1916 (Anti-1, Anti-3 and Anti-5) compared to wild-type plants (Fig. 2A). Finally, these positive plants were used for further experiments. After withholding water for 14 d, the miR1916OE transgenic tobacco plants exhibited obviously more dehydration and lower
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Fig. 1. Drought tolerance analysis of WT and transgenic tomato plants. (A) Five-week old WT, overexpression and silencing miR1916 transgenic tomato subjected to drought by withholding water. (B) Leaf survival rate. (C) The seedlings grown on 1/2 MS agar medium containing 300 mM mannitol for 14 d. (D) Root length. Data in B and D are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
Fig. 2. Drought tolerance assay of WT and transgenic tobacco plants. (A) The expression of miR1916 in WT and transgenic tobacco plants. (B) Five-week old WT, overexpression and silencing miR1916 transgenic tobacco subjected to drought by withholding water. (C) Leaf survival rate. Data in A and C are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
leaf survival rate (Fig. 2B and C), resembling the results obtained from transgenic tomato plants OE-1 (Fig. 1A and B). In contrast, a majority of the STTM1916 and Anti-miR1916 transgenic tobacco plants appeared to be more healthier and higher leaf survival rate than wild-type plants (Fig. 2B and C). Taken together, these data showed that the negative roles of miR1916 in regulating drought stress resistance in tobacco, which function may be conserved among Solanaceae plants.
3.3. Changes in the physiological indexes and ROS level in transgenic tobacco after drought treatment To further investigate the possible mechanism of the different tolerance to drought stress caused by miR1916 overexpressing or silencing, the RWC and chlorophyll content, and the accumulated levels of proline, soluble sugars and ROS were measured. After drought treatment, the RWC and chlorophyll levels in miR1916OE transgenic plants decreased more than in wild-type plants, while
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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less loss in the STTM1916 and Anti-miR1916 transgenic plants (Fig. 3A and B). Conversely, drought stress resulted in increased proline and soluble sugars concentrations in all plants, but under drought conditions higher proline and soluble sugars contents were detected in STTM1916 and Anti-miR1916 transgenic plants than in wild-type plant, with miR1916OE Plants having the lowest levels (Fig. 3C and D). These results suggested the involvement of miR1916 in the water retention capacity after drought treatment, probably as a result of changes in the osmoregulation of proline and soluble sugars in the transgenic plants. Furthermore, after exposure to drought stress, NBT and DAB staining indicated that the accumulation of H2O2 and O2- were higher level in the leaves of transgenic plants overexpressing miR1916 than in wild-type plants (Fig. 3E). Unlike the miR1916OE plants, the lower levels of ROS were observed in the leaves of STTM1916 and Anti-miR1916 transgenic plants relative to wildtype (Fig. 3E). 3.4. miR1916 negatively regulates the expression of putative target genes HDAC and STR in tobacco Using the psRNATarget software, four putative target genes of
miR1916 were selected in this study (Table S1). Theoretically, miR1916 oversupply or deficiency should downregulate or upregulate of HDAC and STR expression in tobacco (Fig. 4A). To further determine whether the changed level of miR1916 could affect these two genes, we compared the expression of HDAC and STR in miR1916OE, STTM1916 and Anti-miR1916 transgenic plants and wild-type plants by qPCR analysis. We observed a reduction in the transcript levels of HDAC and STR in miR1916OE transgenic plants in comparison with wild-type plants (Fig. 4B and C), whereas increase in STTM1916 and Anti-miR1916 transgenic plants (Fig. 4B and C), indicating that HDAC and STR are negatively regulated by miR1916 and are putative targets of miR1916 in tobacco. 4. Discussion miR1916 has previously been identified in Solanaceae, prickly pear cactus, Triticum dicoccoides and Phaseolus vulgaris [17e21]. Previous studies indicated that the expression of miR1916 was detected after drought stress [22]. Interestingly, in this study, overexpression of miR1916 reduced tolerance to drought in tomato, whereas the miR1916 silenced plants were more able to adapt to drought stress in view of the reduction and increase of leaf survival
Fig. 3. Biochemical analysis of WT and transgenic tobacco plants. (A) RWC of leaf. (B) Chlorophyll content. (C) Proline content. (D) Soluble sugar content. (E) DAB and NBT staining. Data are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Fig. 4. Target genes of miR1916 were predicted and identified. (A) Putative target genes of miR1916 were predicted in tobacco. Expression of HDAC (B) and STR (C) in WT and transgenic tobacco plants by qRT-PCR. Data in B and C are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic tobacco plants at the P < 0.05 level revealed by Duncan's multiple-range test.
ratio. Also, the miR1916-overexpressing plants grew worse and have shorter root than wild-type in the media containing mannitol, whereas the opposite is found in miR1916-silenced plants. The function of miRNAs is similar between tomato and tobacco. For example, previous studies indicated that overexpression of miR396 in tomato and tobacco both decreases resistance to pathogens [14,32]. Indeed, overexpression and silencing of miR1916 in tobacco reduces and improves drought resistance, respectively. Our results together with previous research suggest that miR1916 play a negative role in the regulation of drought stress and the expression level of miR1916 may differ in different drought stress periods. The leaf RWC is a relevant parameter for monitoring water status in plant [27]. Our results clearly show that miR1916-silenced plants had enhanced water retention capacity in comparison with wild-type. Water deficit limits the photosynthetic capacity of leaf, while chlorophyll content is indicator of photosynthetic capability of plant tissues [28]. In this study, we show that under drought stress, miR1916-silenced plants remained higher chlorophyll
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content compared to wild-type, suggesting increases in chlorophyll content contributed to the enhanced drought tolerance of the miR1916-silenced transgenic plants. Proline and soluble sugar act as major osmoprotectants to improve water retention by osmoregulation in the plant. As is known, the accumulation of proline and soluble sugar can be contributed to the stabilization of cellular structures, scavenging reactive oxygen and buffering cellular redox to counteract the effects of osmotic stress [6e8]. In this study, miR1916-silenced transgenic plants were significantly accumulated higher level of free proline and soluble sugar than wild-type. Our data also showed that the levels of ROS were significantly lower in miR1916-silenced transgenic plants than in the wild-type when exposed to drought stress, which also implies more tolerance in those transgenic plants. Therefore, miR1916 can reduce plant drought resistance through multiple ways, including suppressing proline accumulation in miR1916-overexpressing transgenic plants, and decreasing the content of soluble sugar to break energy and soluble osmotic sugar to damage cells, as well as promoting ROS accumulation toxic to cells. To further elucidate the underlying mechanisms of the enhanced drought stress tolerance in miR1916-silencing transgenic plants, we analyzed the potential target genes of miR1916 using online software, and found it targets SGN-U376418 mRNAs that encode hydroxyproline-rich glycoprotein family protein. Considering that HRGP play important roles in controlling accumulate proline in cell to adjust osmotic pressure and adapt to drought in plants [9], these results together indicate that the impact of miR1916 on proline maybe via targeting HRGP. Previous studies indicated that miR1916 regulates anthocyanins biosynthesis negatively regulating UGT in tomato [25]. Overexpression of UGT79B2/B3 significantly increased the anthocyanin accumulation and enhanced drought tolerance in Arabidopsis [33]. Of these targets, UGT, a sugar residue transfer, is an important enzyme in maintaining cell homeostasis, improving plant responses to abiotic stresses [33]. Ectopic expression of UGT85A5 in tobacco has been reported to promote the soluble sugars accumulation and enhance tolerance to the salt stress in the transgenic plants [10]. Here, we speculated that the reduction or induction of soluble sugars contents in transgenic plants after drought treatment might be due to miR1916 affecting the expression of UGT. In addition, HDACs have been previously shown to positively regulate stress responses [34]. Thus, the ectopically expressed miR1916 may be regulating HDAC expression at the transcriptional level in response to drought stress. According to previous research, the expression of STR was higher than control in early and late stage PEG drought [35]. Therefore, An increase in STR expression in miR1916-silencing plants is another reason, which means the increase of STR could also contribute to the increase of drought tolerance. Taken together, our result indicated that miR1916 negatively regulate drought tolerance in tomato and tobacco transgenic plants may be by decreasing osmoregulation and ROS-scavenging. However, the molecular mechanisms underlying the involvement of miR1916 to drought or other abiotic stresses (e.g., salt, cold and heavy metal stress) still need to be investigated. Conflicts of interest The authors declare that there is no conflict of interest in the present investigation. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China, China (Nos. 31872116 and 61872055).
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco Lei Chen a, Jun Meng b, Yushi Luan a, * a b
School of Life Sciences and Biotechnology, Dalian University of Technology, Dalian, 116024, China School of Computer Science and Technology, Dalian University of Technology, Dalian, 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2018 Accepted 27 November 2018 Available online xxx
MicroRNAs (miRNAs) play important roles in regulating plant responses to various environmental stresses. In our study, overexpression of miR1916 in tomato (OE-1) reduced its tolerance to drought. The miR1916-silenced transgenic plants (ST-1 and Anti-7) significantly increased resistance to drought stress. The transgenic tobacco plants also have a similar result in displaying the tolerance of drought. Physiological analysis revealed that miR1916 affected the osmoregulation and reactive oxygen species (ROS) accumulation. In addition, transcript levels of the miR1916 target genes, histone deacetylases (HDAC) and strictosidine synthase (STR), decreased in miR1916-overexpressing transgenic tobacco plants. Our results suggested that miR1916 is a passive regulator in the plant resistance to drought stress and has potentially impacting on abiotic stress responses in Solanaceae. © 2018 Elsevier Inc. All rights reserved.
Keywords: mir1916 Drought stress Tomato Tobacco
1. Introduction Drought is a major abiotic stress factor that affects crops growth and productivity [1]. To cope up with this adverse challenge from environment, the plants have evolved multiple mechanisms to activate stress responses at the molecular, biochemical, and physiological levels [2]. Previous studies have demonstrated that high relative water content and chlorophyll content are two typical mechanisms of resistance to drought [3e5]. Moreover, osmolyte accumulation is also a part of these adaptive mechanisms. Soluble sugars and proline can act as osmoprotectants for plants to counteract the effects drought stress by protecting the cell structures and scavenging the reactive oxygen species [6e8]. An abundance of stress-responsive genes are known to involve the responses to abiotic stress by modulating proline and soluble sugars accumulation, such as hydroxyproline-rich glycoproteins (HRGPs) and UDP-glycosyltransferases (UGTs) genes [9,10]. MicroRNAs (miRNAs) are 18e25 nucleotides small non-coding RNA molecules and negatively regulate the expression of their target genes [11]. Plant miRNAs have been shown to play a crucial
Abbreviations: HDAC, histone deacetylases; HRGP, Hydroxyproline-rich glycoproteins; miR1916, microRNA1916; ROS, reactive oxygen species; S. lycopersicum, Solanum lycopersicum; STR, strictosidine synthase; UGT, UDP-glycosyltransferases. * Corresponding author. Tel.: þ86 411 84706365; fax: 86 411 84706365. E-mail address: [email protected] (Y. Luan).
role in the regulation of switches the response to abiotic stresses [12,13]. For instance, transgenic plants that overexpress miR396a are more tolerance to abiotic stresses by negatively regulating GRFs transcription in tobacco [14]. Similarly, overexpression of miR393a in creeping bentgrass results in reduced the expressions of AsAFB2 and AsTIR1 to improve multiple stresses [15]. Unlike the other miRNAs showing positive modulation of abiotic stresses tolerance, miR417 are overexpressed in Arabidopsis and have been demonstrated to retard seed germination under salt stress [16]. Currently, miR1916 is a nonconserved miRNAs that has been discovered in tomato, tobacco, potato, eggplant, pepper, petunia, prickly pear cactus and bean [17e21]. And according to the previous relevant study, the expression of miR1916 is detected in leaf tissue only in the sample of drought stress, but not in the control sample by miRNA microarray [22]. In tomato, miR1916 in leaves are increased under cold stress treatments [23]. Similarly, treatment with ethylene induces expression of miR1916 in tomato pedicels [24]. Our previous study also found that miR1916 acts as a negative regulator in the defense responses of tomato against Phytophthora infestans and Botrytis cinerea infection [25]. Although some miR1916 expressions have been studied, the biological function of miR1916 in plants response to abiotic remains unknown. In this study, we investigated whether change in the expression of miR1916 in tomato and tobacco affect tolerance against drought stress. To explore the function of miR1916, we examined these
https://doi.org/10.1016/j.bbrc.2018.11.165 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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transgenic plants that altered water, chlorophyll, proline and soluble sugars content, and reactive oxygen species accumulation. We also revealed two target genes of miR1916, HDAC and STR, whose expression is inhibited in the transgenic plants. This finding implies that miR1916 is an underlying molecular in regulatory network of Solanaceae responds to drought stress. 2. Materials and methods
was determined using the Shan et al. method [29]. The sugar content was measured with the Li et al. method [30]. 2.7. ROS quantification After 14 d of treatment with drought stress, the tobacco leaves were harvested. These leaves were treated with nitro blue tetrazolium (NBT) and diamino benzidine (DAB) staining solution at 28 C in the dark as previously described [31].
2.1. Plant material and growth conditions 2.8. Prediction the targets Tomato, including one wild-type (Solanum lycopersicum cv. Zaofen No.2), one transgenic plant that overexpressed sly-miR1916 (OE-1) and two silenced transgenic plants (ST-3 and Anti-7) were grown in a greenhouse at 23e27 C/18e22 C under 16 h photoperiod. Tobacco (Nicotiana tabacum cv. NC89) was used for heterologous transformation.
The miR1916 putative targets were predicted in tobacco using psRNATarget software (version 2017) (http://plantgrn.noble.org/ psRNATarget/) and the library (Nicotiana tabacum(tobacco), unigene, SGN unigene). The expectation and top target genes adjusted to 5.0 and 250, respectively.
2.2. Tobacco transformation and identification
2.9. Statistical analysis
The Agrobacterrium tumefaciens strain GV3101 harboring the vectors (pBI121-MIR1916, pBI121-STTM1916 and pBI121-AntimiR1916) were used for transformation. Positive transgenic plants were obtained on 1/2 MS medium containing 50 mg l1 kanamycin and identified by PCR.
The results were analyzed using Gradpad Prism 5.0 software. The data are expressed as means ± SD. All experiments were repeated three times. The statistical significances were considered at the P < 0.05 level revealed by Duncan's multiple-range test. 3. Results
2.3. Drought stress tolerance assays of tomato 3.1. miR1916 negatively regulates drought tolerance in tomato For drought tolerance tests, 4-week-old wild-type, overexpression of miR1916 and miR1916-silenced plants were withheld from watering for 14 d. The phenotypes and the leaf survival rate were recorded. The wild-type and transgenic tomato seedlings were grown on 1/2 MS medium for 7 d, and then transferred into 1/ 2 MS medium with 300 mM mannitol, and the root length was calculated after 14 d. 2.4. RNA extraction and qRT-PCR Total RNA was prepared from the leaves of tobacco seedlings using the RNAiso Plus (Takara Dalian, China). For miRNA analysis, 5 mg of total RNA was reverse-transcribed into cDNA using the MirX miRNA First-Strand Synthesis Kit (Takara Dalian, China) according to the supplier's protocol. For target genes analysis, the first strand cDNA was synthesized from 300 ng RNA with the PrimeScript™ RT Master Mix (Perfect Real Time) (Takara Dalian, China). The relative expression levels were analyzed using the deltaedelta Ct method [26]. The data were collected in an ABI7500 thermal cycler with Real-time PCR System using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Dalian, China). The primers used in this study are listed in Table S1. 2.5. Transgenic tobacco drought stress treatments The wild-type and transgenic tobacco plants were grown on 1/2 MS medium, and then transferred them into sterilized soil in a greenhouse during root formation (16 h light/8 h dark, 25 ± 2/ 20 ± 2 C). After 2 weeks, they were treated without water for 14 d. The phenotypes and the leaf survival rate were recorded. 2.6. Determination of relative water, chlorophyll, proline and soluble sugars content The relative water content (RWC) of the leaf was determined according to previously described method [27]. Chlorophyll content was measured as previously described [28]. The proline content
The three independent transgenic tomato plants (OE 1, ST-3 and Anti-7), which are shown to change the expression of miR1916 and to display affected plant tolerance to P. infestans and B.cinerea infection in our previous studies [25]. The expression of miR1916 was detected in leaf tissue after drought stress [22]. To investigate whether miR1916 plays a role in the drought-stress tolerance, we selected these transgenic plants for use in this study. After 14 d of growth in the absence of irrigation, OE 1 displayed more wilt symptoms and lower leaf survival rate than wild-type plants (Fig. 1A and B). However, higher levels of leaf survival rates were observed in the ST-3 and Anti-7 transgenic plants compared to wild-type plants after drought stress (Fig. 1A and B). Concomitantly, wild-type and transgenic tomato seedlings were cultured in 1/2 MS agar medium with 300 mM mannitol. After 14 d treatment, the root growth was analyzed for evaluate the drought stress. The roots length of OE-1 transgenic plants was shorter than wild-type, while the ST-3 and Anti-7 transgenic plants was observed a significant longer (Fig. 1C and D). These results revealed that by regulating the expression of miR1916 in tomato, the plant tolerance and resistance to drought stress could be altered. 3.2. Overexpression and silencing of miR1916 affects the tolerance of tobacco to drought stresses To elucidate miR1916 functions in Solanaceae, transgenic tobacco plants overexpressing or silencing miR1916 were generated. The expression of miR1916 in all three transgenic plants miR1916OE (OE-1, OE-4 and OE-5) that overexpressed sly-miR1916 were upregulated at least by 15-fold compared to wild-type plants (Fig. 2A). As expected, the expression of miR1916 was significantly reduced in transgenic plants expressing STTM1916 (ST-1, ST-2 and ST-3) and Anti-miR1916 (Anti-1, Anti-3 and Anti-5) compared to wild-type plants (Fig. 2A). Finally, these positive plants were used for further experiments. After withholding water for 14 d, the miR1916OE transgenic tobacco plants exhibited obviously more dehydration and lower
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Fig. 1. Drought tolerance analysis of WT and transgenic tomato plants. (A) Five-week old WT, overexpression and silencing miR1916 transgenic tomato subjected to drought by withholding water. (B) Leaf survival rate. (C) The seedlings grown on 1/2 MS agar medium containing 300 mM mannitol for 14 d. (D) Root length. Data in B and D are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
Fig. 2. Drought tolerance assay of WT and transgenic tobacco plants. (A) The expression of miR1916 in WT and transgenic tobacco plants. (B) Five-week old WT, overexpression and silencing miR1916 transgenic tobacco subjected to drought by withholding water. (C) Leaf survival rate. Data in A and C are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
leaf survival rate (Fig. 2B and C), resembling the results obtained from transgenic tomato plants OE-1 (Fig. 1A and B). In contrast, a majority of the STTM1916 and Anti-miR1916 transgenic tobacco plants appeared to be more healthier and higher leaf survival rate than wild-type plants (Fig. 2B and C). Taken together, these data showed that the negative roles of miR1916 in regulating drought stress resistance in tobacco, which function may be conserved among Solanaceae plants.
3.3. Changes in the physiological indexes and ROS level in transgenic tobacco after drought treatment To further investigate the possible mechanism of the different tolerance to drought stress caused by miR1916 overexpressing or silencing, the RWC and chlorophyll content, and the accumulated levels of proline, soluble sugars and ROS were measured. After drought treatment, the RWC and chlorophyll levels in miR1916OE transgenic plants decreased more than in wild-type plants, while
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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less loss in the STTM1916 and Anti-miR1916 transgenic plants (Fig. 3A and B). Conversely, drought stress resulted in increased proline and soluble sugars concentrations in all plants, but under drought conditions higher proline and soluble sugars contents were detected in STTM1916 and Anti-miR1916 transgenic plants than in wild-type plant, with miR1916OE Plants having the lowest levels (Fig. 3C and D). These results suggested the involvement of miR1916 in the water retention capacity after drought treatment, probably as a result of changes in the osmoregulation of proline and soluble sugars in the transgenic plants. Furthermore, after exposure to drought stress, NBT and DAB staining indicated that the accumulation of H2O2 and O2- were higher level in the leaves of transgenic plants overexpressing miR1916 than in wild-type plants (Fig. 3E). Unlike the miR1916OE plants, the lower levels of ROS were observed in the leaves of STTM1916 and Anti-miR1916 transgenic plants relative to wildtype (Fig. 3E). 3.4. miR1916 negatively regulates the expression of putative target genes HDAC and STR in tobacco Using the psRNATarget software, four putative target genes of
miR1916 were selected in this study (Table S1). Theoretically, miR1916 oversupply or deficiency should downregulate or upregulate of HDAC and STR expression in tobacco (Fig. 4A). To further determine whether the changed level of miR1916 could affect these two genes, we compared the expression of HDAC and STR in miR1916OE, STTM1916 and Anti-miR1916 transgenic plants and wild-type plants by qPCR analysis. We observed a reduction in the transcript levels of HDAC and STR in miR1916OE transgenic plants in comparison with wild-type plants (Fig. 4B and C), whereas increase in STTM1916 and Anti-miR1916 transgenic plants (Fig. 4B and C), indicating that HDAC and STR are negatively regulated by miR1916 and are putative targets of miR1916 in tobacco. 4. Discussion miR1916 has previously been identified in Solanaceae, prickly pear cactus, Triticum dicoccoides and Phaseolus vulgaris [17e21]. Previous studies indicated that the expression of miR1916 was detected after drought stress [22]. Interestingly, in this study, overexpression of miR1916 reduced tolerance to drought in tomato, whereas the miR1916 silenced plants were more able to adapt to drought stress in view of the reduction and increase of leaf survival
Fig. 3. Biochemical analysis of WT and transgenic tobacco plants. (A) RWC of leaf. (B) Chlorophyll content. (C) Proline content. (D) Soluble sugar content. (E) DAB and NBT staining. Data are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic plants at the P < 0.05 level revealed by Duncan's multiple-range test.
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Fig. 4. Target genes of miR1916 were predicted and identified. (A) Putative target genes of miR1916 were predicted in tobacco. Expression of HDAC (B) and STR (C) in WT and transgenic tobacco plants by qRT-PCR. Data in B and C are the means ± SD from three independent experiments. Letters on top of the bar charts indicate significant differences between WT and transgenic tobacco plants at the P < 0.05 level revealed by Duncan's multiple-range test.
ratio. Also, the miR1916-overexpressing plants grew worse and have shorter root than wild-type in the media containing mannitol, whereas the opposite is found in miR1916-silenced plants. The function of miRNAs is similar between tomato and tobacco. For example, previous studies indicated that overexpression of miR396 in tomato and tobacco both decreases resistance to pathogens [14,32]. Indeed, overexpression and silencing of miR1916 in tobacco reduces and improves drought resistance, respectively. Our results together with previous research suggest that miR1916 play a negative role in the regulation of drought stress and the expression level of miR1916 may differ in different drought stress periods. The leaf RWC is a relevant parameter for monitoring water status in plant [27]. Our results clearly show that miR1916-silenced plants had enhanced water retention capacity in comparison with wild-type. Water deficit limits the photosynthetic capacity of leaf, while chlorophyll content is indicator of photosynthetic capability of plant tissues [28]. In this study, we show that under drought stress, miR1916-silenced plants remained higher chlorophyll
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content compared to wild-type, suggesting increases in chlorophyll content contributed to the enhanced drought tolerance of the miR1916-silenced transgenic plants. Proline and soluble sugar act as major osmoprotectants to improve water retention by osmoregulation in the plant. As is known, the accumulation of proline and soluble sugar can be contributed to the stabilization of cellular structures, scavenging reactive oxygen and buffering cellular redox to counteract the effects of osmotic stress [6e8]. In this study, miR1916-silenced transgenic plants were significantly accumulated higher level of free proline and soluble sugar than wild-type. Our data also showed that the levels of ROS were significantly lower in miR1916-silenced transgenic plants than in the wild-type when exposed to drought stress, which also implies more tolerance in those transgenic plants. Therefore, miR1916 can reduce plant drought resistance through multiple ways, including suppressing proline accumulation in miR1916-overexpressing transgenic plants, and decreasing the content of soluble sugar to break energy and soluble osmotic sugar to damage cells, as well as promoting ROS accumulation toxic to cells. To further elucidate the underlying mechanisms of the enhanced drought stress tolerance in miR1916-silencing transgenic plants, we analyzed the potential target genes of miR1916 using online software, and found it targets SGN-U376418 mRNAs that encode hydroxyproline-rich glycoprotein family protein. Considering that HRGP play important roles in controlling accumulate proline in cell to adjust osmotic pressure and adapt to drought in plants [9], these results together indicate that the impact of miR1916 on proline maybe via targeting HRGP. Previous studies indicated that miR1916 regulates anthocyanins biosynthesis negatively regulating UGT in tomato [25]. Overexpression of UGT79B2/B3 significantly increased the anthocyanin accumulation and enhanced drought tolerance in Arabidopsis [33]. Of these targets, UGT, a sugar residue transfer, is an important enzyme in maintaining cell homeostasis, improving plant responses to abiotic stresses [33]. Ectopic expression of UGT85A5 in tobacco has been reported to promote the soluble sugars accumulation and enhance tolerance to the salt stress in the transgenic plants [10]. Here, we speculated that the reduction or induction of soluble sugars contents in transgenic plants after drought treatment might be due to miR1916 affecting the expression of UGT. In addition, HDACs have been previously shown to positively regulate stress responses [34]. Thus, the ectopically expressed miR1916 may be regulating HDAC expression at the transcriptional level in response to drought stress. According to previous research, the expression of STR was higher than control in early and late stage PEG drought [35]. Therefore, An increase in STR expression in miR1916-silencing plants is another reason, which means the increase of STR could also contribute to the increase of drought tolerance. Taken together, our result indicated that miR1916 negatively regulate drought tolerance in tomato and tobacco transgenic plants may be by decreasing osmoregulation and ROS-scavenging. However, the molecular mechanisms underlying the involvement of miR1916 to drought or other abiotic stresses (e.g., salt, cold and heavy metal stress) still need to be investigated. Conflicts of interest The authors declare that there is no conflict of interest in the present investigation. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China, China (Nos. 31872116 and 61872055).
Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165
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Please cite this article as: L. Chen et al., miR1916 plays a role as a negative regulator in drought stress resistance in tomato and tobacco, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.165