Genome-wide analysis of SnRK gene family in Brachypodium distachyon and functional characterization of BdSnRK2.9.

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ARTICLE IN PRESS

PSL 9190 1–13

Plant Science xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Genome-wide analysis of SnRK gene family in Brachypodium distachyon and functional characterization of BdSnRK2.9

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Lianzhe Wang 1 , Wei Hu 1 , Jiutong Sun, Xiaoyu Liang, Xiaoyue Yang, Shuya Wei, Xiatian Wang, Yi Zhou, Qiang Xiao, Guangxiao Yang ∗ , Guangyuan He ∗ The Genetic Engineering International Cooperation Base of Chinese Ministry of Science and Technology, The Key Laboratory of Molecular Biophysics of Chinese Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

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Article history: Received 16 March 2015 Received in revised form 11 May 2015 Accepted 12 May 2015 Available online xxx

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Keywords: SnRK Expression Protein interaction Evolution Abiotic stress B. distachyon

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1. Introduction

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The sucrose non-fermenting 1 (SNF1)-related protein kinases (SnRKs) play key roles in plant signaling pathways including responses to biotic and abiotic stresses. Although SnRKs have been systematically studied in Arabidopsis and rice, there is no information concerning SnRKs in the new Poaceae model plant Brachypodium distachyon. In the present study, a total of 44 BdSnRKs were identified and classified into three subfamilies, including three members of BdSnRK1, 10 of BdSnRK2 and 31 of BdSnRK3 (CIPK) subfamilies. Phylogenetic reconstruction, chromosome distribution and synteny analyses suggested that BdSnRK family had been established before the dicot-monocot lineage parted, and had experienced rapid expansion during the process of plant evolution since then. Expression analysis of the BdSnRK2 subfamily showed that the majority of them could respond to abiotic stress and related signal molecules treatments. Protein–protein interaction and co-expression analyses of BdSnRK2s network showed that SnRK2s might be involved in biological pathway different from that of dicot model plant Arabidopsis. Expression of BdSnRK2.9 in tobacco resulted in increased tolerance to drought and salt stresses through activation of NtABF2. Taken together, comprehensive analyses of BdSnRKs would provide a basis for understanding of evolution and function of BdSnRK family. © 2015 Published by Elsevier Ireland Ltd.

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During the processes of growth and development, plants are constantly confronted with multiple adverse stresses, such as high

Abbreviations: ABA, Abscisic acid; ABI, ABA-insensitive-clade protein phosphatases PP2Cs; ABRE, ABA-responsive element; AREB/ABF, ABA-responseelement-binding proteins; BAP, Benzylaminopurine; CIPK, CBL-interacting protein kinases; DRE, Dehydration-responsive element; HAB, Homology to ABI; IAA, Indole3-acetic acid; IL, Ion leakage; LTRE, Low temperature-responsive element; MDA, Malonaldehyde; PP2C, 2C-type protein phosphatase; qRT-PCR, Quantitative realtime polymerase chain reaction; SAPK, Osmotic stress/abscisic acid-activated protein kinases, SnRK2; SCS, SnRK2-interacting calcium sensor; SnRK, Sucrose nonfermenting 1 (SNF1)-related protein kinases; VC, Vector control; WT, Wild-type. ∗ Corresponding authors. Tel.: +86 27 87792271; fax: +86 27 87792272. E-mail addresses: [email protected] (L. Wang), [email protected] (W. Hu), [email protected] (J. Sun), [email protected] (X. Liang), [email protected] (X. Yang), [email protected] (S. Wei), [email protected] (X. Wang), [email protected] (Y. Zhou), [email protected] (Q. Xiao), [email protected] (G. Yang), [email protected] (G. He). 1 These authors contributed equally to this work.

salinity, drought, extreme temperatures and pathogens, which seriously affect their growth, development and productivity. To survive and complete life cycle, plants have developed various complicated mechanisms to deal with these biotic and abiotic stresses [1]. Protein kinases and phosphatases, the major components of intracellular signal transduction, play important roles in stress responses [2]. Of them, sucrose non-fermenting 1 (SNF1)related protein kinases (SnRKs) are highly conserved in organisms and involved in various physiological processes. Plant SnRKs that belong to Ser/Thr protein kinase are grouped into three subfamilies (SnRK1, SnRK2 and SnRK3) based on the sequence similarity and gene structures [3]. The SnRK1 subfamily, which contains three domains: N-terminal protein kinase (Pkinase) domain, ubiquitinassociated (UBA) domain and kinase-associated 1 (KA1) domain, is highly conserved with SNF1 in yeast and AMP-activated protein kinases (AMPKs) in animals, and is involved in carbon metabolism regulation [4]. SnRK1s have also been reported to be induced by ABA, implying their roles in cross-talk with metabolic signaling and stress pathways [5,6]. Unlike SnRK1, the other two subfamilies are unique in plants and participate in stress signaling pathways. The SnRK2s harbor

http://dx.doi.org/10.1016/j.plantsci.2015.05.008 0168-9452/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: L. Wang, et al., Genome-wide analysis of SnRK gene family in Brachypodium distachyon and functional characterization of BdSnRK2.9, Plant Sci. (2015), http://dx.doi.org/10.1016/j.plantsci.2015.05.008

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two domains including the N-terminal Pkinase domain and the C-terminal regulatory domain. Further, C-terminal domain consists of two subdomains, i.e. Domain I and Domain II [7]. Domain I with about 30 amino acid residues is characteristic for all SnRK2 subfamily members while Domain II with about 40 amino acid residues is specific to the 2c group SnRK2s (AtSnRK2.2, AtSnRK2.3 and AtSnRK2.6) and mediates interaction with the clade A type 2C protein phosphatases (PP2Cs) [8,9]. To date, a large number of studies have focused on the involvement of SnRK2s in stress signaling pathways. All 10 SnRK2s in Arabidopsis thaliana (AtSnRK2.1-2.10) except AtSnRK2.9 as well as all 10 SnRK2s in Oryza sativa (OsSAPK1-10) could be activated by hyperosmotic stress. Five AtSnRK2s (SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7 and SnRK2.8) and three OsSAPKs (OsSAPK8, OsSAPK9 and OsSAPK10) were activated by abscisic acid (ABA). Some members of AtSnRKs and OsSAPKs have been confirmed to play crucial roles in response to salinity and water stresses [8,10–12]. For example, overexpression of AtSnRK2.8 and OsSAPK4 significantly enhanced tolerance to drought and salt respectively in transgenic plants [13,14]. In other species such as maize (Zea mays L.) and wheat (Triticum aestivum L.), most SnRK2s could also respond to one or more abiotic stresses and overexpression of some SnRKs could increase plant tolerance to abiotic stresses [15–17]. These studies suggest that SnRK2s are activated by abiotic stresses and play important roles in enhancing tolerance to multi-environmental stresses in plants. SnRK3 kinases, designated as CIPKs (CBL-interacting protein kinases), interact with calcium sensor calcineurin B-like proteins (CBLs) to mediate calcium signaling pathway. Generally, CIPKs consist of a conserved Pkinase domain in N-terminal region, a NAF domain and a PPI domain in C-terminal regulatory region [18]. The NAF domain has been identified to mediate the CBL interaction and the PPI domain has been shown to mediate the interaction with PP2C [19]. To date, CIPKs have been demonstrated to participate in regulating the Na+ , K+ and NO3 − transportation, abiotic stress responses and some developmental processes in Arabidopsis [20–22]. The first identified CIPK in Arabidopsis is CIPK24 (SOS3) which interacts with CBL4 (SOS2) to function on Na+ /H+ antiporter (SOS1) and H+ -ATPase, improving plant tolerance to salt stress [23]. The expression patterns and functions of the CIPKs have been widely studied in other species besides the Arabidopsis, including rice [24–26]; maize [27,28] and wheat [29]. These researches emphasize the importance of SnRKs function in the stress response and nutritional efficiency, and ultimately improve crop tolerance to stress by genetically manipulating these proteins. Bioinformatic analysis of SnRK family has identified a total of 39 SnRKs in Arabidopsis [3,30–32], and 48 SnRKs in rice [8,31,33,34]. However, genome-wide identification of SnRKs has not been reported in Brachypodium distachyon, which is the first sequenced Poaceae grass and has close relationships with important crops such as wheat (T. aestivum L.), barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) [35]. In this study, we identified 44 BdSnRK genes from B. distachyon, and analyzed their genomic structures, chromosomal locations, phylogenetic expansion and evolutionary mechanism. Additionally, expression patterns and interaction analyses of BdSnRK2 subfamily were preformed to detect their responses to abiotic stress and their interaction network responding to abiotic stress in B. distachyon. Further, functional analysis of BdSnRK2.9 revealed its positive role in response to drought and salt stresses. These systematical analyses will be helpful for understanding the roles of SnRK family in B. distachyon under abiotic stress and provides valuable information for further functional characterization of SnRKs in other monocot crops.

2. Materials and methods 2.1. Identification and phylogenetic analysis of SnRK gene family in B. distachyon The non-redundant amino acid sequences of the Arabidopsis and rice SnRKs were collected from TAIR v10 (http://www. arabidopsis.org/) and RGAP v7 databases (http://rice.plantbiology. msu.edu/) respectively. More than 100 SnRKs sequences belonging to some other plant species were downloaded from Uniprot (http://www.uniprot.org/) (Table S1). The whole protein sequences of B. distachyon were downloaded from the Brachypodium Genome Database v1.2 (http://www.brachypodium.org/). To identify the predicted SnRKs in B. distachyon, the local Hidden Markov Modelbased searches in the protein sequence dataset were performed separately with HMM profiles built from each subfamily of known SnRKs as queries using HMMER software [36]. In addition, BLAST searches with all SnRK sequences of rice as queries were performed to identify the predicted SnRKs in the Brachypodium Genome Database. All the potential BdSnRK proteins identified from HMM search and BLAST search were validated for the presence of conserved domain with the PFAM (http://pfam.sanger.ac.uk/) and CDD databases (http://www.ncbi.nlm.nih.gov/cdd/). Further, the sequences were reciprocally searched against the Arabidopsis and rice database to identify the best hit among all the SnRK genes. The proteins that did not contain the known conserved domains and motifs or the best hits of reciprocally searches were not the SnRKs, were removed manually. Due to high variation in the C-terminal sequences of the SnRK proteins, the conserved Pkinase domain regions of SnRK family from Arabidopsis, rice and B. distachyon were selected to perform multiple alignment using Clustal X 2.0 [37]. The amino acid substitution model was calculated by the ModelGenerator v0.85 and the optimal model of “JTT + I + G + F” was selected [38]. The maximumlikelihood (ML) tree was constructed using MEGA5 program with bootstrap values for 1000 replicates [39]. 2.2. Protein properties and sequence analyses The relative molecular mass and isoelectric points of putative proteins were obtained by the ExPASy proteomics server (http://expasy.org/). The motifs were identified using the MEME program (http://meme.sdsc.edu/meme/intro.html), with optimum motif width ≥6 and ≤200 bp, maximum number of motifs 23. The motifs were annotated by InterProScan (http://www.ebi.ac.uk/ Tools/pfa/iprscan/). The gene information of BdSnRKs was retrieved from the Brachypodium database and the gene structures were drawn with the GSDS (http://gsds.cbi.pku.edu.cn/). To analyze the cis-element in promoter regions, the 1.5 kb upstream regions of the coding sequence region were selected from the Brachypodium Genome Database and analyzed with the PLACE (http://www. dna.affrc.go.jp/PLACE/) and PlantCARE (http://bioinformatics.psb. ugent.be/webtools/plantcare/html/) databases. Among them, the ABA-responsive element (ABRE; ACGTGG/TC) [40], dehydrationresponsive element (DRE)/C-repeat (DRE; TACCGACAT) [41], and low temperature-responsive element (LTRE) [42] were selected for analyses. 2.3. Chromosomal location, genome synteny and gene duplication analyses The chromosomal location information of BdSnRK family was downloaded from Brachypodium database. The syntenic blocks information of Arabidopsis, rice and B. distachyon was downloaded from the Plant Genome Duplication Database (http://chibba.agtec.


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uga.edu/duplication/index/home). Tandem duplication genes were defined as the genes tightly linked within 200 kb, and the identity of the genes ≥70% [43]. The chromosomal locations and syntenic diagrams were drawn using Circos software based on gene position in the genome [44]. 2.4. Expression profiles of the SnRK genes in rice Rice microarray data were downloaded from Rice Oligonucleotide databases (http://www.ricearray.org/). The log2 foldchange data in response to drought (GSE6901), salt (GSE6901), cold (GSE6901) and data for hormone treatment (GSE5167) from the Affymetrix data were integrated for the 46 of the 48 OsSnRKs, and heat map of expression profiles was drawn using TM4 software [45]. 2.5. Plant material, RNA extraction and qRT-PCR analysis

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B. distachyon strain Bd21, which is a community standard diploid inbred line, was used in this study. After germination, the seedlings were planted in soil at 22 ◦ C and with 16 h light/8 h night photoperiod condition. For abiotic stress and signal molecule treatments, 2-week-old seedlings were subjected to 4 ◦ C, 20% polyethylene glycol (PEG6000), 200 mM NaCl, 100 ␮M ABA, 10 mM H2 O2 and 100 ␮M ethylene (ETH) treatments, respectively. The leaves of all the samples for RNA extractions were collected at different times (0, 1, 3, 6, 12 and 24 h) after treatments. Organs including the roots, stems, leaves and spikelets were collected from adult plants separately for RNA extraction and used for organ-specific expression analysis. Total RNA was isolated according to the method described previously [46]. qRT-PCR was performed using SYBR green (Thermo Scientific) on a CFX real time PCR machine (Bio-Rad, Hercules, CA, USA). Gene-specific primers information of the tested genes was listed in Table S2 and the BdUBC18 (ubiquitin-conjugating enzyme 18) and NtActin were used as an internal control. Each reaction was done in three biological replicates. The obtained values for each genes were then normalized according to the expression of the internal control, and fold changes were calculated using the 2−CT method to indicate expression levels [47].

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2.6. Yeast two-hybrid assay

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Because AtABIs (ABA-insensitive-clade protein phosphatases, PP2Cs), AtHABs (homology to ABI), AtPP2CA, AtAREB1/ABF2 (ABAresponse-element-binding proteins, bZIP transcript factor family) and AtSCS (SnRK2-interacting calcium sensor) have been reported to interact with AtSnRK2s and mediate stress or ABA signaling [48–53], the homologs of these proteins in B. distachyon and tobacco were selected and identified by reciprocal best BLASTP with Arabidopsis interacting proteins as queries, and the best hit sequence of each protein was selected for following study. Due to the best hits of AtABI1/2 and AtHAB1/2 were the same proteins in B. distachyon, top three best hits (coverage >70% and identity >50%) of ABI/HABs (BdPP2C6, BdPP2C50, BdPP2C53) were chosen for further examining their interactions with BdSnRK2s. All cDNAs of cloned genes were submitted to NCBI and accession numbers were from KJ850308 to KJ850323 (Tables S3 and S4). The CDS of the BdSnRK2s and genes that encode putative interacting proteins were cloned into the GAL4 activation and DNA-binding domain plasmids independently. These recombinant vectors were then transformed into the yeast strain AH109. Yeast two-hybrid analysis was performed as described in the previous study [18] for at least three times. The 3-amino-1, 2, 4-triazole (3-AT), which works as an inhibitor of histidine synthetase, was used to eliminate the non-uniform

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background when the putative protein had transcriptional activation (More detailed information in Table S4). 2.7. Tobacco transformation and stress tolerance assay The recombinant plasmids pCAMBIA1304-BdSnRK2.9 under the control of the CaMV 35S promoter and the vacant vector pCAMBIA1304 control (VC) were introduced into Agrobacterium tumefaciens strain EHA105, and then transformed to the tobacco (Nicotiana tabacum L. cv Samsun) using an Agrobacteriummediated leaf disc transformation, as described by Horsch et al. [54]. Seedlings of three independent transgenic lines (OE1, OE4 and OE7) that almost all survived on MS medium containing 30 mg/L of hygromycin and were confirmed by RT-PCR, were used for the further experiments. The wild-type (WT), VC and transgenic lines were cultured in MS medium for one week and transferred to MS medium supplied with 150 or 300 mM mannitol and 100 or 200 mM NaCl for 1 week, then the root length were measured, and the whole young seedlings were collected for RNA extraction. Three-week-old transgenic tobacco plants were deprived of water for 20 days and then re-watered for 5 days for the drought tolerance assay. The plants were irrigated with 300 mM NaCl solution three times a week for 30 days for the salt tolerance assay. The malonaldehyde (MDA) and Ion leakage (IL) of WT, VC and transgenic plants under abiotic stress were determined as described in the previous study [55]. H2 O2 content was measured by using a Detection Kit (A0641, Jiancheng, China) according to the manufacturer’s instructions. All of the experiments were performed in triplicate. All data were analyzed by multiple range test at P ≤ 0.05.

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3. Results

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3.1. Identification of the SnRK family in B. distachyon

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To extensively identify the BdSnRK proteins from B. distachyon, both Hidden Markov Model searches and BLAST searches were performed. Results showed that all 44 identified BdSnRKs had a Ser/Thr kinase domain. Three proteins, which had the Pkinase (PF00069 of Pfam), UBA (PF00627) and KA1 (PF02149) domains, were grouped into SnRK1 subfamily; 10 proteins containing Pkinase domain and a conserved motif (Motif 17 of Table S5), and exhibiting high similarity with rice SAPK family were grouped into SnRK2 subfamily; the remaining 31 proteins harboring Pkinase and NAF (PF03822) domains were assigned to SnRK3 subfamily. Through this approach, the BdSnRKs were renamed and numbered according to their closest homologs of rice SnRKs, therefore, the SnRK3 subfamily was renamed as CIPK (Table S6). Additionally, Bradi1g20160 and Bradi3g05790 were renamed as BdCIPK21-1 and BdCIPK21-2 respectively because they had high similarity with OsCIPK21 and AtCIPK21 respectively. Due to the occurrence of alternative mRNA splicing in some genes, the longest protein for each gene was selected for further analyses. The amino acid residues of 44 predicted SnRKs ranged from 342 to 525, and the relative molecular mass varied from 38.7 to 58.2 kDa (Table S6). 3.2. Phylogenetic analysis of SnRK family in B. distachyon, rice and Arabidopsis In order to gain an insight into the evolutionary relationships of the SnRK proteins among B. distachyon, rice and Arabidopsis, ML tree was constructed based on conserved Pkinase domain regions (Fig. 1). The 131 SnRKs from B. distachyon, Arabidopsis and rice were grouped into three major clusters of SnRK1, SnRK2 and SnRK3 subfamilies, which is congruent with previous studies of Arabidopsis [3]. In detail, SnRK1 subfamily is divided into two subgroups of


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Fig. 1. Phylogenetic tree of SnRK families in B. distachyon, rice and Arabidopsis. The Maximum-Likelihood tree (JTT + I + G + F model) was constructed using MEGA5 with conserved protein kinase domain regions of 131 SnRKs. Signs of different shapes represent SnRK proteins from B. distachyon (red solid round, Bd), rice (blue hollow round, Os) and Arabidopsis (green solid rhombus, At).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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1a and 1b. SnRK1a genes were expressed throughout the developmental period of the plant, but the more divergent SnRK1bs are cereal-specific and mostly specifically expressed in the seed [4,56]. As in the case of Arabidopsis and rice, the BdSnRK2 subfamily could be classified into three subgroups (2a, 2b and 2c) based on the relationships with ABA. It was found that 2a group was not induced by ABA, 2b group was not induced or induced weakly by ABA (plant species dependent), while 2c was strongly induced by ABA [7]. The BdSnRK3 subfamily could be grouped into five subgroups (3a–e) depending on the sequence similarity. As expected, SnRK proteins from B. distachyon generally exhibited closer relationships with the proteins from rice rather than that from Arabidopsis, which is consistent with the current understanding of plant evolutionary history. This phylogenetic tree was further supported by the motif analysis. The deduced amino acid sequences of the whole BdSnRK proteins were submitted to the MEME software, and the eight motifs of the 23 identified motifs were annotated from InterPro (Table S5). The results showed that all the SnRKs had the conserved Pkinase domain containing the motif 1, 2, 3, 4, and the motif 8, 10 were functionally associated with NAF domain only in CIPK

subfamily. Moreover, the majority of the conserved motifs were found in the same subgroups (Fig. S1), which suggested that the similarity of amino acid residue corresponded with the classification of subgroups.

3.3. Analyses of gene structures and cis-elements in promoters To compare all 44 BdSnRKs directly, a phylogenetic tree was constructed based on the full-length amino acid sequences and the exon-intron structures of BdSnRKs were also analyzed (Fig. S2). It was found that each of the three members of SnRK1 subfamily had 10 introns, nine genes of the total 10 SnRK2 subfamily had introns varied from seven to nine and the BdSnRK2.5 had only two introns. For the CIPK subfamily, there was an interesting phenomenon, the exon-rich CIPK genes with more than 10 exons were clustered to the subgroup 3a, while the exon-poor members with each gene containing less than four exons were gathered to the other four subgroups (3b, 3c, 3d and 3e). This conserved exon numbers in each subfamily supports their close evolutionary relationship and the classification of subgroups.


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Q5 Fig. 2. Chromosomal location and synteny analyses of SnRK genes between B. distachyon, rice and Arabidopsis. (A) Chromosomal location and synteny analyses of SnRKs in B. distachyon (Bd). (B) Synteny analysis between B. distachyon and rice (Os). (C) Synteny analysis between B. distachyon and Arabidopsis (At). The syntenic gene pairs were parsed from the Plant Genome Duplication Database (PGDD) (Tables S7–S9). The positions of all the SnRK genes are depicted in the chromosomes of B. distachyon (red bands of Bdchr), rice (blue bands of Oschr) and Arabidopsis (green bands of Atchr), respectively. The different color lines indicate the synteny of SnRK1 (yellow), SnRK2 (red) and CIPK (blue) subfamily genes. The picture was drawn by the Circos program.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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To identify putative abiotic stress-responsive cis-elements in promoters, 1500 bp sequences upstream of the BdSnRK CDS were selected, and the cis-elements of ABRE, DRE and LTRE which were involved in the abiotic stress response were analyzed. The results suggested that all the BdSnRK1s, 80% of the SnRK2s and 77% of the CIPKs contained more than two kinds of tested elements in their promoter regions; while SnRK2.3, CIPK3 and CIPK20 harbored only DRE element, and CIPK10, CIPK14, CIPK21-1 and CIPK26 had only ABRE element, CIPK21-2 had only LTRE element, while SnRK2.2 had no tested elements (Table S6). 3.4. Analyses of chromosomal location, genome synteny and gene duplication According to the chromosomal location analysis, the 44 BdSnRK genes were distributed throughout the five chromosomes of B. distachyon (Fig. 2A). Three BdSnRK1 subfamily genes were located on

the chromosome 1 and 2; BdSnRK2 subfamily members were separately located on each of the five chromosomes while some of the BdCIPK subfamily genes were in clusters closely, and their distributions were non-random, because none of BdCIPKs were present on chromosome 5. Because gene duplication events play crucial roles in the amplification of gene family members in the genome, the gene duplication events were analyzed to further understand the expansion mechanism of the BdSnRK genes. As shown in Fig. 2A and Table S7, 24 pairs of BdSnRKs were identified in the same syntenic blocks, including 13 segmental duplication events between different chromosomes and the other 11 duplication events within the same chromosome. The results suggested that the segmental duplication events played vital roles in expansion of SnRK genes in B. distachyon genome. For the tandem duplication, some tightly linked BdCIPK genes such as the BdCIPK16/27, BdCIPK12/30, BdCIPK5/13 and BdCIPK2/29 were located less than 20 kb, however, the identities of the gene pairs


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were less than 50%, therefore they could not considered as tandem duplication event. Since Arabidopsis and rice are the two most important dicot and monocot model plant species with the functions of most SnRK genes have been well-characterized, genome synteny of the SnRK family among B. distachyon, rice and Arabidopsis were preformed to further explore the origin and evolutionary process of BdSnRKs. The results showed that 95% of BdSnRK genes were identified to exhibit synteny with their homologs of rice (Fig. 2B and Table S8), while only 18% of this family genes were found having synteny between B. distachyon and Arabidopsis (Fig. 2C and Table S9). These results indicated that SnRK genes might have evolved from a common origin and have rapid differentiation after the separation of the monocot and dicot species. 3.5. Expression profiles of B. distachyon SnRK2 genes in different organs and after abiotic stress and signal molecule treatments As B. distachyon had closer relationships with rice, we performed a meta-analysis with OsSnRKs microarray data under abiotic stress and hormone treatments to better understand the expression profiles of SnRK family (Fig. S3 and Table S10). From the rice expression profiles, we found that nearly all the OsSAPK (SnRK2) subfamily

members had significant response to multiple abiotic stresses, thus the BdSnRK2 subfamily was chosen for further expression analysis by qRT-PCR. The expression profiles of BdSnRK2 genes in roots, stems, leaves and spikelets were examined (Fig. S4). According to their expression patterns, the BdSnRK2 genes were divided into two groups. Eight genes had the global expression values in all the tested organs with varied abundance, such as BdSnRK2.2 and BdSnRK2.3 had relatively higher expression level in leaves and spikelets compared to the other organs. On the contrary, two genes derived from the gene duplication showed organ-specific expression. BdSnRK2.6 expressed only in the stems and leaves, while BdSnRK2.7 expressed only in the stems and roots. SnRK2s have been confirmed to participate widely in abiotic stress response. The expression patterns of BdSnRK2 subfamily genes under abiotic stress treatments were thus examined and the results showed that all the BdSnRK2s were responsive to at least two stress treatments at mRNA levels, including up- or down-regulation of more than two fold abundances (Fig. 3 and Table S11). All the BdSnRK2s, except BdSnRK2.10, were induced after cold treatment, in which eight genes were up-regulated and the BdSnRK2.8 was down-regulated (Fig. 3A). Under PEG6000 treatment, eight genes showed increased expressions, whereas two genes (BdSnRK2.3 and

Fig. 3. Expression levels of BdSnRK2 genes following short-term abiotic stresses of cold (A), PEG (B) and NaCl (C) treatments. BdUBC18 was used as an internal control for qRT-PCR. Fold changes were used to indicate expression levels in treated leaves compared to negative controls, which were set to 1. In addition, as BdSnRK2.7 had almost no detectable expression in leaves without treatments, the expression after 1 h of cold, 3 h of PEG and 1 h of NaCl treatments were set to 1 respectively. Data are means ± SD calculated from three replicates.


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Fig. 4. Expression levels of BdSnRK2 genes following short-term ABA (A), ETH (B) and H2 O2 (C) treatments. BdUBC18 was used as an internal control for qRT-PCR and fold changes were used to indicate expression levels in treated leaves compared to negative controls, which were set to 1. In addition, as BdSnRK2.7 had almost no detectable expression in leaves without treatments, the expression after 1 h of ABA treatment was set to 1. Data are means ± SD calculated from three replicates.

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BdSnRK2.5) had decreased expressions (Fig. 3B). Following NaCl treatment, five genes (BdSnRK2.1, BdSnRK2.2, BdSnRK2.4, BdSnRK2.7 and BdSnRK2.9) showed up-regulation, two genes (BdSnRK2.5 and BdSnRK2.10) showed down-regulation, and the other genes (BdSnRK2.3, BdSnRK2.6, and BdSnRK2.8) had no significant expression changes (Fig. 3C). These results suggested that the majority of BdSnRK2s could respond to various abiotic stress treatments. Signal molecules such as ABA, ETH and H2 O2 play important roles in regulating plant developmental processes and signaling networks involved in the responses to a wide range of biotic and abiotic stresses [57]. Therefore, the responses of BdSnRK2s to stress-related signal molecules (ABA, ETH and H2 O2 ) were tested in the leaves of B. distachyon. The results showed that all the SnRK2s except BdSnRK2.9 could be induced by one or more signal molecules (Fig. 4 and Table S11). In detail, under the ABA treatment, except that BdSnRK2.8 was down-regulated as well as SnRK2.9 had no response to the treatment, all the other genes were up-regulated (Fig. 4A). After ETH treatment, five genes (BdSnRK2.2, BdSnRK2.4, BdSnRK2.5, BdSnRK2.6 and BdSnRK2.10) were up-regulated while the others had no change in expression (Fig. 4B). Following H2 O2 treatment, four genes (BdSnRK2.2, BdSnRK2.4, BdSnRK2.5 and BdSnRK2.10) showed increased expression, BdSnRK2.8 showed decreased expression, and the other genes

had no significant expression changes (Fig. 4C). Taken together, these results indicated that the majority of BdSnRK2s could respond to these signal molecules at transcriptional levels. 3.6. The BdSnRK2 interaction network As genes implement their biological function typically through the interaction networks, the study of potential networks associated with a gene family is a very useful method for understanding the putative genes function [58]. The SnRK2 interaction network of Arabidopsis was well studied previously, such as AtSnRK2s involved in ABA signaling via PP2C-SnRK2 and SnRK2-ABF pathway [48–52], while there were not enough studies of that network in monocot plants. Whether the SnRK2 network in B. distachyon works in the same way as that of Arabidopsis? To clarify this, we studied the interactions between SnRK2 subfamily with the putative interacting proteins using yeast two-hybrid assay. From the Fig. 5A, BdSCS showed significant interactions with BdSnRK2.1, BdSnRK2.2, BdSnRK2.4, BdSnRK2.6 and BdSnRK2.7, while BdABF2 exhibited significant interactions with BdSnRK2.1, BdSnRK2.2, BdSnRK2.9, BdSnRK2.10 and moderate interactions with BdSnRK2.3, BdSnRK2.6, BdSnRK2.7 and BdSnRK2.8. For the PP2C proteins, BdPP2C30, the homolog of AtPP2CA, could


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Fig. 5. The protein–protein interaction and co-expression of BdSnRK2s and putative interacting protein genes. (A) Comparative yeast two-hybrid interaction analysis of ten BdSnRK2s with six predicted interacting proteins. The yeast strain AH109 containing the indicated plasmid combinations were grown on nutritional selective medium minus His, Leu, Trp, Ade and plus X-␣-Gal. 40 mM 3-AT which works as an inhibitor of histidine synthetase was used to inhibit the transcriptional activation of putative proteins if they had self-activation (For more information, see Table S4). Positive or negative control on the right corner showed the interactions between SV40 large T-antigen and murine p53 or SV40 large T-antigen and human lamin C (Lam), respectively. (B) Co-expression patterns of interacting gene pairs under the abiotic stress and signal molecule treatments. BdUBC18 was used as an internal control for qRT-PCR. Fold changes were used to indicate expression levels in treated leaves compared to negative controls, which were set to 1. Data are means ± SD calculated from three replicates.

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interact with BdSnRK2.1, while the other three homologs (BdPP2C6, BdPP2C53 and BdPP2C50) of AtABI/HABs did not show any affinity toward the BdSnRK2s. To further assess the possible roles of BdSnRK2 mediated interaction network in response to abiotic stress and signal molecules, qRT-PCR was carried out to test the co-expression of BdSnRK2 and interacting protein genes in leaves upon abiotic stress and signal molecule treatments (Fig. 5B and Figs. S5 and S6). From the results, BdPP2C30, which interacted with BdSnRK2.1, had co-expression with uniformly up-regulation after ABA treatment. In contrast, they had negative correlation in the gene expression under PEG6000 and NaCl treatments, with BdPP2C30 down-regulated and BdSnRK2.1 up-regulated. Besides, BdABF2 and BdSnRK2.10 had interaction and also showed down-regulation after NaCl treatment. The BdSCS interacting with BdSnRK2.1, BdSnRK2.2 and BdSnRK2.4 had negative correlation in the gene expression, among which BdSCS showed decreased expression while BdSnRK2.1, BdSnRK2.2 and BdSnRK2.4 showed increased expression after NaCl treatment (Fig. 5B). These protein–protein interaction and co-expression analyses suggested that some interactions between BdSnRK2s and their partner might be involved in the special signal pathways.

3.7. Overexpression of BdSnRK2.9 enhances tolerance to drought and salt stresses in transgenic tobacco BdSnRK2.9, which belongs to 2c subgroup of SnRK family, was remarkably induced by PEG and NaCl treatments (Figs. 1 and 3), and the protein exhibited a significant interaction with transcriptional factor BdABF2 and had no interaction with negative regulators such as BdPP2Cs and BdSCS (Fig. 5A). Because the SnRKABF pathway could positively regulate abiotic stress response in plants, they might have a potential effect for future application in agriculture [59]. Therefore, BdSnRK2.9 which may have a positive effect in response to stress, was chosen for further functional characterization with its overexpression in tobacco. Roots of young transgenic seedlings overexpressing BdSnRK2.9 were significantly longer than that of WT and VC after 150 mM mannitol and 100–200 mM NaCl treatments, although no apparent differences were observed between WT, VC and transgenic lines under normal growth conditions (Fig. 6A and B). To characterize the performance of BdSnRK2.9-overexpression seedlings under abiotic stress treatments, 3-week-old seedlings of transgenic and control plants were subjected to drought and salt treatments. All of the seedlings


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Fig. 6. Analyses of enhanced drought and salt stress tolerance of BdSnRK2.9 overexpressing tobacco lines. (A) Root length of WT, VC and transgenic tobacco lines on MS medium, containing different concentration of mannitol or NaCl. (B) Root length was calculated. (C) Phenotype of WT, VC and transgenic tobacco lines under normal, drought and salt stresses. (D) Survival rate. (E) MDA content. (F) Ion leakage content. (G) H2 O2 content. Data are means ± SD calculated from three replicates. Significant differences between the OE and control lines are indicated as *P < 0.05; **P < 0.01. 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

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performed well with no significant differences between the WT, VC and the transgenic plants under normal growth conditions. After withholding water for 20 d, all leaves in WT and VC plants were severely wilted, while only some of the transgenic plants showed yellow leaves with no curled phenotype. After recovery, transgenic plants retained remarkably higher survival rates than the WT and VC (Fig. 6C and D). For salt treatment, leaves of transgenic plants turned yellow and wilted significantly, but continued to grow when irrigated with 300 mM NaCl solution for 30 days, whereas the WT and VC plants exhibited chlorosis or died (Fig. 6C and D). These results indicated that overexpression of BdSnRK2.9 enhanced drought and salt stresses tolerance in transgenic tobaccos. To investigate the physiological mechanisms underlying the function of BdSnRK2.9 in response to abiotic stresses, the IL, MDA and H2 O2 levels, which reflected the membrane damage and the intracellular oxidative stress, were measured in the transgenic plants and controls. The results showed that transgenic plants exhibited lower levels of IL, MDA and H2 O2 compared with the controls under drought and salt conditions (Fig. 6E–G). These physiological indices demonstrated that the transgenic lines were more resistant to environmental stress. 3.8. BdSnRK2.9 interacts with NtABF2 and induces the expression of ABF-regulated genes in tobacco plants To further understanding the molecular mechanisms of BdSnRK2.9 function in drought and salt stress tolerances, the interactions between BdSnRK2.9 and NtABFs and the expression of ABF-regulated genes were investigated (More information of NtABFs can be seen in Table S4). For the interaction test, the yeast transformed with recombinant plasmid selected by media lacking histidine, leucine, tryptophan, adenine hemisulfate (SDLTHA). As shown in Fig. 7A, BdSnRK2.9 interacted with NtABF2

grew on the selective medium with 40 mM of 3-AT to inhibit the transcriptional action of NtABF2. Then the transcript levels of NtABF2 and ABF-related genes were detected in WT, VC and the transgenic lines (Fig. 7B). Under normal and osmotic conditions, expression of NtABF2, NtMYB102 and NtLTP1 were higher in transgenic plants than in controls. Under salt stress condition, besides NtABF2, NtMYB102, NtLTP1, another gene, NtLEA5, was also found to be up-regulated in transgenic plants than in WT and VC. These results suggested that BdSnRK2.9 improves tolerance to osmotic and salt stresses through interaction with NtABF2 and up-regulation of ABF-regulated genes in transgenic tobacco. 4. Discussion 4.1. Evolutionary patterns and functional diversification of BdSnRK gene family Recent studies suggest that SnRK2s and SnRK3s originated by duplication of SnRK1s and then had rapid differentiations during the process of plant evolution to fulfill new functions that enable plants to develop networks linking abiotic stress and ABA signaling with metabolic signaling [5]. Segmental duplication, tandem duplication and transposition events are the main reasons for gene family expansion [60]. Synteny analysis suggested that 72.7% BdSnRK genes were involved in syntenic blocks (Fig. 2A), indicating that segmental duplication events play major roles in the expansion of the SnRKs in B. distachyon. This result is consistent with previous studies that gene duplication contributed to SnRKs expansion in Arabidopsis [3]. Furthermore, the synteny analysis among B. distachyon, rice and Arabidopsis supports the hypothesis that the main subfamily of the plant SnRK gene family had been established before the dicot-monocot lineage parted and had experienced rapid expansion during plant evolution (Figs. 1 and 2).


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Fig. 7. Interaction analysis of BdSnRK2.9-NtABFs and expression patterns of ABF-regulated genes under normal, osmotic and salt conditions. (A) Yeast two-hybrid interaction analysis of BdSnRK2.9 and three NtABFs. The yeast strain AH109 containing the indicated plasmid combinations were grown on nutritional selective medium minus His, Leu, Trp, Ade plus X-␣-Gal and 40 mM 3-AT (More information in Table S4). (B) Expression patterns ABF-regulated genes of the WT, VC and transgenic plants under normal, osmotic and salt condition. Data are means ± SD calculated from three replicates. Significant differences between the OE and control lines are indicated as *P < 0.05; **P < 0.01.

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4.2. BdSnRKs respond to multiple stresses at transcriptional levels Increasing evidences have revealed the crucial roles of SnRKs in response to various stimuli, especially to plant hormone and abiotic stress signal response [8,10–12]. According to the expression profiles of SnRKs in rice, more than 70% OsSAPKs and 30% OsCIPKs were up-regulated by abiotic stress including drought, salt and cold, which indicated that SnRK2 subfamily members are highly responsive to the abiotic stresses (Fig. S3). Therefore, BdSnRK2 subfamily members were chosen for further study. In previous study, all 10 SnRK2s in Arabidopsis except AtSnRK2.9 as well as all 10 OsSAPKs could be activated by hyperosmotic stress [8,10–12]. In the present study, a more thorough expression analysis of BdSnRK2s showed that all of them could significantly respond to several stresses and signal molecules. Notably, BdSnRK2.2, BdSnRK2.4 and BdSnRK2.5 responded to all the tested treatments and BdSnRK2.1, BdSnRK2.2, BdSnRK2.9 and BdSnRK2.10 had more than 10 fold expression changes upon special stress (Figs. 3 and 4). These results suggest that the BdSnRK2 genes are extensively involved in the stress responses. In addition, some genes grouped in the same clade had similar expression patterns after defined treatments. For example, the duplicated pair of BdSnRK2.1/2.2 grouped in 2b showed up-regulation after cold, PEG6000, NaCl, and ABA treatments; the gene pairs of BdSnRK2.4/2.5 and BdSnRK2.6/2.7 grouped in 2a were induced after cold, PEG6000 and ABA treatments; BdSnRK2.8/2.9/2.10 grouped in 2c were up-regulated after PEG6000 treatment (Figs. 1, 3 and 4). These results suggest that these gene pairs arose by recent gene duplication events probably have similar functions. 4.3. SnRK2 subfamily interaction networks in B. distachyon are different from that in Arabidopsis Previous studies showed that SnRK2 subfamily could interact with proteins to mediate the response to multiple abiotic stresses and ABA signaling. These SnRK2 interacting proteins

mainly include PP2Cs, ABFs and SCS. PP2C-type phosphatases can bind to SnRK2s and act as negative modulators of ABA pathway [52,61,62]. SnRK2s can phosphorylate AREB/ABFs which belong to leucine zipper (bZIP) transcription factors family to participate in diverse environmental stress responses [48,49]. The PP2C-SnRK2 and SnRK2-AREB/ABF pathways have been confirmed to function in osmotic stress and ABA signaling in plants [59]. In addition, SCS, a plant-specific calcium-binding protein, act as another negative regulator of SnRK2s, through protein interaction to mediate the Ca2+ signaling pathway [53]. The SnRK2s interaction network has been well studied in Arabidopsis, whereas there is little interaction network analysis on SnRK2s in monocot. Therefore, there is a need to comprehensively investigate the interaction and coexpression patterns between SnRK2 subfamily and the putative interacting proteins, including PP2Cs, ABF and SCS in B. distachyon. Interestingly, we found that the protein–protein interaction and co-expression patterns of SnRK2s in B. distachyon were different from those in dicot Arabidopsis (Fig. 5). The AtSCS interacted with SnRK2.4 (group 2a), SnRK2.6 (group 2c) and SnRK2.8 (group 2b) in Arabidopsis [53], whereas the homolog of AtSCS in B. distachyon could interact with five BdSnRK2s (BdSnRK2.1, BdSnRK2.2, BdSnRK2.4, BdSnRK2.6 and BdSnRK2.7) that belong to the 2a and 2b group (Figs. Fig. 11 and Fig. 55A). The gene pairs of BdSCS/BdSnRK2.1, BdSCS/BdSnRK2.2 and BdSCS/BdSnRK2.4 had negative correlation in expression under NaCl treatment (Fig. 5B), suggesting that BdSCS may be a negative regulator of SnRK2s, which is consistent with the SCS study in Arabidopsis [53]. According to the basic model of ABA signal pathway in Arabidopsis, in the absence of ABA, 2c group AtSnRK2 (AtSnRK2.2, AtSnRK2.3, AtSnRK2.6) proteins were consistently bound and inactivated through direct dephosphorylation by clade A PP2Cs, including ABI1, ABI2, HAB1, HAB2 and PP2CA [51,52], however, there was only one interaction (BdPP2C30 and BdSnRK2.1) between BdSnRK2s and four homologs of the PP2Cs in B. distachyon. This result is consistent with the previous study in rice, the OsSAPK2 grouped


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in 2b could interact with OsPP2C30 [63]. For the co-expression analysis, BdPP2C30 transcript decreased while BdSnRK2.1 transcript increased after PEG and NaCl treatments, which consists with the basic model that PP2C worked as the negative regulator of SnRK2s. However, BdPP2C30 and BdSnRK2.1 had co-expression pattern in ABA treatment with uniformly up-regulation, and this result is consistent with previous studies that expression levels of clade A PP2Cs and AtSnRK2.7 grouped in 2b were uniformly up-regulated after ABA treatment in Arabidopsis, which explained that posttranslational modification of PP2Cs might play an important role in ABA signaling and stress responses in Arabidopsis [64,65]. AtAREB1/ABF2 could interact with group 2c AtSnRKs (SnRK2.2, SnRK2.3, SnRK 2.6) and most AtABFs and AtSnRK2s uniformly induced both by ABA and osmotic stress [48,65]. In the present study, BdABF2 (AREB1) interacted with all the BdSnRK2s except BdSnRK2.4/2.5, and the gene pairs of BdABF2 and BdSnRK2.10 showed co-expression after the NaCl treatment. Although in some situations co-regulation occurs mostly on the protein level, almost independent of cellular mRNA levels, co-expression data could provide some valuable information for the gene function analysis [66]. Although there are still some unknown mechanisms in coexpression patterns and interactions between BdSnRK2 and their interacting proteins, BdSnRK2 interaction network is involved in special stress signal pathway. Additionally, protein–protein interactions of BdSnRK2 changed along with gene expansion and were different from that of the Arabidopsis. Together, BdSnRK2s have a rapid evolution and functional innovations in the regulatory network during the separation of the monocot and dicot species. 4.4. BdSnRK2.9 is involved in SnRK-ABF pathway to enhance tolerance to drought and salt stresses in transgenic tobacco plants It has been well established that the SnRKs, especially SnRK2s, play important roles in various stress responses. Overexpression of AtSnRK2s, OsSAPKs and TaSnRK2s could enhances tolerance to abiotic stresses [13,14,16,17]. However, a little is known about the specific roles of BdSnRK2s in response to abiotic stress. The expression profiles and interaction network of SnRK2s provide a baseline for their further functional characterization in relation to stress tolerance. The BdSnRK2.9, which had remarkable up-regulation to the drought and salt treatments and involved in SnRK-ABF pathway, was chosen to further investigate its roles in transgenic plants. Due to the SnRK-ABF pathway could positively regulate abiotic stress response in plants, they might have more effect for future application [59]. It was found that overexpression of BdSnRK2.9 enhances tolerance to drought and high salinity stresses in transgenic tobacco (Fig. 6). SnRK-ABF pathway plays important role in diverse environmental stress in plants because the ABF transcription factors can positively regulate many genes encoding transcription factors, protein kinases, and phosphatases, and functional proteins to protect plant under abiotic stress, including MYB transcription factor genes (MYB102), ABAregulated genes (LTP1), LEA class genes (LEA5) and so on [67]. In the present study, BdSnRK2.9 could interact with NtABF2 and BdSnRK2.9 overexpression in tobacco increased the expression of NtABF2 and ABF-regulated genes including NtMYB102, NtLTP1 and NtLEA5 (Fig. 7). The up-regulated MYB102, which belongs to MYB TFs family and perform a variety of functions in plant biological processes and stress response [67,68], indicated that there were more MYB102 in the transgenic lines to play roles in the osmotic and salt stresses. The increased expression of NtLTP, which belongs to the lipid transfer protein family [69], suggested that BdSnRK2.9 affects the expression of some lipid-transfer protein genes that are responsive to reduce the membrane damage. Late embryogenesis abundant (LEA) proteins play roles in binding water, stabilizing

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labile enzymes, and protecting cellular and macromolecular structures during tolerance from abiotic stress [70]. Stronger induction of NtLEA5 under salt stress suggested that more LEA proteins may be synthesized in transgenic plants to salt stress. Together, the increase of positive ABF-regulated genes enhanced the tolerance of BdSnRK2 transgenic tobacco to drought and salt stresses. It has been well documented that the ABFs activated by SnRK2 are involved in ABA signaling pathway in Arabidopsis and rice. Although the expression of BdSnRK2.9 does not respond to ABA treatment (Fig. 4), the BdSnRK2.9-ABF may still function through ABA signaling because post-translational modification such as phosphorylation/dephosphorylation plays crucial roles in ABA signal transduction. It was reported that OsSAPK2, OsSAPK4 and OsSAPK6 which interact with OREB1 (a rice ABF) were not induced by ABA at transcriptional level, they were still involved in ABA signaling [8,63,71]. Because SnRK2 kinases can phosphorylate AREB1/ABF2 and TRAB1 (a rice ABF) in Arabidopsis and rice in the presence of ABA, respectively, and ABA plays a crucial role in the phosphorylation activation of ABFs, which suggests that the ABFs may be activated by ABA-dependent posttranslational phosphorylation modification [48,49]. In conclusion, this study provides the first report for identification of SnRK from B. distachyon in different phylogenetic depths to indicate evolution and expansion of this gene family. Comparative analyses of expression profiles and interaction network provides valuable information for further exploration of the functions of BdSnRK2 subfamily members in various stress responses and biological pathways, and BdSnRK2.9 was functionally characterized as a target gene for genetic engineering approaches to improve plants’ tolerance to multiple abiotic stresses. However, these results were lab-based experiments, and the test of improved stress tolerance in the field should be done in the future. These analyses will provide valuable information regarding the roles of SnRK family in stresses and the underlying molecular mechanism. Acknowledgments This work was supported by International S & T Cooperation Key Projects of MoST (Grant no. 2009DFB30340), National Genet- Q3 ically Modified New Varieties of Major Projects of China (Grant no. 2015ZX08002004-007 and 2015ZX08010004), Research Fund for the Doctoral Program of Higher Education of China (Grant no. 2012014211075) and Open Research Fund of State Key Laboratory of Hybrid Rice in Wuhan University (Grant no. KF201302). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci.2015.05. 008 References [1] H.J. Bohnert, Q. Gong, P. Li, S. Ma, Unraveling abiotic stress tolerance mechanisms-getting genomics going, Curr. Opin. Plant Biol. 9 (2006) 180–188. [2] T. Hunter, Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling, Cell 80 (1995) 225–236. [3] E.M. Hrabak, C.W. Chan, M. Gribskov, J.F. Harper, J.H. Choi, N. Halford, J. Kudla, S. Luan, H.G. Nimmo, M.R. Sussman, M. Thomas, K. Walker-Simmons, J.K. Zhu, A.C. Harmon, The Arabidopsis CDPK-SnRK superfamily of protein kinases, Plant Physiol. 132 (2003) 666–680. [4] N.G. Halford, D.G. Hardie, SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37 (1998) 735–748. [5] N.G. Halford, S.J. Hey, Snf1-related protein kinases (SnRKs) act within an intricate network that links metabolic and stress signalling in plants, Biochem. J. 419 (2009) 247–259. [6] P. Coello, E. Hirano, S.J. Hey, N. Muttucumaru, E. Martinez-Barajas, M.A. Parry, N.G. Halford, Evidence that abscisic acid promotes degradation of SNF1-related protein kinase (SnRK) 1 in wheat and activation of a putative calciumdependent SnRK2, J. Exp. Bot. 63 (2012) 913–924.


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Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

Identification of the ASR gene family from Brachypodium distachyon and functional characterization of BdASR1 in response to drought stress.

Systematic analysis and identification of stress-responsive genes of the NAC gene family in Brachypodium distachyon.

Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon.

Genome-wide evolutionary characterization and expression analyses of WRKY family genes in Brachypodium distachyon.

Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon.

Genomic architecture and functional relationships of intronless, constitutively- and alternatively-spliced genes in Brachypodium distachyon.

Molecular Cloning and Functional Characterization of Two Brachypodium distachyon UBC13 Genes Whose Products Promote K63-Linked Polyubiquitination.

A Member of the 14-3-3 Gene Family in Brachypodium distachyon, BdGF14d, Confers Salt Tolerance in Transgenic Tobacco Plants.

Ultrastructure of microsporogenesis and microgametogenesis in Brachypodium distachyon.

Selection and phenotypic characterization of a core collection of Brachypodium distachyon inbred lines.

Identification and Characterization of Carboxylesterases from Brachypodium distachyon Deacetylating Trichothecene Mycotoxins.

Genome-wide Analysis and Expression Profile of the MYB genes in Brachypodium distachyon.

Genetic Architecture of Flowering-Time Variation in Brachypodium distachyon.

Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

The arrangement of Brachypodium distachyon chromosomes in interphase nuclei.

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon.

Changes in Rubisco activase gene expression and polypeptide content in Brachypodium distachyon.

Comparative analysis and modeling of superoxide dismutases (SODs) in Brachypodium distachyon L.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

Long noncoding RNAs in the model species Brachypodium distachyon.

Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L.

Quantitative Trait Loci Associated with Drought Tolerance in Brachypodium distachyon.