Wheat Brassinosteroid-Insensitive1 (TaBRI1) Interacts with Members of TaSERK Gene Family and Cause Early Flowering and Seed Yield Enhancement in Arabidopsis

Brassinosteroids (BRs) hormones are important for plant growth, development and immune responses. They are sensed by the transmembrane receptor kinase Brassinosteroid-Insensitive 1 (BRI1) when they bind to its extracellular Leu-rich repeat (LRR) domain. We cloned and characterized the TaBRI1 from T. aestivum and raised overexpression transgenics in Arabidopsis to decipher its functional role. TaBRI1 protein consists of a putative signal peptide followed by 25 leucine rich repeats (LRR), a transmembrane domain and a C-terminal kinase domain. The analysis determined the interaction of TaBRI1 with five members of the wheat Somatic Embryogenesis Receptor Kinase (TaSERKs) gene family (TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5), at the plasma membrane. Furthermore, overexpression of TaBRI1 in Arabidopsis leads to the early flowering, increased silique size and seed yield. Root growth analysis of TaBRI1 overexpressing transgenic plants showed hypersensitivity to epi-brassinolide (epi-BL) hormone in a dose-dependent manner. Interestingly, transgenic Arabidopsis plants show thermotolerance phenotype at the seedling stages as revealed by chlorophyll content, photosystem II activity and membrane stability. The transcriptome profiling on the basis of microarray analysis indicates up-regulation of several genes related to brassinosteroid signaling pathway, abiotic stress response, defense response and transcription factors. These studies predict the possible role of TaBRI1 gene in plant growth and development imparting tolerance to thermal stress.


Introduction
Plant growth regulators brassinosteroids (BRs) are ubiquitously present throughout the plant kingdom and play a pivotal role in plant growth and development with regulatory functions during cell elongation, cell division, vascular differentiation, biotic and abiotic stress response and senescence [1,2]. BRs are sensed by the brassinosteroid-insensitive 1 (BRI1), a member of the leucine rich repeat receptor kinase (LRR-RK) family, at the cell surface for the coordinated action in response to changing environment [3]. BRI1 gene was first identified and characterized in Arabidopsis as a BR insensitive mutant [4]. In recent years, it was isolated from various plant species, including dicots viz., Pisum sativum [5], Lycopersicon esculentum [6], Gossypium hirsutum [7] and Glycine max [8] and monocots such as Oryza sativa [9] and Hordeum vulgare [10]. BRI1 is ubiquitously expressed in almost all organs in Arabidopsis and rice and is localized to the plasma membrane [9,11]. The strongest evidence for the indispensable nature of BRs as plant hormones comes from the discovery of Arabidopsis thaliana mutants in the mid 1990s resulting in the loss of BR biosynthesis and having characteristic dwarf stature with curled leaves, short stems, delayed senescence and flowering and de-etiolated phenotypes [12][13][14]. In the past few years, studies on BRI1 highlights its valuable role in imparting plant architecture and yield enhancement [8]. In O. sativa, the BRI1 loss of function mutant depicted reduction in height significantly with little effect on fertility [15]. In H. vulgare, the BRI1 mutant showed similar BR insensitive mutant phenotype which leads to lodging resistance [10]. All these investigations develops our interest to study the BRI1 function in wheat which is one of the major crop plant.
Brassinosteroid signal transduction involves several upstream and downstream signaling components which have been studied in detail. A small group of RLKs namely Somatic Embryogenesis Receptor Kinases (SERKs) show close homology with the Brassinosteroid-Insensitive1 (BRI1) Associated Receptor Kinase 1 (BAK1) and BAK1-like 1 (BKK1) which act as co-receptors in the BR signal transduction. Besides its BR function, BAK1 plays a role in several pathways independently by enhancing the signal output of varied LRR-RLK partners through binding with different ligands [16,17]. In the absence of BR, BRI1 appear to exist in plasma membranes as a ligand independent homodimer whose cytoplasmic domain interacts with BRI1 Kinase Inhibitor 1 (BKI1) inhibiting the interaction between the BRI1 and its coreceptor Somatic Embryogenesis Receptor Kinase (SERK) subfamily of LRR-RLKs/ BRI1-Associated Receptor Kinase I (BAK1) [17][18][19]. The binding of ligand-receptor induces conformational change leading to autophosphorylation of the BRI1 kinase domain which further creates a docking platform for the interaction of BRI1 with its co-receptor SERKs/BAK1 [20][21][22]. Signal transduction to BR responses is mediated by plasma membrane-associated BR-signaling kinases (BSKs) which inhibit the kinase activity of a major downstream negative regulator BIN2 by promoting the BSU1 (BRI1 suppressor 1) phosphatase [23,24]. BIN2 inactivation causes the activation of two closely related transcription factors, Brassinazole-Resistant 1 (BZR1) and BRI1-Ems Suppressor 1 (BES1) in nuclei, eliciting a wide range of gene expression changes of BR responses [25,26].
We report here the characterization of Brassinosteroid insensitive 1, TaBRI1 gene isolated from Triticum aestivum cDNA library [27]. In this report we analyze its structural organization and create a phylogenetic tree. We explored the protein-protein interactions of TaBRI1 with its coreceptors, TaSERKs. Our results show that TaBRI1 overexpressing transgenic lines in Arabidopsis display faster germination, early flowering, longer siliques and higher seed yield. TaBRI1 results in enhanced root growth which is inhibited in a concentration dependent manner in presence of BR. To provide additional evidence we checked its response during thermal stresses.

Plant material and growth conditions
Arabidopsis thaliana ecotype Col-0 plants was used as wild-type (WT) as genetic background to raise transgenic plants and for all experiments. Seeds were obtained from Arabidopsis Biological Resource Centre, USA. Plants were grown under 16 h light (~100-125 μmol m -2 s -1 photoperiod) and 8 h dark at 22±1°C, in pots containing soilrite supplemented with nutrient medium [28]. Arabidopsis seeds were surface sterilized with 2% sodium hypochlorite and 0.01% Triton X-100, washed with sterile RO water and inoculated in petriplates containing MS medium (pH 5.8) supplemented with 2% sucrose and 0.8% agar which were then placed to cold room (4°C) for stratification for two days followed by transfer to culture room. For expression analysis, 3-week-old seedlings were treated with different hormones (BL, auxin, BAP and ABA) at various intervals, frozen in liquid nitrogen and stored at -70°C until further use.

Sequence analysis of TaBRI1
The full length cDNA sequence was used to search homologous sequences and percentage sequence similarity via BLASTX and BLASTP in NCBI database (http://www.ncbi.nlm.nih. gov). For domain analysis TaBRI1 protein sequence was analyzed by CDD (Conserved domain database) and SMART (Simple Modular Architecture Research Tool). Multiple sequence alignment was carried out by CLUSTALW program and phylogenetic tree was constructed using MEGA6 program by neighbor-joining method.

Subcellular localization of TaB2 protein coding gene
The complete open reading frame (ORF) of TaB2 protein coding gene was fused to yellow fluorescent protein (YFP) reporter gene in frame under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) in pSITE-3CA vector. About 2μg of the plasmid construct was used to coat gold particles. Inner epidermal peels of white onion were placed inside-up on MS medium. Onion (Allium cepa) peels were bombarded by using PDS-1000/ He system (Bio-Rad, Canada) at 1,100 p.s.i. with DNA coated gold particles and 6cm of target distance using 1.0μm of gold micro-carriers. After bombardment, the petriplates were sealed with parafilm and incubated overnight at 28°C before observation. The onion epidermal cells were observed by using confocal microscope (Leica 1 TCS SP5II).

TaBRI1 cloning and overexpression in Arabidopsis
For generating overexpression construct of TaBRI1 gene to characterize its function, a 3.4 Kb ORF was amplified with gene specific primer (S1 Table) using cDNA as a template isolated from 13-d-old leaf base of T. aestivum. The amplified product was then cloned in an entry vector (pENTR™/ D-TOPO) and then in destination vector pMDC32 under CaMV 35S promoter following Gateway ™ cloning strategy (Directional TOPO Cloning kit and LR clonase Enzyme mix II kit, Invitrogen Inc. USA). The AGL1 strain of Agrobacterium tumefaciens harboring pMDC32-TaBRI1 was used for transformation in Arabidopsis through floral dip method [29]. The T1 seeds were selected on MS-agar plates supplemented with 50 μg/μl hygromycin and the resistant plants transferred to pots. All further analysis was conducted using best three transgenic lines (T4 seeds) homozygous for the transgene.

Yeast two hybrid analysis
From pENTR/D-TOPO vector the ORF of TaBRI1 was cloned into bait vector pDEST-GBKT7 (DNA binding domain, BD) and all five TaSERKs (activation domain, AD) into prey vector pDEST-GADT7 using Gateway™ LR Clonase™ enzyme mix (Invitrogen). Both the construct were co-transformed into yeast strain AH109 and selected on SD medium lacking Trp and Leu, respectively, as per the manufacturer protocol. Further, inoculum from primary culture containing colonies of SD (-L,T) plates were used in the secondary culture (3 mL) and grown at 30°C, 200 rpm for 3 h till the OD at 600 nm reached 0.5. Each culture was then serially diluted (10 −1 , 10 −2 , 10 −3 and 10 −4 ) and droplets of 10μL of each dilution including the undiluted culture were placed on the selection media (SD/-LT and SD/-HLT supplemented with 1mM 3-aminotriazole) and incubated at 30°C for 3-5 days till the formation of colonies.

Bimolecular fluorescence complementation (BiFC) assay and intracellular localization
To visualize protein-protein interaction BiFC assay was performed. Cloning of TaBRI1 and TaSERKs was done in Gateway™ vectors pSITE-nEYFP and pSITE-cEYFP, complete ORF (without stop codon) were amplified using Phusion High Fidelity Taq Polymerase (Finnzymes) and inserted into the entry vector (pENTR™/D-TOPO) following the above BiFC destination vector using Gateway ™ LR Clonase enzyme mix (Invitrogen Inc. USA) as per the manufacturer protocol. For visualization of YFP fluorescence these constructs particle bombarded on onion epidermal cells using Biolistic PDS-1000/He particle delivery system (Bio-Rad, USA) according to the protocol described earlier [30]. 3 μg of DNA was used for each construct to coat 0.2 mg gold particles per shot in a chamber vaccum of 27 in Hg pressure where particles accelerated with 1100 psi from the cylinder containing helium (He) gas pressure and the onion peel plate was loaded on to the target platform (6 cm from the launch assembly). The plates were incubated at 28°C in dark conditions for 12-16 h for stabilization. The onion peels were then observed for YFP expression under the Leica 1 TCS SP5II confocal microscope.

Root growth inhibition assays
For root growth analysis in presence of brassinosteroid as well as abiotic stresses, treatment with epi-brassinolide (24 epi-BL) purchased from Sigma (St Louis, MO, USA) was used. Twenty seedlings were grown on half-strength MS medium for 3-days and then transferred on fresh MS plates supplemented with different working concentrations of epi-BL which were added from 10 −2 M stock solution in 95% ethanol before the media solidified. Petriplates containing seedlings were grown vertically in culture room under 16 h light and 8 h dark at 22 ±1°C for 4 days. The root length was measured on fifth day of transfer and compared with respect to control (MS medium without hormone). In addition to determine the effect of heat and cold stress, 7-day old seedlings grown on control conditions were given heat stress at 40°C for 2 h and cold stress at 4°C for 24 h. Plates were then kept vertically for five days in culture room and root length, plant height as well as rosette diameter examined after fifth day. All experiments were done in triplicates and the values presented in the data are mean of these experiments. Standard error and student's t-test was used to display significant difference between WT and transgenic and a P-value 0.05.

Physiological analysis of overexpression transgenics
Estimation of total chlorophyll content. Chlorophyll content was estimated from the non-stressed and stressed 21-d old seedlings (twenty seedlings each) by using 100 mg of tissue for both WT and transgenic lines in each tube containing 2.5 mL of DMSO. The tubes were then incubated overnight in dark for chlorophyll leaching. Absorbance was recorded at 645 and 663 nm in a UV-Vis spectrophotometer (Agilent Cary 60). Chlorophyll content was calculated according to the formula of Arnon [31].
Photosynthetic Yield (Fv/Fm). Photosystem II activity was recorded of WT and transgenic leaves under stressed and non-stressed conditions using a pulse amplitude modulation fluorometer (Junior PAM-210, H. Waltz, Germany) at room temperature. Chlorophyll fluorescence measurements were made from the upper surface of the leaves which were dark-adapted for 20 min before measuring the induction of fluorescence [32]. The intensity of the measuring, modulated red light was~0.1 μmol.m -2 s -1 to induce the minimum fluorescence (F 0 ). Saturating flashes were provided to completely reduce the PSII acceptor site Q A -and to measure the maximum fluorescence yield (F m ). The intensity of the saturating light flash (1s) used for the measurements of Fm was 3000 μmol.m -2 s -1 . Photosynthetic yield was measured using the following formula: Fv/Fm where Fv = Fm-Fo [33]. The ratio F v : F m reflects the potential yield of the photochemical reaction of PSII.
Membrane Stability Index (MSI). MSI was determined by recording the electrical conductivity according to the Sairam et al. [34] protocol. 21-d old stressed and non-stressed seedling tissue (0.1g) was taken in double distilled water and initial conductivity (C1) measured at 30°C for 30 min. The samples were then autoclaved for 15 min. and again readings were measured termed as C2 by conductivity meter (Eutech instrument, Singapore). MSI was calculated using formula: Real Time PCR analysis Total RNA was isolated by RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer protocol followed by on-column DNase-I treatment for removal of genomic DNA contamination. For real time PCR analysis, first strand cDNA was synthesized with 2μg of total RNA using the High Capacity cDNA Archive kit (Applied Biosystems, USA) and 200nM of each primer mixed with SYBR Green PCR Master Mix (Applied Biosystems) for real-time PCR reactions, using the ABI Prism 7000 Sequence Detection System and Software (PE Applied Biosystems) according to the manufacturer's instruction. Primer Express 2.0 (Applied Biosystems 1 ) was used for designing primers from a unique region, specific to the gene and each pair was confirmed by BLAST program in NCBI and TAIR database (S1 Table). The relative mRNA levels in different RNA samples were normalized with respect to internal control gene, Actin. The Ct (threshold cycles) values were averaged for two biological replicates and three technical replicates.

Transcriptome analysis of TaBRI1 overexpression transgenics
For microarray analysis, approximately 20-30 Arabidopsis seedlings of twelve-day old WT overexpression transgenic was harvested, weighed and frozen in liquid nitrogen. The RNA was isolated using RNeasy plant mini kit (Qiagen) and quality and quantity of RNA were checked by Agilent Bioanalyzer (Agilent Technologies, North Carolina, USA). Microarray hybridization was performed using Affymetrix GeneChip 3 0 -IVT express kit according to manufacturer's instructions (Affymetrix, Santa Clara, CA, USA) with 500 ng RNA. Affymetrix gene chip data files (.CEL files) were generated and analysed by ARRAY ASSIST software where normalization of data and probe summarization for all the genes on the chip was done by Gene Chip Robust Multiarray Analysis (GCRMA) algorithm. This was followed by log transformation of data and finally average log signal intensity values of three biological replicates were computed with overall correlation coefficient values of ! 0.95 were used for analyzing differential gene expression. Genes showing up-and down-regulation with a fold change ! 2 and a p-value cut off 0.05 was selected with respect to the wild-type. The heat maps showing the expression profiles of varied genes were generated using signal intensity values via Multi Experiment Viewer (v4.8).

Statistical analysis
Statistical analysis was done by calculating mean value and standard error (SE) for all the three replicates. Student's t-test was performed to reveal significant differences between WT and transgenic lines. A P-value of 0.05 was considered significant.

Sequence, structural analysis and sub-cellular localization of TaBRI1
With the aim to elucidate the molecular and functional role of TaBRI1, an EST of 1.5 kb of TaBRI1 gene was identified from 13-day-old leaf base, auxin induced, cDNA library from wheat, T. aestivum [27]. Full length of TaBRI1 (DQ655711.1 Accession no.) 3,834 bp was amplified and the ORF of 3.4 Kb was then cloned in overexpression vector pMDC32 under CaMV 35S promoter. The deduced protein comprises of 1,124 amino acid residues with a predicted molecular weight of 119.81 kDa and an isoelectric point of 5.69. TaBRI1 protein sequence revealed that its polypeptide (Fig 1a) harbours 25 leucine rich repeat (LRR) from position 199 to 514 amino acid (green boxes) among which eight LRR domains are highly conserved that are a characteristic feature of BR receptor, PFAM LRR_1 domain from position 633 to 655 amino acid (yellow box) suggested to be involved in diverse protein-protein interactions, followed by a transmembrane region from position 721 to 743 amino acid (purple box) and a long stretch of kinase _Tyr domain from position 810 to 1083 amino acid (pink box) comprising of serine/threonine kinase and tyrosine protein kinase at C-terminal end that possibly takes part in most of the cellular activities. The multiple sequence alignment of Triticum aestivum, TaBRI1 showed high homology to other BRI1 of various plant species (S1 Fig). Based on the BLASTP from NCBI database TaBRI1 showed 96% similarity with HvBRI1 homolog of Hordeum vulgare (Accession No. BAD01654.1) highlighting the close relatedness, 83% identity with homolog of Oryza sativa, OsBRI1 (Accession No. NP001044077.1), 79% similarity with the homolog of Zea mays, ZmBRI1 (Accession No.XP008656807.1) and 49% identity with the putative homolog of Brachypodium distachyon, BdBRI1 (Accession No. XP003577946.1). It also showed homology with dicot plant species where TaBRI1 shares 57% similarity with the putative homolog of Solanum lycopersicum, SlBRI1 (Accession No. XP004237477.1), 55% similarity with homologs of other two dicots Populus trichocarpa, PtBRI1 (Accession No. XP002307140.2) and Arabidopsis thaliana, AtBRI1 (Accession No. NP195650.1).
The phylogenetic tree constructed revealed that T. aestivum, TaBRI1 shares maximum closeness with H. vulgare, HvBRI1 (Fig 1b). As observed the tree does not show a clear demarcation between monocots and dicots clustering. Among the dicot plant species, S. lycopersicum, SlBRI1 and P. trichocarpa, PtBRI1 grouped in separate clade and clustered together showing close relatedness with each other. However, A. thaliana, AtBRI1 arise from the same branch node as observed in TaBRI1 and HvBRI1 which suggests they might be closely related with respect to their evolution. Indeed, from the sequence alignment and phylogenetic tree of T. aestivum, TaBRI1 depicts high degree of conservation implicating this gene might play an important role as a BR receptor in the brassinosteroid transduction pathway.
Transient expression assays in onion epidermal cells by confocal microscopy revealed that 35S:TaBRI1-YFP fusion protein was mainly expressed in the nucleus and plasma membrane as compared to the YFP alone which was found to be distributed throughout the cell (Fig 1c).

In vivo identification of TaBRI1 interaction with TaSERKs
Several reports showed hetero-oligomerization of BRI1 receptor and BAK1 (SERK3), SERK1 and SERK4 required in BR signaling mechanism in Arabidopsis [35,36]. We examined the protein-protein interactions between T. aestivum, TaBRI1 and TaSERKs (TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5) using yeast two-hybrid system. The interactions of TaBRI1 with the TaSERK family members were specific as revealed by the presence of viable colonies (Fig 2a) on the selection medium (SD -H,L,T). Also, visualization of fluorescence in the plasma membrane performed by BiFC assay (Fig 2b) showed the positive interaction of TaBRI1 with the five TaSERKs. These results, therefore conclude that TaBRI1 heterodimerizes with the TaSERKs viz. TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5.

Overexpression of TaBRI1 causes early flowering in Arabidopsis transgenics
Expression of transgenic lines were examined in seven independent lines (1.5, 2.5, 3.4, 7.2, 8.3, 10.1 and 11.5) by real-time PCR analysis (Fig 3) where the transgenic lines showed varying transcript levels relative to the wild-type (WT). Three transgenic lines (1.5, 2.5 and 3.4) which exhibit an increased expression levels were selected for further analysis. To explore the role of TaBRI1, Arabidopsis transgenic TaBRI1-OE and Col-0 WT were grown under long day culture conditions of 16 h light and 8 h dark. Transgenic TaBRI1 plants displayed early flowering as compared to the WT (Fig 4a). Flowering time was recorded as the number of days from the transfer of plants to the culture room to the day of the appearance of floral buds. TaBRI1 overexpression transgenic plants started flowering 10-12 days earlier than the WT as monitored and recorded in Table 1. Also, the constitutive expression of TaBRI1 in Arabidopsis resulted in larger silique size (Fig 4b and 4c) and increased number of siliques per plant than that of WT (Fig 4d) which leads to the enhancement of total seed yield per plant in TaBRI1 overexpression transgenic plants (Fig 4e). The increased seed production in transgenics was mainly due to a greater number of seeds per plant since no significant difference was observed in seed size as compared to the WT. In addition, enhanced plant height was also observed at 35 days after germination in transgenic TaBRI1 overexpression plants than the WT (Table 1). However, less number of rosette leaves was monitored in TaBRI1-OE plants at the time of bolting as compared to the WT, while, rosette diameter in transgenic lines was similar to the WT which inferred that the flowering time was affected by the transgene activity. Similarly, leaf length and leaf width of TaBRI1-OE was comparable with that of WT and no significant difference could be deduced.
TaBRI1 overexpression leads to hypersensitivity to brassinosteroid mediated root growth At lower concentrations BRs play an indispensable role in the growth and development of roots [37]. In order to determine how exogenously applied epi-brassinolide (BL) affects root growth, we investigated the response of WT and TaBRI1-OE Arabidopsis seedlings on different concentrations of BL. TaBRI1 overexpression Arabidopsis seedlings were more sensitive to the brassinosteroid hormone and result in root length inhibition in a concentration dependent manner (Fig 5a). The overexpression TaBRI1 lines showed enhanced primary root growth under control conditions (without BR treatment) as compared to the WT (Fig 5b) however, root length gradually decreased as the concentration of BL increases from 0.1 nM to 100 nM from 100% to 20% at 100 nM epi-BL whereas in WT the decrease in primary root length was from 100% to 40% (Fig 5c). These results imply hypersensitivity of overexpression TaBRI1 transgenics in presence of exogenously supplied BL.

TaBRI1 imparts heat and cold stress tolerance to transgenic Arabidopsis
To analyze the response of transgenics to temperature extremes, the TaBRI1 transgenics and WT were grown on half-strength MS medium for 7 days under control conditions. For heat stress, the plates were incubated at 40°C for 2 h and at 4°C for 24 h for cold stress and transferred to the culture room for 5 days. Data presented in Fig 6a and 6b shows, the transgenic TaBRI1-OE lines exhibited elongated roots as compared to the WT, and the inhibition in root growth in different transgenic lines was lower under heat and cold stress as compared to WT. Also, an increase in plant height and rosette diameter of TaBRI1-OE transgenic lines was observed (Fig 6b) resulting in an overall tolerant phenotype (remained green, healthier and decreased senescence) under heat and cold stress conditions. In addition to the morphological analysis, we also monitored the stress tolerance levels quantitatively by analyzing physiological parameters like chlorophyll content, photosynthetic efficiency and membrane stability. Under non-stressed conditions, the level of chlorophyll content in overexpression lines of TaBRI1 showed higher levels as compared to the WT (Fig 6c) however, transgenics under heat and cold stress conditions had higher chlorophyll levels as compared to WT. Additionally, we observed the Fv/Fm ratio to check the photosynthetic efficiency (PSII), where the activity increases in overexpression lines under non-stressed and stressed conditions (Fig 6c) and also significant differences were observed on membrane stability index (MSI) (Fig 6c) where the percentage of MSI under heat stress and cold stress conditions increased to 70-80% as compared to the WT, respectively. The above results implicate enhanced expression of TaBRI1 leads to better temperature tolerance than the WT plants via maintaining membrane integrity, hence this gene can be exploited for heat and cold stress tolerance.

Whole genome transcriptome profiling of TaBRI1 overexpression transgenics
In an attempt to understand the fundamental mechanism causing the above mentioned morphological changes and study the hypersensitive response towards brassinolide hormone, whole genome microarray analysis of WT and transgenic seedlings (line 1.5) was performed. Data analysis revealed that a total of 119 genes (! 2 fold change and p value 0.05) were differentially expressed in transgenic line constitutively expressing TaBRI1 with respect to the WT, out of which 45 were up-regulated and 74 down-regulated (S2 and S3 Tables). This included the genes encoding proteins which acts as transcription factor families like F-Box, Zinc finger (C2H2 and C3HC4 type) and jumonji (jmjc), stress responsive proteins like glutathione S-transferase (GST tau and phi type), putative expressed proteins, LTP protein family, genes involved in enzymatic activity like lipooxygenase (LOX2), carboxymethyl transferase, protease, reductase, peroxidase, dehydrogenase, cytochrome P450 (CP450), putative thionine and trypsin inhibitor, transporter proteins like sulphate and mannitol transporter, senescence and defense response related genes (CC-NBS-LRR and LRR) and ubiquitin proteins (Fig 7). Amongst these many play an important biological role in growth and development of plants, abiotic stress response, disease resistance and proteasomal degradation.
To validate the results obtained from the microarray analysis we checked the transcript levels of above mentioned transcription factors in presence of either both brassinosteroid (epi-BL) or auxin (2,4-D) and also investigated some of the other interesting up-and down-regulated genes influenced by brassinosteroid (epi-BL) through real-time PCR analysis. The real-time PCR data validated the observation by microarray analysis thus reconfirming the differential regulation of genes in response to BR and 2,4-D. Among the transcription factors, only zinc finger was found to be auxin mediated showing a fold change of >20 in transgenics, whereas no significant difference was observed in presence of BR. However, transcript levels of other transcription factors viz., jumonji and F-Box do not show a significant difference in presence of hormones (Fig 8). Further, genes which are related to the other biological functions such as plant development, stress response, defense related and enzymatic activity also showed varied transcript levels in control as well as in BL-treated seedlings (Fig 9). The transcript levels for LTP3 and LTP4 were higher in TaBRI1-OE seedlings while other putative LTPs show downregulation in transgenics with no significant difference as compared to the WT. Additionally, the results obtained for transcripts of LOX2, GST (tau), CP450 (Accession no. AT5G47990) and TPR shows upregulation in transgenics when compared with WT, were consistent with the microarray data. In contrast, the transcript levels of GST (phi), senescence related gene, other CP450 genes, CC-NBS-LRR and LRR down-regulated in TaBRI1-OE seedlings whereas in presence of BR overall decrease in the transcript levels of selected genes was observed as compared to the control conditions, respectively. Values are mean ± SD for ten seedlings each. Asterisk and double asteriks (**P 0.01 and *P 0.05) indicate statistically significant differences between WT and overexpression transgenic lines. doi:10.1371/journal.pone.0153273.g005

Discussion
We describe here the isolation and characterization of TaBRI1 gene from T. aestivum, most closely related to the H. vulgare, HvBRI1. Sequence analysis revealed that the TaBRI1 protein contains 25 LRR among which eight LRRs are highly conserved and kinase domains at its carboxyl terminal which is a characteristic feature of BR receptors required for BRI1 function in BR signal transduction pathway [4]. TaBRI1 interacts with five TaSERKs isolated from T. aestivum which acts as a homolog of BAK1 coreceptor in the BR signaling pathway. Ectopic expression of TaBRI1 leads to early flowering in Arabidopsis and enhances root growth. TaBRI1 gene expression was differentially regulated by hormones and is hypersensitive in presence of BR in a dose-dependent manner. Transgenic Arabidopsis plants overexpressing TaBRI1 showed considerable tolerance to adverse temperature stresses. These results imply that TaBRI1 is a novel gene which plays crucial role in plant growth and development and participate in abiotic stress tolerance.

TaBRI1 shares conserved domains with other plant species
The predicted TaBRI1 protein shares conserved domain structure with other BRI1 polypeptides which include leucine rich repeats (LRR) followed by PFAM LRR_1 domain and a serine (Ser)/ threonine (Thr) kinase domain at the carboxyl terminal which is responsible for BR reception in the BR signal transduction pathway [4]. TaBRI1 shows strong sequence similarity with H. vulgare, HvBRI1 (96%) and OsBRI1 of O. sativa, (83%) and phylogenetic relationship displayed that TaBRI1 group together with HvBRI1 in the same clade. Brassinosteroid-Insensitive 1 (BRI1) was originally identified from Arabidopsis mutant analysis which was then cloned and demonstrated for the presence of leucine rich repeat receptor like kinase [4] located specifically in the plasma membrane [11,38]. Similarly, in the present study also TaBRI1 is also found to be localized to the plasma membrane.

Protein-protein interaction of TaBRI1 with TaSERKs
Many studies have shown the in vivo interaction between BRI1, a ligand dependent receptor of BR signaling with members of the Somatic Embryogenesis Receptor like Kinase (SERK) subfamily [39]. SERK3 homolog of BRI1-Associated Receptor Kinase 1 (BAK1) acts as a coreceptor show interaction with BRI1 both in vitro and in vivo in Arabidopsis [18,40,41]. SERK1, also heterodimerizes with BRI1 to enhance BR signaling [42]. SERK4, also known as BAK1-LIKE (BKK1), shows interaction with BRI1 in vivo in a BR-dependent manner [43]. Our study shows the in vivo interaction of TaBRI1 with all five identified TaSERKs (TaSERK1, TaSERK2, TaSERK3, TaSERK4 and TaSERK5) at the plasma membrane where TaBRI1 oligomerizes with respective TaSERKs independent of the BR ligand. Since interaction of both the receptors is found to be necessary in the initiation of BR signaling pathway, it would be interesting to explore further the co-localization of TaBRI1 and TaSERKs in presence of BRs.
Ectopic overexpression of TaBRI1 causes early flowering and root growth development BR's have a pleiotropic effect on the vegetative and reproductive development of plants which is mediated by BZR1 regulation by reducing transcript levels of a potent floral repressor [44].
The present results shed light on reproductive development and root growth. BR insensitive mutants and bri1 mutants were reported to be involved in late flowering [4,12]. Moreover, BRI1 was found to be an important player participating as a flowering time enhancer by repressing FLOWERING LOCUS C (FLC) expression, a MADS-box transcription factor [45]. Also, other components of BR signal transduction, BES1 interacting proteins, ELF6 (early flowering 6), its homolog REF6 (relative of early flowering 6) and JMJ30 which belongs to the Jumonji N/C (jmjN/C) domain containing proteins were found to regulate time of flowering [46,47]. These studies imply a strong connection between BR signal transduction and pathways controlling floral initiation [48]. The present study provides evidence that ectopic expression of TaBRI1 in Arabidopsis results in early flowering of transgenics and enhances the seed yield via increase in number of siliques. Roots are one of the major sources of BRs which play an important role in plant growth and development. Several BR-deficient mutants display impaired root growth and display a short root phenotype than the wild-type. However, effect of BR on root growth depends on its concentration being stimulative at low levels and inhibitory at high levels [49,50]. In the present study, we demonstrate that overexpression of TaBRI1 enhances primary root growth as compared to the WT while, inhibition of root length occurs in overexpressing lines as the concentration of BL increases indicating hypersensitivity of primary root in a dose dependent manner. In support of our results, transgenic plants ectopically overexpressing the BAK1-associating receptor-like kinase 1(BARK1) have enhanced primary root growth and show hypersensitivity to BR in a concentration dependent manner [51].

TaBRI1 imparts abiotic stress tolerance
Besides the well characterized function of BRI1 during growth and development their biological role in response to abiotic stress tolerance remain to be explored. The expression of OsBRI1 found to be upregulated under salinity stress conditions [52]. In contrast to our finding, mutant bri1-9 was found to be more resistant to cold stress than the BRI1 overexpressing Arabidopsis plants [53]. To gain insights on the role of TaBRI1 gene during environmental stress we investigated the root length phenotype of TaBRI1 overexpressing transgenics in presence of heat and cold stress. Root growth assays provide both qualitative and quantitative data of plants to various stresses [54]. In our study we observed decreased root growth inhibition, enhanced plant height and high chlorophyll content, photosynthetic activity and better membrane stability in transgenic lines under heat and cold stress as compared to WT. These results suggested that TaBRI1 might be involved in thermal stress tolerance and can be considered as a suitable gene to improve crop plants under extreme environmental stress conditions. Transcriptome analysis of TaBRI1 transgenics identify differentially expressed genes The whole-genome expression profiling of TaBRI1 overexpression revealed differential expression of several transcription factors, amongst which jumonji (jmjc), zinc finger (C3HC4 type) and zinc finger (C2H2 type) were up-regulated while F-box transcription factor was down-regulated. One of the reports showed that putative zinc finger transcription factor (AT4G39070) was repressed by BR hormone, thus negatively regulating the BR signaling pathway. Our real time PCR analysis also showed that the putative zinc finger transcription factor (AT1G27730) showed down-regulation of transcript level in presence of BR while in presence of auxin it was upregulated, which means this zinc-finger response is auxin-mediated. In contrast, one putative zinc finger transcription factor (AT2G01150) was upregulated both in the presence of BR and auxin and a jumonji N/C domain-containing transcription factor was involved in reproductive development by directly interacting with BES1. RELATIVE OF EARLY FLOWERING (REF6), a jumonji transcriptional regulator, represses FLOWERING LOCUS C (FLC) thereby leading to early flowering by BR-mediated signal transduction [47,46]. In the present study, the expression of jumonji transcription factor in transgenics decreases in presence of BR and auxin as compared to the control condition (without hormone treatment). Further, Fbox transcription factor which acts as a receptor and signaling component in plant hormone signaling pathway showed downregulation in transgenics and almost negligible expression in presence of BR and auxins.
We also analyzed several development, stress and defense related genes like Lipid Transfer Protein (LTP) family protein, Lipoxygenase (LOX2), Glutathione S-transferase (GST, tau and phi class), senescence associated family protein, Cytochrome P450, CC-NBS-LRR, LRR and TPR. Earlier reports showed that LTP gene family plays an important role in plant growth and development, defense against pathogens, as well as adaptation of plants towards various environmental stresses [55]. In this study, five putative LTPs were differentially expressed, where LTP3 and LTP4 were up-regulated in transgenics, other putative LTPs were down-regulated. These were selected and validated in presence of BL by real-time PCR analysis. Influence of hormones was observed in only one of the putative LTP (AT4G12470) whereas no significant change was apparent in other LTPs which suggests that their regulation is not BR mediated. LOX2, encodes a chloroplast lipoxygenase plays a key role in wound induced synthesis of the plant growth regulator jasmonic acid, JA [56]. Analysis of LOX2 done by Müssig et al. [57] revealed that transcript levels enhanced on application of BR in WT and BR-deficient mutants (cbb1/dwf1) which explains BR and JA interaction might be conditional during plant development. However, in our studies LOX2 gene showed higher fold change in transgenics and is down-regulated on application of BL. The Cytochrome P450, implicated to be an essential player of BR biosynthesis, however, accession number AT5G57220 encodes CYP81F2 which is involved in aphid resistance [58] downregulated in TaBRI1 transgenics and At5G47990 encoding CYP705A5 upregulated in transgenics was known to be involved in regulating gravitropism [59]. Moreover, the TaBRI1 overexpression may be detrimental in R-gene mediated resistance since the expression of CC-NBS-LRR class gene [60] was downregulated in transgenics. Tetratricopeptide repeat (TPR) domains domains which are involved in mediating protein-protein interactions are present in many stress related genes and in steroid receptor complexes [61] found to be upregulated in TaBRI1 transgenic. TPR domains are present at the C-terminus of BSK1 and BSK2 (BR signaling kinases) sequence which might act as a substrate of BRI1 or its coreceptor BAK1 [62].
Overall, our results demonstrate that constitutive expression of TaBRI1 leads to early flowering, modulates root development and confer tolerance under adverse temperature stress. In addition, TaBRI1 shows in vivo interaction with all five TaSERKs isolated from wheat at the plasma membrane. In future, it will be of interest to investigate protein-protein interaction between TaBRI1 and TaSERKs in presence of BR and to understand the intricacies of BRdependent and BR-independent pathways in higher plants.