Identification of FtGRAS genes in Tartary buckwheat
In this study, 47 FtGRAS genes were identified from the Tartary buckwheat genome. They were then renamed FtGRAS1 to FtGRAS47 according to their chromosomal location (Additional file 3: Table S1). The basic characteristics were analyzed, including the coding sequence length (CDS), protein molecular weight (Mw), isoelectric point (pI) and subcellular localization (http://cello.life.nctu.edu.tw/) (Additional file 3: Table S1). Of the 47 FtGRAS proteins, FtGRAS7 was the smallest protein with 44 amino acids, and the largest protein was FtGRAS1 with 755 amino acids. The Mws of the proteins ranged from 5.19 kDa (FtGRAS1) to 83.5 kDa (FtGRAS7), and the pI ranged from 4.85 (FtGRAS19) to 9.72 (FtGRAS39), with a mean of 6.45. The CDSs of the FtGRAS genes varied greatly, ranging from 132 to 2265 bp. The CDS of FtGRAS7 was the shortest at 132 bp, and the CDS of FtGRAS1 was the longest, reaching 2265 bp. The predicted subcellular localization results showed that 19 FtGRAS proteins were located in the nuclear region, 14 in the cytoplasm, 8 in the plasma membrane, 3 in the chloroplast, and 3 in the mitochondria.
Phylogenetic analysis and classification of FtGRAS genes
To explore the phylogenetic relationship of GRAS protein in Tartary buckwheat, we constructed a phylogenetic tree using the Maximum Likelihood (ML) method based on the amino acid sequences of 47 FtGRAS and 31 AtGRAS proteins (Fig. 1). According to their homology with GRAS proteins in Arabidopsis thaliana, the 47 GRAS genes of Tartary buckwheat were divided into 10 subfamilies: LAS, SCL4/7, HAM, SCR, DLT, SCL3, DELLA, PAT1, SHR, and LISCL. The LISCL subfamily had the largest number of members, with 19 FtGRAS genes. The SCL4/7, LAS, and DLT subfamilies all contained only one member, and there were 10, 6, 3, 2, 2, and 2 FtGRAS genes in HAM, PAT1, DELLA, SCR, SHR, and SCL3, respectively (Fig. 1).
Gene structure and motif composition of the FtGRAS gene family
To understand the structural components of the FtGRAS genes, the exon and intron structures of the FtGRAS genes were obtained by comparing the corresponding genomic DNA sequences (Fig. 2b). Forty-seven FtGRAS genes all contained the GRAS domain, and most of the FtGRAS genes (41, ~ 87%) contained no introns; FtGRAS18, FtGRAS31, FtGRAS34, FtGRAS37 and FtGRAS38 contained one intron, and only FtGRAS20 contained two introns. In general, members of the same subfamily had similar gene structures.
To further study the characteristic region of the FtGRAS proteins, the motifs of 47 FtGRAS proteins were analyzed using an online MEME (Fig. 2c). A total of 10 distinct conserved motifs (named Motif 1–10) were found (Fig. 2c; Additional file 4: Table S2). The motifs were arranged according to the sequence of domains, with motif 6 belonging to the LHRI domain, motif 7 and 1 belonging to the VHIID domain, motif 4 and 9 belonging to the LHRII domain, motif 3 belonging to the RFYRE domain, motif 2, 8, 5 belong to the SAW domain. Motif 10 was distributed between motif 1 and motif 4. Most of the FtGRAS proteins (89%) contained motif 3 and motif 5. FtGRAS7 did not contain any motif, FtGRAS8 contained only motif 3, and FtGRAS18 contained only motif 5. Simultaneously, we found that some motifs were only present in specific subfamilies. For instance, motif 4 was only present in LISCL, SHR and PAT1, and motif 10 only in HAM. When the FtGRAS gene family members were compared, the results showed that most of the closely related members had similar motifs. For example, the SCL3 group contained motifs 6, 7, 1, 9, 3 2, and 5, but the DELLA group contained motifs 6, 7, 1, 4, 9, 3, and 2.
Protein models of all the 47 FtGRAS were built using SWISS-MODEL (Additional file 1: Figure S1), and the results showed that the tertiary structures of FtGRAS protein mainly contained α-helices and random coils. The six proteins (FtGRAS7, 8, 18, 34, 41, 42) contained fewer α-helices and random coils (Additional file 1: Figure S1). Overall, the conserved motif composition and similar gene structures within the same groups of GRAS members, coupled with the results of the phylogenetic analysis, supported the reliability of the population classification.
Chromosomal distribution and synteny analysis of FtGRAS genes
A map of the physical position of the FtGRAS genes was created based on the physical location information of the Tartary buckwheat genome (Fig. 3). According to the result, the FtGRAS genes were unevenly distributed on 8 chromosomes of Tartary buckwheat. Ft1 had the most FtGRAS genes (12, ~ 26%), followed by Ft2 (10, ~ 21%), Ft7 (8, ~ 17%), Ft3 (6, ~ 13%), Ft5 (5, ~ 11%), Ft4 (3, ~ 6%), Ft6 (2, ~ 4%) and Ft8 containing only one GRAS gene (~ 2%). Interestingly, the number of GRAS genes distributed in the middle of the 8 chromosomes in Tartary buckwheat was relatively low, and the distribution of the GRAS gene on the chromosomes was similar to ATGRAS and OsGRAS [10].
In addition, we analyzed the duplication events of the FtGRAS genes because gene duplication plays an important role in the occurrence of new functions and the amplification of the gene family (Fig. 3; Fig. 4). Chromosomal regions within a 200 kb range of two or more genes were defined as tandem duplication events [39]. Twelve FtGRAS genes were clustered into eight tandem duplication event regions in Tartary buckwheat chromosomes 1, 2, and 3, indicating that they were hot spots for FtGRAS gene distributions (Fig. 3). Ft1 had three clusters (FtGRAS1/FtGRAS2, FtGRAS4/FtGRAS5, FtGRAS5/FtGRAS6), Ft2 also had three clusters (FtGRAS13/FtGRAS14, FtGRAS14/FtGRAS16, FtGRAS15/FtGRAS16), and Ft3 had two clusters (FtGRAS26/FtGRAS27, FtGRAS27/FtGRAS28). At the same time, five pairs of segmental duplication events were detected between 5 chromosomes: Ft1 (FtGRAS112)/Ft2 (FtGRAS21), Ft1 (FtGRAS11)/Ft3 (FtGRAS26), Ft1 (FtGRAS3)/Ft5 (FtGRAS33), Ft3 (FtGRAS25)/Ft7 (FtGRAS43) and Ft7 (FtGRAS43)/Ft7 (FtGRAS46) (Fig. 4). There were no segmental duplication gene pairs on Ft4, 6, 8. In conclusion, the FtGRAS gene tandem duplication and segmental duplication events occurred mainly in HAM and LISCL. Simultaneously, we carried out a synteny analysis of the Tartary buckwheat GRAS genes (Fig. 4). Most of the genes in Tartary buckwheat were kept in collinear blocks, suggesting that the GRAS gene family of Tartary buckwheat had a high degree of retention on the corresponding chromosomes during evolution [40]. Concisely, these results suggested that certain FtGRAS genes may have been produced by gene duplication and that tandem duplication events may have been the main driving force of FtGRAS evolution.
Evolutionary analysis of FtGRAS genes and GRAS genes of several different species
Based on the existing Tartary buckwheat GRAS genes, the diversity of the GRAS gene family during evolution Was further studied. A phylogenetic tree was constructed using the GRAS protein sequences of seven dicotyledonous plants (Arabidopsis thaliana, beet, soybean, grape, tomato, sunflower and Tartary buckwheat) and one monocotyledonous plant (rice). Concurrently, the motifs of the 8 plant GRAS proteins were determined (Fig. 5; Additional file 4: Table S2).
The number of GRAS gene and genome size of seven species were soybean (139, 1.025 Gb) [41], rice (60, 389.77 Mb) [42], tomato (53, 900 Mb) [43], Tartary buckwheat (47, 489.3 Mb) [38], grape (43, 427.2 Mb) [44], Arabidopsis thaliana, (32, 125 Mb) [45], beet (28, 394.6 Mb) [46], and sunflower (9, 3.6 Gb) [47], respectively. Among the seven species, Arabidopsis thaliana has the smallest genome, but the number of GRAS genes was not the least; sunflower had the largest genome, but the number of GRAS genes was not the largest. Therefore, there is no positive correlation between genome size and the number of GRAS genes of these species. We also used MEME web servers to search for conserved motifs that were shared among the GRAS proteins, and ten different conserved motifs were found (Motif 1–10) (Fig. 5; Additional file 4: Table S2). Arrangement of the motifs according to the sequence of domains showed that motif 7 belonged to the LHRI domain, motifs 6, 2, and 8 to the VHIID domain, motif 9 to the LHRII domain, motifs 3 and 1 to the RFYRE domain, and motif 5 to the SAW domain. Motif 10 was distributed between motif 9 and motif 3, and motif 4 was distributed between motif 1 and motif 5. Almost all GRAS proteins contained motif 7. GRAS members in the same clade, especially the most closely related members, usually shared common motifs, indicating potential functional similarities between GRAS proteins.
To further deduce the phylogenetic mechanism of the Tartary buckwheat GRAS gene family, we constructed seven representative comparative systematic maps with Tartary buckwheat, including six dicotyledonous plants (Arabidopsis thaliana, soybean, grape, tomato, beet and sunflower) and one monocotyledonous plant (rice) (Fig. 6; Additional file 5: Table S3). A total of 27 FtGRAS genes showed syntenic relationships with those in soybean, followed by tomato (21), grape (18), beet (16), Arabidopsis thaliana (8), sunflower (6) and rice (5). The number of homologous pairs of the other 7 species (soybean, tomato, grape, beet, Arabidopsis thaliana, sunflower and rice) were 57, 29, 28, 19, 13, 6 and 6, respectively. The FtGRAS gene had the most syntenic gene pairs with soybean, FtGRAS19, FtGRAS31, FtGRAS34, and FtGRAS40 had four syntenic gene pairs with soybean, and FtGRAS21 had six syntenic gene pairs with soybean. In addition, FtGRAS21 had syntenic genes with GRAS genes in another five plants (tomato, grape, beet, sunflower and rice), suggesting an important role of FtGRAS21 in gene evolution.
Expression patterns of the FtGRAS genes in different plant tissues
An evolutionary analysis of the FtGRAS gene of several different species was carried out, and 28 genes that may have potential research value were selected (Fig. 7; Additional file 5: Table S4;). To investigate the physiological role of these FtGRAS genes, real-time PCR was used to analyze the transcription products of the 28 FtGRAS genes in the root, stem, leaf and flower (Fig. 7a). Most of the genes were highly expressed in root, 4 genes (FtGRAS9, FtGRAS22, FtGRAS25, FtGRAS35) were highly expressed in both stem and flower, 2 genes (FtGRAS12 and FtGRAS32) were highly expressed in fruit, and 2 genes (FtGRAS21 and FtGRAS23) were highly expressed in flower. We also found that FtGRAS10 was not expressed in stem and FtGRAS37 was not expressed in leaf and fruit. The results showed diverse transcriptional abundance of FtGRAS genes in different tissues and organs, indicating that the FtGRAS genes had multiple functions in the growth and development of Tartary buckwheat.
Concomitantly, we analyzed the correlations among the FtGRAS gene expression patterns (Fig. 7b). A large proportion of FtGRAS gene expression was positively correlated, and some FtGRAS genes, such as FtGRAS24/FtGRAS27 (0.921), FtGRAS12/FtGRAS24 (0.980), and FtGRAS1/FtGRAS22 (0.947), were significantly correlated.
Differential expression of FtGRAS genes during fruit development of Tartary buckwheat
The main edible part of Tartary buckwheat is the fruit, which is known for its high content of rutin. Rutin can effectively prevent liver damage and cardiovascular and cerebrovascular diseases [48]. A few reports have examined the gene regulatory networks that regulate the physiological changes during the development of Tartary buckwheat fruit that are supported by the genome of Tartary buckwheat. Therefore, it is important to study the expression patterns of FtGRAS genes during the development of Tartary buckwheat fruit. By exploring the expression patterns of the FtGRAS gene in different plant tissues, we further selected 26 genes that might be related to fruit development (Fig. 8a). According to previous reports, the green fruit stage (8–14 DAP), discoloration stage (14–22 DAP), and initial maturity stage (22–26 DAP) represent the early, middle and late stages of buckwheat fruit development, respectively [49]. We used real-time PCR to detect the expression of the 26 FtGRAS genes at 13, 19 and 25 days after pollination (DAP) (Fig. 8a). The results showed that most of the genes were highly expressed at 13DAP, and 4 genes (FtGRAS5, FtGRAS12, FtGRAS29, FtGRAS32) were highly expressed at 25DAP. Two genes (FtGRAS4, FtGRAS46) maintained a relatively stable expression level during fruit development.
FtGRAS gene expression was negatively correlated with fruit development, except for FtGRAS5, FtGRAS12, FtGRAS29, FtGRAS32, FtGRAS38, and FtGRAS46. By analyzing the correlations among the FtGRAS gene expression patterns (Fig. 8b), we found that most the FtGRAS gene expression was positively correlated, and some FtGRAS genes, such as FtGRAS24/FtGRAS27 (0.997), FtGRAS24/FtGRAS40 (1.000), and FtGRAS9/FtGRAS22 (1.000) were significantly correlated.
Expression of DELLA subfamily genes after paclobutrazol treatment
DELLA protein, as the main negative regulator of GA signal transduction, may play an important role in the development of Tartary buckwheat fruit [22, 50]. To further study the relationship between DELLA subfamily genes (FtGRAS22, FtGRAS24, FtGRAS29) and fruit development of Tartary buckwheat, we first measured the changes in endogenous GA. Then, we applied paclobutrazol, a triazole plant growth regulator and an inhibitor of endogenous GA synthesis, to affect fruit development [51, 52].
We found that the endogenous GA content decreased from 13 to 19 DAP and increased at 19–25 DAP (Fig. 9a). Different concentrations of paclobutrazol (80, 120, 160, 120, and 240 mg L− 1) were sprayed on Tartary buckwheat at the bud stage (Fig. 9b). The results showed that the fresh weight of mature fruit increased significantly to 24.58 mg after 160 mg L− 1 paclobutrazol treatment, which was 106% of the blank group (23.22 mg). When the concentration of paclobutrazol was higher or less than 200 mg L− 1, there was no significant effect on fruit weight gain, and concentration that too high would reduce fruit weight (Fig. 9b). After spraying 160 mg L− 1 paclobutrazol, the fruit size increased during the whole fruit development stage (Fig. 9c). We then further explored the effect of exogenous application of 160 mg L− 1 paclobutrazol on the expression of the DELLA subfamily genes (FtGRAS22, FtGRAS24, FtGRAS29). Paclobutrazol treatment (5 mL) was used as the experimental group and the same amount of water treatment as the control group. The changes in expression of DELLA genes (FtGRAS22, FtGRAS24, FtGRAS29) under different treatments were compared (Fig. 9d). The expression levels of the three genes in the fruit development stage changed greatly after treatment with exogenous paclobutrazol. Compared with the control group, FtGRAS29 expression decreased at 13DAP, increased significantly at 19DAP, and decreased significantly at 25DAP. FtGRAS24 expression increased at 13 DAP but decreased at 19 DAP and 25 DAP. It is worth noting that after treatment with exogenous paclobutrazol, the expression of FtGRAS22 increased significantly during the whole fruit development stage. In summary, among the three genes, the responses of FtGRAS22 and FtGRAS29 to external paclobutrazol were more obvious, especially FtGRAS22.