Mol Biol Rep DOI 10.1007/s11033-014-3440-y
Cloning and characterization of SnRK2 subfamily II genes from Nicotiana tabacum Hongying Zhang • Hongfang Jia • Guoshun Liu • Shengnan Yang • Songtao Zhang • Yongxia Yang Peipei Yang • Hong Cui
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Received: 27 October 2013 / Accepted: 28 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract SnRK2 is a plant-specific protein kinase family involved in abiotic stress signalling. In this study, NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3, were cloned from tobacco by in silico cloning and reverse transcription PCR. The three protein kinases were classed into subfamily II of the SnRK2 family using a phylogenetic tree and C-terminus analysis. Subcellular localization revealed NtSnRK2s in the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco plants that were exposed to drought, salt, or cold stressors were characterised using quantitative real-time PCR. It was revealed that the three genes showed similar patterns of transcription under abiotic stress responses; there was evidence NtSnRK2s participated in abscisic acid-dependent signalling pathways. NtSnRK2.1–3 responded much faster to drought and salt than to cold stress. To investigate the role of NtSnRK2s under abiotic stresses, NtSnRK2.1 gene was over-expressed in tobacco. A stress tolerance assay showed that tobacco plants that over-expressed NtSnRK2.1 plants had greater salt tolerance. The results indicate that NtSnRK2s are involved in abiotic stress response pathways. Keywords NtSnRK2s Gene expression Subcellular localization Abiotic stress Nicotiana tabacum
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3440-y) contains supplementary material, which is available to authorized users. H. Zhang H. Jia G. Liu S. Yang S. Zhang Y. Yang P. Yang H. Cui (&) Key Laboratory for Cultivation of Tobacco Industry, College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China e-mail: [email protected]
Introduction Abiotic stresses, such as high salinity, drought and extreme temperatures, impose osmotic stresses on plants, leading to imbalances in ionic homeostasis, oxidative damages, and growth inhibition. To cope with multiple adverse stresses, plants have developed a range of molecular mechanisms to perceive stress signals and to manifest adaptive responses [1, 2]. Protein phosphorylation/dephosphorylation is a major mechanism for mediating intracellular responses, including responses to hormonal, pathogenic, and environmental stimuli; it also plays a role in control of metabolism [3]. Many studies in plants have indicated that protein kinases are involved in stress signalling via phosphorylation. The sucrose non-fermenting1 (SNF1) kinase family is known as SNF1 protein kinases in yeasts, mammalian AMP-activated protein kinases, and plant SNF1-related protein kinases (SnRKs) [4]. Compelling evidence shows that all the kinases in yeasts and mammals play pivotal roles in carbohydrate metabolism, whereas plant SnRKs may function as interfaces between stress and metabolic signalling pathways [5–7]. A relatively small plant-specific gene family, SnRK2s encode serine/threonine kinases containing an N-terminal region and a relatively shot C-terminal region. The divergent C-terminal domain is rich in acidic amino acids (Asp/ Glu). According to the C-terminal region, SnRK2 kinases are further grouped into two subfamilies: SnRK2a and SnRK2b [5]. It was suggested that the C-terminal region may function in kinase activation, participate in protein– protein interactions, and be involved in abscisic acid signalling pathways [8–10]. Increasing evidence suggests that SnRK2 kinases are convergence points for many signalling pathways, and
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function in diverse environmental stress responses [5, 7]. The first member of the SnRK2s to be described, PKABA1 (also named TaW55), was isolated from wheat and was activated by hyperosmotic stress and ABA treatment [11]. Subsequent work indicated that it acts as an intermediate in the ABA suppression of GA-responsive gene transcription [12–14]. In our recent study, four SnRK2 members of wheat were isolated and characterised. Over-expression of these protein kinases could significantly enhance the tolerance of abiotic stressors in Arabidopsis. Experimental evidence suggested that TaSnRK2s were involved in stress signalling pathways and carbohydrate metabolism [15–18]. Ten SnRK2 kinases were cloned from Arabidopsis, among which nine were induced by salinity and hyperosmotic stresses, and five of the nine were induced by ABA, while none was induced by cold stress [19, 20]. In rice, 10 SnRK2 members (SAPK1–10) have been identified. All these kinases were induced by osmotic stress, and three of them (SAPK8, SAPK9, and SAPK10) were also induced by ABA treatment [21]. Over-expression of SAPK4 in rice could significantly enhance the salt tolerance [22]. Similarly, 10 SnRK2s were characterised in maize, and all these kinases were activated by osmotic stresses [23]. In tobacco, osmotic stress-activated protein kinase (NtOSAK) has been assigned to the SnRK2 family [24]. A detailed analysis revealed that a complex of a GAPDH isoform and NtOSAK was partially localised in the nucleus in BY2 cells after salt stress. This complex seemed to be regulated by NO:GAPDH and was directly S-nitrosylated, which could promote its translocation into the nucleus of the complex [25]. Thus, solid evident suggests that plant SnRK2 family is involved in the abiotic stress responses, each member fulfilling distinct functions. However, the specific functions of tobacco SnRK2s and the molecular mechanism of their activation have seldom been studied. In this study, we isolated members of the tobacco SnRK2 family and characterised their expression patterns under diverse environmental stresses. Transgenic experiments indicated that the over-expression of NtSnRK2.1 in tobacco significantly increased the plant’s tolerance of salt stress.
were stressed by exposure to a PEG 6000 solution (0.5 MPa water potential), a 300 mM NaCl solution, 4 °C, or a 50 lM ABA spray, all of which were shown to constitute significant stress in pilot experiments. Control plants were cultured normally. All the true leaves, sampled at 0, 1, 3, 6, 12, 24, 48, and 72 h after treatments, were removed at the main midribs. One leaf from each plant was used for RNA extraction. The other was analysed to determine the amounts of total soluble sugars and reducing sugars according to tobacco industrial standard methods YC/T 159-2002. Isolation of the cDNA sequences of NtSnRK2s The amino acid sequences of Arabidopsis SnRK2s were used as query probes to screen the tobacco expressed sequence tag (EST) database. These candidate ESTs were assembled into contiguous fragments by in silico cloning. Based on the putative cDNA sequence, specific primers were designed to amplify the NtSnRK2s cDNA sequences (Supplementary Table S1). Similarity searches and sequence alignments were conducted using BLAST and DNAStar’s Megalign program, respectively. The functional region and activity sites were predicted with SignalP and PROSITE servers. To determine the relationship between NtSnRK2s and SnRK2 members in other plant species, ClustalW and PHYLIP soft package (version 3.69) were used to construct a phylogenetic tree, which was viewed using TREEVIEW software. Quantitative real-time PCR (qRT-PCR) The cDNA was synthesized using the M-MuLVreverse transcriptase (New England BioLabs) according to the instruction manual. The qRT-PCR was performed with SYBR Premix Ex TaqTM (Takara) using an ABI PRISMÒ 7000 system (Applied Biosystems). Specific primers are listed in Supplementary Table S2. The transcription of ribosomal protein gene L25 was used as an internal control. Relative expression changes of NtSnRK2s were calculated using the 2-DDCT method [26].
Materials and methods
Subcellular localization of NtSnRK2s
Stress experiments and amounts of total soluble sugars and reducing sugars
To construct an NtSnRK2s-GFP fusion protein, the coding sequence of NtSnRK2s without a termination codon was fused upstream of GFP and downstream of 35S of the pJIT163-GFP expression vector. The recombinant pJIT16335S-NtSnRKs-GFP construct was transferred into living onion (Allium cepa) epidermal cells with a GeneGun (Biorad HeliosTM). The transformed cells were incubated and observed as described previously [16].
Seeds of tobacco (Kentucky 326) were directly sown in mixed soil (1:1 vermiculite: humus) saturated with water in salver. Seedlings were cultured in a growth chamber at 22 °C under a 16 h light/8 h dark cycle for 8 weeks. Next, the plants were assigned to a treatment group in which they
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Mol Biol Rep Table 1 Three SnRK2s in tobacco and some characteristics of them Gene name
ORF (bp)
Protein length (aa)
Protein molecular mass (kd)
pI
NtSnRK2.1
1020
339
38.2
5.85
NtSnRK2.2
1035
344
39.0
5.23
NtSnRK2.3
1020
339
38.3
5.59
differentiation media containing 150 mM NaCl, which was shown to cause significant stress in pilot experiments. When symptoms of salt stress were evident, the percentage of green explants was recorded. Results Molecular characterization of NtSnRK2s
Transformation of NtSnRK2.1 in tobacco The coding region of NtSnRK2.1 was cloned into the vector pCAMBIA-NPT-GUS as a b-glucuronidase (GUS)-fused fragment using the primers 50 -GAGAGAATTCCCCTTG ATTATGGAGCGT-30 (EcoR I site underlined) and 50 -GA GAACTAGTTTACATCACAATTGACCAACA-30 (Spe I site underlined). The vectors p35S-NtSnRK2.1-GUS and p35S-GUS were separately transformed into Agrobacterium tumefaciens (strain LBA4404) cells and then transferred into tobacco. NtSnRK2.1 plants (T0) were firstly identified on kanamycin plates (50 lg/mL) and further verified by GUS histochemical staining assays and RTPCR. The RT-PCR was performed using the primers 50 -GC CAGTGGAACTGATGGAAGGAGG-30 and 50 -GGGTGA ACGTGTAGTGCGCTGTATT-30 , which were designed according to NtSnRK2.1 and GUS genes, respectively. Abiotic stress tolerance assays Leaves from untransformed and transgenic plants were removed, and leaf discs measuring 1.2 cm2 in area were aseptically excised from them, in each case from the same part of the leaf. These explants were cultured on
The cDNA sequences of three NtSnRK2s were identified from tobacco through in silico cloning and RT-PCR (Table 1). These protein kinase genes were designated NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3 on the basis of their similarity to their orthologous counterparts in rice and maize. Prediction of the functional region and activity sites indicated that NtSnRK2s have the potential for serine/ threonine kinase activities and, like other SnRK2 members, have two domains (an N-terminal catalytic domain and a C-terminal regulatory domain). The C-terminal regions of NtSnRK2s are quite divergent. However, the N-terminal regions of NtSnRK2s are highly conserved and contain 11 subdomains that are identified in serine/threonine kinases (Fig. 1). In addition, a protein kinase-activating site and an ATP-binding site are detected in the catalytic domain. Phylogenetic analysis based on the putative amino acid sequences of NtSnRK2s and SnRK2 members of rice, maize, and Arabidopsis, showed that these sequences were grouped into three subfamilies (subfamily I, II, and III); NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3 are assigned to the same clade, subfamily II, whereas the previously reported gene NtOSAK [24] was assigned to subfamily I. As shown in Fig. 2a.
Fig. 1 Primary structure analysis of NtSnRK2.1–3 cDNA. Roman numerals indicate the 11 conserved subdomains of serine/threonine kinases. The conserved prosite motifs are underlined: 1 the ATP binding site, 2 protein kinase activating signature, and 3 trans-membrane spanning region. Sequence alignments were conducted using the DNAStar’s Megalign program. Shared residues are shown in black background, and gaps are indicated by dashes
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A phylogenetic tree created on the basis of the sequences at the C-terminus was essentially the same. A comparison of the C-terminal domain (Fig. 2b) suggested that there were more sequence similarities between subfamilies II and III than between other pairs. For example, the acidic patches of subfamilies II and III are abundant in Asp, while those of subfamily I are rich in Glu; the C-terminus blocks in subfamilies II and III are conserved; and there are fewer amino acids in these subfamilies than in subfamily I.
amino acid sequence of NtSnRK2.1–3 contains a transmembrane spanning region, suggesting that NtSnRK2.1–3 might mediate the association between nuclear and cell membrane system. We investigate the cellular distribution of NtSnRK2s proteins in living onion epidermal cells through transient expression. As shown in Fig. 3, NtSnRK2s proteins were localized to the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco under various abiotic stresses
Subcellular localization of NtSnRK2 proteins Protein kinases are localised in specific cell compartments in order to perform their proper function. The deduced Fig. 2 Tobacco, rice and Arabidopsis SnRK2 kinases. a Phylogenetic analysis of tobacco, rice and Arabidopsis SnRK2 protein kinase. Three groups are presented in grey. The tobacco and Arabidopsis kinases are separately indicated with black and white backgrounds. The phylogenetic tree was constructed with amino acid sequences using the ClustalW and PHYLIP soft package. b C-terminus alignment. Shared sequences are shown in black background. Dashed lines indicate gaps in the sequences to allow maximal alignment
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The qRT-PCR was used to analyse the transcription changes of NtSnRK2s after exposure to PEG (to induce water deficit), high salinity, cold, and ABA. As shown in
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Fig. 4a, although various expression patterns were upregulated in response to these treatments, NtSnRK2.1–3 transcription was induced with a similar expression pattern in each treatment group. In response to PEG treatment, the transcripts peaked at 1 h, decreased sharply, and got a second peak at 24 h. Under NaCl stress, the transcription of NtSnRK2.1–3 was induced rapidly, remained at a steady level until 6 h, and decreased at 12 h. After this time the patterns diverged: NtSnRK2.1 remained at a relatively low level, two to four fold; NtSnRK2.2 increased gradually from two fold to eight fold; and NtSnRK2.3 declined gradually from seven fold to four fold. Under cold stress, the transcription of NtSnRK2s increased gradually and got its maximum at 48 h. ABA treatment induced only a slight increase in transcription of NtSnRK2.1–3 compared with the other abiotic stresses. Three hours after treatment, the transcription levels were no more than twofold, and the peak levels following ABA treatment were 2.8, 4.1, and 5.4-fold in the three experimental replicates. These dynamic expression profiles provided evidence that NtSnRK2.1–3 are involved in the abiotic stress responses of tobacco. Under various stresses, the total
soluble sugars and the amount of reducing sugar in tobacco leaves increased significantly (Fig. 4b). The transcription of NtSnRK2.1 in transgenic plants NtSnRK2.1 plants (T0) were firstly identified on kanamycin plates and re-verified by RT-PCR and GUS histochemical staining assays (Fig. 5a–c). Four NtSnRK2.1 plants were randomly selected for detection of gene transcription levels. Compared with untransformed plants (CK) and GUS controls, the expression levels of NtSnRK2.1 increased significantly in transgenic plants and varied in different transgenic plants. Notably, NtSnRK2.1 was over-expressed strongly in NtSnRK2.1–3 and relatively weakly overexpressed in NtSnRK2.1-1 (Fig. 5d). The two NtSnRK2.1 plants were used in stress tolerance assays. NtSnRK2.1 plants exhibit enhanced stress tolerance To investigate the function of NtSnRK2s under stress, explants of NtSnRK2.1 plants were cultured on differentiation media containing 150 mM NaCl. Two weeks after
Fig. 3 Subcellular localization of NtSnRK2.1–3. Onion epidermal cells were separately bombarded with GFP or NtSnRK2s-GFP constructs. 1 images of green fluorescence, 2 bright field images, and 3 overlaid images. Bar = 100 lm
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Fig. 4 Expression patterns of NtSnRK2s (a), total soluble sugars and reducing sugar contents (b) in tobacco leaves under various treatments. Relative expression changes of NtSnRK2s were calculated using the 2-DDCT method. The transcripts, total soluble sugars and
reducing sugar contents in non-stressed seedling leaves were used as the control. Values are mean of three samples ± SE. *Significantly different from the control with F-test (*P \ 0.05, **P \ 0.01)
salinity treatment, most of the CK and GFP controls stopped differentiating and began to bleach and die (Fig. 6a). By comparison, some explants of the NtSnRK2.11 group and most explants of the NtSnRK2.1–3 group were vigorous and continued to proliferate under the salinity treatment. As shown in Fig. 6b, significantly more explants of the NtSnRK2.1 group survived than did plants of controls. Furthermore, the NtSnRK2.1–3 plant that showed higher transcription levels exhibited more effective salt tolerance than did plants in the NtSnRK2.1-1 group.
maintained the original gene structure after expansion. Moreover, phylogenetic tree analysis showed that the orthologous genes from rice, maize, Arabidopsis, and tobacco were clustered in the same clade (Fig. 2), implying that SnRK2s originated before the divergence of monocots and dicots. Examining the fusion protein in living onion epidermis cells is a credible method to investigate the cellular distribution of GFP/protein [28]. Using this method, four SnRK2 members in wheat were distributed in the nuclear and cytoplasmic compartments [15–18]. The rice SnRK2 genes were found in similar locations [29]. In the current study, NtSnRK2.1–3 kinases were also present in the nuclear and cytoplasmic compartments, implying that they may fulfil similar functions in plant. The detailed locations of NtSnRK2s remain to be determined. Numerous studies have demonstrated that SnRK2 is involved in multiple abiotic stress responses. This study focused on determining the dynamic transcription changes of NtSnRK2s under various stresses and ABA treatments (Fig. 4a) and suggested that NtSnRK2.1–3 kinases were involved in an intricate network for responses to multiple environmental stressors. A comparison of these expression patterns showed that induction of NtSnRK2.1–3 (clustered in subfamily II) was more rapid following stresses than
Discussion Over the course of evolutionary time, some duplicated genes involved in plant development may have diverged and evolved rapidly through selection or acquisition to cope with various external stressors [27]. It was suggested that SnRK2 originated from SnRK1 duplication and then evolved rapidly to acquire new functions that enabled plants to develop networks linking stress signalling with metabolism [7]. Sequence alignments showed that NtSnRK2s had highly conserved structure (Fig. 1), indicating that the SnRK2 genes in tobacco might have developed from a same evolutionary process and thus
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Mol Biol Rep Fig. 5 Identification of the NtSnRK2.1 transformed tobacco plants. a Screened positive transgenic plants on kanamycin plates. 1 Non-transgenic explants differentiated on kanamycin plates (50 lg/mL) as negative control, 2 Nontransgenic explants differentiated on MS medium as positive control, 3 Transformed leaf discs differentiated on kanamycin plates (50 lg/mL). b Identification of transgenic NtSnRK2.1 plants by RT-PCR. M, 200-bp ladder; 1, positive control (p35S-NtSnRK2.1-GUS construct); 2, negative control (transgenicp35S-GUS plants); 3–11, transgenic p35SNtSnRK2.1-GUS plants. c Identification of positive transgenic plants by GUS histochemical staining assays. CK untransformed plants, GUS p35S-GUS-NOS transformed plants, 1–4 four individual NtSnRK2.1 transgenic plants. d Expression levels of NtSnRK2.1 in untransformed and transgenic plants
following ABA treatment. This finding suggests that ABA does not induce NtSnRK2.1–3 directly, and some other factors might be involved in induction. Among these stimuli, NtSnRK2.1–3 responded much more quickly to PEG and NaCl stresses than to cold stress, suggesting that NtSnRK2.1–3 kinases were more sensitive to osmotic stress. These results indicated that SnRK2s in subfamily II may have similar functions in tobacco. In Arabidopsis, SnRK2 subfamily II and III members were reported to be involved in ABA dependent signalling pathways [19, 20], Furthermore, SAPK8-10 (subfamily III) were induced significantly by ABA treatment [21]. Supporting these findings, NtSnRK2.1–3 kinases, clustered in subfamily II, were induced by ABA treatment. Genetic analyses in ABA signaling indicated that PP2C functions as a negative regulator of SnRK2 in the absence of ABA.
When ABA accumulates, it binds to PYR/PYL/RCAR receptor. The complex could repress PP2C, and permits auto-activation of SnRK2 [30, 31]. However, these studies only used subfamily III of SnRK2; the molecular mechanisms of ABA-dependent and independent pathways of other subfamilies remain to be elucidated. Despite the fact that the SnRK2 family is related to the evolutionarily conserved Snf1/AMPK/SnRK1 kinases, which are active in metabolic functions and energy sensing, little is known about the roles of SnRK2 in carbohydrate metabolism. A recent report found that AtSnRK2.6 protein was involved in plant growth through regulating sucrose metabolism in Arabidopsis [20]. Here, the amounts of total soluble sugars and reducing sugars in tobacco leaves significantly increased under various stresses and ABA treatment (Fig. 4b). Soluble sugar accumulation may lower
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However, the stress tolerance assay primarily used the T0NtSnRK2.1 plants. Further comprehensive investigations of the morphological and physiological features of pure NtSnRK2.1 lines under adverse conditions are ongoing. The results of these studies may enable us to strengthen the abiotic stress tolerance of crop plants and to dissect the actual molecular mechanisms by which NtSnRK2s functions in tobacco. Acknowledgments This work was supported by the grants from State Tobacco Monopoly Administration of China (No. 110200902045) and the research program on the metabolism and molecular basis of strong-flavor characteristic high-quality tobacco formation (No. TS-01-2011004).
References
Fig. 6 NtSnRK2.1 plants exhibit enhanced salt tolerance on differentiation media. a Explants of untransformed and transgenic plants were cultured in differentiation media containing 150 mM NaCl. CK untransformed plants, GUS p35S-GUS-NOS transformed plants, 1, 3 individual NtSnRK2.1 transgenic plants. b Comparison of the survival explants (%) of NtSnRK2.1 plants and the two controls under stress. Values are mean ± SE, n = 33. Significant difference between NtSnRK2.1 plants and controls with F-test (**P \ 0.01)
the osmotic potential in the plant sap that helps to maintain regular cell turgor under osmotic stress, and thus enhance stress tolerance of plants. In our previous study, the overexpression of wheat SnRK2s in Arabidopsis resulted in lower osmotic potential and enhanced tolerance to stresses [15–18]. Future research should use transgenic analysis to decipher the relationship between the transcription levels of NtSnRK2s and increased amounts of sugars under osmotic stresses. To examine the in vivo role of NtSnRK2s in plants under stress responses, the construct NtSnRK2.1-GUS was transferred into tobacco. In accordance with the expression patterns, over-expressing NtSnRK2.1 plants tolerated severe osmotic stress better, and higher transcription levels were associated with greater stress tolerance (Fig. 6).
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Cloning and characterization of SnRK2 subfamily II genes from Nicotiana tabacum Hongying Zhang • Hongfang Jia • Guoshun Liu • Shengnan Yang • Songtao Zhang • Yongxia Yang Peipei Yang • Hong Cui
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Received: 27 October 2013 / Accepted: 28 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract SnRK2 is a plant-specific protein kinase family involved in abiotic stress signalling. In this study, NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3, were cloned from tobacco by in silico cloning and reverse transcription PCR. The three protein kinases were classed into subfamily II of the SnRK2 family using a phylogenetic tree and C-terminus analysis. Subcellular localization revealed NtSnRK2s in the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco plants that were exposed to drought, salt, or cold stressors were characterised using quantitative real-time PCR. It was revealed that the three genes showed similar patterns of transcription under abiotic stress responses; there was evidence NtSnRK2s participated in abscisic acid-dependent signalling pathways. NtSnRK2.1–3 responded much faster to drought and salt than to cold stress. To investigate the role of NtSnRK2s under abiotic stresses, NtSnRK2.1 gene was over-expressed in tobacco. A stress tolerance assay showed that tobacco plants that over-expressed NtSnRK2.1 plants had greater salt tolerance. The results indicate that NtSnRK2s are involved in abiotic stress response pathways. Keywords NtSnRK2s Gene expression Subcellular localization Abiotic stress Nicotiana tabacum
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3440-y) contains supplementary material, which is available to authorized users. H. Zhang H. Jia G. Liu S. Yang S. Zhang Y. Yang P. Yang H. Cui (&) Key Laboratory for Cultivation of Tobacco Industry, College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China e-mail: [email protected]
Introduction Abiotic stresses, such as high salinity, drought and extreme temperatures, impose osmotic stresses on plants, leading to imbalances in ionic homeostasis, oxidative damages, and growth inhibition. To cope with multiple adverse stresses, plants have developed a range of molecular mechanisms to perceive stress signals and to manifest adaptive responses [1, 2]. Protein phosphorylation/dephosphorylation is a major mechanism for mediating intracellular responses, including responses to hormonal, pathogenic, and environmental stimuli; it also plays a role in control of metabolism [3]. Many studies in plants have indicated that protein kinases are involved in stress signalling via phosphorylation. The sucrose non-fermenting1 (SNF1) kinase family is known as SNF1 protein kinases in yeasts, mammalian AMP-activated protein kinases, and plant SNF1-related protein kinases (SnRKs) [4]. Compelling evidence shows that all the kinases in yeasts and mammals play pivotal roles in carbohydrate metabolism, whereas plant SnRKs may function as interfaces between stress and metabolic signalling pathways [5–7]. A relatively small plant-specific gene family, SnRK2s encode serine/threonine kinases containing an N-terminal region and a relatively shot C-terminal region. The divergent C-terminal domain is rich in acidic amino acids (Asp/ Glu). According to the C-terminal region, SnRK2 kinases are further grouped into two subfamilies: SnRK2a and SnRK2b [5]. It was suggested that the C-terminal region may function in kinase activation, participate in protein– protein interactions, and be involved in abscisic acid signalling pathways [8–10]. Increasing evidence suggests that SnRK2 kinases are convergence points for many signalling pathways, and
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function in diverse environmental stress responses [5, 7]. The first member of the SnRK2s to be described, PKABA1 (also named TaW55), was isolated from wheat and was activated by hyperosmotic stress and ABA treatment [11]. Subsequent work indicated that it acts as an intermediate in the ABA suppression of GA-responsive gene transcription [12–14]. In our recent study, four SnRK2 members of wheat were isolated and characterised. Over-expression of these protein kinases could significantly enhance the tolerance of abiotic stressors in Arabidopsis. Experimental evidence suggested that TaSnRK2s were involved in stress signalling pathways and carbohydrate metabolism [15–18]. Ten SnRK2 kinases were cloned from Arabidopsis, among which nine were induced by salinity and hyperosmotic stresses, and five of the nine were induced by ABA, while none was induced by cold stress [19, 20]. In rice, 10 SnRK2 members (SAPK1–10) have been identified. All these kinases were induced by osmotic stress, and three of them (SAPK8, SAPK9, and SAPK10) were also induced by ABA treatment [21]. Over-expression of SAPK4 in rice could significantly enhance the salt tolerance [22]. Similarly, 10 SnRK2s were characterised in maize, and all these kinases were activated by osmotic stresses [23]. In tobacco, osmotic stress-activated protein kinase (NtOSAK) has been assigned to the SnRK2 family [24]. A detailed analysis revealed that a complex of a GAPDH isoform and NtOSAK was partially localised in the nucleus in BY2 cells after salt stress. This complex seemed to be regulated by NO:GAPDH and was directly S-nitrosylated, which could promote its translocation into the nucleus of the complex [25]. Thus, solid evident suggests that plant SnRK2 family is involved in the abiotic stress responses, each member fulfilling distinct functions. However, the specific functions of tobacco SnRK2s and the molecular mechanism of their activation have seldom been studied. In this study, we isolated members of the tobacco SnRK2 family and characterised their expression patterns under diverse environmental stresses. Transgenic experiments indicated that the over-expression of NtSnRK2.1 in tobacco significantly increased the plant’s tolerance of salt stress.
were stressed by exposure to a PEG 6000 solution (0.5 MPa water potential), a 300 mM NaCl solution, 4 °C, or a 50 lM ABA spray, all of which were shown to constitute significant stress in pilot experiments. Control plants were cultured normally. All the true leaves, sampled at 0, 1, 3, 6, 12, 24, 48, and 72 h after treatments, were removed at the main midribs. One leaf from each plant was used for RNA extraction. The other was analysed to determine the amounts of total soluble sugars and reducing sugars according to tobacco industrial standard methods YC/T 159-2002. Isolation of the cDNA sequences of NtSnRK2s The amino acid sequences of Arabidopsis SnRK2s were used as query probes to screen the tobacco expressed sequence tag (EST) database. These candidate ESTs were assembled into contiguous fragments by in silico cloning. Based on the putative cDNA sequence, specific primers were designed to amplify the NtSnRK2s cDNA sequences (Supplementary Table S1). Similarity searches and sequence alignments were conducted using BLAST and DNAStar’s Megalign program, respectively. The functional region and activity sites were predicted with SignalP and PROSITE servers. To determine the relationship between NtSnRK2s and SnRK2 members in other plant species, ClustalW and PHYLIP soft package (version 3.69) were used to construct a phylogenetic tree, which was viewed using TREEVIEW software. Quantitative real-time PCR (qRT-PCR) The cDNA was synthesized using the M-MuLVreverse transcriptase (New England BioLabs) according to the instruction manual. The qRT-PCR was performed with SYBR Premix Ex TaqTM (Takara) using an ABI PRISMÒ 7000 system (Applied Biosystems). Specific primers are listed in Supplementary Table S2. The transcription of ribosomal protein gene L25 was used as an internal control. Relative expression changes of NtSnRK2s were calculated using the 2-DDCT method [26].
Materials and methods
Subcellular localization of NtSnRK2s
Stress experiments and amounts of total soluble sugars and reducing sugars
To construct an NtSnRK2s-GFP fusion protein, the coding sequence of NtSnRK2s without a termination codon was fused upstream of GFP and downstream of 35S of the pJIT163-GFP expression vector. The recombinant pJIT16335S-NtSnRKs-GFP construct was transferred into living onion (Allium cepa) epidermal cells with a GeneGun (Biorad HeliosTM). The transformed cells were incubated and observed as described previously [16].
Seeds of tobacco (Kentucky 326) were directly sown in mixed soil (1:1 vermiculite: humus) saturated with water in salver. Seedlings were cultured in a growth chamber at 22 °C under a 16 h light/8 h dark cycle for 8 weeks. Next, the plants were assigned to a treatment group in which they
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Mol Biol Rep Table 1 Three SnRK2s in tobacco and some characteristics of them Gene name
ORF (bp)
Protein length (aa)
Protein molecular mass (kd)
pI
NtSnRK2.1
1020
339
38.2
5.85
NtSnRK2.2
1035
344
39.0
5.23
NtSnRK2.3
1020
339
38.3
5.59
differentiation media containing 150 mM NaCl, which was shown to cause significant stress in pilot experiments. When symptoms of salt stress were evident, the percentage of green explants was recorded. Results Molecular characterization of NtSnRK2s
Transformation of NtSnRK2.1 in tobacco The coding region of NtSnRK2.1 was cloned into the vector pCAMBIA-NPT-GUS as a b-glucuronidase (GUS)-fused fragment using the primers 50 -GAGAGAATTCCCCTTG ATTATGGAGCGT-30 (EcoR I site underlined) and 50 -GA GAACTAGTTTACATCACAATTGACCAACA-30 (Spe I site underlined). The vectors p35S-NtSnRK2.1-GUS and p35S-GUS were separately transformed into Agrobacterium tumefaciens (strain LBA4404) cells and then transferred into tobacco. NtSnRK2.1 plants (T0) were firstly identified on kanamycin plates (50 lg/mL) and further verified by GUS histochemical staining assays and RTPCR. The RT-PCR was performed using the primers 50 -GC CAGTGGAACTGATGGAAGGAGG-30 and 50 -GGGTGA ACGTGTAGTGCGCTGTATT-30 , which were designed according to NtSnRK2.1 and GUS genes, respectively. Abiotic stress tolerance assays Leaves from untransformed and transgenic plants were removed, and leaf discs measuring 1.2 cm2 in area were aseptically excised from them, in each case from the same part of the leaf. These explants were cultured on
The cDNA sequences of three NtSnRK2s were identified from tobacco through in silico cloning and RT-PCR (Table 1). These protein kinase genes were designated NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3 on the basis of their similarity to their orthologous counterparts in rice and maize. Prediction of the functional region and activity sites indicated that NtSnRK2s have the potential for serine/ threonine kinase activities and, like other SnRK2 members, have two domains (an N-terminal catalytic domain and a C-terminal regulatory domain). The C-terminal regions of NtSnRK2s are quite divergent. However, the N-terminal regions of NtSnRK2s are highly conserved and contain 11 subdomains that are identified in serine/threonine kinases (Fig. 1). In addition, a protein kinase-activating site and an ATP-binding site are detected in the catalytic domain. Phylogenetic analysis based on the putative amino acid sequences of NtSnRK2s and SnRK2 members of rice, maize, and Arabidopsis, showed that these sequences were grouped into three subfamilies (subfamily I, II, and III); NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3 are assigned to the same clade, subfamily II, whereas the previously reported gene NtOSAK [24] was assigned to subfamily I. As shown in Fig. 2a.
Fig. 1 Primary structure analysis of NtSnRK2.1–3 cDNA. Roman numerals indicate the 11 conserved subdomains of serine/threonine kinases. The conserved prosite motifs are underlined: 1 the ATP binding site, 2 protein kinase activating signature, and 3 trans-membrane spanning region. Sequence alignments were conducted using the DNAStar’s Megalign program. Shared residues are shown in black background, and gaps are indicated by dashes
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A phylogenetic tree created on the basis of the sequences at the C-terminus was essentially the same. A comparison of the C-terminal domain (Fig. 2b) suggested that there were more sequence similarities between subfamilies II and III than between other pairs. For example, the acidic patches of subfamilies II and III are abundant in Asp, while those of subfamily I are rich in Glu; the C-terminus blocks in subfamilies II and III are conserved; and there are fewer amino acids in these subfamilies than in subfamily I.
amino acid sequence of NtSnRK2.1–3 contains a transmembrane spanning region, suggesting that NtSnRK2.1–3 might mediate the association between nuclear and cell membrane system. We investigate the cellular distribution of NtSnRK2s proteins in living onion epidermal cells through transient expression. As shown in Fig. 3, NtSnRK2s proteins were localized to the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco under various abiotic stresses
Subcellular localization of NtSnRK2 proteins Protein kinases are localised in specific cell compartments in order to perform their proper function. The deduced Fig. 2 Tobacco, rice and Arabidopsis SnRK2 kinases. a Phylogenetic analysis of tobacco, rice and Arabidopsis SnRK2 protein kinase. Three groups are presented in grey. The tobacco and Arabidopsis kinases are separately indicated with black and white backgrounds. The phylogenetic tree was constructed with amino acid sequences using the ClustalW and PHYLIP soft package. b C-terminus alignment. Shared sequences are shown in black background. Dashed lines indicate gaps in the sequences to allow maximal alignment
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The qRT-PCR was used to analyse the transcription changes of NtSnRK2s after exposure to PEG (to induce water deficit), high salinity, cold, and ABA. As shown in
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Fig. 4a, although various expression patterns were upregulated in response to these treatments, NtSnRK2.1–3 transcription was induced with a similar expression pattern in each treatment group. In response to PEG treatment, the transcripts peaked at 1 h, decreased sharply, and got a second peak at 24 h. Under NaCl stress, the transcription of NtSnRK2.1–3 was induced rapidly, remained at a steady level until 6 h, and decreased at 12 h. After this time the patterns diverged: NtSnRK2.1 remained at a relatively low level, two to four fold; NtSnRK2.2 increased gradually from two fold to eight fold; and NtSnRK2.3 declined gradually from seven fold to four fold. Under cold stress, the transcription of NtSnRK2s increased gradually and got its maximum at 48 h. ABA treatment induced only a slight increase in transcription of NtSnRK2.1–3 compared with the other abiotic stresses. Three hours after treatment, the transcription levels were no more than twofold, and the peak levels following ABA treatment were 2.8, 4.1, and 5.4-fold in the three experimental replicates. These dynamic expression profiles provided evidence that NtSnRK2.1–3 are involved in the abiotic stress responses of tobacco. Under various stresses, the total
soluble sugars and the amount of reducing sugar in tobacco leaves increased significantly (Fig. 4b). The transcription of NtSnRK2.1 in transgenic plants NtSnRK2.1 plants (T0) were firstly identified on kanamycin plates and re-verified by RT-PCR and GUS histochemical staining assays (Fig. 5a–c). Four NtSnRK2.1 plants were randomly selected for detection of gene transcription levels. Compared with untransformed plants (CK) and GUS controls, the expression levels of NtSnRK2.1 increased significantly in transgenic plants and varied in different transgenic plants. Notably, NtSnRK2.1 was over-expressed strongly in NtSnRK2.1–3 and relatively weakly overexpressed in NtSnRK2.1-1 (Fig. 5d). The two NtSnRK2.1 plants were used in stress tolerance assays. NtSnRK2.1 plants exhibit enhanced stress tolerance To investigate the function of NtSnRK2s under stress, explants of NtSnRK2.1 plants were cultured on differentiation media containing 150 mM NaCl. Two weeks after
Fig. 3 Subcellular localization of NtSnRK2.1–3. Onion epidermal cells were separately bombarded with GFP or NtSnRK2s-GFP constructs. 1 images of green fluorescence, 2 bright field images, and 3 overlaid images. Bar = 100 lm
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Fig. 4 Expression patterns of NtSnRK2s (a), total soluble sugars and reducing sugar contents (b) in tobacco leaves under various treatments. Relative expression changes of NtSnRK2s were calculated using the 2-DDCT method. The transcripts, total soluble sugars and
reducing sugar contents in non-stressed seedling leaves were used as the control. Values are mean of three samples ± SE. *Significantly different from the control with F-test (*P \ 0.05, **P \ 0.01)
salinity treatment, most of the CK and GFP controls stopped differentiating and began to bleach and die (Fig. 6a). By comparison, some explants of the NtSnRK2.11 group and most explants of the NtSnRK2.1–3 group were vigorous and continued to proliferate under the salinity treatment. As shown in Fig. 6b, significantly more explants of the NtSnRK2.1 group survived than did plants of controls. Furthermore, the NtSnRK2.1–3 plant that showed higher transcription levels exhibited more effective salt tolerance than did plants in the NtSnRK2.1-1 group.
maintained the original gene structure after expansion. Moreover, phylogenetic tree analysis showed that the orthologous genes from rice, maize, Arabidopsis, and tobacco were clustered in the same clade (Fig. 2), implying that SnRK2s originated before the divergence of monocots and dicots. Examining the fusion protein in living onion epidermis cells is a credible method to investigate the cellular distribution of GFP/protein [28]. Using this method, four SnRK2 members in wheat were distributed in the nuclear and cytoplasmic compartments [15–18]. The rice SnRK2 genes were found in similar locations [29]. In the current study, NtSnRK2.1–3 kinases were also present in the nuclear and cytoplasmic compartments, implying that they may fulfil similar functions in plant. The detailed locations of NtSnRK2s remain to be determined. Numerous studies have demonstrated that SnRK2 is involved in multiple abiotic stress responses. This study focused on determining the dynamic transcription changes of NtSnRK2s under various stresses and ABA treatments (Fig. 4a) and suggested that NtSnRK2.1–3 kinases were involved in an intricate network for responses to multiple environmental stressors. A comparison of these expression patterns showed that induction of NtSnRK2.1–3 (clustered in subfamily II) was more rapid following stresses than
Discussion Over the course of evolutionary time, some duplicated genes involved in plant development may have diverged and evolved rapidly through selection or acquisition to cope with various external stressors [27]. It was suggested that SnRK2 originated from SnRK1 duplication and then evolved rapidly to acquire new functions that enabled plants to develop networks linking stress signalling with metabolism [7]. Sequence alignments showed that NtSnRK2s had highly conserved structure (Fig. 1), indicating that the SnRK2 genes in tobacco might have developed from a same evolutionary process and thus
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Mol Biol Rep Fig. 5 Identification of the NtSnRK2.1 transformed tobacco plants. a Screened positive transgenic plants on kanamycin plates. 1 Non-transgenic explants differentiated on kanamycin plates (50 lg/mL) as negative control, 2 Nontransgenic explants differentiated on MS medium as positive control, 3 Transformed leaf discs differentiated on kanamycin plates (50 lg/mL). b Identification of transgenic NtSnRK2.1 plants by RT-PCR. M, 200-bp ladder; 1, positive control (p35S-NtSnRK2.1-GUS construct); 2, negative control (transgenicp35S-GUS plants); 3–11, transgenic p35SNtSnRK2.1-GUS plants. c Identification of positive transgenic plants by GUS histochemical staining assays. CK untransformed plants, GUS p35S-GUS-NOS transformed plants, 1–4 four individual NtSnRK2.1 transgenic plants. d Expression levels of NtSnRK2.1 in untransformed and transgenic plants
following ABA treatment. This finding suggests that ABA does not induce NtSnRK2.1–3 directly, and some other factors might be involved in induction. Among these stimuli, NtSnRK2.1–3 responded much more quickly to PEG and NaCl stresses than to cold stress, suggesting that NtSnRK2.1–3 kinases were more sensitive to osmotic stress. These results indicated that SnRK2s in subfamily II may have similar functions in tobacco. In Arabidopsis, SnRK2 subfamily II and III members were reported to be involved in ABA dependent signalling pathways [19, 20], Furthermore, SAPK8-10 (subfamily III) were induced significantly by ABA treatment [21]. Supporting these findings, NtSnRK2.1–3 kinases, clustered in subfamily II, were induced by ABA treatment. Genetic analyses in ABA signaling indicated that PP2C functions as a negative regulator of SnRK2 in the absence of ABA.
When ABA accumulates, it binds to PYR/PYL/RCAR receptor. The complex could repress PP2C, and permits auto-activation of SnRK2 [30, 31]. However, these studies only used subfamily III of SnRK2; the molecular mechanisms of ABA-dependent and independent pathways of other subfamilies remain to be elucidated. Despite the fact that the SnRK2 family is related to the evolutionarily conserved Snf1/AMPK/SnRK1 kinases, which are active in metabolic functions and energy sensing, little is known about the roles of SnRK2 in carbohydrate metabolism. A recent report found that AtSnRK2.6 protein was involved in plant growth through regulating sucrose metabolism in Arabidopsis [20]. Here, the amounts of total soluble sugars and reducing sugars in tobacco leaves significantly increased under various stresses and ABA treatment (Fig. 4b). Soluble sugar accumulation may lower
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However, the stress tolerance assay primarily used the T0NtSnRK2.1 plants. Further comprehensive investigations of the morphological and physiological features of pure NtSnRK2.1 lines under adverse conditions are ongoing. The results of these studies may enable us to strengthen the abiotic stress tolerance of crop plants and to dissect the actual molecular mechanisms by which NtSnRK2s functions in tobacco. Acknowledgments This work was supported by the grants from State Tobacco Monopoly Administration of China (No. 110200902045) and the research program on the metabolism and molecular basis of strong-flavor characteristic high-quality tobacco formation (No. TS-01-2011004).
References
Fig. 6 NtSnRK2.1 plants exhibit enhanced salt tolerance on differentiation media. a Explants of untransformed and transgenic plants were cultured in differentiation media containing 150 mM NaCl. CK untransformed plants, GUS p35S-GUS-NOS transformed plants, 1, 3 individual NtSnRK2.1 transgenic plants. b Comparison of the survival explants (%) of NtSnRK2.1 plants and the two controls under stress. Values are mean ± SE, n = 33. Significant difference between NtSnRK2.1 plants and controls with F-test (**P \ 0.01)
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