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Genome-wide identification of GRAS genes in Brachypodium distachyon and functional characterization of BdSLR1 and BdSLRL1

Abstract

Background

As one of the most important transcription factor families, GRAS proteins are involved in numerous regulatory processes, especially plant growth and development. However, they have not been systematically analyzed in Brachypodium distachyon, a new model grass.

Results

In this study, 48 BdGRAS genes were identified. Duplicated genes account for 41.7% of them and contribute to the expansion of this gene family. 33, 39, 35 and 35 BdGRAS genes were identified by synteny with their orthologs in rice, sorghum, maize and wheat genome, respectively, indicating close relationships among these species. Based on their phylogenic relationships to GRAS genes in rice and maize, BdGRAS genes can be divided into ten subfamilies in which members of the same subfamily showed similar protein sequences, conserved motifs and gene structures, suggesting possible conserved functions. Although expression variation is high, some BdGRAS genes are tissue-specific, phytohormones- or abiotic stresses-responsive, and they may play key roles in development, signal transduction pathways and stress responses. In addition, DELLA genes BdSLR1 and BdSLRL1 were functionally characterized to play a role in plant growth via the GA signal pathway, consistent with GO annotations and KEGG pathway analyses.

Conclusions

Systematic analyses of BdGRAS genes indicated that members of the same subfamily may play similar roles. This was supported by the conserved functions of BdSLR1 and BdSLRL1 in GA pathway. These results laid a foundation for further functional elucidation of BdGRAS genes, especially, BdSLR1 and BdSLRL1.

Background

Transcription factors play key roles in plant growth, development and stress responses. Among them, GRAS proteins are an important family. The acronym, GRAS, originates from the first three functionally characterized gibberellic acid insensitive (GAI), repressor of GAI (RGA) and scarecrow (SCR) genes [1,2,3]. Subsequently, many GRAS genes have been functionally characterized to participate in a number of processes during plant growth and development, including radial organization [3, 4], root development [5, 6], formation and maintenance of meristems [7,8,9,10,11,12,13], anther microsporogenesis [14] phytochrome transduction [15,16,17], gibberellin signaling [1, 2, 18,19,20], brassinosteroid signaling [21], and responses to stresses [22,23,24], and other processes.

Most GRAS proteins share a highly conserved GRAS domain in the C-terminal that is composed of five motifs: LHRI (leucine heptads repeat I), VHIID, LHRII (leucine heptads repeat II), PFYRE and SAW [25, 26]. With a leucine-rich repeat, LHRI and LHRII are vital for protein dimerization; VHIID may interact with other proteins [27]. VHIID, PFYRE and SAW are also important for stabilizing the structure of the GRAS domain and maintaining protein function [28,29,30,31]. In contrast, the N-terminal is highly variable and can act as bait during molecular recognition events [32]. For example, the N-terminal domain of SCR is required for interactions with LHP1 and other partners and is essential for repression of asymmetric cell divisions [33]. Other GRAS proteins contain an additional conserved DELLA domain and a TVHYNP motif at the N-terminal and are thus referred to as DELLA proteins. Both the DELLA domain and TVHYNP motif are essential for interaction with GID1 in GA-induced ubiquitination and proteasome-mediated degradation of DELLA proteins [34,35,36,37,38].

According to phylogenic analyses, GRAS members are initially divided into eight subfamilies in Arabidopsis thaliana: SCR, SHR, DELLA, SCL3, PAT1, LlSCL (SCL9), SCL4/7 and HAM [39]. Since then, members of LAS and DLT subfamilies have been identified [21, 26]. To this point, genome-wide analyses of GRAS genes have been reported in several species, for example, Arabidopsis [26], tomato (Solanum lycopersicum) [40], Populus (Populus euphratica) [41], and grape (Vitis vinifera L.) [42], et al. However, no systemic analyses of GRAS genes have been reported for Brachypodium distachyon, one model grass plant with sequenced genome [43].

In this study, we identified and analyzed BdGRAS genes at genome-wide. Meanwhile, we characterized the functions of two DELLA genes in plant growth. Our study lays a foundation for further study of GRAS genes.

Results

Identification of BdGRAS genes

A total of 48 BdGRAS genes were identified. This number is more than 33 in Arabidopsis [26], and less than 57 in rice (Oryza sativa) [26], and 86 in maize (Zea mays) [44]. These genes distribute unevenly on 5 chromosomes (Fig. 1a). Of them, 22 were validated by expressed sequence tags (ESTs) (Additional file 1: Table S1). The length of putative proteins varies from 150 to 805 amino acids with molecular weights (MW) ranging from 16.98 to 88.93 kDa (Additional file 1: Table S1). The grand average of hydropathicity (GRAVY) from 43 BdGRAS proteins was negative while the value of other 5 proteins is close to zero (Additional file 1: Table S1), suggesting that most of BdGRAS proteins were hydrophilic, similar to that in Arabidopsis and Prunus mume [45]. The isoelectric point (pI) of BdGRAS proteins varies from 4.77 to 9.95 with an average of 6.27 (Additional file 1: Table S1), implying that most were faintly acidic and different BdGRAS proteins might function in different microenvironments.

Fig. 1
figure1

Chromosome location of BdGRAS genes and their collinearity with OsGRAS, ZmGRAS, SbGRAS and TaGRAS genes. a Chromosome location and duplication of BdGRAS genes on Brachypodium distachyon chromosomes. (B-E) Duplicated GRAS genes between Brachypodium distachyon, rice (b), maize (c), sorghum (d), wheat (e) genomes. Genes in red were tandem duplicated genes. Chromosomes of Brachypodium distachyon were colored in red (BdChr1), orange (BdChr2), blue (BdChr3), green (BdChr4) and yellow (BdChr5). Connecting lines indicate duplicated genes wherein colored lines represent GRAS genes while grey lines signify collinear blocks in whole genome

Duplication events and synteny of BdGRAS genes

As duplication events have contributed to the expansion of the GRAS genes in other plants [41, 42, 44,45,46], we analyzed the tandem and segmental duplication events of BdGRAS genes. 7 (14.6%) BdGRAS genes on chromosome 4 were found in a tandem repeat (BRADI4G09155 through BRADI4G09197; Fig. 1a and Additional file 1: Table S2). 14 genes (29.2%, forming 7 segmental duplicated pairs) were identified on four chromosomes (Fig. 1 and Additional file 1: Table S3). Other 28 BdGRAS genes showed no corresponding relatives. In total, 41.7% (20/48) of BdGRAS genes came from either tandem or segmental duplicated events, indicating an important role for duplication in the expansion of BdGRAS genes.

We additionally analyzed the synteny to explore the origin and evolution of BdGRAS genes using MCScanX [47]. A total of 32653, 35423, 35346 and 58494 syntenic gene pairs were identified as anchors of collinear blocks between Brachypodium distachyon and rice, sorghum, maize and wheat, respectively (Additional file 11: text 1, Additional file 12: text 2, Additional file 13: text 3, and Additional file 14: text 4). This suggests that Brachypodium distachyon has significant synteny with these four Poaceae and functional studies of genes in Brachypodium distachyon may provide information for their homologs. Among them, 33, 39, 35 and 35 BdGRAS genes were identified to have orthologs in the corresponding syntenic blocks of rice (Fig. 1b and Additional file 1: Table S4), sorghum (Fig. 1c and Additional file 1: Table S5), maize (Fig. 1d and Additional file 1: Table S6) and wheat (Fig. 1e and Additional file 1: Table S7), respectively. Intriguingly, TraesCS4A01G176700/TraesCS4A01G176600 and TraesCS4D01G135900/TraesCS4D01G136000, which were homologs of two tandem duplicated gene pairs in Brachypodium distachyon, BRADI4G09155/BRADI4G09160 and BRADI4G09170/BRADI4G09197, respectively, were still tightly linked in the chromosomes of wheat. However, we found no such homologs in other three species, indicating higher conservation of these blocks and a closer relationship between Brachypodium distachyon and wheat.

In the grass family, the Bambusoideae, Ehrhartoideae and Pooideae clade split with the Panicoideae about 50 Mya [48]. Subsequently, rice in the Ehrhartoideae, wheat and Brachypodium distachyon in the Pooideae split about 46 Mya [48]. Then Brachypodium distachyon and wheat diverged about 38 Mya from a common progenitor [49] while maize and sorghum in the Panicoideae diverged about 12 Mya [50]. According to the Ks values [51], average divergent time of tandem and segmental duplicated BdGRAS genes was about 59.6 Mya and 71.0 Mya, respectively, earlier than the diversification of the grasses (50Mya) [48]. GRAS genes in Brachypodium distachyon split with those in rice, sorghum, maize and wheat about 58.6, 85.3, 74.4 and 46.0 Mya. These results indicate that large-scale duplications predated the divergence of these species and play a role in the expansion of GRAS gene family.

Phylogenic trees, conserved motifs and gene structures

To study the evolutionary relationships of BdGRAS genes, we constructed an un-rooted Neighbor-Joining phylogenic tree (MEGA v6.0 software [52]) based on multiple alignment of 449 GRAS proteins (Additional file 15: text 5) in five grasses including Brachypodium distachyon (48), wheat, sorghum (Sorghum bicolor) (80) [44], maize (86) [44] and rice (56) [26] (Fig. 2). We also built a second tree based on the multiple alignment of BdGRAS proteins (Fig. 3). Both phylogenic trees showed similar classifications. According to the clade support values and the classification of orthologs in rice and maize [26, 41, 44], BdGRAS genes were divided into ten known subfamilies: DELLA (consisting of 6 BdGRAS genes), HAM (7), LISCL (14), PAT1 (5), LAS (2), SCR (4), SHR (4), DLT (1), SCL3 (3) and SCL4/7 (2) (Fig. 2 and Fig. 3). Duplicated gene pairs were in the same subfamily. All 7 tandem duplicated genes belonged to subfamily LlSCL, similar to those in grapevine and Prunus mume [42, 45]. Segmental duplicated gene pairs were also distributed in the same subfamilies (BRADI3G32890 and BRADI3G50930, BRADI4G24867 and BRADI4G41880 in subfamily HAM; BRADI1G36180 and BRADI3G07160 in LAS; BRADI1G00220 and BRADI4G09155, BRADI1G15123 and BRADI4G03867 in LlSCL; BRADI4G26520 and BRADI4G43200 in SCL3; BRADI2G22010 and BRADI4G44090 in SCR).

Fig. 2
figure2

Un-rooted phylogenic tree of GRAS transcription factors in Brachypodium distachyon, rice, maize, sorghum and wheat. The GRAS proteins of rice, maize, sorghum, wheat and Brachypodium distachyon were marked by yellow, blue, orange, red and purple stars, respectively. Different subfamilies were marked with different background colors

Fig. 3
figure3

Conserved motifs and gene structures of BdGRAS transcription factors. Different motifs were displayed in different colors; exons and introns were indicated by dark grey boxes and dark grey lines respectively; UTRs were indicated by light grey boxes. The length of motifs, exons, introns and UTRs was drawn in proportion

In each subfamily, amino acid sequences of BdGRAS proteins showed high identities. Similar to their homologs in other plants [26, 40, 44, 53], all BdGRAS proteins possess a GRAS domain consisting of LHRI, VHIID, LHRII, PFYRE and SAW at the C-terminal (Additional file 2: Figure S1). In contrast to the conserved C-terminal, the N-terminal of BdGRAS proteins varied substantially while members of the same subfamily possess relatively conserved motifs that might be correlated to different functions [32]. Fourteen subfamily-specific motifs, identified by Sun et al. [32] containing hydrophobic or aromatic residues repeat at the N-terminal, were also found in particular subfamilies of BdGRAS proteins except for LAS proteins with too short N-terminal. These were named after their most conserved amino acids except DELLA, TVHYNP and LR/KXI which were already known (Additional file 2: Figure S1). Motifs NLMAIA and WMESLI exist in BRADI1G10330 in SCL4/7. Motif FLNWI was identified in HAM members except for BRADI1G67340. Motif NVREII was found in BRADI4G44090 and motif DEEG with high proportion of positively and negatively charged residues was detected in BRADI2G22010, BRADI1G24310 and BRADI4G44090 which all belong to subfamily SCR. Motif RAKRT is located in the DLT protein BRADI1G49630. Motif LRSDERG lies in in SCL3 members. Motifs DELLA, TVHYNP and LR/KXI are exclusive to the DELLA member BRADI1G11090. Motif ELEXXLL was detected in PAT1 proteins while motif MDEDF was identified in SHR. Motif YISRMLM and motif FDKVLL were found in most LlSCL members. These motifs at the N-terminal may contain molecular recognition features essential for protein interactions [32]. For example, motif DELLA and TVHYNP, which were exclusive to DELLA subfamily, might directly interact with the GA-receptor GID1a to accept GA signals [38, 54]. Besides, some of these motifs showed rich acidic residues alongside the hydrophobic or aromatic residues, such as motif DELLA, ELEXXLL and YISRMLM, which suggests a connection with transcriptional activation [14, 55]. The distribution of conserved motifs in the N-termini further supported our classification of BdGRAS proteins.

The conserved motifs of full length BdGRAS proteins were identified by MEME. As shown in Figs. 3, 20 conserved motifs were identified (Additional file 3: Figure S2). The majority of the motifs were located in the GRAS conserved domain [26], except for motifs LMEED, FFYQYP and ANKFLP (named after their most conserved amino acids) which were found only in the N-terminal of LlSCL members and appear to be related to transcriptional co-activation functions [32]. Motifs LHRII-A and SAW were found in almost all BdGRAS proteins, indicating high conservation. LHRII-A contained three leucine heptad repeats (LX6LX6L; L leucine and X any amino acid) that play essential roles in protein interactions [1, 2, 26, 42, 56]. Motif SAW is part of the SAW domain [26] and may be related to stabilizing the structure of the GRAS domain [29].

Other 15 motifs were found only in some BdGRAS proteins. The entire LHRI motif occurred in subfamily LlSCL and PAT1. LHRI-A and LHRI-B were found in other eight subfamilies, indicating a discrepancy in the connected parts between 2 units in these subfamilies. Although both VHIID-A1 and VHIID-A2 were unit A of the VHIID domain, they showed different amino acids and were found in different subfamilies: VHIID-A2 was prominently found in LlSCL and PAT1 while VHIID-A1 was found in other eight subfamilies. VHIID-B was found in nine subfamilies, but not PAT1. Motif VHIID-B/C was found in LlSCL, PAT1, and DELLA, suggesting a closer relationship between these subfamilies. LHRII-B was identified in eight subfamilies except for SCR and HAM. Both P1 and P2 corresponded to the P part of the PFYRE domain; P2 was found in members of subfamily LlSCL, PAT1, DELLA and some members of SCL3 and SCL4/7, while P1 was mainly identified in SHR, PAT1, LAS, SCR, HAM and partial proteins of SCL3 and SCL4/7. Motif FY/RE/RVER/W-G-A contained the FYRE, the RVER and half of the W-G part of the SAW domain, while motifs FY and RE were only part of the PFYRE domain. These three motifs were differently distributed in 10 subfamilies; PAT1, DELLA, SCR and SCL3 contained all three motifs, SHR members contained both FY and RE, proteins in LAS possessed FY/RE/RVER/W-G-A and RE, LlSCL and DLT proteins contained only FY/RE/RVER/W-G-A, and HAM and SCL4/7 members had only FY. As the former and latter part of W-G in the SAW domain, W-G-A was found in nine subfamilies except LlSCL which possessed W-G-B exclusively. In total, GRAS proteins in the same subfamily showed similar motif components and distribution.

Apart from motifs, the gene structures of BdGRAS are also quite conserved (Fig. 3). Most BdGRAS genes (41/48) are mono-exonic, similar to other reported plant species [40,41,42, 45]. These intron-less genes could be inherited from ancient prokaryotes [57]. Among the other 7 genes, 4 (2 in subfamily SCR, 1 in subfamily LlSCL and 1 in subfamily SHR), 2 (1 each in subfamily DELLA and SHR), and 1 (in subfamily SCL3) contain two, three and four exons, respectively (Fig. 3). Both tandem duplicated and segmental duplicated BdGRAS gene pairs showed similar intron-exon structures except for 2 genes in LlSCL, BRADI1G15123 (without intron) and BRADI4G03867 (one intron), which might result from intron gain or loss events [58].

Expression profiles of BdGRAS genes

As gene function are related to expression, we analyzed BdGRAS genes expression profiles in roots, stems, leaves and inflorescences during the filling stage using qPCR (Fig. 4a). Transcripts of 38 genes were detected (primers are listed in Additional file 1: Table S8). In general, the transcription levels of BdGRAS genes in different tissues varied greatly. Transcripts of 14 genes in seven subfamilies were detected in all four tissues while some genes displayed some tissue-specific expression. For example, BRADI1G23350 (PAT1) was mainly expressed in roots, BRADI1G32070 (DELLA) and BRADI5G19190 (PAT1) were predominantly detected in stems, BRADI1G52240 was highly accumulated in leaves, while BRADI4G26520 and BRADI4G43200 (SCL3), BRADI2G57940 and BRADI4G18390 (DELLA), BRADI4G09160, BRADI4G09170, and BRADI4G09180 (LlSCL), were specifically expressed in inflorescences. Remarkably, the expression of almost all BdGRAS genes were detected in inflorescences (except BRADI1G47900) which is similar to the expression profiles of SlGRAS genes [40]. Tissue specific expression imply that these genes might be involved in these tissues development.

Fig. 4
figure4

Expression profiles of BdGRAS genes in different tissues and under treatments of different abiotic stresses and phytohormones. a Expression of BdGRAS genes in roots, stems, leaves and inflorescences. (b-d) Expression of BdGRAS genes in two-week-old seedling leaves (b and c), roots (d and e) treated with various phytohormones or under abiotic stresses. Standard errors are indicated by vertical bars

Some BdGRAS genes on the same branches showed similar expression profiles. For instance, BRADI1G23350 and BRADI3G24210 (PAT1) are mainly expressed in roots and leaves. BRADI1G25370 and BRADI2G56910 (PAT1) are expressed in all four tissues with high level in leaves. BRADI4G26520 and BRADI4G43200 (SCL3), BRADI2G57940 and BRADI4G18390 (DELLA) are specifically expressed in inflorescences. In particular, duplicated gene pairs showed similar expression profiles. For example, tandem duplicated genes (LlSCL) BRADI4G09160, BRADI4G09170, BRADI4G09180 are specifically expressed in inflorescences, while BRADI4G09190 and BRADI4G09197 are mainly expressed in roots and inflorescences. As segmental duplicated genes, BRADI4G24867 and BRADI4G41880 (HAM), BRADI4G26520 and BRADI4G43200 (SCL3) accumulated significantly in inflorescences, while BRADI1G00220 and BRADI4G09155 (LlSCL) are expressed in leaves, stems and inflorescence. Similar expression patterns may indicate similar functions of these genes in the development of these tissues.

We also investigated gene expression under abiotic stresses and phytohormone treatments. The expression of 40 BdGRAS genes was detected. In general, there is no regularity (Fig. 4b-e).

In seedling leaves, 6-BA induced the expression of most BdGRAS genes in subfamily DELLA, DLT, SCR, HAM and LlSCL while inhibited the expression of members in subfamily PAT1. In seedling roots, 6-BA induced most BdGRAS genes (except BRADI2G45117 in DELLA, BRADI1G10330 in SCL4/7, BRADI3G07160 in LAS, BRADI4G24867 in HAM, and BRADI3G24210 in PAT1). Of them, the most up-regulated gene was BRADI4G43200 in leaves, and the most down-regulated gene was BRADI2G45117 in roots. In seedling leaves, ABA inhibited the expression of 5 BdGRAS genes while up regulated 7 BdGRAS genes but have no significant effect on the other 28 genes. In seedling roots, ABA slightly induced 26 BdGRAS genes while reduced the expression of 9 genes. Of them, BRADI4G43200 and BRADI3G07160 were the most up-regulated and down-regulated genes, respectively. SA up-regulated the expression of 35 leaf and 32 root BdGRAS genes, in which BRADI4G09155 increased a surprising 11287 times in roots. GA suppressed the expression of 23 leaf and 22 root BdGRAS genes, wherein BRADI4G18390 and BRADI4G09170 declined to zero in both roots and leaves. Some genes expressed differently responding to the same hormone in different tissues. For example, MeJA suppressed the transcription of 21 BdGRAS genes in leaves while induced 30 BdGRAS genes in roots. GA intensively suppressed the expression of BRADI3G07160 in roots but induced it in leaves by approximately 16 times. 6-BA promoted BRADI4G24867 in leaves whereas strongly suppressed it in roots. These results indicated that BdGRAS genes might participate in the crosstalk among phytohormones.

The effects of abiotic stresses including salt, drought, oxidation, cold and heat on the expression of BdGRAS genes were also detected. NaCl slightly up regulated the expression of 28 leaf and 22 root BdGRAS genes. The most up-regulated gene was BRADI4G43200 in leaves while the most down-regulated gene was BRADI2G57940 in leaves, BRADI1G32070 in roots and BRADI4G09235 in both tissues. PEG promoted the transcription of 21 and 26 BdGRAS genes in leaves and roots, respectively. Among these, BRADI4G09190 in roots increased the most, while BRADI4G24867 in roots, BRADI4G03867, BRADI4G43680 and BRADI5G19190 in leaves, and BRADI2G57940 in both tissues dropped to zero. H2O2 dramatically increased the expression of 22 BdGRAS genes in leaves with the most up-regulated gene expression (3831 times) in BRADI4G43200. Twenty genes were slightly induced in roots. Thirty-five leaf and 27 root BdGRAS members were induced by cold. In this case, the highest expression was found in BRADI3G50930 in roots while the lowest in BRADI2G45117, BRADI4G18390, BRADI4G43200, BRADI4G03867 and BRADI5G19190 in roots. Heat stress up-regulated the expression of 28 BdGRAS genes in leaves while inhibited 26 BdGRAS genes in roots. The expression of BRADI4G41880 in roots was the most elevated while BRADI3G07160, BRADI4G24867 and BRADI4G09170 in roots declined the greatest. Although they differed greatly, the expression patterns of BdGRAS genes identified some tissue-specific genes, phytohormone- and abiotic stress-responsive genes and provided useful information for functional studies.

cis-elements of BdGRAS genes

We also analyzed the cis-elements of BdGRAS genes (Additional file 1: Table S11). Ten cis-elements that were related to plant growth and development were identified. Among them, three are light responsive and two are involved in endosperm expression. The remaining are related to meristem expression, circadian control, meristem specific activation, zein metabolism regulation, and cell cycle regulation. Especially, all BdGRAS genes contain light responsive cis-elements and 43 BdGRAS genes contain at least one cis-element related to endosperm expression. 19 BdGRAS genes contain meristem expression-related cis-element CAT-box. 28 BdGRAS genes have the meristem specific activation element CCGTCC-box. 34 BdGRAS genes contain circadian control element These results indicate that BdGRAS genes participate extensively in plant growth and development.

Ten cis-elements were identified to be responsive to different phytohomones including ABA (ABRE was found in 30 BdGRAS genes), MeJA (TGACG-motif in 32 BdGRAS genes), SA (TCA-element and SARE in 24 BdGRAS genes), auxin (AuxRR-core and TGA-element in 17 BdGRAS genes), gibberellin (GARE-motif, P-box and TATC-box in 29 BdGRAS genes), ethylene (ERE in 8 BdGRAS genes). These cis-elements may be associated with the expression profile. For example, BRADI1G32070, BRADI1G49630, and BRADI4G09190 possessing TCA-element or SARE are strongly induced by SA. ABA positively regulates the expression of BRADI4G18390 and BRADI2G13560 that contain ABRE elements. MeJA noticeably inhibits the expression of BRADI2G45117, BRADI1G10330 and BRADI2G52227 that possess TGAC-motifs. GA inhibits the expression of genes in subfamily DELLA (BRADI1G11090, BRADI4G18390, and BRADI2G57940) and PAT1 (BRADI1G25370 and BRADI1G23350) which have the gibberellin responsive elements TATC-box, P-box or GARE. In total, cis-elements correspond with the expression of many BdGRAS genes.

GO annotations and KEGG pathways of BdGRAS proteins and conserved functions of BdSLR1 and BdSLRL1 in plant growth

Only one BdGRAS gene BdSHR has been functionally characterized in Brachypodium distachyon. It plays a similar role with its orthologs AtSHR and OsSHR in the regulation of meristem and root growth [59]. The functions of most BdGRAS genes still remain to be studied.

We analyzed the gene ontology of 48 BdGRAS genes. Although they were all GO annotated and presumed to be involved in DNA-templated transcription (Additional file 1: Table S9 and S10), no conclusive results were found.

We also performed KEGG pathway analyses of BdGRAS genes. Only two genes BRADI1G11090 (BdSLR1) and BRADI2G45117 (BdSLRL1) were identified with the same annotation K14494. Congruent with GO analyses, both genes may be involved in GA mediated signaling transduction pathway.

Based on phylogenic analyses, both BdSLR1 and BdSLRL1 were DELLA genes whose orthologs in Arabidopsis, rice, maize and wheat play key roles in plant growth via negatively regulating GA signal [1, 2, 20, 28, 34]. However, such functions have not yet been reported in Brachypodium distachyon. Here, we characterized the functions of these two BdDELLA genes by ectopic expressing them in Arabidopsis.

Twenty-three transgenic Arabidopsis lines over-expressing BdSLR1 were obtained. Sixteen lines showed later flowering (Fig. 5a, b) and dwarfism (Fig. 5c-e) compared with the wild type. According to the expression level (Fig. 5f), Lines 4 and 7 were selected for further analyses. After maturation, the height of control and the transgenic plants were measured. The average height of lines 35S-BdSLR1–4 and 35S-BdSLR1–7 was 23.04 ± 4.89 cm (n = 33) and 25.47 ± 5.10 cm (n = 32) respectively, while the average height of control was 30.34 ± 3.66 cm (n = 36) (Fig. 5g). The hypocotyls of transgenic Arabidopsis were clearly shorter than the control (Fig. 5h). These phenotypes are similar to some GA-deficient mutants. When treated with 10 μM GA3, the hypocotyl lengths of both transgenic and wild type Arabidopsis increased noticeably (P < 0.05), and the rate of transgenic Arabidopsis (58.72%) was higher than that of wild type (38.08%) (Fig. 5h).

Fig. 5
figure5

Phenotypes of transgenic Arabidopsis over-expressing BdSLR1 under normal conditions and GA3 treatment. a 4-week-old wild type (left) and transgenic Arabidopsis (middle and right). b Rosette leaf numbers of wild type and transgenic Arabidopsis at bolting stage. For 35S-BdSLR1–4, n = 33; for 35S-BdSLR1–7, n = 32. c-e 6-week wild type (c) and two severely dwarf transgenic Arabidopsis (d and e). f Relative expression level of BdSLR1. g Final height. h 7-day-old seedlings and hypocotyl length with 0 μM or 10 μM GA3, n = 30. i Expression levels of GA related genes in wild type and transgenic Arabidopsis. Scale bars = 1 cm. ** indicates that p<0.01 by Student’s t test. Standard errors are indicated by vertical bars

We also detected the expression of GA related genes. GA20-oxidase 1 and 2 catalyze the sequential oxidation of active GAs [60], GA3-oxidase 1 catalyzes the last step for the synthesis of bioactive GAs [61], and GA2-oxidase 1 inactivates GA [62]. These four genes are under the feedback regulation of GA; the expression of GA20ox1, GA20ox2 and GA3ox1 is down-regulated while the expression of GA2ox1 is up-regulated by GA3 [63]. qRT-PCR results showed that the expression level of GA20ox1, GA20ox2, and GA3ox1 was higher than control, while the expression of GA2ox1 in transgenic Arabidopsis was lower than the control (Fig. 5i). When treated by GA3, the expression level of these four genes in transgenic plants recovered to normal levels and was indistinguishable from that of the wild type (Fig. 5i). These results indicated that, as with the GO annotation, BdSLR1 participates in plant growth via negatively regulating GA signals like its orthologs in other plants [1, 2, 20, 28, 34]..

Seventeen transgenic Arabidopsis lines over-expressing BdSLRL1 were acquired with Fourteen lines displayed late flowering (Fig. 6a, b) and dwarfism (Fig. 6c-f). Among these, four showed a severe dwarf phenotype with a height of less than 3.5 cm (Fig. 6d). Three showed mild dwarfism with a height between 3.5 and 10 cm (Fig. 6e). The height of seven lines was more than 10 cm yet slightly shorter than that of control (Fig. 6f). Additionally, the severe and mild dwarf plants had shorter stamen filaments (Fig. 6g) resulting in sterile flowers. So, the two slightly dwarf lines (Lines 5 and 6) were selected for further analyses due to their expression levels (Fig. 6h). After maturation, the average height of 35S-BdSLRL1–5 and 35S-BdSLRL1–6 transgenic Arabidopsis was 20.61 ± 3.81 cm (n = 36) and 16.22 ± 3.48 cm (n = 31) respectively, while the average height of control was 30.24 ± 3.80 cm (n = 33) (Fig. 6i). The hypocotyls of transgenic Arabidopsis were also significantly shorter (Fig. 6j). The transcription levels of GA-related genes were also similar to those in transgenic Arabidopsis over-expressing BdSLR1. Under normal conditions, the expression of GA20ox1, GA20ox2, and GA3ox1 was higher, while the expression of GA2ox1 was lower in transgenic Arabidopsis than that in the wild type. But different from BdSLR1, transgenic Arabidopsis that over expressed BdSLRL1 was insensitive to exogenous GA3 (Fig. 6j). Consistent with other findings, exogenous GA3 had no obvious effect on the expression of those 4 genes in transgenic plants over-expressing BdSLRL1 (Fig. 6k). The phenotypes of dwarfism and insensitivity to exogenous GA3 in transgenic Arabidopsis over-expressing BdSLRL1 were also found in transgenic plants over-expressing its orthologous OsSLRL1 [64] and OsSLRL2 [65].

Fig. 6
figure6

Phenotypes of transgenic Arabidopsis over-expressing BdSLRL1 under normal conditions and GA3 treatment. a 4-week-old wild type (left) and transgenic Arabidopsis (middle and right). b Rosette leaf numbers of wild type and transgenic Arabidopsis at flowering time. For 35S-BdSLRL1–5, n = 36; for 35S-BdSLRL1–6, n = 31. c-f 6-week-old wild type (C) and three typical transgenic Arabidopsis lines (d-f). g Fower of wild type (left) and short-stamen transgenic Arabidopsis (right). h Relative expression level of BdSLRL1. i Final height. j 7-day-old seedlings and hypocotyl length with 0 μM or 10 μM GA3, n = 30. k Expression levels of GA related genes in wild type and transgenic Arabidopsis. Scale bars = 1 cm in A-F; scale bar = 2 mm in G; ** indicates that p<0.01 by Student’s t test. Standard errors are indicated by vertical bars

These results indicated that both BdSLR1 and BdSLRL1 play a conserved role in plant growth via negatively regulating GA signal like their orthologs in Arabidopsis, rice, maize and wheat [1, 2, 20, 28, 34, 64, 65] and verified the GO and KEGG pathway annotations.

Conserved functional mechanisms of BdSLR1 and BdSLRL1

Although BdSLR1 and BdSLRL1 regulate plant growth via inhibiting GA mediated signaling pathway, transgenic plants over-expressing two genes showed different sensitivity to exogenous GA3, implying some differences between the two genes. As protein is the main manifestation of gene function, we further analyzed both BdSLR1 and BdSLRL1 proteins. Sequence alignment showed that BdSLR1 and BdSLRL1 were highly homologous with OsSLR1 (identity of full length sequence = 85.37%) and OsSLRL1 (identity of full length sequence = 73.93%), respectively (Fig. 7). All four proteins contain a conserved GRAS domain at the C-terminal. In addition, BdSLR1 and OsSLR1 contain an additional DELLA domain and TVHYNP motif at the N-terminal while BdSLRL1 and OsSLRL1 do not. As there may be annotation errors that could lead to a truncated gene sequence, we performed the 5′-RACE of BdSLRL1 and acquired a single product of 233 bp (Additional file 4: Figure S3 and Additional file 5: Figure S4). A PCR of a full length of BdSLRL1 with 5′-UTR using BdSLRL1FLPF (the first 21 bp of 5′-UTR) and BdSLRL1FLPR (BdSLRL1Y2H reverse primer without a restriction enzyme cutting site) also generated a single band (Additional file 6: Figure S5, Additional file 7: Figure S6, and Additional file 8: Figure S7). Sequence analyses showed that, BdSLRL1 generates a single transcript of 1604 bp including 89 bp 5′-UTR (Additional file 9: Figure S8), indicating that BdSLRL1 has no DELLA domain or TVHYNP domain.

Fig. 7
figure7

Multiple alignment of BdSLR1, BdSLRL1, OsSLR1 and OsSLRL1 protein sequences. Black, pink and blue background color indicate the homology level of amino acid residues is 100%, above 75, and 50%, respectively. Conserved DELLA domain and TVHYNP motifs were marked with red square

In rice, SLR1 showed transcriptional activation activity and interaction with GID1 depending on the presence of the DELLA domain and TVHYNP motif [34]. We further investigated whether BdSLR1 and BdSLRL1 have similar activities. Experiment with yeast have shown that BdSLR1 have transcriptional activation activity (Additional file 10: Figure S9). Similar to OsSLR1, after deleting the DELLA domain and TVHYNP motif (1–142 amino acids at the N-terminal), the truncated protein (BdSLR1D) lost the activity, suggesting that the DELLA domain and TVHYNP motif are essential for transcriptional activation of BdSLR1 [34]. Whereas, BdSLRL1 which lacks the DELLA domain and the TVHYNP motif did not show transcriptional activation (Additional file 10: Figure S9).

RGA (an ortholog of BdSLR1 in Arabidopsis), which can be degraded by interacting with GID1 [66, 67], negatively controls PIFs-mediated hypocotyl elongation through physically interacting with phytochrome-interacting factors (PIFs) AtPIF3 and ZmPIF4 [35, 68]. We hypothesized that, BdSLR1 and BdSLRL1 could interact with BdGID1 (BRADI2G25600), BdPIF3 (BRADI2G11100) and BdPIF4 (BRADI1G13980) due to their high identities. Results suggested that both BdSLR1D and BdSLRL1 could interact with BdPIF3 and BdPIF4 (Fig. 8a, b). There were also weak interactions between BdGID1 with full, but not the truncated, BdSLR1 that could be strengthened by GA3 (Fig. 8c, d). This suggests that the interaction between BdSLR1 and BdGID1 also depends on the DELLA domain and the TVHYNP motif like OsSLR1 and OsGID1 [34, 69, 70]. Consistent with this hypothesis, BdSLRL1 did not interact with BdGID1 (Fig. 8b). As well, BdSLR1 and BdSLRL1 can form homo-dimers (Fig. 8a), but they could not interact with each other (Additional file 10: Figure S9). Bimolecular florescence complementation (BiFC) assay further verified these interactions (Fig. 8e). The protein interaction activity and transactivation activity of BdSLR1 and BdSLRL1 were similar to their homologs in Arabidopsis, rice and maize [34, 35, 68,69,70], indicating that orthologs with same motifs may have conserved functions among different species. Using this enables us to predict the functions of unknown genes in a variety of plant species.

Fig. 8
figure8

Protein interaction assays of BdSLR1 and BdSLRL1 with BdGID1, BdPIF3 and BdPIF4 and between themselves. a, b protein interactions in yeast cells (c, d) enhanced interaction between BdSLR1 and GID1 by GA3. Values for ±standard errors were determined by three replicates and ** indicates that p<0.01 by Student’s t test. Standard errors are indicated by horizontal bars. e Further assay of protein interactions by BiFC in tabacoo leaf cells

Discussion

GRAS transcription factors have been investigated widely among plants [71]. In this study, 48 GRAS genes were identified from Brachypodium distachyon genome. Among them, 7 (14.6%) and 14 (29.2%) genes were identified as tandem duplicated and segmental duplicated genes respectively. This corresponded with those in Arabidopsis (2 tandem duplicated genes/16 segmental duplicated genes/34 genes in total) [72], rice (10/8/45) [72], maize (11/22/86) [44] and Prunus mume (10/14/46) [45] and indicated that gene duplications play a role in the expansion of GRAS gene family in both monocots and dicots [40, 45, 53, 73]. These duplicated genes might undergo non-functionalization, neo-functionalization or sub-functionalization during evolution process that could generate alternative functions [74].

Genome-wide identification of GRAS genes has been reported in many plants including dicots such as Arabidopsis [26], tomato [40], and Chinese cabbage [53], monocots including rice [26, 41] and maize [44], and so on. According to phylogenic analyses, GRAS genes were divided into ten main subfamilies: SCR, SHR, DELLA, SCL3, PAT1, LlSCL (SCL9), SCL4/7, HAM, DLT and LAS [44, 75]. Based on the clade support values and the classification of homologs in rice and maize, BdGRAS genes were also divided into the same ten subfamilies.

The expression of BdGRAS genes in some subfamilies were similar to their homologs in other species. For example, in subfamily PAT1, most genes showed higher expression in leaves and roots that matched their orthologs in Arabidopsis [76], rice [26], castor bean [77] and sacred lotus [58]. Members of SCL3 were mainly expressed in inflorescence which is consistent with their homologs in Arabidopsis [76] and castor beans [77]. LAS gene was expressed in all four tissues, similar to its homologs in Arabidopsis [76]. BdSCR genes showed higher expressed in roots and leaves, consistent with those in rice [26], sacred lotus [58] and castor beans [77]. Conserved expression profiles of GRAS genes in different species indicate that homologous genes might have related functions in the development of these tissues. While the expression levels of BdGRAS genes in responses to different abiotic stresses and phytohormones were quite different even among members of the same subfamily. This is consistent with those in tomato [40], Populus [41] and Prunus mume [45], suggesting their different roles in abiotic stress responses and hormone-mediated signal pathways.

Similar to their homologs in Arabidopsis [26], rice [26], Chinese cabbage [53] and maize [44], BdGRAS genes in the same subfamilies showed conserved amino acid sequences and conserved motifs, which might imply conserved functions. Based on reports of functionally characterized genes, this might enable us to predict the functions of unknown proteins.

In most cases, GRAS members on the same branches showed similar functions (Additional file 1: Table S12). For example, SCARECROW genes in Arabidopsis, rice and maize function in the radial patterning of roots and shoots [3, 4, 78, 79]. Arabidopsis LAS and tomato Lateral suppressor are members of the LAS subfamily. Their mutations led to strong defects in axillary shoot meristem initiation [8, 10]. Similarly, mutations in the rice ortholog MOC1, resulted in few tillers, rachis branches and spikelets [9]. Arabidopsis, pepper, tomato and petunia HAM genes showed conserved function in the maintenance and organization of shoot apical meristem [7, 11,12,13]. In Brachypodium distachyon, the GRAS gene BdSHR was reported to play similar roles with its orthologs, AtSHR and OsSHR, in the regulation of meristem and root growth [59]. However, functions of other BdGRAS genes have not been reported. More effort needs to be made to characterize the functions of BdGRAS genes.

According to GO analyses and the KEGG pathway, two DELLA genes BdSLR1 and BdSLRL1 likely participate in the GA mediated signaling pathway. As reported previously, DELLA genes negatively regulate GA signals, including GAI, RGA and RGL1/2/3 in Arabidopsis [1, 2], SLR1 in rice [18], SLN1 in barley [19], DWARF-8 and DWARF-9 [20, 80] in maize, and Rht-B1/Rht-D1 in wheat [20]. Nevertheless, the functions of DELLAs in Brachypodium distachyon have not been verified. In this study, we showed the conserved functions of two DELLA genes BdSLR1 and BdSLRL1 in plant growth via GA signals.

Both transgenic plants over-expressing BdSLR1 and BdSLRL1 displayed dwarfism and late-flowering phenotypes which was consistent with their orthologs [1, 2, 20, 28, 34, 64, 65]. In addition, over-expression of BdSLRL1 resulted in transgenic plants insensitive to exogenous GA3 like their orthologs in rice OsSLRL1 [64] and OsSLRL2 [65]. Sequence analyses showed that BdSLR1 contains a DELLA domain and a TVHYNP motif at the N-terminal, and a conserved GRAS domain at the C-terminal. BdSLRL1 has only a conserved GRAS domain at the C-terminal (Fig. 7). In rice, the suppressive function of SLR1 depends on the conserved GRAS domain; the N-terminal including the DELLA domain and the TVHYNP motif acts as the GA signal perception domain [28]. The repressive function of both BdDELLA proteins may also be due to the conserved GRAS domain while the difference between BdSLR1 and BdSLRL1 in GA signal perception may depend on the existence of the DELLA domain and the TVHYNP motif at the N-terminal of BdSLR1.

In Arabidopsis, RGA can physically interact with AtPIF3 and ZmPIF4 in the absence of GA and negatively controls PIFs-mediated hypocotyl elongation [35, 68]. After application of GA3, GIBBERELLIN INSENSITIVE DWARF1 (GID1), a GA receptor, binds to GA3 and then blocks the repression activity of DELLA proteins by directly interacting with the DELLA domain [36] or starting DELLA ubiquitylation by SCFSLY1 E3 ligase to initiate their degradation [66, 67, 69]. Thus, PIFs were released to promote plant growth. BdSLR1 and BdSLRL1 could also interact with BdPIF3 and BdPIF4 (Fig. 8). In addition, BdSLR1 could interact with BdGID1 and this interaction could be strengthened by GA3 (Fig. 8).

In 35S-BdSLR1 transgenic Arabidopsis, exogenous GA3 strengthened the interaction between GID1 and BdSLR1 (Fig. 8). This could restrain repressor activity and release PIFs from BdSLR1-PIF complexes. As a result, the phenotypes were recovered by application of exogenous GA3. As opposed to BdSLR1, BdSLRL1 could not interact with GID1, but could interact with BdPIFs (Fig. 8). When BdSLRL1 is excessively accumulated, the BdPIFs are restrained and affect growth. Since GID1 does not interact with BdSLRL1, the GA3-GID1 complex could not compete BdSLRL1 with PIFs. Thus, exogenous GA3 could not recover the phenotype. Further research should be able to verify these predictions and reveal vital mechanisms.

Our results showed that BdSLR1 and BdSLRL1 play a role in plant growth via negatively regulating the GA signal in a conserved manner similar to their orthologs [1, 2, 18,19,20, 80]. This supported the prediction that genes in the same branch may play conserved functions.

Conclusions

We identified 48 GRAS genes in the Brachypodium distachyon genome and classified them into ten subfamilies using phylogenic analyses. Bioinformatics analyses and expression profiles indicate different GRAS proteins have different functions, while the members in same subfamily likely have similar functions. This was supported by the conserved functions of both BdSLR1 and BdSLRL1 genes in plant development via negatively regulating GA signals.

Methods

Genome-wide identification of BdGRAS genes

Sequences of genome DNA, CDS and proteins of Brachypodium distachyon (Bd21) (assembly v2.0) and Triticum aestivum (Chinese Spring) (assembly iwgsc_refseqv1.0) were obtained from http://www.gramene.org/. Protein sequences of the GRAS family in maize and sorghum were downloaded from the Plant Transcription Factor Database (PlantTFDB v4.0) [81]. Multiple alignments were performed using MEGA software (v6.0) (choosing ‘align by clustalW’ option with default parameters) [52].

The GRAS family Hidden Markov Model (HMM) profile (PF03514) was downloaded from the Pfam database (http://pfam.xfam.org/, v31.0). A second GRAS HMM profile was built by HMMER (v3.0) based on the multiple sequence alignment of conserved GRAS domains of AtGRAS proteins (downloaded from http://www.Arabidopsis.org/, release 10.0) and OsGRAS proteins (downloaded from http://rice.plantbiology.msu.edu/, release 7.0) [75]. Both GRAS HMM profiles were used as the query to perform the hmmsearch by HMMER (v3.0) against the annotated protein database of Brachypodium distachyon and wheat with a cut-off expected value (E-value) of 10− 5.

All hits identified by two HMM profiles were compared, and consensus sequences were retained. SMART sequence analysis [82] with a threshold of E-value less than 10− 5 was conducted among these candidate proteins to exclude those lacking the GRAS domain. BlastN in expressed sequence tags (EST) with a threshold of E-value less than 10− 5 and identity above 50% was also applied to support our identifications (http://www.ncbi.nlm.nih.gov).

Analyses of protein properties, GO annotations, KEGG pathways, and phylogenetic relationship

Molecular weight (MW) and the theoretical isoelectric point (PI) values were calculated by ExPASy (https://web.expasy.org/protparam/). The GO (gene ontology) annotations were obtained from Monocots PLAZA v4.0 (https://bioinformatics.psb.ugent.be/plaza/) and Gramene v3.0 and then analyzed by BGIWEGO (v2.0) [83]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of BdGRAS genes were analyzed online using protein sequences (https://www.genome.jp/kegg/pathway.html). An un-rooted neighbor joining (NJ) tree of GRAS proteins from rice, wheat, maize, sorghum and Brachypodium distachyon was constructed using MEGA (v6.0) [52] with 1000 bootstrap replications and annotated by Evolview (v2.0) [84].

Synteny, cis-elements, gene structures and conserved motifs analyses of BdGRAS genes

Syntenic gene pairs among Brachypodium distachyon, and between rice, maize, sorghum, wheat and Brachypodium distachyon were identified using the Multiple Collinearity Scan toolkit (MCScanX) with default parameters [47]. The Brachypodium gene set was used as the chromosomal reference. Chromosomal distributions of GRAS genes were obtained from genome annotations and visualized using Circos (v 0.69) along with duplicated gene pairs.

The synonymous substitution (Ks) and non-synonymous substitution (Ka) rates were calculated by KaKs_calculator (v2.0) using the NG method [85]. Ks values were used to calculate the dates of duplication events (T) using the formula T = Ks/2λ × 10− 6 (millions of year, Mya) [51] assuming universal clock-like rate for Brachypodium distachyon was 6.1 × 10− 9 substitutions per synonymous site per year [86].

The 1.5 kb genomic DNA sequences in the 5′ flanking region of BdGRAS genes were downloaded from NCBI and then submitted to the PlantCARE for cis-elements analysis. The intron-exon organizations were analyzed through the Gene Structure Display Server v2.0 (http://gsds.cbi.pku.edu.cn/). MEME server v5.0.4 was applied to detect the conserved motifs with maximum number of 20 and optimum width of 5–200 amino acids. Gene structures and conserved motifs were visualized using Evolview (v2.0) [84].

Stress and phytohormone treatments of Brachypodium distachyon and quantity RT-PCR

Two-week-old Bd21 seedlings were put in a Murashige and Skoog (MS) liquid medium containing 200 mM NaCl, 20% PEG6000, 10 mM H2O2, 1 mM SA, 100 μM MeJA, 100 μM ABA, 20 μM 6-BA and 3 μM GA for 2 h, respectively, to mimic salt, drought, oxidative stresses and phytohormone stimulation. Seedlings were placed in a 45 °C or 4 °C climate chamber for 2 h to imitate heat or cold stresses. Seedlings with no treatment served as control. The leaves and roots of Bd21 were collected separately after treatment. Roots, stems, leaves and inflorescences were acquired from plants during the heading period. All materials were flash frozen by liquid nitrogen and stored at − 80 °C until analysis.

Total RNA was extracted using the TRIZOL reagent (TAKARA) and treated with RNase-free DNase I (TAKARA) according to the manufacturer’s instructions. A reverse transcription reaction using total RNA (above) was carried out as described previously with a Transcriptor First Strand cDNA Synthesis Kit (Roche) [87]. qRT-PCR reaction were performed by a QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific) in triplicate with 15 μl reaction mixture consisting of 7.5 μl SYBR® Premix Ex Taq (TAKARA), 0.5 μl cDNA (5.0 ng/μl), 0.3 μl ROX reference Dye (50×),1.5 μl (10 pmol/μl) forward primer, 1.5 μl (10 pmol/μl) reverse primer, and 3.7 μl ddH2O. The qRT-PCR event sequence was: preheat at 50 °C for 2 min, predenaturation at 95 °C for 10 min, 40 cycles of PCR reactions at 95 °C for 15 s and 60 °C for 1 min with fluorescence being measured at the end of each cycle, melt curve at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s with fluorescence being measured during the heating period from 60 °C to 95 °C. Relative expression levels of target genes (primers used in this study were designed by Primer Premier v5.0 [88] and listed in Additional file 1: Table S8) were calculated by the 2(−ΔΔCt) analysis method [89]. Ct means were normalized with the expression of GAPDH in Brachypodium distachyon (BRADI3G14120) [90] and Arabidopsis (AT1G13440) [91].

5′-rapid amplification of cDNA ends (5′-RACE)

Total RNA extracted from inflorescences of Brachypodium distachyon was used to produce the first strand cDNA with Rapid Amplification of cDNA Ends kit (TAKARA) using the manufacturer’s instructions. Primers for 5′-RACE (Rapid Amplication of cDNA Ends) of BRADI2G45117 were designed using Primer Premier v5.0 [88] based on its CDS sequence downloaded from Gramene. 5′-RACE was conducted using primers RACEPR1 (used in the first PCR) and RACEPR2 (second PCR) (Additional file 1: Table S8) with the following PCR conditions: 95 °C for 5 min, 40 cycles (95 °C for 30s, 58 °C for 30s, 72 °C for 1 min), 72 °C for 10 min. Then the full length sequence of BRADI2G45117 containing the 5′-UTR and CDS was amplified using rTaq (Sangon) according to the manufacturer’s protocols with cDNA from inflorescence of Brachypodium distachyon as the template and BdSLRL1FLPF and BdSLRL1FLPR as primers. The PCR process was as follows: 98 °C for 5 min, 40 cycles (98 °C for 45 s, 58 °C for 45 s, 72 °C for 2 min), 72 °C for 10 min.

Plasmid construction, yeast two-hybrid assay, BiFC and plant transformation

The amplified fragments with additional Nde I and EcoR I sites through corresponding primers (Additional file 1: Table S8) were cloned separately into the DNA binding vector pGBKT7 and activation domain vector pGADT7. Recombined vectors were transformed into the yeast strain Y2H using the LiAc transformation method [92] and coated on synthetic dextrose (SD) -Trp or SD-Trp-Leu for growth tests. Yeast clones were plated on SD-His-Trp-Ade and SD-His-Trp-Leu-Ade medium for 3 days at 30 °C to assay for self-activation and protein interaction.

The coding sequence were amplified using primers listed in Additional file 1: Table S8 and then introduced into vectors p1302-eYFP-N and p1302-eYFP-C using recombination reactions. Recombined plasmids were transformed into the Agrobacterium tumefaciens strain GV3101 and then co-infiltrated with Agrobacterium carrying the p19 silencing plasmid into leaves of 1-month-old Nicotiana benthamiana plants. Two days after infiltration, eYFP signals were observed with a fluorescence microscopy (Olympus IX83-FV1200).

The coding sequence were cloned with primers in Additional file 1: Table S8 and ligated into the over-expression vector pCAMBIA1300 using recombination reactions. Recombined plasmids were introduced into GV3101 and then transformed into Arabidopsis Col-0 via the flowerer-dipping method [93].

Arabidopsis materials and treatments

Plants were grown in long-day conditions of 22 °C, 16 h light/20 °C, 8 h dark cycles. Transgenic lines were selected using 1/2MS medium containing 40 mg/L hygromycin B. For the GA treatment, surface-sterilized seeds were vertically cultivated on 1/2MS medium with or without 10 μM GA3 for 7 days. Photographs were then taken and seedlings were collected for gene expression. Hypocotyls of at least 30 seedlings were measured via ImageJ and data was analyzed with SPSS (IBM SPSS Statistics 20).

Availability of data and materials

The GRAS gene family datasets analyzed in this article are included within this article and supplementary files (the Additional files 11-15), and the rests are available from the corresponding author on reasonable request.

References

  1. 1.

    Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP, Harberd NP. The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev. 1997;11(23):3194–205.

  2. 2.

    Silverstone AL, Ciampaglio CN, Sun T. The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell. 1998;10(2):155.

  3. 3.

    Di LL, Wysockadiller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN. The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell. 1996;86(3):423.

  4. 4.

    Wysocka-Diller JW, Helariutta Y, Fukaki H, Malamy JE, Benfey PN. Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development. 2000;127(3):595.

  5. 5.

    Helariutta Y, Fukaki H, Wysockadiller J, Nakajima K, Jung J, Sena G, Hauser MT, Benfey PN. The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell. 2000;101(5):555.

  6. 6.

    Heo JO, Estelle M. Funneling of gibberellin signaling by the GRAS transcription regulator scarecrow-like 3 in the Arabidopsis root. Proc Natl Acad Sci U S A. 2011;108(5):2166.

  7. 7.

    Stuurman J, Jaggi F, Kuhlemeier C. Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells. Genes Dev. 2002;16(17):2213–8.

  8. 8.

    Greb T, Clarenz O, Schafer E, Muller D, Herrero R, Schmitz G, Theres K. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev. 2003;17(9):1175–87.

  9. 9.

    Li X, Qian Q, Fu Z, Wang Y, Xiong G, Zeng D, Wang X, Liu X, Teng S, Hiroshi F. Control of tillering in rice. Nature. 2003;422(6932):618.

  10. 10.

    Schumacher K, Schmitt T, Rossberg M, Schmitz G, Theres K. The Lateral suppressor (ls) gene of tomato encodes a new member of the VHIID protein family. Proc Natl Acad Sci U S A. 1999;96(1):290.

  11. 11.

    Engstrom EM, Andersen CM, Gumulaksmith J, Hu J, Orlova E, Sozzani R, Bowman JL. Arabidopsis homologs of the Petunia HAIRY MERISTEM gene are required for maintenance of shoot and root indeterminacy. Plant Physiol. 2011;155(2):735–50.

  12. 12.

    Davidschwartz R, Borovsky Y, Zemach H, Paran I. CaHAM is autoregulated and regulates CaSTM expression and is required for shoot apical meristem organization in pepper. Plant Sci. 2013;203-204(2):8.

  13. 13.

    Hendelman A, Kravchik M, Ran S, Frank W, Arazi T. Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis. J Exp Bot. 2016;67(21):6187–200.

  14. 14.

    Morohashi K, Minami M, Takase H, Hotta Y, Hiratsuka K. Isolation and characterization of a novel GRAS gene that regulates meiosis-associated gene expression. J Biol Chem. 2003;278(23):20865.

  15. 15.

    Bolle C, Koncz C, Chua NH. PAT1, a new member of the GRAS family, is involved in phytochrome a signal transduction. Genes Dev. 2000;14(10):1269–78.

  16. 16.

    Torresgalea P, Huang LF, Chua NH, Bolle C. The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light signaling, but can also modulate phytochrome a responses. Mol Gen Genomics. 2006;276(1):13–30.

  17. 17.

    Torresgalea P, Hirtreiter B, Bolle C. Two GRAS proteins, SCARECROW-LIKE21 and PHYTOCHROME a SIGNAL TRANSDUCTION1, function cooperatively in phytochrome a signal TRANSDUCTION. Plant Physiol. 2013;161(1):291.

  18. 18.

    Sato T, Miyanoiri Y, Takeda M, Naoe Y, Mitani R, Hirano K, Takehara S, Kainosho M, Matsuoka M, Ueguchitanaka M. Expression and purification of a GRAS domain of SLR1, the rice DELLA protein. Protein Expr Purif. 2014;95(3):248–58.

  19. 19.

    Fu X, Richards DE, Aitali T, Hynes LW, Ougham H, Peng J, Harberd NP. Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor. Plant Cell. 2002;14(12):3191–200.

  20. 20.

    Peng J, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE, Beales J, Fish LJ, Worland AJ, Pelica F, et al. Green revolution' genes encode mutant gibberellin response modulators. Nature. 1999;400(6741):256–61.

  21. 21.

    Tong H, Jin Y, Liu W, Li F, Fang J, Yin Y, Qian Q, Zhu L, Chu C. DWARF AND LOW-TILLERING, a new member of the GRAS family, plays positive roles in brassinosteroid signaling in rice. Plant J. 2009;58(5):803–16.

  22. 22.

    Ma HS, Liang D, Shuai P, Xia XL, Yin WL. The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot. 2010;61(14):4011–9.

  23. 23.

    Czikkel BE, Maxwell DP. NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes to the nucleus. J Plant Physiol. 2007;164(9):1220–30.

  24. 24.

    Chen K, Li H, Chen Y, Zheng Q, Li B, Li Z. TaSCL14, a novel wheat (Triticum aestivum L.) GRAS gene, regulates plant growth, photosynthesis, tolerance to photooxidative stress, and senescence. J Genet Genomics. 2015;42(1):21–32.

  25. 25.

    Pysh LD, Wysockadiller JW, Camilleri C, Bouchez D, Benfey PN. The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J. 1999;18(1):111.

  26. 26.

    Tian C, Wan P, Sun S, Li J, Chen M. Genome-wide analysis of the GRAS gene family in Rice and Arabidopsis. Plant Mol Biol. 2004;54(4):519–32.

  27. 27.

    Gao M, Parkin I, Lydiate D, Hannoufa A. An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase. Plant Mol Biol. 2004;55(3):417.

  28. 28.

    Itoh H, Ueguchi-Tanaka M, Sato Y, Ashikari M, Matsuoka M. The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei. Plant Cell. 2002;14(1):57–70.

  29. 29.

    Hofmann NR. A Structure for Plant-Specific Transcription Factors: the GRAS Domain Revealed. Plant Cell. 2016;28(5):tpc.00309.02016.

  30. 30.

    Li S, Zhao Y, Zhao Z, Wu X, Sun L, Liu Q, Wu Y. Crystal structure of the GRAS domain of SCARECROW-LIKE7 in Oryza sativa. Plant Cell. 2016;28(5):1025–34.

  31. 31.

    Smit P, Geurts R. NSP1 of the GRAS protein family is essential for Rhizobial nod factor-induced transcription. Science. 2005;308(5729):1789.

  32. 32.

    Sun X, Xue B, Jones WT, Rikkerink E, Dunker AK, Uversky VN. A functionally required unfoldome from the plant kingdom: intrinsically disordered N-terminal domains of GRAS proteins are involved in molecular recognition during plant development. Plant Mol Biol. 2011;77(3):205–23.

  33. 33.

    Cui HC, Benfey PN. Interplay between SCARECROW, GA and LIKE HETEROCHROMATIN PROTEIN 1 in ground tissue patterning in the Arabidopsis root. Plant J. 2009;58(6):1016–27.

  34. 34.

    Hirano K, Kouketu E, Katoh H, Aya K, Ueguchitanaka M, Matsuoka M. The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity. Plant J. 2012;71(3):443–53.

  35. 35.

    Feng S, Martinez C, Gusmaroli G, Wang Y, Zhou J, Wang F, Chen L, Yu L, Iglesiaspedraz JM, Kircher S. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature. 2008;451(7177):475–9.

  36. 36.

    Ariizumi T, Murase K, Sun TP, Steber CM. Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the GIBBERELLIN receptor GIBBERELLIN INSENSITIVE DWARF1. Plant Cell. 2008;20(9):2447–59.

  37. 37.

    Björn C, Willige SG, Nill C, Zourelidou M, Dohmann EMN, Maier A, Schwechheimer C. The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell. 2007;19(4):1209–20.

  38. 38.

    Kohji M, Yoshinori H, Tai-Ping S, Toshio H. Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature. 2008;456(7221):459–63.

  39. 39.

    Bolle C. The role of GRAS proteins in plant signal transduction and development. Planta. 2004;218(5):683–92.

  40. 40.

    Huang W, Xian Z, Kang X, Tang N, Li Z. Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato. BMC Plant Bio. 2015;15(1):209.

  41. 41.

    Liu X, Widmer A. Genome-wide Comparative Analysis of the GRAS Gene Family in Populus , Arabidopsis and Rice. Plant Mol Biol Rep. 2014;32(6):1129–45.

  42. 42.

    Jérôme G, Patricia AR, Teixeira RT, Martinez-Zapater JM, Fortes AM. Structural and functional analysis of the GRAS gene family in grapevine indicates a role of GRAS proteins in the control of development and stress responses. Front Plant Sci. 2016;7(e39547):353.

  43. 43.

    Bevan MW, Garvin DF, Vogel JP. Brachypodium distachyon genomics for sustainable food and fuel production. Curr Opin Biotech. 2010;21(2):211–7.

  44. 44.

    Guo Y, Wu H, Li X, Li Q, Zhao X, Duan X, An Y, Lv W, An H. Identification and expression of GRAS family genes in maize (Zea mays L.). PLoS One. 2017;12(9):e0185418.

  45. 45.

    Lu J, Wang T, Xu Z, Sun L, Zhang Q. Genome-wide analysis of the GRAS gene family in Prunus mume. Mol Gen Genomics. 2015;290(1):303.

  46. 46.

    Liu B, Yan S, Xue J, Li R. Genome-wide characterization and expression analysis of GRAS gene family in pepper (Capsicum annuumL.). Peerj. 2018;6(1):4796.

  47. 47.

    Yupeng W, Haibao T, Debarry JD, Xu T, Jingping L, Xiyin W, Tae-Ho L, Huizhe J, Barry M, Hui G. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.

  48. 48.

    Gaut BS. Evolutionary dynamics of grass genomes. New Phytol. 2002;154(1):15–28.

  49. 49.

    Huo N, Vogel JP, Lazo GR, You FM, Ma Y, Mcmahon S, Dvorak J, Anderson OD, Luo MC, Gu YQ. Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat. Plant Mol Biol. 2009;70(1–2):47–61.

  50. 50.

    Swigonova Z, Lai J, Ma J, Ramakrishna W, Llaca V, Bennetzen JL, Messing J. Close split of sorghum and maize genome progenitors. Genome Res. 2004;14(10A):1916–23.

  51. 51.

    Lynch M, ., Conery JS: The evolutionary fate and consequences of duplicate genes. Science 2000, 290(5494):1151–1155.

  52. 52.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.

  53. 53.

    Song XM, Liu TK, Duan WK, Ma QH, Ren J, Wang Z, Li Y, Hou XL. Genome-wide analysis of the GRAS gene family in Chinese cabbage (Brassica rapa ssp. pekinensis). Mol Gen Genomics. 2014;103(1):135–46.

  54. 54.

    Xiaolin S, Jones WT, Dawn H, Edwards PJB, Pascal SM, Christopher K, Thérèse C, Sheerin DJ, Jasna R, Oldfield CJ. N-terminal domains of DELLA proteins are intrinsically unstructured in the absence of interaction with GID1/gibberellic acid receptors. J Biol Chem. 2010;285(15):11557–71.

  55. 55.

    Triezenberg SJ. Structure and function of transcriptional activation domains. Curr Opin Genet Dev. 1995;5(2):190–6.

  56. 56.

    Heery DM, Kalkhoven E, Hoare S, Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature. 1997;387(6634):733–6.

  57. 57.

    Zhang D, Iyer LM, Aravind L: Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms. Bioinformatics. 2012;28(19):2407.

  58. 58.

    Wang Y, Shi S, Zhou Y, Zhou Y, Yang J, Tang X. Genome-wide identification and characterization of GRAS transcription factors in sacred lotus (Nelumbo nucifera). Peerj. 2016;4(8):e2388.

  59. 59.

    Wu S, Lee CM, Hayashi T, Price S, Divol F, Henry S, Pauluzzi G, Perin C, Gallagher KL. A plausible mechanism, based upon short-root movement, for regulating the number of cortex cell layers in roots. Proc Natl Acad Sci U S A. 2014;111(45):16184–9.

  60. 60.

    Xu YL, Li L, Wu K, Peeters AJ, Gage DA, Zeevaart JA. The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression. Proc Natl Acad Sci U S A. 1995;92(14):6640–4.

  61. 61.

    Williams J, Gaskin P, Hedden P. Function and substrate specificity of the gibberellin 3β-hydroxylase encoded by the Arabidopsis GA4 gene. Plant Physiol. 1998;117(2):559.

  62. 62.

    Thomas SG, Phillips AL, Hedden P. Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc Natl Acad Sci U S A. 1999;96(8):4698–703.

  63. 63.

    Olszewski N, Sun TP, Gubler F. Gibberellin signaling: biosynthesis, catabolism, and response pathways. Plant Cell. 2002;14(90001):S61.

  64. 64.

    Itoh H, Shimada A, Ueguchi-Tanaka M, Kamiya N, Hasegawa Y, Ashikari M, Matsuoka M. Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice. Plant J. 2010;44(4):669–79.

  65. 65.

    Liu T, Gu JY, Xu CJ, Gao Y, An CC. Overproduction of OsSLRL2 alters the development of transgenic Arabidopsis plants. Biochem Biophys Res Commun. 2007;358(4):983–9.

  66. 66.

    Mcginnis KM, Thomas SG, Soule JD, Strader LC, Zale JM, Sun TP, Steber CM. The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. Plant Cell. 2003;15(5):1120–30.

  67. 67.

    Dill A, Thomas S, Hu J, Steber C, Sun T. The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. Plant Cell. 2004;16(6):1392–405.

  68. 68.

    Shi Q, Zhang H, Song X, Jiang Y, Liang R, Li G. Functional characterization of the maize Phytochrome-interacting factors PIF4 and PIF5. Front Plant Sci. 2017;8:2273.

  69. 69.

    Ueguchitanaka M, Nakajima M, Katoh E, Ohmiya H, Asano K, Saji S, Xiang H, Ashikari M, Kitano H, Yamaguchi I. Molecular interactions of a soluble gibberellin receptor, GID1, with a Rice DELLA protein, SLR1, and gibberellin. Plant Cell. 2007;19(7):2140.

  70. 70.

    Eckardt NA. GA perception and signal transduction: molecular interactions of the GA receptor GID1 with GA and the DELLA protein SLR1 in Rice. Plant Cell. 2007;19(7):2095–7.

  71. 71.

    Engstrom EM. Phylogenetic analysis of GRAS proteins from moss, lycophyte and vascular plant lineages reveals that GRAS genes arose and underwent substantial diversification in the ancestral lineage common to bryophytes and vascular plants. Plant Signal Behav. 2011;6(6):850–4.

  72. 72.

    Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, Guo J, Liang W, Chen L, Yin J. Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol. 2006;141(4):1167–84.

  73. 73.

    Wu ZY, Wu PZ, Chen YP, Li MR, Wu GJ, Jiang HW. Genome-wide analysis of the GRAS gene family in physic nut (Jatropha curcas L.). Genetics & Molecular Research Gmr. 2015;14(4):19211.

  74. 74.

    Prince VE, Pickett FB. Splitting pairs: the diverging fates of duplicated genes. Nat Rev Genet. 2002;3(11):827–37.

  75. 75.

    Sun X, Jones W, Rikkerink E. GRAS proteins: the versatile roles of intrinsically disordered proteins in plant signalling. Biochem J. 2012;442(1):1–12.

  76. 76.

    Lee MH, Kim B, Song SK, Heo JO, Yu NI, Lee SA, Kim M, Kim DG, Sohn SO, Lim CE. Large-scale analysis of the GRAS gene family in Arabidopsis thaliana. Plant Mol Biol. 2008;67(6):659.

  77. 77.

    Xu W, Chen Z, Ahmed N, Han B, Cui Q, Liu A. Genome-wide identification, evolutionary analysis, and stress responses of theGRASGene family in Castor beans. Int J Mol Sci. 2016;17(7):1004.

  78. 78.

    Kamiya N, Itoh J, Morikami A, Nagato Y, Matsuoka M. The SCARECROW gene's role in asymmetric cell divisions in rice plants. Plant J. 2003;36(1):45–54.

  79. 79.

    Lim J, Benfey PN. Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems. Plant Cell. 2000;12(8):1307.

  80. 80.

    Lawit SJ, Wych HM, Xu D, Kundu S, Tomes DT. Maize DELLA proteins dwarf plant8 and dwarf plant9 as modulators of plant development. Plant Cell Physiol. 2010;51(11):1854.

  81. 81.

    Jin J, Tian F, Yang DC, Meng YQ, Kong L, Luo J, Gao G. PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res. 2017;45(Database issue):D1040–5.

  82. 82.

    Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018;46(Database issue):D493–6.

  83. 83.

    Ye J, Zhang Y, Cui H, Liu J, Wu Y, Cheng Y, Xu H, Huang X, Li S, Zhou A. WEGO 2.0: a web tool for analyzing and plotting GO annotations, 2018 update. Nucleic Acids Res. 2018;46(W1):W71–75.

  84. 84.

    He Z, Zhang H, Gao S, Lercher MJ, Chen WH, Hu S. Evolview v2: an online visualization and management tool for customized and annotated phylogenetic trees. Nucleic Acids Res. 2016;44(W1):W236–41.

  85. 85.

    Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteomics Bioinformatics. 2010;8(1):77–80.

  86. 86.

    Vogel JP, Garvin DF, Mockler TC, Schmutz J, Dan R, Bevan MW, Barry K, Lucas S, Harmonsmith M, Lail K. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature. 2010;463(7282):763–8.

  87. 87.

    Xin N, Guan Y, Chen S, Li H. Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon. BMC Genomics. 2017;18(1):619.

  88. 88.

    Singh VK, Mangalam AK, Dwivedi S, Naik S. Primer premier: program for design of degenerate primers from a protein sequence. BioTechniques. 1998;24(2):318–9.

  89. 89.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.

  90. 90.

    Hong S-Y, Seo PJ, Yang M-S, Xiang F, Park C-M. Exploring valid reference genes for gene expression studies in Brachypodium distachyonby real-time PCR. BMC Plant Bio. 2008;8(1):112.

  91. 91.

    Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 2005;139(1):5–17.

  92. 92.

    Gietz RD, Woods RA. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 2002;350(350):87.

  93. 93.

    Clough SJ, Bent AF. Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 2010;16(6):735–43.

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (No. 31571657) for the design and execution of the study.

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HL and WJ designed the experiments and revised the manuscript; XN, SC, JL and YL conducted the experiments and drafted the manuscript; Manuscript preparation: HL, XN. All the authors have read and approved the final manuscript.

Correspondence to Wanquan Ji or Haifeng Li.

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Additional files

Additional file 1

: Table S1. The chromosome location and physicochemical characteristics of BdGRAS genes. Table S2. The Ka and Ks values and estimated divergence time for tandemly duplicated BdGRAS genes. Table S3. The Ka and Ks values and estimated divergence time for segmentally duplicated BdGRAS genes. Table S4. The chromosome location, Ka and Ks values, and estimated divergence time for orthologous GRAS genes between Brachypodium and rice. Table S5. The chromosome location, Ka and Ks values, and estimated divergence time for orthologous GRAS genes between Brachypodium and sorghum. Table S6. The chromosome location, Ka and Ks values, and estimated divergence time for orthologous GRAS genes between Brachypodium and maize. Table S7. The chromosome location, Ka and Ks values, and estimated divergence time for orthologous GRAS genes between Brachypodium and wheat. Table S8. The primers used in this study. Table S9. GO annotations of BdGRAS proteins. Table S10. GO descriptions for BdGRAS proteins. Table S11. Numbers of known cis-elements in the promoter regions of BdGRAS genes. Table S12. Functions of GRAS genes in other species. (XLSX 163 K) (XLSX 162 kb)

Additional file 2:

Figure S1. Alignment of BdGRAS proteins to show conserved domains and amino acids. (JPG 9.5 M) (JPG 9755 kb)

Additional file 3:

Figure S2. Amino acid sequence of conserved motifs identified by MEME. The font size represents the frequency of each amino acid. (JPG 3.8 M) (JPG 3895 kb)

Additional file 4:

Figure S3. Agarose gel electrophoresis results of BRADI2G45117 5′-RACE (second PCR). (TIF 1.03 M) (TIF 1055 kb)

Additional file 5:

Figure S4. DNA sequencing results of BRADI2G45117 5′-UTR. (TIF 1.9 M) (TIF 1970 kb)

Additional file 6:

Figure S5. Agarose gel electrophoresis results of BRADI2G45117 full length (including 5′-UTR and CDS) PCR. (JPG 406 K) (JPG 406 kb)

Additional file 7:

Figure S6. DNA sequencing results of BRADI2G45117 full length (including 5′-UTR and CDS) PCR using forward primer BdSLRL1FLPF. (JPG 2.3 M) (JPG 2311 kb)

Additional file 8:

Figure S7. DNA sequencing results of BRADI2G45117 full length (including 5′-UTR and CDS) PCR using reverse primer BdSLRL1FLPR. (JPG 2.4 M) (JPG 2456 kb)

Additional file 9:

Figure S8. Sequence analyses of BRADI2G45117 (including 5′-UTR and CD). 5′-UTR are in grey. Start codon and stop codon are in red and blue, respectively. Full length PCR primers, 5′-RACE GSP outer primer and inner primer are underlined with dashed lines, full lines and wavy lines, respectively. (PDF 17 K) (PDF 16 kb)

Additional file 10:

Figure S9. Yeast two hybrid and transactivation activities assays of BdSLR1 and BdSLRL1. (TIF 14.7 M) (TIF 15054 kb)

Additional file 11:

Text 1. Synteny gene pairs between rice and Brachypodium distachyon. (TXT 904 K) (TXT 903 kb)

Additional file 12:

Text 2. Synteny gene pairs between rice and Brachypodium distachyon. (TXT 1.77 M) (TXT 1818 kb)

Additional file 13:

Text 3. Synteny gene pairs between rice and Brachypodium distachyon. (TXT 1.28 M) (TXT 1320 kb)

Additional file 14:

Text 4. Synteny gene pairs between rice and Brachypodium distachyon. (TXT 2.59 M) (TXT 2661 kb)

Additional file 15:

Text 5. Protein sequences of GRAS in rice, maize, sorghum, wheat and Brachypodium distachyon. (TXT 270 K) (TXT 270 kb)

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Keywords

  • GRAS
  • Brachypodium distachyon
  • Genome-wide analyses
  • DELLA
  • GA