- Email: [email protected]
Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis
Journal Pre-proof Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis Yongheng Zhang, Siqin Wan, Xianghong Liu, Jingyuan He, Long Cheng, Mengsha Duan, Huan Liu, Weidong Wang, Youben Yu PII:
S0981-9428(20)30089-9
DOI:
https://doi.org/10.1016/j.plaphy.2020.02.035
Reference:
PLAPHY 6069
To appear in:
Plant Physiology and Biochemistry
Received Date: 23 December 2019 Revised Date:
9 February 2020
Accepted Date: 25 February 2020
Please cite this article as: Y. Zhang, S. Wan, X. Liu, J. He, L. Cheng, M. Duan, H. Liu, W. Wang, Y. Yu, Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2020.02.035. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.
Contribution: Youben Yu, Yongheng Zhang and Siqing Wan were responsible for design of and conducting the experiments and preparation of the manuscript. Xianghong Liu, Jingyuan He and Long Cheng were responsible for analysis of data. Mengsha Duan, Huan Liu, Weidong Wang and Youben Yu edited and revised the manuscript.
Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis Yongheng Zhang
1
1 Siqin Wan , Xianghong Liu, Jingyuan He, Long Cheng,
Mengsha Duan, Huan Liu, Weidong Wang, Youben YU* College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China.
1
These authors contributed equally to this work.
*Author for correspondence: Youben Yu E-mail: [email protected]; Phone: +86 187-2956-5376
Acknowledgements This work was supported by the earmarked fund for the Modern Agro-industry Technology Research System (CARS-19), the Agricultural Special Fund Project of Shaanxi Province, and the special fund for the University-Supported Extension Model (XTG2019-04).
Conflict of interest statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abstract: Drought is a major factor limiting crop productivity and quality. Sucrose non-fermenting-1 (SNF1)-related protein kinase 2s (SnRK2s) play critical roles in plant abiotic stress responses, especially in drought stress. However, knowledge regarding the functional roles of SnRK2s in drought stress and their underlying
mechanisms is relatively limited in tea plant. In this study, CsSnRK2.5, a PEG 6000and ABA-induced SnRK2 gene from tea plant, was overexpressed in Arabidopsis to investigate its potential function in drought stress response. The results showed that overexpression of CsSnRK2.5 resulted in enhanced drought tolerance, as indicated by an amelioration of the changes in various physiological indexes, including a decreased rate of water loss and decreased accumulation of ROS and MDA. In addition, CsSnRK2.5 overexpression conferred hypersensitivity to exogenous ABA, and transgenic plants exhibited improved ABA-mediated stomatal closure compared to WT plants. Moreover, the expression of some stress response genes, including AtRAB18 and AtRD29b, was more strongly induced in transgenic plants than in the WT when subjected to ABA and drought treatments. Taken together, our results indicate that CsSnRK2.5 is a positive regulator of ABA-regulated drought stress responses.
Keywords: SnRK2 gene, Camellia sinensis, drought stress, abscisic acid (ABA)
1. Introduction Drought is a widely distributed environmental stress that can greatly limit plant growth and development, and plants have evolved a series of intricate strategies to survive this stress (Blum. 1996). In these complex regulatory pathways, the regulation of protein phosphorylation and dephosphorylation is mediated by protein kinases and protein phosphatases, respectively, and these processes significantly influence stress signal transduction (Fujita et al., 2006). Drought often induces hyperosmotic stress (also known as osmotic stress). When plants are exposed to osmotic stress, many protein kinases are rapidly activated, such as mitogen-activated protein kinases (MAPKs),
calcium-dependent
protein
kinases
(CDPKs),
calcineurin
B-like
(CBL)-interacting protein kinases (CIPKs), and members of the sucrose non-fermenting-1 (SNF1)-related protein kinase 2 (SnRK2) family (Boudsocq et al., 2004, Boudsocq et al., 2005, Droillard et al., 2004, Fujii et al., 2012, Ichimura et al., 2000, Kobayashi et al., 2004).
SnRK2s, which are widespread in many plants, are serine/threonine protein kinases with a molecular weight of approximately 40 kDa (Kulik et al., 2011). In recent decades, the SnRK2 gene family has been identified in Arabidopsis (Boudsocq et al., 2004), Oryza sativa (Kobayashi et al., 2004), Zea mays (Huai et al., 2008), grapevine (Liu et al., 2016), wheat (Zhang et al., 2016), etc. A series of studies of SnRK2 members indicated that they were divided into three groups based on phylogenetic analysis, and members of these different groups showed different responses to ABA. Specifically, the members of group I were not activated by ABA, members of group II were not activated or were only weakly activated by ABA, while the members of group III were strongly activated by ABA (Boudsocq et al., 2004, Kobayashi et al., 2004, Kulik et al., 2011). Recently, ABA-activated SnRK2s (mainly referred to as members of group III) have gained a great deal of attention due to the connection between ABA receptors (PYR/PYL/RCAR), protein phosphatase 2Cs (PP2Cs) and SnRK2s (Fujii et al., 2009, Ma et al., 2009, Park et al., 2009, Umezawa et al., 2009). In the absence of ABA, the phosphatase activities of SnRK2s were suppressed by clade A PP2Cs in an interactive manner, while in the presence of ABA, the inhibition of PP2Cs by PYR/PYL/RCARs releases SnRK2s, and active SnRK2s phosphorylate a series of substrate proteins, including transcription factors (Kobayashi et al., 2005, Yoshida et al., 2015), ion channels (Geiger et al., 2009, Sato et al., 2009) and other stress-related proteins (Sirichandra et al., 2009), as part of the osmotic stress response. For example, AtSnRK2.2, AtSnRK2.3 and AtSnRK2.6 phosphorylate AREB/ABF transcription factors in response to osmotic stress in Arabidopsis (Fujita et al., 2013, Furihata et al., 2006, Yoshida et al., 2015). The remaining SnRK2s, which are not activated by ABA, are thought to function in plant ABA-independent osmotic responses as well, as evidenced by a clear disruption of normal osmotic stress responses in the Arabidopsis snrk2 decuple mutant lacking all ten SnRK2s (Fujii et al., 2011). Furthermore, in duodecuple Arabidopsis ABA receptor mutants, the activation of SnRK2 protein kinases by ABA was blocked, but osmotic stress activation of SnRK2s was enhanced (Zhao et al., 2018). These findings suggest that SnRK2s play vital roles in plant
osmotic responses in both the ABA-dependent and ABA-independent pathways. Although SnRK2s are mainly regulated at the posttranslational level by phosphorylation, there is a large amount of evidence indicating that the expression of genes encoding these kinases is also regulated in response to osmotic stress (Kulik et al., 2011). In wheat, TaSnRK2.3 was induced by PEG and ABA, and TaSnRK2.3 overexpression significantly improved the drought tolerance of transgenic Arabidopsis (Tian et al., 2013). Similarly, in apple, MpSnRK2.10 was induced by drought and ABA treatment, and enhanced drought tolerance was found in both transgenic apples and Arabidopsis (Shao et al., 2019). ZmSAPK8 (ZmOST1), a SnRK2 from Zea mays, was found to be induced by drought and ABA and to function in response to drought stress (Wu et al., 2019). These studies imply the potential value of SnRK2s for engineering abiotic stress tolerance across crop species. The tea plant [Camellia sinensis (L.) O. Kuntze], a commercially important perennial evergreen woody crop widely cultivated throughout the world, can easily suffer drought stress (Parmar et al., 2019). In our previous research, SnRK2 members in tea plant were identified, and several CsSnRK2s were found to be induced by stress treatments (Zhang et al., 2018). One of these, CsSnRK2.5, was highly similar to AtSnRK2.6 (AtOST1) in predicted polypeptides and significantly induced by PEG 6000 and ABA treatment, implying a positive role in the drought response of tea plant. In this study, to further investigate the function of CsSnRK2.5, the drought stress tolerance and ABA sensitivity of both CsSnRK2.5-overexpressing and WT plants were investigated, and our results revealed that CsSnRK2.5 positively regulates drought response in transgenic Arabidopsis via ABA-mediated stomatal closure.
2. Materials and Methods 2.1 Plant materials and growing conditions Seeds of Arabidopsis wild-type (WT) Columbia (Col-0) and transgenic lines were surface-sterilized with 20% NaClO (with 0.1% Triton X-100), sown on MS agar medium and then placed in a controlled growth chamber at 16/8 h (25/22 °C) day/night conditions with a relative humidity of approximately 65%. Seedlings at the
four-leaf stage were transferred to pots with soil for transformation or abiotic stress tolerance assays. 2.2. Sequence and phylogenetic analysis of CsSnRK2.5 and its homologues Multiple amino acid alignment was performed using DNAMAN 6 software, and a phylogenetic tree was constructed using the neighbour-joining method with 1000 bootstraps using MEGA7 software. 2.3 Plasmid construction and transformation The coding region of CsSnRK2.5 was amplified and cloned into the Spe I sites of the 3302Y vector to generate the 35S::CsSnRK2.5 construct. Then, the construct was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into WT by the floral dip method (Zhang et al., 2006). The T3 generation homozygous lines (L2 and L3) with highly expressed CsSnRK2.5 genes and WT were used for further analysis. The primers used are listed in Supplementary Table S1. 2.4 Yeast Two-Hybrid Analysis For yeast interaction assays, the full-length cDNAs of CsAHG3 and CsABI1 were cloned into the pGADT7 vector, and CsSnRK2.5 was cloned into the pGBKT7 vector. The pairs of vectors were cotransformed into yeast strain Y2H and grown on SD/-Leu-Trp medium at 30 °C for 3 days, and the clones were transferred into SD/-Leu-Trp-His-Ade medium at 30 °C for 3–5 days. To confirm the results, positive yeast clones were adjusted to OD 0.6, spotted in serial dilutions (1:1, 1:10, 1:100 and 1:1000) and cultured on SD/-Leu-Trp-His-Ade medium. The primers used for gene amplification are included in the primer list (Table S1). 2.5 Stress tolerance assays For the drought treatment in soil, WT and transgenic seeds were germinated and grown to the four-leaf stage on MS agar medium. Then, the seedlings were transferred into soil and grown under unstressed conditions. After one week, watering was withheld until stress symptoms occurred, and the plants were rewatered three days after wilting was observed. Survival rate was counted three times, and 48 seedlings of each genotype were treated intotal. The sensitivity of seed germination to mannitol and ABA was assayed on MS
agar medium containing mannitol (0 and 150 mM) or ABA (0 and 1 µM). To break seed dormancy, seeds were sterilized and incubated at 4 °C for 2 days, sown on MS agar medium and placed in a growth chamber. Seeds in the mannitol treatment were considered germinated when the radicle completely penetrated the seed coat, and the germination rate was calculated four days after the seeds were sown on MS agar medium. The cotyledon greening rate of seedlings in the ABA treatment was calculated ten days after the seeds were sown on MS agar medium. Root extension was also analysed under mannitol and ABA treatment. Seedlings were transferred to MS medium containing mannitol and ABA when their cotyledons turned green and their roots began to extend on MS medium. The root length was measured three days after vertical growth was observed. 2.6 Measurement of reactive oxygen species (ROS) and malondialdehyde (MDA) ROS and MDA contents were detected in transgenic and WT plants subjected to drought. Drought stress was carried out in soil as described above, and the leaves from at least three individual plants were sampled. Then, 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining was performed to detect the accumulation of hydrogen peroxide (H2O2) and superoxide (O2−) according to previously described methods (Yang et al., 2018). To determine the MDA content, a thiobarbituric acid (TBA) reaction was performed according to a previously described method (Draper et al., 1993) 2.7 Water loss rate analysis Ten leaves from five individual four-week-old plants of each genotype were detached, and the fresh weight (W1) was measured immediately. Then, the leaves were placed in an indoor environment, and the weight (W2) was measured at 1, 2, 4, 6, and 8 h. The water loss rate was calculated as follows: water loss rate = (W1-W2)/W1 × 100%. 2.8 Measurement of stomatal movement Four-week-old Arabidopsis plants were grown under mild conditions. To encourage the stomata to open, plants were exposed to light for 3 h. Then, rosette leaves were detached from three individual plants of each genotype and immediately
incubated in stomatal closure buffer (25 mM MES, pH 7.0, 10 mM KCl, and 1 mM CaCl2) containing different concentrations of ABA (0, 0.5 and 1.0 µM) for 2 h. Afterward, leaves were immediately fixed with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS, pH 6.8) to avoid any alterations during sample preparation. After being rinsed five times with PBS, the leaves were dehydrated through a graded ethanol series, vacuum-dried, and gold-coated. Observations were made with a JSM- 6360LV microscope (JEOL Ltd., Tokyo, Japan). Six to ten stomatal cells per leaf abaxial peel were captured randomly, and the lengths and widths of the stomata were measured from 30 stomata using ImageJ software. Then, the width/length ratio (W/L) was calculated. 2.9. Quantitative real-time PCR (qPCR) analysis Arabidopsis leaves of WT and CsSnRK2.5-overexpressing genotypes under normal, ABA (20 µM) and drought conditions were collected. Total RNA was extracted using the SimplyP Total RNA Extraction Kit (#BSC52S1, BioFlux, Hangzhou, China). First-strand cDNAs were synthesized from RNA using 5× All-in-one RT MasterMix (#G485, Applied Biological Materials, Canada). Afterward, the reaction product was diluted to 200 ng µL−1 with sterile water. Quantitative real-time RT-PCR (qRT-PCR) was performed on a QuantStudio®5 (Life Technologies, USA) using EvaGreen 2× qPCR MasterMix-ROX (#MasterMix-R, Applied Biological Materials, Canada) according to the manufacturer’s instructions. Three independent biological replicates were performed for each experiment. The relative expression levels of these genes were analysed by the 2−∆∆Ct method (Livak et al., 2001). The primers used for qRT-PCR are listed in Table S1.
3. Results 3.1 CsSnRK2.5 is a homologue of AtOST1 AtSnRK2.6 (AtOST1), a group III member in Arabidopsis, has been well studied in regard to ABA-mediated stomatal movement and drought responses. In this study, we found that CsSnRK2.5 was a homologue of AtOST1, sharing 89.01% amino acid identity, especially in some function-related domains, such as the ATP binding loop,
activation loop, SnRK2 box and ABA box, which were almost entirely conserved (Fig. 1A). OST1 is involved in the ABA response in Arabidopsis, and clade A PP2C, a vital element of the ABA regulation network, interacts with and inhibits the phosphatase activities of AtOST1. Therefore, we examined the interaction between CsSnRK2.5 and two clade A PP2Cs in tea plant using the yeast two-hybrid system, and as expected, CsSnRK2.5 was found to interact with CsAHG3 (Fig. 1B). 3.2 Overexpression of CsSnRK2.5 enhances drought tolerance in Arabidopsis To investigate the role of CsSnRK2.5 in the plant response to drought stress, the CsSnRK2.5 gene was overexpressed in Arabidopsis. T3 generation homozygous lines (L2 and L3) with highly expressed CsSnRK2.5 genes were selected for further analysis (Fig. S1). Under long-term water shortage, the WT exhibited a more severely wilted phenotype than the L2 and L3 transgenic lines. After watering was resumed, the growth of the WT was weaker than that of the L2 and L3 transgenic lines (Fig. 2A, B). Additionally, the water loss rate of the detached leaves showed that the L2 and L3 transgenic lines had a stronger water-holding ability than the WT (Fig. 2C). Taken together, our results indicate that overexpression of CsSnRK2.5 improved the drought tolerance of transgenic Arabidopsis. To further confirm the drought tolerance of the transgenic lines, mannitol treatment was employed to mimic drought stress, and germination and root extension under normal conditions and mannitol treatment were examined. As expected, in the absence of mannitol, the germination rates and root lengths of the two overexpression lines were indistinguishable from those of the WT (Fig. 3A, B). The application of mannitol significantly inhibited the germination and root elongation of both the WT and transgenic lines; however, the germination and root growth of the two overexpression lines was less inhibited than that of the WT plants, indicating that CsSnRK2.5 overexpression in Arabidopsis enhanced tolerance to mannitol-induced drought stress. 3.3 Overexpression of CsSnRK2.5 alleviates ROS and MDA accumulation in Arabidopsis Drought usually induces the excessive accumulation of ROS, which eventually
leads to oxidative stress (Verslues et al., 2006). To explore whether the enhanced drought tolerance was associated with the rescue of ROS levels in transgenic lines, the accumulation of H2O2 and O2− in leaves under normal and drought conditions was measured using DAB and NBT staining. The results show that under normal conditions, ROS levels in WT and transgenic lines were insignificant, while when exposed to drought stress, the L2 and L3 transgenic lines accumulated less ROS than the WT (Fig. 4A, B). MDA is considered a marker of membrane lipid peroxidation caused by excessive ROS (Jambunathan. 2010), so we measured the MDA contents of WT and transgenic lines under drought stress. The WT consistently accumulated more MDA than did the transgenic lines under drought conditions (Fig. 4C, D), implying that CsSnRK2.5 overexpression in Arabidopsis alleviates ROS and MDA accumulation under drought stress. 3.4 Overexpression of CsSnRK2.5 increases ABA sensitivity in transgenic Arabidopsis The ABA signalling pathway is central to the drought response in plants, and ABA accumulates rapidly in the roots and leaves when a plant is subjected to drought stress (Kuromori et al., 2018). CsSnRK2.5, which is an important element in the ABA signalling pathway, was found to interact with a clade A PP2C member in tea plant, which prompted us to explore whether CsSnRK2.5 overexpression in Arabidopsis affects seedling growth under ABA treatment. As the result shows, at day 10 under treatment with 1.0 µM ABA, the cotyledon greening rate of the transgenic lines was significantly higher than that of the WT (Fig. 5A, B). Moreover, the root extension length of the WT three days after transfer to the ABA treatment was greater than those of the transgenic lines (Fig. 5C, D). Taken together, our data indicate that CsSnRK2.5 overexpression in Arabidopsis increases plant sensitivity to ABA. 3.5 Overexpression of CsSnRK2.5 promotes ABA-mediated stomatal closure in Arabidopsis ABA-mediated stomatal closure plays a vital role in plant stress responses by reducing water loss (Kuromori et al., 2018). To investigate whether the ABA-mediated stomatal closure of transgenic lines was distinct from that of the WT,
detached leaves of transgenic and WT lines were treated with exogenous ABA, and the results showed that the stomatal apertures of overexpression lines were comparable to those of the WT when the stomatal closure buffer contained 0 µM ABA (Fig. 6A, B). The application of ABA significantly promoted stomatal closure in a dose-dependent manner in both the WT and transgenic lines, and the stomatal apertures of the transgenic lines under both the 0.5 and 1.0 µM ABA treatments were smaller than that of the WT (Fig. 6A, B), suggesting that CsSnRK2.5 overexpression in Arabidopsis promotes ABA-mediated stomatal closure. 3.6 Overexpression of CsSnRK2.5 activates the expression of stress-responsive genes in Arabidopsis Previous
studies
demonstrated
that
the
elevated
stress
tolerance
of
SnRK2-overexpressing plants was accompanied by the upregulation of a series of stress-responsive genes (Feng et al., 2018, Shao et al., 2019, Ying et al., 2011). Thus, to further understand the role of CsSnRK2.5 overexpression in drought response pathways, the expression of drought-responsive genes in Arabidopsis, including AtABF1, AtRAB18, AtRD29a and AtRD29b, was evaluated in both the CsSnRK2.5-overexpressing and WT lines using qRT-PCR. As shown in Fig. 7, under normal conditions, the expression of these analysed genes showed no difference, while not coincidentally, all these genes were significantly induced by drought stress in both genotypes; however, notably higher expression levels of these genes, with the exception of AtABF1, were observed in both overexpression lines than in the WT. In addition, higher expression levels of AtRD29b and AtRAB18 were observed in both overexpression lines than in the WT under ABA treatment. These results indicated that overexpression of CsSnRK2.5 leads to additional upregulation of some stress-responsive genes in Arabidopsis under drought and ABA treatment.
4. Discussion A series of studies have indicated that SnRK2s play a critical role in the response to drought stress in both the ABA-dependent and ABA-independent pathways in plants (Lou et al., 2017, Wang et al., 2018, Zhao et al., 2018). For example, OsSAPK2
(Lou et al., 2017) and OsSAPK9 (Dey et al., 2016) were found to play critical roles in the ABA-mediated drought response, while AtSnRK2.10 and TaSnRK2.7 regulate the drought response in an ABA-independent manner (Maszkowska et al., 2019, Zhang et al., 2011). In our previous research, CsSnRK2.5 was found to be significantly induced by PEG 6000 and ABA treatment (Zhang et al., 2018), which prompted us to investigate its role in the drought response and ABA signalling pathway. In the current study, transgenic Arabidopsis plants overexpressing CsSnRK2.5 exhibited a stress-tolerant phenotype compared to the WT when subjected to drought and showed a decreased water loss rate. In addition, CsSnRK2.5 overexpression significantly decreased the inhibition of germination and root growth by mannitol. These results were similar to those of the corresponding MdSnRK2.10 (Shao et al., 2019), BdSnRK2.9 (Wang et al., 2015), TaSnRK2.8 (Zhang et al., 2010), and OsSAPK4-overexpressing plants (Diedhiou et al., 2008), indicating that CsSnRK2.5 overexpression confers tolerance to drought stress in Arabidopsis. Physiological and biochemical changes in plants to some extent reflect the capacity of plants to cope with adversity (Jambunathan. 2010). Drought stress often causes excessive accumulation of ROS and results in oxidative damage in plants (Verslues et al., 2006). In particular, lipid peroxidation is an indicator of oxidative damage at the cellular level and has often been estimated by the content of MDA (Verslues et al., 2006). Similar to studies of other SnRK2 genes (Shao et al., 2019, Tian
et
al.,
2013,
Zhang
et
al.,
2010),
the
ROS
contents
of
two
CsSnRK2.5-overexpressing lines were notably lower than that of the WT under drought stress. Simultaneously, the MDA contents of the two transgenic lines followed the same trends. These results suggested that transgenic plants suffered milder oxidative damage than WT plants under drought stress and that overexpression of CsSnRK2.5 conferred tolerance to drought in Arabidopsis plants at the cellular level, which constituted a foundation for their stress-tolerant phenotype. Drought-induced ABA biosynthesis rapidly activates ABA-dependent SnRK2s; thus, a series of stress-related genes downstream of SnRK2 kinases are regulated, leading to enhanced drought stress tolerance (Fujita et al., 2011). In this study,
CsSnRK2.5 was found to interact with clade A PP2Cs in tea plant, and CsSnRK2.5 overexpression in Arabidopsis increased ABA sensitivity. These findings were consistent with those for its counterparts in other plants (Shao et al., 2019, Umezawa et al., 2009, Wang et al., 2015, Zhang et al., 2016), implying that CsSnRK2.5 was involved in the ABA signal transduction pathway. In Arabidopsis, group III SnRK2s were reported as critical regulators in ABA signalling and can phosphorylate AREB/ABFs to regulate the expression of a number of genes, including AtRAB18, RD29a and RD29b (Fujita et al., 2005, Kim et al., 2004, Msanne et al., 2011, Yoshida et al., 2015). Thus, we examined whether the expression of the genes mentioned above differed in WT and CsSnRK2.5 overexpression lines under drought and ABA treatment, and it was not surprising that, under drought stress, the expression levels of AtRAB18, AtRD29a and AtRD29b in the CsSnRK2.5 overexpression lines were significantly higher than those in the WT, indicating that drought stress responses were strengthened in CsSnRK2.5 overexpression lines. Furthermore, these genes, with the exception of AtRD29a, showed additional upregulation under ABA treatment, which means that CsSnRK2.5 overexpression elevated ABA signal output in transgenic Arabidopsis and thus may work in concert with the increased ABA sensitivity of transgenic plants. We noticed that the expression of the AREB/ABF transcription factor gene AtABF1 showed no difference between CsSnRK2.5 overexpression lines and WT under ABA and drought conditions, and similar results were observed in the study of MdSnRK2.10 (Shao et al. 2019). Though the expression of AtABF1 was not altered, AtABF1 may still be involved in the drought response in transgenic plants through CsSnRK2.5-mediated phosphorylation, because such posttranslational modifications of AtABF1 are critical for its function in regulating downstream gene expression and may be responsible for the additional upregulation of AtRD29b and AtRAB18 under ABA and drought conditions in CsSnRK2.5 overexpression lines. Stomatal closure provides a critical strategy for drought resistance by decreasing water loss (Brodribb et al., 2003). In recent decades, numerous elements involved in stomatal closure have been characterized; in particular, SnRK2 members were found
to play a positive role in ABA-mediated stomatal closure (Belin et al., 2006, Mustilli et al., 2002, Wu et al., 2019). In previous studies, AtOST1 was reported as a positive regulator in ABA-dependent stomatal closure (Acharya et al., 2013, Yoshida et al., 2002); furthermore, ZmOST1, a homologue of AtOST1, also positively regulated ABA-dependent stomatal closure (Wu et al., 2019). In the current study, CsSnRK2.5 overexpression in Arabidopsis resulted in increased sensitivity to ABA and promoted ABA-mediated stomatal closure. These results indicate that CsSnRK2.5 positively regulates stomatal closure in an ABA-dependent manner in transgenic Arabidopsis plants. Correspondingly, a decreased water loss rate was detected in transgenic detached leaves, together with elevated ABA-mediated stomatal closure of transgenic lines, implying that the enhanced drought tolerance of the transgenic plants could be attributed, at least partially, to elevated ABA-mediated stomatal closure. In summary, our results suggest that CsSnRK2.5 positively regulates drought responses, such as drought symptoms, germination and root growth, in transgenic Arabidopsis, as evidenced by a drought stress-tolerant phenotype. In addition, the detection of stress-related indexes, including the water loss rate, ROS content, MDA content and expression patterns of stress-related genes, further confirmed the improved drought tolerance of the transgenic plants. Furthermore, hypersensitivity and improved stomatal closure were found in transgenic plants subjected to ABA treatment, indicating a positive role for CsSnRK2.5 in the ABA pathway in transgenic Arabidopsis, which was further demonstrated by the additional upregulation of some ABA-related drought stress genes, such as AtRAB18 and AtRD29b, under ABA treatment. Overall, our results indicated that CsSnRK2.5 positively regulates the drought response in transgenic Arabidopsis through the ABA signal transduction pathway, providing a foundation for further understanding the drought response mechanism of tea plant.
References Acharya, B R., Jeon, B W., Zhang, W., Assmann, S M. 2013. Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells. The New
phytologist. 200, 1049-1063. https://doi.org/10.1111/nph.12469. Belin, C., de Franco, P O., Bourbousse, C., Chaignepain, S., Schmitter, J M., Vavasseur, A., Giraudat, J., Barbier-Brygoo, H., Thomine, S. 2006. Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant physiology. 141, 1316-1327. https://doi.org/10.1104/pp.106.079327. Blum, A. 1996. Crop responses to drought and the interpretation of adaptation. Plant Growth Regulation. 20, 135-148. https://doi.org/10.1007/BF00024010. Boudsocq, M., Barbier-Brygoo, H., Lauriere, C. 2004. Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. The Journal of biological chemistry. 279, 41758-41766. https://doi.org/10.1074/jbc.M405259200. Boudsocq, M., Lauriere, C. 2005. Osmotic signaling in plants: multiple pathways mediated by emerging kinase families. Plant physiology. 138, 1185-1194. https://doi.org/10.1104/pp.105.061275. Brodribb, T J., Holbrook, N M. 2003. Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant physiology. 132, 2166-2173. https://doi.org/10.1104/pp.103.023879. Dey, A., Samanta, M K., Gayen, S., Maiti, M K. 2016. The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression. BMC plant biology. 16, 158. https://doi.org/ 10.1186/s12870-016-0845-x. Diedhiou, C J., Popova, O V., Dietz, K J., Golldack, D. 2008. The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice. BMC plant biology. 8, 49. https://doi.org/ 10.1186/14712229-8-49. Draper, H H., Squires, E J., Mahmoodi, H., Wu, J., Agarwal, S., Hadley, M. 1993. A comparative evalution of thiobarbituric acid methods for the determination of malondialdehyde in biologicalmaterials. Free Radical Biology & Medicine. 15, 353-363. https://doi.org/10.1016/0891-5849(93)90035-s. Droillard, M J., Boudsocq, M., Barbier-Brygoo, H., Lauriere, C. 2004. Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance. FEBS letters. 574, 42-48. https://doi.org/10.1016/ j.febslet.2004.08.001. Feng, J L., Wang, L Z., Wu, Y N., Luo, Q C., Zhang, Y., Qiu, D., Han, J P., Su, P P., Xiong, Z Y., Chang, J L., Yang, G X., He, G Y. 2018. TaSnRK2.9, a Sucrose Non-fermenting 1-Related Protein Kinase Gene, Positively Regulates Plant Response to Drought and Salt Stress in Transgenic Tobacco. Frontiers in plant science. 9, 2003. https://doi.org/10.3389/fpls.2018.02003. Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., Antoni, R., Park, S Y., Cutler, S R., Sheen, J., Rodriguez, P L., Zhu, J K. 2009. In vitro reconstitution of an abscisic acid signalling pathway. Nature. 462, 660-664. https://doi.org/10.1038/ nature08599.
Fujii, H., Verslues, P E., Zhu, J K. 2011. Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo. Proceedings of the National Academy of Sciences of the United States of America. 108, 1717-1722. https://doi.org/10.1073/pnas.1018367108. Fujii, H., Zhu, J K. 2012. Osmotic stress signaling via protein kinases. Cellular and molecular life sciences : CMLS. 69, 3165-3173. https://doi.org/10.1007/s00018012-1087-1. Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K., Shinozaki, K. 2006. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current opinion in plant biology. 9, 436-442. https://doi.org/10.1016/j.pbi. 2006.05.014. Fujita, Y., Fujita, M., Satoh, R., Maruyama, K., Parvez, M M., Seki, M., Hiratsu, K., Ohme-Takagi, M., Shinozaki, K., Yamaguchi-Shinozaki, K. 2005. AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. The Plant cell. 17, 3470-3488. https://doi.org/10.1105/tpc.105.035659. Fujita, Y., Fujita, M., Shinozaki, K., Yamaguchi-Shinozaki, K. 2011. ABA-mediated transcriptional regulation in response to osmotic stress in plants. Journal of plant research. 124, 509-525. https://doi.org/10.1007/s10265-011-0412-3. Fujita, Y., Yoshida, T., Yamaguchi-Shinozaki, K. 2013. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiologia plantarum. 147, 15-27. https://doi.org/ 10.1111/j.1399-3054.2012.01635.x. Furihata, T., Maruyama, K., Fujita, Y., Umezawa, T., Yoshida, R., Shinozaki, K., Yamaguchi-Shinozaki, K. 2006. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proceedings of the National Academy of Sciences of the United States of America. 103, 1988-1993. https://doi.org/10.1073/pnas.0505667103. Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., Ache, P., Matschi, S., Liese, A., Al-Rasheid, K A., Romeis, T., Hedrich, R. 2009. Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings of the National Academy of Sciences of the United States of America. 106, 21425-21430. https://doi.org/10.1073/pnas. 0912021106. Huai, J., Wang, M., He, J., Zheng, J., Dong, Z., Lv, H., Zhao, J., Wang, G. 2008. Cloning and characterization of the SnRK2 gene family from Zea mays. Plant cell reports. 27, 1861-1868. https://doi.org/10.1007/s00299-008-0608-8. Ichimura, K., Mizoguchi, T., Yoshida, R., Yuasa, T., Shinozaki, K. 2000. Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. The Plant Journal. 24, 655-665. https://doi.org/ 10.1046/j.1365313x.2000.00913.x. Jambunathan, N. 2010. Determination and detection of reactive oxygen species (ROS), lipid peroxidation, and electrolyte leakage in plants. Methods in molecular
biology. 639, 292-298. https://doi.org/10.1007/978-1-60761-702-0_18. Kim, S., Kang, J y., Cho, D I., Park, J H., Kim, S Y. 2004. ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. The Plant Journal. 40, 75-87. https://doi.org/10.1111/j.1365-313X.2004.02192.x. Kobayashi, Y., Murata, M., Minami, H., Yamamoto, S., Kagaya, Y., Hobo, T., Yamamoto, A., Hattori, T. 2005. Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors. The Plant journal : for cell and molecular biology. 44, 939-949. https://doi.org/10.1111/j.1365313X.2005.02583.x. Kobayashi, Y., Yamamoto, S., Minami, H., Kagaya, Y., Hattori, T. 2004. Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid. The Plant cell. 16, 1163-1177. https://doi.org/10.1105/tpc.019943. Kulik, A., Wawer, I., Krzywinska, E., Bucholc, M., Dobrowolska, G. 2011. SnRK2 protein kinases--key regulators of plant response to abiotic stresses. Omics : a journal of integrative biology. 15, 859-872. https://doi.org/10.1089/omi.2011. 0091. Kuromori, T., Seo, M., Shinozaki, K. 2018. ABA Transport and Plant Water Stress Responses. Trends in plant science. 23, 513-522. https://doi.org/10.1016/j.tplants. 2018.04.001. Liu, J Y., Chen, N N., Cheng, Z M., Xiong, J S. 2016. Genome-wide identification, annotation and expression profile analysis of SnRK2 gene family in grapevine. Australian Journal of Grape and Wine Research. 22, 478-488. https://doi.org/ 10.1111/ajgw.12223. Livak, K J., Schmittgen, T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25, 402-408. https://doi.org/10.1006/meth.2001.1262. Lou, D J., Wang, H P., Liang, G., Yu, D Q. 2017. OsSAPK2 Confers Abscisic Acid Sensitivity and Tolerance to Drought Stress in Rice. Frontiers in plant science. 8, 993. https://doi.org/10.3389/fpls.2017.00993. Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, Y., Christmann, A., Grill, E. 2009. Regulators of PP2C phosphatase activity function as abscisic acid sensors.. Science. 324, 1064-1068. https://doi.org/10.1126/science.1172408. Maszkowska, J., Debski, J., Kulik, A., Kistowski, M., Bucholc, M., Lichocka, M., Klimecka, M., Sztatelman, O., Szymanska, K P., Dadlez, M., Dobrowolska, G. 2019. Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress. Plant, cell & environment. 42, 931-946. https://doi.org/10.1111/pce.13465. Msanne, J., Lin, J., Stone, J M., Awada, T. 2011. Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta. 234, 97-107. https://doi.org/10.1007/s00425-011-1387-y. Mustilli, A C., Merlot, S., Vavasseur, A., Fenzi, F., Giraudat, J. 2002. Arabidopsis
OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. The Plant cell. 14, 3089-3099. https://doi.org/10.1105/tpc.007906. Park, S Y., Fung, P., Nishimura, N., Jensen, D R., Fujii, H., Zhao, Y., Lumba, S., Santiago, J., Rodrigues, A., Chow, T F., Alfred, S E., Bonetta, D., Finkelstein, R., Provart, N J., Desveaux, D., Rodriguez, P L., McCourt, P., Zhu, J K., Schroeder, J I., Volkman, B F., Cutler, S R. 2009. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 324, 1068-1071. https://doi.org/10.1126/science.1173041. Parmar, R., Seth, R., Singh, P., Singh, G., Kumar, S., Sharma, R K. 2019. Transcriptional profiling of contrasting genotypes revealed key candidates and nucleotide variations for drought dissection in Camellia sinensis (L.) O. Kuntze. Scientific reports. 9, 7487. https://doi.org/10.1038/s41598-019-43925-w. Sato, A., Sato, Y., Fukao, Y., Fujiwara, M., Umezawa, T., Shinozaki, K., Hibi, T., Taniguchi, M., Miyake, H., Goto, D B., Uozumi, N. 2009. Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. The Biochemical journal. 424, 439-448. https://doi.org/10.1042/BJ20091221. Shao, Y., Zhang, X., van Nocker, S., Gong, X., Ma, F. 2019. Overexpression of a protein kinase gene MpSnRK2.10 from Malus prunifolia confers tolerance to drought stress in transgenic Arabidopsis thaliana and apple. Gene. 692, 26-34. https://doi.org/10.1016/j.gene.2018.12.070. Sirichandra, C., Gu, D., Hu, H C., Davanture, M., Lee, S., Djaoui, M., Valot, B., Zivy, M., Leung, J., Merlot, S., Kwak, J M. 2009. Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase. FEBS letters. 583, 2982-2986. https://doi.org/10.1016/j.febslet.2009.08.033. Tian, S J., Mao, X G., Zhang, H Y., Chen, S S., Zhai, C C., Yang, S M., Jing, R L. 2013. Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat. Journal of experimental botany. 64, 2063-2080. https://doi.org /10.1093/jxb/ert072. Umezawa, T., Sugiyama, N., Mizoguchi, M., Hayashi, S., Myouga, F., Yamaguchi-Shinozaki, K., Ishihama, Y., Hirayama, T., Shinozaki, K. 2009. Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America. 106, 17588-17593. https://doi.org/10.1073/pnas.0907095106. Verslues, P E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., Zhu, J K. 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. The Plant journal : for cell and molecular biology. 45, 523-539. https://doi.org/10.1111/j.1365-313X.2005.02593.x. Wang, J N., Song, Z P., Jia, H F., Yang, S., Zhang, H Y. 2018. Characterization of wheat TaSnRK2.7 promoter in Arabidopsis. Planta. 248, 1393-1401. https://doi. org/10.1007/s00425-018-2984-9. Wang, L Z., Hu, W., Sun, J T., Liang, X Y., Yang, X Y., Wei, S Y., Wang, X T., Zhou, Y., Xiao, Q., Yang, G X., He, G Y. 2015. Genome-wide analysis of SnRK gene
family in Brachypodium distachyon and functional characterization of BdSnRK2.9. Plant science : an international journal of experimental plant biology. 237, 33-45. https://doi.org/10.1016/j.plantsci.2015.05.008. Wu, Q., Wang, M., Shen, J., Chen, D., Zheng, Y., Zhang, W. 2019. ZmOST1 mediates abscisic acid regulation of guard cell ion channels and drought stress responses. Journal of integrative plant biology. 61, 478-491. https://doi.org/10.1111/jipb. 12714. Xia, E H., Zhang, H B., Sheng, J., Li, K., Zhang, QJ., Kim, C., Zhang, Y., Liu, Y., Zhu, T., Li, W., Huang, H., Tong, Y., Nan, H., Shi, C., Shi, C., Jiang, J J., Mao, S Y., Jiao, J Y., Zhang, D., Zhao, Y., Zhao, Y J., Zhang, L P., Liu, Y L., Liu, B Y., Yu, Y., Shao, S F., Ni, D J., Eichler, E E., Gao, L Z. 2017. The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis. Mol Plant. 10:866–877. https://doi.org/10.1016/j.molp.2017.04. 002. Yang, X W., He, K., Chi, X Y., Chai, G H., Wang, Y P., Jia, C L., Zhang, H P., Zhou, G K., Hu, R B. 2018. Miscanthus NAC transcription factor MlNAC12 positively mediates abiotic stress tolerance in transgenic Arabidopsis. Plant science : an international journal of experimental plant biology. 277, 229-241. https://doi.org/ 10.1016/j.plantsci.2018.09.013. Ying, S., Zhang, D F., Li, H Y., Liu, Y H., Shi, Y S., Song, Y C., Wang, T Y., Li, Y. 2011. Cloning and characterization of a maize SnRK2 protein kinase gene confers enhanced salt tolerance in transgenic Arabidopsis. Plant cell reports. 30, 1683-1699. https://doi.org/10.1007/s00299-011-1077-z. Yoshida, R., Hobo, T., Ichimura, K., Mizoguchi, T., Takahashi, F., Aronso, J., Ecker, J., Shinozaki, K. 2002. ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant & cell physiology. 43, 1473-1483. https://doi.org/10.1093/pcp/pcf188. Yoshida, T., Fujita, Y., Maruyama, K., Mogami, J., Todaka, D., Shinozaki, K., Yamaguchi-Shinozaki, K. 2015. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress. Plant, cell & environment. 38, 35-49. https://doi.org/10.1111/pce.12351. Zhang, H Y., Li, W Y., Mao, X G., Jing, R L., Jia, H F. 2016. Differential Activation of the Wheat SnRK2 Family by Abiotic Stresses. Frontiers in plant science. 7, 420. https://doi.org/10.3389/fpls.2016.00420. Zhang, H Y., Mao, X G., Jing, R L., Chang, X P., Xie, H M. 2011. Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses. Journal of experimental botany. 62, 975-988. https://doi.org /10.1093/jxb/erq328. Zhang, H Y., Mao, X G., Wang, C S., Jing, R L. 2010. Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis. PloS one. 5, e16041. https://doi.org/10.1371/journal.pone. 0016041. Zhang, X., Henriques, R., Lin, S S., Niu, Q W., Chua, N H. 2006.
Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature protocols. 1, 641-646. https://doi.org/10.1038/nprot.2006.97. Zhang, Y H., Wan, S Q., Wang, W D., Chen, J F., Huang, L L., Duan, M S., Yu, Y B. 2018. Genome-wide identification and characterization of the CsSnRK2 family in Camellia sinensis. Plant physiology and biochemistry. 132, 287-296. https://doi. org/10.1016/j.plaphy.2018.09.021. Zhao, Y., Zhang, Z J., Gao, J H., Wang, P C., Hu, T., Wang, Z G., Hou, Y J., Wan, Y Z., Liu, W S., Xie, S J., Lu, T J., Xue, L., Liu, Y J., Macho, A P., Tao, W A., Bressan, R A., Zhu, J K. 2018. Arabidopsis Duodecuple Mutant of PYL ABA Receptors Reveals PYL Repression of ABA-Independent SnRK2 Activity. Cell reports. 23, 3340-3351 e3345. https://doi.org/10.1016/j.celrep.2018.05.044.
Figure Legends Fig. 1 Sequence alignment and yeast two-hybrid analysis. (A) Alignment of the predicted AtOST1 (NP_001320129.1) and CsSnRK2.5 (ALL97706.1) polypeptides. (B) The yeast two-hybrid assay confirms the interaction between CsSnRK2.5 and PP2Cs. The PP2Cs of Arabidopsis were taken from NCBI, and the PP2Cs of Camellia sinensis were taken from the genome database of Camellia sinensis according to Xia (Xia et al., 2017). Fig. 2 Overexpression of CsSnRK2.5 improves drought stress tolerance in transgenic Arabidopsis. (A) Performance of CsSnRK2.5 overexpression lines and the WT during drought stress treatment in soil. Water was withheld from two-week-old plants for 10 d (D0) and 17 d (D7), followed by rewatering and recovery for 5 d (R5). (B) Survival rates of the WT and transgenic lines at R5. (C) Water loss rate of detached leaves for the WT and transgenic lines at 16 h. Values represent the mean of three biological replicates. Error bars indicate the SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 3 CsSnRK2.5 overexpression lines exhibit increased tolerance to mannitol stress. (A, B) Germination assay under 150 mM mannitol treatment. (C, D) Root length assay under 150 mM mannitol treatment, Bar = 1 cm. Values represent the mean of three biological replicates. Error bars indicate the SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 4 Overexpression of CsSnRK2.5 alleviates ROS and MDA accumulation under drought stress. (A, B) Histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2−) using DAB staining (A) and NBT staining (B), respectively. (C) Colour phenotype triggered by reactions between TBA and MDA. (D) Quantification of MDA content. Values represent the mean of three biological replicates. Error bars indicate SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test.
Fig. 5 Overexpression of CsSnRK2.5 increases ABA sensitivity in transgenic Arabidopsis. (A, B) Cotyledon greening rates of transgenic lines and the WT under 1.0 mM ABA treatment. (C, D) Root length assay under 1.0 µM ABA treatment. Values represent the mean of three biological replicates. Bar = 1 cm, error bars indicate SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 6 CsSnRK2.5 overexpression in Arabidopsis promotes ABA-mediated stomatal closure. (A) Stomatal apertures of transgenic lines and the WT under 0, 0.5 and 1.0 µM ABA conditions. (B) Measurements of stomatal apertures (width/length) of transgenic and WT plants under ABA treatment, bar = 10 µm. Error bars indicate SD. Values not followed by the same letter are significantly different according to Duncan’s multiple range test (P < 0.05). Fig. 7 Overexpression of CsSnRK2.5 activates the expression of stress-responsive genes. The relative expression of six abiotic stress-responsive genes in transgenic lines and the WT under normal, ABA or drought stress conditions was analysed by qRT-PCR. Values represent the means of three biological replicates. Error bars represent the SD. Significant differences between transgenic lines and the WT are indicated by asterisks (* P < 0.05).
Highlights (1) Overexpression of CsSnRK2.5 enhances drought tolerance in Arabidopsis. (2) CsSnRK2.5 positively regulates stomatal closure in transgenic Arabidopsis. (3) Overexpression of CsSnRK2.5 activates the expression of ABA-responsive genes.
Conflict of interest statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
S0981-9428(20)30089-9
DOI:
https://doi.org/10.1016/j.plaphy.2020.02.035
Reference:
PLAPHY 6069
To appear in:
Plant Physiology and Biochemistry
Received Date: 23 December 2019 Revised Date:
9 February 2020
Accepted Date: 25 February 2020
Please cite this article as: Y. Zhang, S. Wan, X. Liu, J. He, L. Cheng, M. Duan, H. Liu, W. Wang, Y. Yu, Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/j.plaphy.2020.02.035. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.
Contribution: Youben Yu, Yongheng Zhang and Siqing Wan were responsible for design of and conducting the experiments and preparation of the manuscript. Xianghong Liu, Jingyuan He and Long Cheng were responsible for analysis of data. Mengsha Duan, Huan Liu, Weidong Wang and Youben Yu edited and revised the manuscript.
Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis Yongheng Zhang
1
1 Siqin Wan , Xianghong Liu, Jingyuan He, Long Cheng,
Mengsha Duan, Huan Liu, Weidong Wang, Youben YU* College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China.
1
These authors contributed equally to this work.
*Author for correspondence: Youben Yu E-mail: [email protected]; Phone: +86 187-2956-5376
Acknowledgements This work was supported by the earmarked fund for the Modern Agro-industry Technology Research System (CARS-19), the Agricultural Special Fund Project of Shaanxi Province, and the special fund for the University-Supported Extension Model (XTG2019-04).
Conflict of interest statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abstract: Drought is a major factor limiting crop productivity and quality. Sucrose non-fermenting-1 (SNF1)-related protein kinase 2s (SnRK2s) play critical roles in plant abiotic stress responses, especially in drought stress. However, knowledge regarding the functional roles of SnRK2s in drought stress and their underlying
mechanisms is relatively limited in tea plant. In this study, CsSnRK2.5, a PEG 6000and ABA-induced SnRK2 gene from tea plant, was overexpressed in Arabidopsis to investigate its potential function in drought stress response. The results showed that overexpression of CsSnRK2.5 resulted in enhanced drought tolerance, as indicated by an amelioration of the changes in various physiological indexes, including a decreased rate of water loss and decreased accumulation of ROS and MDA. In addition, CsSnRK2.5 overexpression conferred hypersensitivity to exogenous ABA, and transgenic plants exhibited improved ABA-mediated stomatal closure compared to WT plants. Moreover, the expression of some stress response genes, including AtRAB18 and AtRD29b, was more strongly induced in transgenic plants than in the WT when subjected to ABA and drought treatments. Taken together, our results indicate that CsSnRK2.5 is a positive regulator of ABA-regulated drought stress responses.
Keywords: SnRK2 gene, Camellia sinensis, drought stress, abscisic acid (ABA)
1. Introduction Drought is a widely distributed environmental stress that can greatly limit plant growth and development, and plants have evolved a series of intricate strategies to survive this stress (Blum. 1996). In these complex regulatory pathways, the regulation of protein phosphorylation and dephosphorylation is mediated by protein kinases and protein phosphatases, respectively, and these processes significantly influence stress signal transduction (Fujita et al., 2006). Drought often induces hyperosmotic stress (also known as osmotic stress). When plants are exposed to osmotic stress, many protein kinases are rapidly activated, such as mitogen-activated protein kinases (MAPKs),
calcium-dependent
protein
kinases
(CDPKs),
calcineurin
B-like
(CBL)-interacting protein kinases (CIPKs), and members of the sucrose non-fermenting-1 (SNF1)-related protein kinase 2 (SnRK2) family (Boudsocq et al., 2004, Boudsocq et al., 2005, Droillard et al., 2004, Fujii et al., 2012, Ichimura et al., 2000, Kobayashi et al., 2004).
SnRK2s, which are widespread in many plants, are serine/threonine protein kinases with a molecular weight of approximately 40 kDa (Kulik et al., 2011). In recent decades, the SnRK2 gene family has been identified in Arabidopsis (Boudsocq et al., 2004), Oryza sativa (Kobayashi et al., 2004), Zea mays (Huai et al., 2008), grapevine (Liu et al., 2016), wheat (Zhang et al., 2016), etc. A series of studies of SnRK2 members indicated that they were divided into three groups based on phylogenetic analysis, and members of these different groups showed different responses to ABA. Specifically, the members of group I were not activated by ABA, members of group II were not activated or were only weakly activated by ABA, while the members of group III were strongly activated by ABA (Boudsocq et al., 2004, Kobayashi et al., 2004, Kulik et al., 2011). Recently, ABA-activated SnRK2s (mainly referred to as members of group III) have gained a great deal of attention due to the connection between ABA receptors (PYR/PYL/RCAR), protein phosphatase 2Cs (PP2Cs) and SnRK2s (Fujii et al., 2009, Ma et al., 2009, Park et al., 2009, Umezawa et al., 2009). In the absence of ABA, the phosphatase activities of SnRK2s were suppressed by clade A PP2Cs in an interactive manner, while in the presence of ABA, the inhibition of PP2Cs by PYR/PYL/RCARs releases SnRK2s, and active SnRK2s phosphorylate a series of substrate proteins, including transcription factors (Kobayashi et al., 2005, Yoshida et al., 2015), ion channels (Geiger et al., 2009, Sato et al., 2009) and other stress-related proteins (Sirichandra et al., 2009), as part of the osmotic stress response. For example, AtSnRK2.2, AtSnRK2.3 and AtSnRK2.6 phosphorylate AREB/ABF transcription factors in response to osmotic stress in Arabidopsis (Fujita et al., 2013, Furihata et al., 2006, Yoshida et al., 2015). The remaining SnRK2s, which are not activated by ABA, are thought to function in plant ABA-independent osmotic responses as well, as evidenced by a clear disruption of normal osmotic stress responses in the Arabidopsis snrk2 decuple mutant lacking all ten SnRK2s (Fujii et al., 2011). Furthermore, in duodecuple Arabidopsis ABA receptor mutants, the activation of SnRK2 protein kinases by ABA was blocked, but osmotic stress activation of SnRK2s was enhanced (Zhao et al., 2018). These findings suggest that SnRK2s play vital roles in plant
osmotic responses in both the ABA-dependent and ABA-independent pathways. Although SnRK2s are mainly regulated at the posttranslational level by phosphorylation, there is a large amount of evidence indicating that the expression of genes encoding these kinases is also regulated in response to osmotic stress (Kulik et al., 2011). In wheat, TaSnRK2.3 was induced by PEG and ABA, and TaSnRK2.3 overexpression significantly improved the drought tolerance of transgenic Arabidopsis (Tian et al., 2013). Similarly, in apple, MpSnRK2.10 was induced by drought and ABA treatment, and enhanced drought tolerance was found in both transgenic apples and Arabidopsis (Shao et al., 2019). ZmSAPK8 (ZmOST1), a SnRK2 from Zea mays, was found to be induced by drought and ABA and to function in response to drought stress (Wu et al., 2019). These studies imply the potential value of SnRK2s for engineering abiotic stress tolerance across crop species. The tea plant [Camellia sinensis (L.) O. Kuntze], a commercially important perennial evergreen woody crop widely cultivated throughout the world, can easily suffer drought stress (Parmar et al., 2019). In our previous research, SnRK2 members in tea plant were identified, and several CsSnRK2s were found to be induced by stress treatments (Zhang et al., 2018). One of these, CsSnRK2.5, was highly similar to AtSnRK2.6 (AtOST1) in predicted polypeptides and significantly induced by PEG 6000 and ABA treatment, implying a positive role in the drought response of tea plant. In this study, to further investigate the function of CsSnRK2.5, the drought stress tolerance and ABA sensitivity of both CsSnRK2.5-overexpressing and WT plants were investigated, and our results revealed that CsSnRK2.5 positively regulates drought response in transgenic Arabidopsis via ABA-mediated stomatal closure.
2. Materials and Methods 2.1 Plant materials and growing conditions Seeds of Arabidopsis wild-type (WT) Columbia (Col-0) and transgenic lines were surface-sterilized with 20% NaClO (with 0.1% Triton X-100), sown on MS agar medium and then placed in a controlled growth chamber at 16/8 h (25/22 °C) day/night conditions with a relative humidity of approximately 65%. Seedlings at the
four-leaf stage were transferred to pots with soil for transformation or abiotic stress tolerance assays. 2.2. Sequence and phylogenetic analysis of CsSnRK2.5 and its homologues Multiple amino acid alignment was performed using DNAMAN 6 software, and a phylogenetic tree was constructed using the neighbour-joining method with 1000 bootstraps using MEGA7 software. 2.3 Plasmid construction and transformation The coding region of CsSnRK2.5 was amplified and cloned into the Spe I sites of the 3302Y vector to generate the 35S::CsSnRK2.5 construct. Then, the construct was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into WT by the floral dip method (Zhang et al., 2006). The T3 generation homozygous lines (L2 and L3) with highly expressed CsSnRK2.5 genes and WT were used for further analysis. The primers used are listed in Supplementary Table S1. 2.4 Yeast Two-Hybrid Analysis For yeast interaction assays, the full-length cDNAs of CsAHG3 and CsABI1 were cloned into the pGADT7 vector, and CsSnRK2.5 was cloned into the pGBKT7 vector. The pairs of vectors were cotransformed into yeast strain Y2H and grown on SD/-Leu-Trp medium at 30 °C for 3 days, and the clones were transferred into SD/-Leu-Trp-His-Ade medium at 30 °C for 3–5 days. To confirm the results, positive yeast clones were adjusted to OD 0.6, spotted in serial dilutions (1:1, 1:10, 1:100 and 1:1000) and cultured on SD/-Leu-Trp-His-Ade medium. The primers used for gene amplification are included in the primer list (Table S1). 2.5 Stress tolerance assays For the drought treatment in soil, WT and transgenic seeds were germinated and grown to the four-leaf stage on MS agar medium. Then, the seedlings were transferred into soil and grown under unstressed conditions. After one week, watering was withheld until stress symptoms occurred, and the plants were rewatered three days after wilting was observed. Survival rate was counted three times, and 48 seedlings of each genotype were treated intotal. The sensitivity of seed germination to mannitol and ABA was assayed on MS
agar medium containing mannitol (0 and 150 mM) or ABA (0 and 1 µM). To break seed dormancy, seeds were sterilized and incubated at 4 °C for 2 days, sown on MS agar medium and placed in a growth chamber. Seeds in the mannitol treatment were considered germinated when the radicle completely penetrated the seed coat, and the germination rate was calculated four days after the seeds were sown on MS agar medium. The cotyledon greening rate of seedlings in the ABA treatment was calculated ten days after the seeds were sown on MS agar medium. Root extension was also analysed under mannitol and ABA treatment. Seedlings were transferred to MS medium containing mannitol and ABA when their cotyledons turned green and their roots began to extend on MS medium. The root length was measured three days after vertical growth was observed. 2.6 Measurement of reactive oxygen species (ROS) and malondialdehyde (MDA) ROS and MDA contents were detected in transgenic and WT plants subjected to drought. Drought stress was carried out in soil as described above, and the leaves from at least three individual plants were sampled. Then, 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining was performed to detect the accumulation of hydrogen peroxide (H2O2) and superoxide (O2−) according to previously described methods (Yang et al., 2018). To determine the MDA content, a thiobarbituric acid (TBA) reaction was performed according to a previously described method (Draper et al., 1993) 2.7 Water loss rate analysis Ten leaves from five individual four-week-old plants of each genotype were detached, and the fresh weight (W1) was measured immediately. Then, the leaves were placed in an indoor environment, and the weight (W2) was measured at 1, 2, 4, 6, and 8 h. The water loss rate was calculated as follows: water loss rate = (W1-W2)/W1 × 100%. 2.8 Measurement of stomatal movement Four-week-old Arabidopsis plants were grown under mild conditions. To encourage the stomata to open, plants were exposed to light for 3 h. Then, rosette leaves were detached from three individual plants of each genotype and immediately
incubated in stomatal closure buffer (25 mM MES, pH 7.0, 10 mM KCl, and 1 mM CaCl2) containing different concentrations of ABA (0, 0.5 and 1.0 µM) for 2 h. Afterward, leaves were immediately fixed with a 4% glutaraldehyde solution in 0.1 M phosphate-buffered saline (PBS, pH 6.8) to avoid any alterations during sample preparation. After being rinsed five times with PBS, the leaves were dehydrated through a graded ethanol series, vacuum-dried, and gold-coated. Observations were made with a JSM- 6360LV microscope (JEOL Ltd., Tokyo, Japan). Six to ten stomatal cells per leaf abaxial peel were captured randomly, and the lengths and widths of the stomata were measured from 30 stomata using ImageJ software. Then, the width/length ratio (W/L) was calculated. 2.9. Quantitative real-time PCR (qPCR) analysis Arabidopsis leaves of WT and CsSnRK2.5-overexpressing genotypes under normal, ABA (20 µM) and drought conditions were collected. Total RNA was extracted using the SimplyP Total RNA Extraction Kit (#BSC52S1, BioFlux, Hangzhou, China). First-strand cDNAs were synthesized from RNA using 5× All-in-one RT MasterMix (#G485, Applied Biological Materials, Canada). Afterward, the reaction product was diluted to 200 ng µL−1 with sterile water. Quantitative real-time RT-PCR (qRT-PCR) was performed on a QuantStudio®5 (Life Technologies, USA) using EvaGreen 2× qPCR MasterMix-ROX (#MasterMix-R, Applied Biological Materials, Canada) according to the manufacturer’s instructions. Three independent biological replicates were performed for each experiment. The relative expression levels of these genes were analysed by the 2−∆∆Ct method (Livak et al., 2001). The primers used for qRT-PCR are listed in Table S1.
3. Results 3.1 CsSnRK2.5 is a homologue of AtOST1 AtSnRK2.6 (AtOST1), a group III member in Arabidopsis, has been well studied in regard to ABA-mediated stomatal movement and drought responses. In this study, we found that CsSnRK2.5 was a homologue of AtOST1, sharing 89.01% amino acid identity, especially in some function-related domains, such as the ATP binding loop,
activation loop, SnRK2 box and ABA box, which were almost entirely conserved (Fig. 1A). OST1 is involved in the ABA response in Arabidopsis, and clade A PP2C, a vital element of the ABA regulation network, interacts with and inhibits the phosphatase activities of AtOST1. Therefore, we examined the interaction between CsSnRK2.5 and two clade A PP2Cs in tea plant using the yeast two-hybrid system, and as expected, CsSnRK2.5 was found to interact with CsAHG3 (Fig. 1B). 3.2 Overexpression of CsSnRK2.5 enhances drought tolerance in Arabidopsis To investigate the role of CsSnRK2.5 in the plant response to drought stress, the CsSnRK2.5 gene was overexpressed in Arabidopsis. T3 generation homozygous lines (L2 and L3) with highly expressed CsSnRK2.5 genes were selected for further analysis (Fig. S1). Under long-term water shortage, the WT exhibited a more severely wilted phenotype than the L2 and L3 transgenic lines. After watering was resumed, the growth of the WT was weaker than that of the L2 and L3 transgenic lines (Fig. 2A, B). Additionally, the water loss rate of the detached leaves showed that the L2 and L3 transgenic lines had a stronger water-holding ability than the WT (Fig. 2C). Taken together, our results indicate that overexpression of CsSnRK2.5 improved the drought tolerance of transgenic Arabidopsis. To further confirm the drought tolerance of the transgenic lines, mannitol treatment was employed to mimic drought stress, and germination and root extension under normal conditions and mannitol treatment were examined. As expected, in the absence of mannitol, the germination rates and root lengths of the two overexpression lines were indistinguishable from those of the WT (Fig. 3A, B). The application of mannitol significantly inhibited the germination and root elongation of both the WT and transgenic lines; however, the germination and root growth of the two overexpression lines was less inhibited than that of the WT plants, indicating that CsSnRK2.5 overexpression in Arabidopsis enhanced tolerance to mannitol-induced drought stress. 3.3 Overexpression of CsSnRK2.5 alleviates ROS and MDA accumulation in Arabidopsis Drought usually induces the excessive accumulation of ROS, which eventually
leads to oxidative stress (Verslues et al., 2006). To explore whether the enhanced drought tolerance was associated with the rescue of ROS levels in transgenic lines, the accumulation of H2O2 and O2− in leaves under normal and drought conditions was measured using DAB and NBT staining. The results show that under normal conditions, ROS levels in WT and transgenic lines were insignificant, while when exposed to drought stress, the L2 and L3 transgenic lines accumulated less ROS than the WT (Fig. 4A, B). MDA is considered a marker of membrane lipid peroxidation caused by excessive ROS (Jambunathan. 2010), so we measured the MDA contents of WT and transgenic lines under drought stress. The WT consistently accumulated more MDA than did the transgenic lines under drought conditions (Fig. 4C, D), implying that CsSnRK2.5 overexpression in Arabidopsis alleviates ROS and MDA accumulation under drought stress. 3.4 Overexpression of CsSnRK2.5 increases ABA sensitivity in transgenic Arabidopsis The ABA signalling pathway is central to the drought response in plants, and ABA accumulates rapidly in the roots and leaves when a plant is subjected to drought stress (Kuromori et al., 2018). CsSnRK2.5, which is an important element in the ABA signalling pathway, was found to interact with a clade A PP2C member in tea plant, which prompted us to explore whether CsSnRK2.5 overexpression in Arabidopsis affects seedling growth under ABA treatment. As the result shows, at day 10 under treatment with 1.0 µM ABA, the cotyledon greening rate of the transgenic lines was significantly higher than that of the WT (Fig. 5A, B). Moreover, the root extension length of the WT three days after transfer to the ABA treatment was greater than those of the transgenic lines (Fig. 5C, D). Taken together, our data indicate that CsSnRK2.5 overexpression in Arabidopsis increases plant sensitivity to ABA. 3.5 Overexpression of CsSnRK2.5 promotes ABA-mediated stomatal closure in Arabidopsis ABA-mediated stomatal closure plays a vital role in plant stress responses by reducing water loss (Kuromori et al., 2018). To investigate whether the ABA-mediated stomatal closure of transgenic lines was distinct from that of the WT,
detached leaves of transgenic and WT lines were treated with exogenous ABA, and the results showed that the stomatal apertures of overexpression lines were comparable to those of the WT when the stomatal closure buffer contained 0 µM ABA (Fig. 6A, B). The application of ABA significantly promoted stomatal closure in a dose-dependent manner in both the WT and transgenic lines, and the stomatal apertures of the transgenic lines under both the 0.5 and 1.0 µM ABA treatments were smaller than that of the WT (Fig. 6A, B), suggesting that CsSnRK2.5 overexpression in Arabidopsis promotes ABA-mediated stomatal closure. 3.6 Overexpression of CsSnRK2.5 activates the expression of stress-responsive genes in Arabidopsis Previous
studies
demonstrated
that
the
elevated
stress
tolerance
of
SnRK2-overexpressing plants was accompanied by the upregulation of a series of stress-responsive genes (Feng et al., 2018, Shao et al., 2019, Ying et al., 2011). Thus, to further understand the role of CsSnRK2.5 overexpression in drought response pathways, the expression of drought-responsive genes in Arabidopsis, including AtABF1, AtRAB18, AtRD29a and AtRD29b, was evaluated in both the CsSnRK2.5-overexpressing and WT lines using qRT-PCR. As shown in Fig. 7, under normal conditions, the expression of these analysed genes showed no difference, while not coincidentally, all these genes were significantly induced by drought stress in both genotypes; however, notably higher expression levels of these genes, with the exception of AtABF1, were observed in both overexpression lines than in the WT. In addition, higher expression levels of AtRD29b and AtRAB18 were observed in both overexpression lines than in the WT under ABA treatment. These results indicated that overexpression of CsSnRK2.5 leads to additional upregulation of some stress-responsive genes in Arabidopsis under drought and ABA treatment.
4. Discussion A series of studies have indicated that SnRK2s play a critical role in the response to drought stress in both the ABA-dependent and ABA-independent pathways in plants (Lou et al., 2017, Wang et al., 2018, Zhao et al., 2018). For example, OsSAPK2
(Lou et al., 2017) and OsSAPK9 (Dey et al., 2016) were found to play critical roles in the ABA-mediated drought response, while AtSnRK2.10 and TaSnRK2.7 regulate the drought response in an ABA-independent manner (Maszkowska et al., 2019, Zhang et al., 2011). In our previous research, CsSnRK2.5 was found to be significantly induced by PEG 6000 and ABA treatment (Zhang et al., 2018), which prompted us to investigate its role in the drought response and ABA signalling pathway. In the current study, transgenic Arabidopsis plants overexpressing CsSnRK2.5 exhibited a stress-tolerant phenotype compared to the WT when subjected to drought and showed a decreased water loss rate. In addition, CsSnRK2.5 overexpression significantly decreased the inhibition of germination and root growth by mannitol. These results were similar to those of the corresponding MdSnRK2.10 (Shao et al., 2019), BdSnRK2.9 (Wang et al., 2015), TaSnRK2.8 (Zhang et al., 2010), and OsSAPK4-overexpressing plants (Diedhiou et al., 2008), indicating that CsSnRK2.5 overexpression confers tolerance to drought stress in Arabidopsis. Physiological and biochemical changes in plants to some extent reflect the capacity of plants to cope with adversity (Jambunathan. 2010). Drought stress often causes excessive accumulation of ROS and results in oxidative damage in plants (Verslues et al., 2006). In particular, lipid peroxidation is an indicator of oxidative damage at the cellular level and has often been estimated by the content of MDA (Verslues et al., 2006). Similar to studies of other SnRK2 genes (Shao et al., 2019, Tian
et
al.,
2013,
Zhang
et
al.,
2010),
the
ROS
contents
of
two
CsSnRK2.5-overexpressing lines were notably lower than that of the WT under drought stress. Simultaneously, the MDA contents of the two transgenic lines followed the same trends. These results suggested that transgenic plants suffered milder oxidative damage than WT plants under drought stress and that overexpression of CsSnRK2.5 conferred tolerance to drought in Arabidopsis plants at the cellular level, which constituted a foundation for their stress-tolerant phenotype. Drought-induced ABA biosynthesis rapidly activates ABA-dependent SnRK2s; thus, a series of stress-related genes downstream of SnRK2 kinases are regulated, leading to enhanced drought stress tolerance (Fujita et al., 2011). In this study,
CsSnRK2.5 was found to interact with clade A PP2Cs in tea plant, and CsSnRK2.5 overexpression in Arabidopsis increased ABA sensitivity. These findings were consistent with those for its counterparts in other plants (Shao et al., 2019, Umezawa et al., 2009, Wang et al., 2015, Zhang et al., 2016), implying that CsSnRK2.5 was involved in the ABA signal transduction pathway. In Arabidopsis, group III SnRK2s were reported as critical regulators in ABA signalling and can phosphorylate AREB/ABFs to regulate the expression of a number of genes, including AtRAB18, RD29a and RD29b (Fujita et al., 2005, Kim et al., 2004, Msanne et al., 2011, Yoshida et al., 2015). Thus, we examined whether the expression of the genes mentioned above differed in WT and CsSnRK2.5 overexpression lines under drought and ABA treatment, and it was not surprising that, under drought stress, the expression levels of AtRAB18, AtRD29a and AtRD29b in the CsSnRK2.5 overexpression lines were significantly higher than those in the WT, indicating that drought stress responses were strengthened in CsSnRK2.5 overexpression lines. Furthermore, these genes, with the exception of AtRD29a, showed additional upregulation under ABA treatment, which means that CsSnRK2.5 overexpression elevated ABA signal output in transgenic Arabidopsis and thus may work in concert with the increased ABA sensitivity of transgenic plants. We noticed that the expression of the AREB/ABF transcription factor gene AtABF1 showed no difference between CsSnRK2.5 overexpression lines and WT under ABA and drought conditions, and similar results were observed in the study of MdSnRK2.10 (Shao et al. 2019). Though the expression of AtABF1 was not altered, AtABF1 may still be involved in the drought response in transgenic plants through CsSnRK2.5-mediated phosphorylation, because such posttranslational modifications of AtABF1 are critical for its function in regulating downstream gene expression and may be responsible for the additional upregulation of AtRD29b and AtRAB18 under ABA and drought conditions in CsSnRK2.5 overexpression lines. Stomatal closure provides a critical strategy for drought resistance by decreasing water loss (Brodribb et al., 2003). In recent decades, numerous elements involved in stomatal closure have been characterized; in particular, SnRK2 members were found
to play a positive role in ABA-mediated stomatal closure (Belin et al., 2006, Mustilli et al., 2002, Wu et al., 2019). In previous studies, AtOST1 was reported as a positive regulator in ABA-dependent stomatal closure (Acharya et al., 2013, Yoshida et al., 2002); furthermore, ZmOST1, a homologue of AtOST1, also positively regulated ABA-dependent stomatal closure (Wu et al., 2019). In the current study, CsSnRK2.5 overexpression in Arabidopsis resulted in increased sensitivity to ABA and promoted ABA-mediated stomatal closure. These results indicate that CsSnRK2.5 positively regulates stomatal closure in an ABA-dependent manner in transgenic Arabidopsis plants. Correspondingly, a decreased water loss rate was detected in transgenic detached leaves, together with elevated ABA-mediated stomatal closure of transgenic lines, implying that the enhanced drought tolerance of the transgenic plants could be attributed, at least partially, to elevated ABA-mediated stomatal closure. In summary, our results suggest that CsSnRK2.5 positively regulates drought responses, such as drought symptoms, germination and root growth, in transgenic Arabidopsis, as evidenced by a drought stress-tolerant phenotype. In addition, the detection of stress-related indexes, including the water loss rate, ROS content, MDA content and expression patterns of stress-related genes, further confirmed the improved drought tolerance of the transgenic plants. Furthermore, hypersensitivity and improved stomatal closure were found in transgenic plants subjected to ABA treatment, indicating a positive role for CsSnRK2.5 in the ABA pathway in transgenic Arabidopsis, which was further demonstrated by the additional upregulation of some ABA-related drought stress genes, such as AtRAB18 and AtRD29b, under ABA treatment. Overall, our results indicated that CsSnRK2.5 positively regulates the drought response in transgenic Arabidopsis through the ABA signal transduction pathway, providing a foundation for further understanding the drought response mechanism of tea plant.
References Acharya, B R., Jeon, B W., Zhang, W., Assmann, S M. 2013. Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells. The New
phytologist. 200, 1049-1063. https://doi.org/10.1111/nph.12469. Belin, C., de Franco, P O., Bourbousse, C., Chaignepain, S., Schmitter, J M., Vavasseur, A., Giraudat, J., Barbier-Brygoo, H., Thomine, S. 2006. Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant physiology. 141, 1316-1327. https://doi.org/10.1104/pp.106.079327. Blum, A. 1996. Crop responses to drought and the interpretation of adaptation. Plant Growth Regulation. 20, 135-148. https://doi.org/10.1007/BF00024010. Boudsocq, M., Barbier-Brygoo, H., Lauriere, C. 2004. Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. The Journal of biological chemistry. 279, 41758-41766. https://doi.org/10.1074/jbc.M405259200. Boudsocq, M., Lauriere, C. 2005. Osmotic signaling in plants: multiple pathways mediated by emerging kinase families. Plant physiology. 138, 1185-1194. https://doi.org/10.1104/pp.105.061275. Brodribb, T J., Holbrook, N M. 2003. Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant physiology. 132, 2166-2173. https://doi.org/10.1104/pp.103.023879. Dey, A., Samanta, M K., Gayen, S., Maiti, M K. 2016. The sucrose non-fermenting 1-related kinase 2 gene SAPK9 improves drought tolerance and grain yield in rice by modulating cellular osmotic potential, stomatal closure and stress-responsive gene expression. BMC plant biology. 16, 158. https://doi.org/ 10.1186/s12870-016-0845-x. Diedhiou, C J., Popova, O V., Dietz, K J., Golldack, D. 2008. The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice. BMC plant biology. 8, 49. https://doi.org/ 10.1186/14712229-8-49. Draper, H H., Squires, E J., Mahmoodi, H., Wu, J., Agarwal, S., Hadley, M. 1993. A comparative evalution of thiobarbituric acid methods for the determination of malondialdehyde in biologicalmaterials. Free Radical Biology & Medicine. 15, 353-363. https://doi.org/10.1016/0891-5849(93)90035-s. Droillard, M J., Boudsocq, M., Barbier-Brygoo, H., Lauriere, C. 2004. Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance. FEBS letters. 574, 42-48. https://doi.org/10.1016/ j.febslet.2004.08.001. Feng, J L., Wang, L Z., Wu, Y N., Luo, Q C., Zhang, Y., Qiu, D., Han, J P., Su, P P., Xiong, Z Y., Chang, J L., Yang, G X., He, G Y. 2018. TaSnRK2.9, a Sucrose Non-fermenting 1-Related Protein Kinase Gene, Positively Regulates Plant Response to Drought and Salt Stress in Transgenic Tobacco. Frontiers in plant science. 9, 2003. https://doi.org/10.3389/fpls.2018.02003. Fujii, H., Chinnusamy, V., Rodrigues, A., Rubio, S., Antoni, R., Park, S Y., Cutler, S R., Sheen, J., Rodriguez, P L., Zhu, J K. 2009. In vitro reconstitution of an abscisic acid signalling pathway. Nature. 462, 660-664. https://doi.org/10.1038/ nature08599.
Fujii, H., Verslues, P E., Zhu, J K. 2011. Arabidopsis decuple mutant reveals the importance of SnRK2 kinases in osmotic stress responses in vivo. Proceedings of the National Academy of Sciences of the United States of America. 108, 1717-1722. https://doi.org/10.1073/pnas.1018367108. Fujii, H., Zhu, J K. 2012. Osmotic stress signaling via protein kinases. Cellular and molecular life sciences : CMLS. 69, 3165-3173. https://doi.org/10.1007/s00018012-1087-1. Fujita, M., Fujita, Y., Noutoshi, Y., Takahashi, F., Narusaka, Y., Yamaguchi-Shinozaki, K., Shinozaki, K. 2006. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current opinion in plant biology. 9, 436-442. https://doi.org/10.1016/j.pbi. 2006.05.014. Fujita, Y., Fujita, M., Satoh, R., Maruyama, K., Parvez, M M., Seki, M., Hiratsu, K., Ohme-Takagi, M., Shinozaki, K., Yamaguchi-Shinozaki, K. 2005. AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. The Plant cell. 17, 3470-3488. https://doi.org/10.1105/tpc.105.035659. Fujita, Y., Fujita, M., Shinozaki, K., Yamaguchi-Shinozaki, K. 2011. ABA-mediated transcriptional regulation in response to osmotic stress in plants. Journal of plant research. 124, 509-525. https://doi.org/10.1007/s10265-011-0412-3. Fujita, Y., Yoshida, T., Yamaguchi-Shinozaki, K. 2013. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiologia plantarum. 147, 15-27. https://doi.org/ 10.1111/j.1399-3054.2012.01635.x. Furihata, T., Maruyama, K., Fujita, Y., Umezawa, T., Yoshida, R., Shinozaki, K., Yamaguchi-Shinozaki, K. 2006. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proceedings of the National Academy of Sciences of the United States of America. 103, 1988-1993. https://doi.org/10.1073/pnas.0505667103. Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., Ache, P., Matschi, S., Liese, A., Al-Rasheid, K A., Romeis, T., Hedrich, R. 2009. Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings of the National Academy of Sciences of the United States of America. 106, 21425-21430. https://doi.org/10.1073/pnas. 0912021106. Huai, J., Wang, M., He, J., Zheng, J., Dong, Z., Lv, H., Zhao, J., Wang, G. 2008. Cloning and characterization of the SnRK2 gene family from Zea mays. Plant cell reports. 27, 1861-1868. https://doi.org/10.1007/s00299-008-0608-8. Ichimura, K., Mizoguchi, T., Yoshida, R., Yuasa, T., Shinozaki, K. 2000. Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. The Plant Journal. 24, 655-665. https://doi.org/ 10.1046/j.1365313x.2000.00913.x. Jambunathan, N. 2010. Determination and detection of reactive oxygen species (ROS), lipid peroxidation, and electrolyte leakage in plants. Methods in molecular
biology. 639, 292-298. https://doi.org/10.1007/978-1-60761-702-0_18. Kim, S., Kang, J y., Cho, D I., Park, J H., Kim, S Y. 2004. ABF2, an ABRE-binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance. The Plant Journal. 40, 75-87. https://doi.org/10.1111/j.1365-313X.2004.02192.x. Kobayashi, Y., Murata, M., Minami, H., Yamamoto, S., Kagaya, Y., Hobo, T., Yamamoto, A., Hattori, T. 2005. Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors. The Plant journal : for cell and molecular biology. 44, 939-949. https://doi.org/10.1111/j.1365313X.2005.02583.x. Kobayashi, Y., Yamamoto, S., Minami, H., Kagaya, Y., Hattori, T. 2004. Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid. The Plant cell. 16, 1163-1177. https://doi.org/10.1105/tpc.019943. Kulik, A., Wawer, I., Krzywinska, E., Bucholc, M., Dobrowolska, G. 2011. SnRK2 protein kinases--key regulators of plant response to abiotic stresses. Omics : a journal of integrative biology. 15, 859-872. https://doi.org/10.1089/omi.2011. 0091. Kuromori, T., Seo, M., Shinozaki, K. 2018. ABA Transport and Plant Water Stress Responses. Trends in plant science. 23, 513-522. https://doi.org/10.1016/j.tplants. 2018.04.001. Liu, J Y., Chen, N N., Cheng, Z M., Xiong, J S. 2016. Genome-wide identification, annotation and expression profile analysis of SnRK2 gene family in grapevine. Australian Journal of Grape and Wine Research. 22, 478-488. https://doi.org/ 10.1111/ajgw.12223. Livak, K J., Schmittgen, T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25, 402-408. https://doi.org/10.1006/meth.2001.1262. Lou, D J., Wang, H P., Liang, G., Yu, D Q. 2017. OsSAPK2 Confers Abscisic Acid Sensitivity and Tolerance to Drought Stress in Rice. Frontiers in plant science. 8, 993. https://doi.org/10.3389/fpls.2017.00993. Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, Y., Christmann, A., Grill, E. 2009. Regulators of PP2C phosphatase activity function as abscisic acid sensors.. Science. 324, 1064-1068. https://doi.org/10.1126/science.1172408. Maszkowska, J., Debski, J., Kulik, A., Kistowski, M., Bucholc, M., Lichocka, M., Klimecka, M., Sztatelman, O., Szymanska, K P., Dadlez, M., Dobrowolska, G. 2019. Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress. Plant, cell & environment. 42, 931-946. https://doi.org/10.1111/pce.13465. Msanne, J., Lin, J., Stone, J M., Awada, T. 2011. Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta. 234, 97-107. https://doi.org/10.1007/s00425-011-1387-y. Mustilli, A C., Merlot, S., Vavasseur, A., Fenzi, F., Giraudat, J. 2002. Arabidopsis
OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. The Plant cell. 14, 3089-3099. https://doi.org/10.1105/tpc.007906. Park, S Y., Fung, P., Nishimura, N., Jensen, D R., Fujii, H., Zhao, Y., Lumba, S., Santiago, J., Rodrigues, A., Chow, T F., Alfred, S E., Bonetta, D., Finkelstein, R., Provart, N J., Desveaux, D., Rodriguez, P L., McCourt, P., Zhu, J K., Schroeder, J I., Volkman, B F., Cutler, S R. 2009. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 324, 1068-1071. https://doi.org/10.1126/science.1173041. Parmar, R., Seth, R., Singh, P., Singh, G., Kumar, S., Sharma, R K. 2019. Transcriptional profiling of contrasting genotypes revealed key candidates and nucleotide variations for drought dissection in Camellia sinensis (L.) O. Kuntze. Scientific reports. 9, 7487. https://doi.org/10.1038/s41598-019-43925-w. Sato, A., Sato, Y., Fukao, Y., Fujiwara, M., Umezawa, T., Shinozaki, K., Hibi, T., Taniguchi, M., Miyake, H., Goto, D B., Uozumi, N. 2009. Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase. The Biochemical journal. 424, 439-448. https://doi.org/10.1042/BJ20091221. Shao, Y., Zhang, X., van Nocker, S., Gong, X., Ma, F. 2019. Overexpression of a protein kinase gene MpSnRK2.10 from Malus prunifolia confers tolerance to drought stress in transgenic Arabidopsis thaliana and apple. Gene. 692, 26-34. https://doi.org/10.1016/j.gene.2018.12.070. Sirichandra, C., Gu, D., Hu, H C., Davanture, M., Lee, S., Djaoui, M., Valot, B., Zivy, M., Leung, J., Merlot, S., Kwak, J M. 2009. Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase. FEBS letters. 583, 2982-2986. https://doi.org/10.1016/j.febslet.2009.08.033. Tian, S J., Mao, X G., Zhang, H Y., Chen, S S., Zhai, C C., Yang, S M., Jing, R L. 2013. Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat. Journal of experimental botany. 64, 2063-2080. https://doi.org /10.1093/jxb/ert072. Umezawa, T., Sugiyama, N., Mizoguchi, M., Hayashi, S., Myouga, F., Yamaguchi-Shinozaki, K., Ishihama, Y., Hirayama, T., Shinozaki, K. 2009. Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America. 106, 17588-17593. https://doi.org/10.1073/pnas.0907095106. Verslues, P E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., Zhu, J K. 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. The Plant journal : for cell and molecular biology. 45, 523-539. https://doi.org/10.1111/j.1365-313X.2005.02593.x. Wang, J N., Song, Z P., Jia, H F., Yang, S., Zhang, H Y. 2018. Characterization of wheat TaSnRK2.7 promoter in Arabidopsis. Planta. 248, 1393-1401. https://doi. org/10.1007/s00425-018-2984-9. Wang, L Z., Hu, W., Sun, J T., Liang, X Y., Yang, X Y., Wei, S Y., Wang, X T., Zhou, Y., Xiao, Q., Yang, G X., He, G Y. 2015. Genome-wide analysis of SnRK gene
family in Brachypodium distachyon and functional characterization of BdSnRK2.9. Plant science : an international journal of experimental plant biology. 237, 33-45. https://doi.org/10.1016/j.plantsci.2015.05.008. Wu, Q., Wang, M., Shen, J., Chen, D., Zheng, Y., Zhang, W. 2019. ZmOST1 mediates abscisic acid regulation of guard cell ion channels and drought stress responses. Journal of integrative plant biology. 61, 478-491. https://doi.org/10.1111/jipb. 12714. Xia, E H., Zhang, H B., Sheng, J., Li, K., Zhang, QJ., Kim, C., Zhang, Y., Liu, Y., Zhu, T., Li, W., Huang, H., Tong, Y., Nan, H., Shi, C., Shi, C., Jiang, J J., Mao, S Y., Jiao, J Y., Zhang, D., Zhao, Y., Zhao, Y J., Zhang, L P., Liu, Y L., Liu, B Y., Yu, Y., Shao, S F., Ni, D J., Eichler, E E., Gao, L Z. 2017. The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis. Mol Plant. 10:866–877. https://doi.org/10.1016/j.molp.2017.04. 002. Yang, X W., He, K., Chi, X Y., Chai, G H., Wang, Y P., Jia, C L., Zhang, H P., Zhou, G K., Hu, R B. 2018. Miscanthus NAC transcription factor MlNAC12 positively mediates abiotic stress tolerance in transgenic Arabidopsis. Plant science : an international journal of experimental plant biology. 277, 229-241. https://doi.org/ 10.1016/j.plantsci.2018.09.013. Ying, S., Zhang, D F., Li, H Y., Liu, Y H., Shi, Y S., Song, Y C., Wang, T Y., Li, Y. 2011. Cloning and characterization of a maize SnRK2 protein kinase gene confers enhanced salt tolerance in transgenic Arabidopsis. Plant cell reports. 30, 1683-1699. https://doi.org/10.1007/s00299-011-1077-z. Yoshida, R., Hobo, T., Ichimura, K., Mizoguchi, T., Takahashi, F., Aronso, J., Ecker, J., Shinozaki, K. 2002. ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant & cell physiology. 43, 1473-1483. https://doi.org/10.1093/pcp/pcf188. Yoshida, T., Fujita, Y., Maruyama, K., Mogami, J., Todaka, D., Shinozaki, K., Yamaguchi-Shinozaki, K. 2015. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress. Plant, cell & environment. 38, 35-49. https://doi.org/10.1111/pce.12351. Zhang, H Y., Li, W Y., Mao, X G., Jing, R L., Jia, H F. 2016. Differential Activation of the Wheat SnRK2 Family by Abiotic Stresses. Frontiers in plant science. 7, 420. https://doi.org/10.3389/fpls.2016.00420. Zhang, H Y., Mao, X G., Jing, R L., Chang, X P., Xie, H M. 2011. Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses. Journal of experimental botany. 62, 975-988. https://doi.org /10.1093/jxb/erq328. Zhang, H Y., Mao, X G., Wang, C S., Jing, R L. 2010. Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis. PloS one. 5, e16041. https://doi.org/10.1371/journal.pone. 0016041. Zhang, X., Henriques, R., Lin, S S., Niu, Q W., Chua, N H. 2006.
Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature protocols. 1, 641-646. https://doi.org/10.1038/nprot.2006.97. Zhang, Y H., Wan, S Q., Wang, W D., Chen, J F., Huang, L L., Duan, M S., Yu, Y B. 2018. Genome-wide identification and characterization of the CsSnRK2 family in Camellia sinensis. Plant physiology and biochemistry. 132, 287-296. https://doi. org/10.1016/j.plaphy.2018.09.021. Zhao, Y., Zhang, Z J., Gao, J H., Wang, P C., Hu, T., Wang, Z G., Hou, Y J., Wan, Y Z., Liu, W S., Xie, S J., Lu, T J., Xue, L., Liu, Y J., Macho, A P., Tao, W A., Bressan, R A., Zhu, J K. 2018. Arabidopsis Duodecuple Mutant of PYL ABA Receptors Reveals PYL Repression of ABA-Independent SnRK2 Activity. Cell reports. 23, 3340-3351 e3345. https://doi.org/10.1016/j.celrep.2018.05.044.
Figure Legends Fig. 1 Sequence alignment and yeast two-hybrid analysis. (A) Alignment of the predicted AtOST1 (NP_001320129.1) and CsSnRK2.5 (ALL97706.1) polypeptides. (B) The yeast two-hybrid assay confirms the interaction between CsSnRK2.5 and PP2Cs. The PP2Cs of Arabidopsis were taken from NCBI, and the PP2Cs of Camellia sinensis were taken from the genome database of Camellia sinensis according to Xia (Xia et al., 2017). Fig. 2 Overexpression of CsSnRK2.5 improves drought stress tolerance in transgenic Arabidopsis. (A) Performance of CsSnRK2.5 overexpression lines and the WT during drought stress treatment in soil. Water was withheld from two-week-old plants for 10 d (D0) and 17 d (D7), followed by rewatering and recovery for 5 d (R5). (B) Survival rates of the WT and transgenic lines at R5. (C) Water loss rate of detached leaves for the WT and transgenic lines at 16 h. Values represent the mean of three biological replicates. Error bars indicate the SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 3 CsSnRK2.5 overexpression lines exhibit increased tolerance to mannitol stress. (A, B) Germination assay under 150 mM mannitol treatment. (C, D) Root length assay under 150 mM mannitol treatment, Bar = 1 cm. Values represent the mean of three biological replicates. Error bars indicate the SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 4 Overexpression of CsSnRK2.5 alleviates ROS and MDA accumulation under drought stress. (A, B) Histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2−) using DAB staining (A) and NBT staining (B), respectively. (C) Colour phenotype triggered by reactions between TBA and MDA. (D) Quantification of MDA content. Values represent the mean of three biological replicates. Error bars indicate SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test.
Fig. 5 Overexpression of CsSnRK2.5 increases ABA sensitivity in transgenic Arabidopsis. (A, B) Cotyledon greening rates of transgenic lines and the WT under 1.0 mM ABA treatment. (C, D) Root length assay under 1.0 µM ABA treatment. Values represent the mean of three biological replicates. Bar = 1 cm, error bars indicate SD. Asterisks indicate significant differences (** P < 0.01) compared to the WT as determined by Student's t-test. Fig. 6 CsSnRK2.5 overexpression in Arabidopsis promotes ABA-mediated stomatal closure. (A) Stomatal apertures of transgenic lines and the WT under 0, 0.5 and 1.0 µM ABA conditions. (B) Measurements of stomatal apertures (width/length) of transgenic and WT plants under ABA treatment, bar = 10 µm. Error bars indicate SD. Values not followed by the same letter are significantly different according to Duncan’s multiple range test (P < 0.05). Fig. 7 Overexpression of CsSnRK2.5 activates the expression of stress-responsive genes. The relative expression of six abiotic stress-responsive genes in transgenic lines and the WT under normal, ABA or drought stress conditions was analysed by qRT-PCR. Values represent the means of three biological replicates. Error bars represent the SD. Significant differences between transgenic lines and the WT are indicated by asterisks (* P < 0.05).
Highlights (1) Overexpression of CsSnRK2.5 enhances drought tolerance in Arabidopsis. (2) CsSnRK2.5 positively regulates stomatal closure in transgenic Arabidopsis. (3) Overexpression of CsSnRK2.5 activates the expression of ABA-responsive genes.
Conflict of interest statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.