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
© The Author(s). 2016
Received: 16 March 2016
Accepted: 5 July 2016
Published: 13 July 2016
Family members of sucrose non-fermenting 1-related kinase 2 (SnRK2), being plant-specific serine/threonine protein kinases, constitute the central core of abscisic acid (ABA)-dependent and ABA-independent signaling pathways, and are key regulators of abiotic stress adaptation in plants. We report here the functional characterization of SAPK9 gene, one of the 10 SnRK2s of rice, through developing gain-of-function and loss-of-function phenotypes by transgenesis.
The gene expression profiling revealed that the abundance of single gene-derived SAPK9 transcript was significantly higher in drought-tolerant rice genotypes than the drought-sensitive ones, and its expression was comparatively greater in reproductive stage than the vegetative stage. The highest expression of SAPK9 gene in drought-tolerant Oryza rufipogon prompted us to clone and characterise the CDS of this allele in details. The SAPK9 transcript expression was found to be highest in leaf and upregulated during drought stress and ABA treatment. In silico homology modelling of SAPK9 with Arabidopsis OST1 protein showed the bilobal kinase fold structure of SAPK9, which upon bacterial expression was able to phosphorylate itself, histone III and OsbZIP23 as substrates in vitro. Transgenic overexpression (OE) of SAPK9 CDS from O. rufipogon in a drought-sensitive indica rice genotype exhibited significantly improved drought tolerance in comparison to transgenic silencing (RNAi) lines and non-transgenic (NT) plants. In contrast to RNAi and NT plants, the enhanced drought tolerance of OE lines was concurrently supported by the upgraded physiological indices with respect to water retention capacity, soluble sugar and proline content, stomatal closure, membrane stability, and cellular detoxification. Upregulated transcript expressions of six ABA-dependent stress-responsive genes and increased sensitivity to exogenous ABA of OE lines indicate that the SAPK9 is a positive regulator of ABA-mediated stress signaling pathways in rice. The yield-related traits of OE lines were augmented significantly, which resulted from the highest percentage of fertile pollens in OE lines when compared with RNAi and NT plants.
The present study establishes the functional role of SAPK9 as transactivating kinase and potential transcriptional activator in drought stress adaptation of rice plant. The SAPK9 gene has potential usefulness in transgenic breeding for improving drought tolerance and grain yield in crop plants.
KeywordsAbscisic acid (ABA) Drought tolerance Gene silencing Grain yield Overexpression Osmotic potential Rice crop SAPK9 Stomatal closure Stress-responsive gene Sucrose non-fermenting 1-related kinase 2 (SnRK2)
Plants, being sessile are incessantly confronted by different environmental stresses, which include drought, high salinity, and extreme temperature, affecting both biomass productivity and grain yield of crops. To cope with such adverse multiple stresses, diverse molecular and physiological mechanisms have been evolved by the plant kingdom in general and a plant species in particular. Therefore, it is indispensable to comprehend the specific mechanism of responses by any crop species to such stresses, with the ultimate aim of improving crop performance. Rice (Oryza sativa L.), being one of the most important cereal crops, feeds more than half of the world population and is adversely affected by drought at the morphological, physiological and molecular level. The phytohormone abscisic acid (ABA), which is produced under the drought stress functions to regulate several developmental and physiological processes including seed maturation, germination, seedling growth and transpiration. The ABA level increases under water deficit condition in plants triggering stomatal closure and responses to stress tolerance . Stress signals are recognized by specialized signaling pathways which transmit them to different cellular compartments, and the numerous evidences demonstrated that protein kinases play vital roles in the responses to such environmental stimuli . The sucrose non-fermenting 1-related kinase 2 (SnRK2) family members, which function in diverse developmental processes in plants [3, 4], have been shown to be the positive regulators of plant response to abiotic stresses [5–8]. The members of SnRK2 family work at the merging point of the ABA-dependent and ABA-independent stress signaling pathways.
In Arabidopsis, ten members of the SnRK2 family have been identified and divided into three subclasses based on amino acid sequence similarity . The subclass I comprises of kinases that are not activated by ABA, subclass II are either not activated or very weakly activated by ABA (depending on plant species). However, the subclass III is strongly activated by ABA. The amino acid sequences of all the SnRK2s can be divided into two regions, the highly conserved N-terminal kinase domain, and the C-terminal regulatory domain. The C-terminal domain contains stretches of acidic amino acids, either glutamic acid (E) (subclass I) or aspartic acid (D) (subclass II and III). Further, the C-terminal regulatory domain consists of two subdomains, subdomain I and subdomain II. The subdomain I which is required for activation by osmotic stress, independent of ABA, is present in all SnRK2 family members; whereas subdomain II is necessary for the ABA response and is specific to the ABA-dependent SnRK2s only. All the SnRK2 members, except SnRK2.9, were found to be rapidly induced by different osmolyte treatments, such as mannitol, sorbitol, sucrose or NaCl and few of them also by ABA . The Arabidopsis OST1/SnRK2.6/SRK2E protein kinase has been shown to regulate the ABA-mediated stomatal closure and act upstream of reactive oxygen species production . However, the srk2e (ost1) mutant in Arabidopsis has been found to be defective in ABA-induced stomatal closure and it showed a wilty phenotype . The srk2d/e/i triple mutant (for SnRK2.2/SRK2D, SnRK2.6/SRK2E and SnRK2.3/SRK2I genes) of Arabidopsis displayed dramatically decreased drought tolerance and extreme insensitivity to ABA, as documented by defects in seed germination and seedling growth as well as decreased expression of ABA- and stress-inducible genes . The knockout of these three genes, which belong to the subclass III of SnRK2 family, therefore, almost completely blocks ABA responses, demonstrating that they are the essential components of ABA-stress signaling pathway in Arabidopsis .
Eleven SnRK2 members designated as ZmSnRK2s have been identified in maize, and most of them are inducible by one or more abiotic stresses . The ZmSnRK2.8 protein, which is highly homologous to Arabidopsis OST1, has been found to be involved in diverse stress signaling pathways, particularly in salt stress tolerance [12, 13]. In wheat, the first characterized SnRK2 member, PKABA1 was reported to be induced by hyperosmotic stress, ABA, and multiple other environmental factors [14, 15]. Afterwards, three more wheat SnRK2 members viz., TaSnRK2.3, TaSnRK2.4 and TaSnRK2.8 have been characterized and found to be involved in development and abiotic stress tolerance [16–18]. Thus, substantial evidence showed that the SnRK2 protein kinases are involved in multiple environmental stress responses and all have potential biotechnological utility for generation of high yielding abiotic stress tolerant crops . In vitro studies have documented that the ABA-activated SnRK2s phosphorylate the downstream target proteins in different plant species; and this phosphorylation is needed for the transcriptional activity of the individual target proteins, which in turn induce the expression of hierarchically downstream genes to mitigate the stress condition. This includes bZIP transcription factors, such as TRAB1 from rice , TaABF from wheat , and AREB1 from Arabidopsis , or RNA-binding VfAKIP1 proteins from Vicia faba .
In rice, the ten SnRK2 members, designated as SAPK1-10 (osmotic stress/ABA-activated protein kinase 1-10) have been identified. All of them were found to be activated by hyperosmotic stress, and SAPK8-10 were also induced by ABA . The overexpression of SAPK4 showed improved salt tolerance in rice . In the domain swapping experiments using rice SAPKs, it has been observed that the grafting of the non-catalytic C-terminal region from SAPK8 (Glu-254 to Met-372), which contains the domain involved in ABA-responsive activation, onto the catalytic domain of SAPK2, which doesn’t contain the domain for ABA-responsive activation, is sufficient to confer ABA responsiveness in SAPK2 . None of the three ABA-dependent and osmotic stress-activated SAPKs, i.e. SAPK8, SAPK9, and SAPK10, belonging to subclass III of SnRK2 family, has been functionally characterized by transgenic approach till date. Therefore, understanding the molecular basis of subclass III SAPK gene function in rice is necessary for the development of drought-tolerant transgenic rice or to design SAPK gene-based marker for molecular breeding of drought-tolerant rice cultivar. We were interested in the detailed characterization of the SAPK9 gene and its functional role in drought tolerance through (i) expression profiling of the gene in selected drought-sensitive and drought-tolerant rice genotypes, (ii) determining the allelic polymorphism in the coding DNA sequence (CDS) of the gene in these genotypes, (iii) cloning the highly expressed SAPK9 CDS from the drought-tolerant rice genotype Oryza rufipogon followed by assaying the kinase activity of the recombinant protein, and (iv) developing transgenic rice lines for overexpression and endogenous silencing of this particular gene. Transgenic experiments demonstrated that in contrast to silencing, the overexpression of SAPK9 increased drought tolerance and grain yield by adjusting osmotic potential and stomatal closure, thereby decreasing the cellular membrane damage and reactive oxygen species activity. In addition, the increased and decreased ABA sensitivity and ABA-responsive gene expression in transgenically overexpressed and silenced lines, respectively, confirmed that the SAPK9 is a positive regulator of ABA-dependent stress-responsive signaling pathway in rice.
Plant materials, growth condition and stress treatments
Nine cultivated indica rice (Oryza sativa L.) genotypes and two wild rice progenitors were selected for the experimental works. The cultivated rice genotypes comprised of drought-tolerant- Manipuri, Nagina22, Vandana and drought-sensitive- Swarna, HRC300, IR20, IR36, IR64, and IR72; while the two wild rice genotypes - O. rufipogon and O. nivara are drought-tolerant. The seed samples of all rice genotypes were provided by Indian Agricultural Research Institute, New Delhi. All the experimental works were performed using the seeds of same harvest and storage conditions, and rice genotypes were usually grown for 30 days inside the glasshouse, which was maintained at 25/28 °C temperature with 70 % relative humidity and 16/8 h light/dark photoperiod. Thereafter, the potted plants were transferred into the net-house. To analyse the effects of drought stress and ABA on the expression of SAPK9 gene, rice seedlings were subjected to dehydration and ABA (100 μM) treatment for a period of 6–48 h and an untreated sample at 0 h was used as control. The transcript level of SAPK9 gene was analysed from leaf tissues of rice genotypes grown under: (i) normal growth condition- designated as before stress (BS), (ii) withholding water supply for 8 days- referred as after stress (AS), and (iii) resuming water supply for 3 days after the drought stress period- referred as after recovery (AR). The experiments were performed at both vegetative and reproductive (or grain filling) stages. For T1 transgenic lines and non-transgenic (NT) plants drought stress treatment at the vegetative and early reproductive (panicle initiation) stages, and analysis of grain yield under drought condition were performed following the reported protocol .
Cellular RNA isolation, first strand cDNA synthesis and real‑time PCR analysis
The total cellular RNA was isolated from rice leaves using RNeasy Mini Kit (Qiagen) following the vendor’s protocol. Total RNA (2 μg) was used to synthesize the first strand cDNA with the help of gene-specific 3'-primer and Transcriptor First Strand cDNA synthesis kit version 6.0 (Roche Diagnostics India Pvt. Ltd.) following the instructions of the manufacturer. For transcript expression profiling, real-time PCR was performed by means of SYBR green-based relative quantification method using RealMasterMix SYBR ROX (5 PRIME) kit in Eppendorf Realplex2 Master Cycler. The relative gene expression level was determined following the reported literature . For each sample, three replicates were taken. In each case, rice polyubiquitin1 gene (OsUbi1) was used as internal reference. The different primers used for the real-time PCR are provided in Additional file 1: Table S1.
Standard protocols were followed to isolate the genomic DNA from rice leaves and to perform Southern blot hybridization . In brief, the HindIII-digested genomic DNA (12 μg) upon electrophoresis on agarose gel overnight, was transferred onto nylon membrane (Hybond-N+). A 470 bp DNA fragment from the middle of SAPK9 coding DNA sequence (CDS) from O. rufipogon was PCR-amplified using gene-specific primers SA9SF-SA9SR (Additional file 1: Table S1) and radiolabelled with P32-dCTP (3500 Ci/mmol) by random priming using rediprime II DNA labelling system (GE Healthcare, USA) following the vendor’s instructions. For autoradiography, the Cylone® Plus phosphor system (Perkin Elmer) was used to scan the multi-sensitive X-ray film (Perkin Elmer).
Cloning and sequence analysis of the SAPK9 CDS
The full-length CDS of SAPK9 gene was PCR amplified using 1st strand cDNA sample as a template from the drought-sensitive and drought-tolerant rice genotypes mentioned in plant materials section. The gene-specific primers SK9F and SK9R (Additional file 1: Table S1) were used for the PCR amplification in a thermocycler (Applied Biosystems) with the following thermal profile: an initial denaturation at 98 °C for 2 min, followed by 30 cycles of 98 °C-for 10 s, 62 °C for 15 s, 72 °C for 1 min and a final extension at 72 °C for 8 min. All the PCR amplicons (~1086 bp size) were individually cloned by blunt-end ligation into pUC18 plasmid digested with SmaI restriction enzyme. Positive clones were sequenced, and nucleotide polymorphism of all the CDSs were analysed. The multiple sequence alignment of the CDS-derived polypeptides obtained from all the genotypes was performed using the Jalview 2 software (http://www.jalview.org/Download).
Multiple amino acid sequence alignment of SnRK2 family proteins from Arabidopsis, rice, and a few other plant species were performed using the ClustalX2 (http://www.clusal.org/clustal2/). The phylogenetic tree was constructed using the neighbor-joining method in MEGA6 (http://www.megasoftware.net/mega.php). To evaluate the reliability of the tree, a bootstrap analysis was performed using 1000 replicates in MEGA6.
The homology model of SAPK9 was made using the MODELLER 9.15 (https://salilab.org/modeller/download_installation.html). The solved crystal structure of the OST1/SnRK2.6 protein from Arabidopsis (PDB ID: 3UC4) was taken as a template for constructing the in silico model of SAPK9. The resulting model was energy minimized using Insight II (2000.1, Accelrys Inc.) followed by stereo-chemical evaluation using MolProbity (http://molprobity.biochem.duke.edu/). The pictorial representations were prepared in PyMol (http://www.pymol.org).
In vitro kinase assay
The recombinant pRSET plasmid (Invitrogen) containing the 1086 bp CDS of SAPK9 in XhoI and EcoRI restriction sites allowed expression of 6xHis N-terminal tagged SAPK9 protein in E. coli BL21 (DE3) pLysE strain (Invitrogen) upon 1 mM IPTG induction. Similarly, first 900 bp (encoding 300-amino acid) from the OsbZIP23 CDS of O. rufipogon  was cloned into a pRSET vector in BamHI and EcoRI restriction sites, transformed and expressed in pLysE cells with 0.5 mM IPTG induction. The expressed proteins were purified in native condition and used for in vitro kinase assay. In vitro phosphorylation of generic substrate histone III (Sigma) was performed as described previously . In vitro phosphorylation of OsbZIP23 was performed by incubating the individual reaction mixture for 5, 25 and 40 min at 25 °C following the above-mentioned protocol. The products were fractionated in 12 % SDS-PAGE and visualized by autoradiography.
Preparation of constructs for overexpression and silencing of SAPK9 gene, and development of transgenic rice plants
For the preparation of SAPK9 overexpression (OE) construct, the full-length 1086 bp CDS of SAPK9 was cloned (as SalI-KpnI fragment) from O. rufipogon under the OsUbi1 promoter (as HindIII-SalI fragment) and NOS transcription terminator (as KpnI-EcoRI fragment) in the pCAMBIA1301 binary plasmid. Thus, the SAPK9 OE genetic construct was prepared (Additional file 2: Figure S5a). On the other hand, the RNAi-mediated gene silencing (RNAi) construct of SAPK9 was prepared (Additional file 2: Figure S5b) by cloning a 605 bp DNA fragment from 5′-part of SAPK9 CDS in sense (as SalI-BamHI fragment) and antisense (as BamHI-KpnI fragment) direction flanked by an arbitrary linker DNA (having BamHI site at both ends) under the OsUbi1 promoter (as HindIII-SalI fragment) and NOS transcription terminator (as KpnI-EcoRI fragment) in pCAMBIA1301 binary plasmid. Drought-sensitive IR20 rice cultivar was genetically transformed separately with these OE and RNAi constructs by Agrobacterium-mediated transfer technique, as reported earlier . The transgenic rice lines developed with the OE and RNAi constructs were designated as SAOE#1, 2, 3 etc. and RNAi#1, 2, 3 etc, respectively. The list of primers used for preparing genetic constructs is given in Additional file 1: Table S1.
Western blot analysis of SAPK9 protein in transgenic lines and non-transgenic rice plants
Protein samples (40 μg each) isolated from the leaf tissues of transgenic lines and NT rice plants were used for western blotting experiment as per the method reported earlier . The custom made 15-amino acid peptide (MERNAAGPLGMEMNC) from the N-terminal end of SAPK9 was used to raise the polyclonal antibody in rabbit (IMGENEX India). Affinity purified polyclonal antibody of SAPK9 was taken as primary antibody (with 1: 1000 dilutions). The monoclonal antibody of plant β-actin (Sigma, catalog no.-A0480) was used (with 1:500 dilutions) to detect actin protein in the samples as a loading control. Immuno-detection was performed using the Lumi-LightPLUS Western Blotting Kit (Roche Diagnostics India Pvt. Ltd.), as per the vendor’s protocol.
Estimation of water loss rate, relative water content, proline content and soluble sugar content in leaf samples of transgenic and non-transgenic rice plants
Water loss rate (WLR) and relative water content (RWC) in the leaves of transgenic and NT rice plants were determined on the basis of the published protocols [30, 31]. From the leaves (50 mg) of transgenic lines and NT rice plants free proline content was estimated as per the published literature . Similarly, total soluble sugar content in rice leaves (100 mg) was determined using anthrone reagent . Consistent results were obtained from two sets of the experiment, and the result from one experimental set is documented here. Average of three replicates was calculated to represent each data point.
Analysis of rice stomata by scanning electron microscopy
Scanning electron microscopy analysis of leaf samples from transgenic and NT rice plants (collected before and after drought stress) were performed following the described method . The samples were mounted onto the sample stages and coating treatment was performed. The stomatal pictures were obtained using a scanning electron microscope (ZEISS, Germany).
Estimation of malondialdehyde (MDA) content and detection of reactive oxygen species (ROS) in leaf samples of rice plants
Rice leaf (100 mg) of transgenic lines and NT plants was used to determine the MDA content following the reported method . In vivo localization of ROS in intact leaves of the transgenic lines and NT plants were carried out following the method described earlier . Consistent results were obtained from two sets of experiment and the result from one experimental set is documented here. Average of three replicates was calculated to represent each data point for MDA content.
Subcellular localization of the SAPK9 protein
The SAPK9 CDS was cloned in frame at the 5′-end of the green fluorescence protein (GFP) gene at BglII and SpeI restriction sites under the control of constitutive CaMV35S promoter in the binary plasmid pCAMBIA1302. The list of primers used is given in Additional file 1: Table S1. Agrobacterium-mediated transformation of onion epidermal cells was carried out following the previously published protocol .
Assay of ABA sensitivity during seed germination and post-germination stages
Sensitivity towards ABA during seed germination and post-germination (seedling) stages was analysed using the different concentrations of ABA (0, 1, 3, 6 μM) in the growth media following the reported protocol .
Estimation of spikelet fertility and pollen maturation
The transgenic lines and NT plants were grown normally in PVC pipes. Upon attaining flowering stage, plants were subjected to drought stress for a period of 10 days followed by recovery until seed maturation occurred. The panicle weight and spikelet fertility were calculated in replicates. For pollen staining, 1 % I2-KI solution based method was followed as described previously .
Sequence information of the genes mentioned in this study is available in the GenBank database having their respective accession numbers: Rice polyubiquitin 1 (OsUbi1) gene (AF184279), OsUbi1 promoter (AY785814), SAPK9 CDS from wild rice O. rufipogon (KT387673), OsRab16B gene (AF333275), OsRab21 gene (Y00842) OsLEA3-1 gene (DQ789359), OsbZIP23 gene (KP779638), TRAB1 gene (BAD09357), OsbZIP46 gene (ADK60888), SAPK8 gene (AB125309), SAPK10 gene (AB125311), OsSLAC1 gene (LOC_Os04g48530.1) and OsSLAC7 gene (LOC_Os01g28840).
Results and discussion
The single copy SAPK9 gene displays differential transcript expression profile and allelic polymorphism in drought-tolerant and drought-sensitive rice genotypes
Single nucleotide polymorphism (SNP) in the CDS is of great importance in the crop improvement programme as the functions of several genes are known to be modulated by the associated SNPs leading to the differences in plant performance. Assessing genetic diversity of candidate gene sequences involved in stress-responsive pathways lead to the identification of specific alleles which are linked to particular agronomic traits of abiotic stress responses. Trait enhancing superior alleles, which are dominant and express better in wild species than cultivated varieties and are left behind during evolution and domestication of present-day commercial cultivars, can be transferred to elite genetic backgrounds for improvement of desirable traits of a particular crop species . Therefore, it was of interest to investigate the presence of naturally occurring polymorphisms in the CDS of SAPK9 gene in all the chosen rice genotypes.
For the analysis of natural allelic polymorphism, the full-length CDS of SAPK9 gene was amplified through RT-PCR from total RNA of leaf tissues using two gene-specific primers. All the genotypes showed positive PCR amplification with an expected product size of ~1086 bp (Additional file 3: Figure S1). The sequence of each PCR amplicon was determined after cloning them individually. Three non-synonymous SNPs were detected among the SAPK9 CDSs obtained from eleven rice genotypes upon multiple sequence alignment. The first non-synonymous SNP was found in the CDS of Swarna, creating the nucleotide modification (T to A) at the 593rd position from the start codon, which led to the 198th amino acid change from isoleucine (I) to asparagine (N) (Additional file 4: Figure S2). The resulted amino acid change was located in the αF-helix of SAPK9 protein (Fig. 1d). The 2nd non-synonymous SNP was noticed in O. rufipogon, causing the nucleotide modification (C to T) at the 746th position from the start codon with the 249th amino acid change from proline (P) to leucine (L) (Additional file 4: Figure S2), and this was observed in the αH-helix of the SAPK9 protein (Fig. 1d). The 3rd non-synonymous SNP, being observed in a few genotypes, resulted in the nucleotide modification (A to C) at the 970th position from the start codon, which changed the 324th amino acid from serine (S) to arginine (R). Serine is present at 324th amino acid position in Swarna, IR20, IR36, IR64, HRC300, and Vandana; whereas arginine occurs at the same position in O. rufipogon, O. nivara, IR72, Nagina22 and Manipuri (Additional file 4: Figure S2). This last amino acid change was found to be located in between the SnRK2 box and ABA-dependent activation box (ABA-box) of SAPK9 protein (Fig. 1d). Various reports of non-synonymous SNPs leading to amino acid substitution in the CDS of drought responsive genes and their correlation with the particular trait have been published earlier . However, the present study showed that the natural occurrence of three non-synonymous SNPs did not affect any important functional domains and also could not be correlated with the differential expression level of the SAPK9 gene in the chosen drought-sensitive and drought-tolerant rice genotypes. Therefore, the highly expressed SAPK9 CDS of wild progenitor O. rufipogon was selected for further functional analyses.
The SAPK9 gene from drought-tolerant O. rufipogon encodes a protein belonging to subclass III of SnRK2 family, exhibits higher expression in leaf tissues and responses to both drought stress and ABA treatment
The tissue specificity of SAPK9 gene was monitored through real-time PCR analyses using the total RNA sample isolated from different tissues, namely root, shoot, stem, leaf, leaf sheath and panicle. The strongest expression of SAPK9 transcript was found in the leaf followed by root (Fig. 2c). Further, to analyse the transcriptional responses of SAPK9 gene under drought stress and ABA treatment, real-time PCR analysis was performed. The SAPK9 expression was observed to be significantly induced by exogenous ABA within 6 h of treatment in comparison to drought stress. However, the SAPK9 expression upon drought stress increased significantly after 24 h (Fig. 2d). The results indicate that the SAPK9 is involved in responses to both drought stress and ABA treatment, confirming the subclass III nature of this SnRK2 member .
The SAPK9 protein has a characteristic kinase fold structure and the recombinant protein possesses autophosphorylation and transphosphorylation activities
To study the kinase activity of recombinant SAPK9 in vitro, the bacterially expressed 6xHis-tagged SAPK9 protein was purified by Ni-NTA chromatography under native condition (Fig. 3c) and kinase assay was performed. It was observed that the generic substrate histone III was phosphorylated by the recombinant kinase (Fig. 3d). Moreover, the occurrence of an extra band corresponding to the size of recombinant SAPK9 (~42 kDa) suggested that it is able to autophosphorylate. This was confirmed by performing the kinase assay in the absence of substrate (Fig. 3d). These results are in accordance with the previous reports on the kinase activity of subclass III SnRK2s, OST1/SnRK2.6 and ZmSnRK2.8 [43, 44]. It is known that the AREB/ABF proteins require the phosphorylation of their multiple conserved sites by SnRK2 protein kinases for ABA-dependent activation [21, 41, 45]. Previous studies have shown that the SAPK9 can phosphorylate the downstream AREB/ABF type transcription factors such as TRAB1 and OsbZIP46 [46, 47]. As OsbZIP23 is phylogenetically very close to OsbZIP46, therefore we investigated the potential phosphorylation activity of SAPK9 on OsbZIP23 substrate. For this, the N-terminal 6xHis-tagged OsbZIP23 was expressed in the bacterial system and the recombinant OsbZIP23 protein was purified by Ni-NTA chromatography under native condition (Fig. 3e) and subsequently, kinase assay was performed for different time intervals. It was found that the SAPK9 phosphorylates OsbZIP23, and the phosphorylation efficiency is increased with the increase in reaction time (Fig. 3f). These results suggest that the SAPK9 protein has both autophosphorylation and transphosphorylation activities in vitro, and has the potentiality of transactivation of the OsbZIP transcription factors in vivo.
Generation of transgenic rice lines for SAPK9 overexpression and endogenous gene silencing
Overexpression of SAPK9 improves drought tolerance in rice by increasing the water retention capacity in transgenic plants through osmotic adjustment and stomatal closure
Globally, water availability dictates the yield of crop plants including rice . Plant with high water holding capacity can sustain the severity of drought stress better than the others. The water status in a plant body is principally determined by two critical parameters- water loss rate (WLR) and relative water content (RWC) [31, 49]. The WLR and RWC were measured in leaf tissues of OE, RNAi lines, and NT plants. It was observed that the OE lines exhibited lower WLR and higher RWC in contrast to the RNAi lines, which showed higher WLR and lower RWC in comparison with NT plants (Fig. 5c). These results indicate that the SAPK9 plays an important functional role in enhancing the water retention capacity of the plants under water deficit condition.
Transgenic rice lines overexpressing SAPK9 show reduced lipid peroxidation and increased antioxidant activity
Overexpression of SAPK9 causes elevated transcript expression of ABA- and stress-responsive genes resulting in increased ABA sensitivity at germination and post-germination stages of transgenic rice
Overexpression of SAPK9 increases grain yield in transgenic rice under drought stress by improving pollen maturation, spikelet fertility, and panicle weight
Possible molecular mechanism of SAPK9 action
Based on the findings of the present study and the information available in the literature, a molecular interactive pathway for SAPK9 has been proposed (Additional file 9: Figure S8). Our work clearly established that the SAPK9 acts as a transactivating kinase and a potential transcriptional activator for modulating the activity of several key components in the ABA-dependent stress adaptation pathway in rice plant. We hypothesize that an unknown factor(s) might be involved in mediating the transcriptional activity of SAPK9, which requires further investigation.
We have characterized the structural, biochemical and physiological functions of SAPK9, a subclass III SnRK2 gene of rice through developing gain-of-function and loss-of-function mutants by transgenesis. The SAPK9 transcript expression has been found to be differentially regulated in the selected drought-tolerant and drought-sensitive rice genotypes, and its expression is comparatively more elevated in reproductive stage than the vegetative stage. The SAPK9 transcript has been observed to be upregulated by drought stress and ABA treatments. The SAPK9 protein has characteristic kinase fold structure, and is able to transactivate its target substrates, including histone III and OsbZIP23; and itself is autophosphorylated. Constitutive overexpression of SAPK9 gene from a drought-tolerant wild rice genotype and RNAi-mediated silencing of endogenous SAPK9 gene in a drought-sensitive cultivar have revealed that the SAPK9 positively regulates drought stress tolerance by boosting the osmotic adjustment and stomatal closure of the plant. The enhanced drought-tolerant transgenic rice lines overexpressing SAPK9 display less cellular oxidative damage resulting from the reduced reactive oxygen species accumulation. The SAPK9 overexpressed lines exhibit increased sensitivity to exogenous ABA and enhanced transcription of other hierarchically downstream ABA-responsive genes, indicating that the SAPK9, indeed, is a positive regulator of ABA-mediated stress signaling pathway in rice plant. The increased functional activity of SAPK9 overexpression in transgenic lines improves grain yield-related traits such as panicle weight and spikelet fertility by increasing the pollen viability. Together, the present findings strengthen our knowledge about the functional role of SAPK9 as transactivating kinase and a probable transcriptional activator, which can be utilized as a promising gene-based molecular marker in transgenic breeding for generating crop plants with improved drought tolerance and grain yield.
ABA, Abscisic acid; CDS, Coding DNA sequence; DAB, 3,3′-diaminobenzidine; MDA, Malondialdehyde; NBT, Nitro blue tetrazolium; NT, Non-transgenic; OE, Overexpression; RNAi, gene silencing by RNA interference; ROS, Reactive oxygen species; RWC, Relative water content; SAPK, Osmotic stress/ABA-activated protein kinase; SLAC, Slow anion channel-associated; SNP, Single nucleotide polymorphism; SnRK2, Sucrose non-fermenting 1-related kinase 2; WLR, Water loss rate.
This work is dedicated to the memory of Late Professor Soumitra K. Sen who was involved with this work from the time it was conceived and designed. We express our gratitude to Prof. Saumen Hajra, Centre of Biomedical Research, Lucknow for his valuable guidance and encouragement throughout this study. We are thankful to Ms. Anuttama Dutta for providing help and support during a few experimental works. The technical help from Mr. Meghnath Prasad, Mr. Partha Das, Mr. Sona Dogra, Mr. Uttam Dogra, Mrs. Gayatri Aditya and Mr. Manoj Aditya are also acknowledged. Department of Science and Technology, Government of India is sincerely acknowledged for providing INSPIRE Fellowship to AD during his research tenure.
This work was supported by Indian Council of Agricultural Research, Government of India (NAIP-PAR-C4/C/30033) to Late Professor Soumitra K. Sen.
Availability of data and materials
All relevant data are within this article and its supporting information files. Gene sequences have been deposited in the GenBank database.
AD designed and performed the major experiments, analysed the results and wrote the manuscript. MKS performed few experiments and interpreted the data. SG designed few experiments and analysed the data. MKM conceived and designed the study, interpreted the results and corrected the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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