Genome-wide characterization of JASMONATE-ZIM DOMAIN transcription repressors in wheat (Triticum aestivum L.)

The structural characteristics of JAZ transcription repressors family in bread wheat

To date, the JAZ gene family has been reported in many plants, such as A. thaliana [24] and H. brasiliensis [49], B. distachyon [45] and O. sativa [43]. The expression patterns, gene and protein characteristics and the function of some JAZ genes have already been unfolded. However, the relative research on JAZ gene family was still infrequent in wheat. Based on the latest draft of wheat genome data, we isolated a JAZ gene family including 14 members from wheat (Table 1).

In plants, the gene construction of JAZ is manifold, and this multiformity is mainly reflected in the length and number of introns [50]. The length of introns in TaJAZ gene family ranged from 54 to 2098 bp. The shortest intron was only 54 bp in TaJAZ7-B, while the longest was 2098 bp in TaJAZ14-D (Fig. 3). Previous study revealed big gaps in the length of introns in JAZ gene family in many plants, ranging from 62 to 4422 bp [50]. We found that the intron length in TaJAZ gene family was in line with this range. In terms of the number of introns, the TaJAZ gene family members had 0–7 introns. TaJAZ5 had the maximum 7 introns, whereas TaJAZ6 and 13 had no introns (Fig. 3). This result was consistent with the number of introns in the JAZ genes of other plants [50]. However, some discrepancies were found in the number of introns among different copies of the same TaJAZ gene. For example, among the three copies of TaJAZ4, TaJAZ4-A and -B had 3 introns each, whereas TaJAZ4-D had 7 introns. TaJAZ14-A and -D had 6 introns each, whereas TaJAZ14-B had 4 introns. The similar characteristics were also found in the copies of TaJAZ9 and 10 (Fig. 3). This characteristic of gene structure was proper in wheat, because wheat has sub-genomes A, B and D. Overall, the results mentioned above suggested that the phenomenon of intron indels or intron lose occurred in TaJAZ genes during the long evolutionary process, resulting in various structures of TaJAZ genes.

Generally, a typical JAZ protein has one TIFY and one Jas domain at its N- and C-terminal, respectively. TIFY domain has two main functions, with one as a medium for the interaction of homomeric and heteromeric complexes formation, and the other as a medium for the interaction between JAZ proteins and MYC transcription factors [24]. The other distinguishing feature of JAZ proteins is the highly conserved Jas domain located near the C-terminus [30]. It is well known that Jas domain participates in protein–protein interaction with both COI and transcription factors, such as MYC2 [26, 51], and the nuclear localization signal (NLS) is included in this domain sequence [52]. In the present study, all TaJAZ proteins contained one TIFY and one Jas domain, but the amino acid composition of TIFY and Jas domain was different. The multiple sequence alignment showed that some amino acid substitutions existed in the core sequence of TIFY domain (Additional file 1: Figure S1). For example, the protein sequence “TIFY” was replaced with “TVFY” in TaJAZ3, “TMFY” in TaJAZ8, and “TLFY” in TaJAZ4. This architectural feature is familiar in JAZ proteins in many other plants [43, 45, 49]. The protein sequences of Jas domain were relatively conserved, and the sequence lengths were almost coincident in Arabidopsis and gramineous rice and maize [24, 43, 44]. But in wheat, the Jas motif sequences of some JAZ proteins, such as TaJAZ4, 5 and 14, were inserted by a short and highly similar NLS-like peptide, respectively (Additional file 1: Figure S1). This structural feature was rare in other plant JAZ protein, and this specific structure was only available in wheat JAZ proteins.

In conclusion, there were some similarities and otherness in the characteristics between TaJAZ gene family and other plant JAZ gene families. The results mentioned above suggested that the TaJAZ gene family might have multiple functions due to the complex structural characteristics as same as JAZ gene families in many other plants.

The putative functions of TaJAZ gene family

In order to gain more insights into the function of TaJAZ genes, we analyzed the cis-acting regulatory elements composition and the expression patterns under various stresses. Cis-acting regulatory elements are important molecular switches involved in the regulation of gene transcription under abiotic or biotic stresses [53]. In this study, we mainly detected the cis-acting regulatory elements that can respond to plant hormones (ABA, IAA, MeJA, GA and SA) and stress tolerances (low temperature, drought and high salinity). Due to the restriction of genomic sequencing data, we failed to gain the promoter region of TaJAZ11. In wheat JAZ gene promoter regions, the differences were mainly in the number and type of cis-acting regulatory elements. For example, the promoter region of TaJAZ1 had 4 types of cis-acting regulatory elements, i.e. ABRE (the number was 4), TGA (the number was 2), box-w1 (the number was 2) and CGTCA-motif (the number was 1), while TaJAZ6 only had 2 ABRE and 2 MBS elements (Additional file 2: Table S3). The promoter sequences of some TaJAZ genes lacked some types of cis-acting regulatory elements, but the expression patterns revealed that all TaJAZ genes could respond to all 8 treatments. For example, we found that all TaJAZ genes had no high salinity response-related cis-acting regulatory elements, but the relative expression levels of all TaJAZ genes were up- or down-regulated under high salinity treatment (Fig. 5). This indicated that the gene expression level under different treatments was not only dependent on the presence of relevant cis-acting regulatory elements, but also might be regulated by other physiological pathways in wheat.

To further investigate the biological functions of TaJAZ genes, we used different phytohormone to treat the wheat seedlings. Under MeJA treatment, the relative expression levels of TaJAZ1, 2, 3, 4, 7, 8, 9, 10 and 12 were obviously increased, while TaJAZ5, 6, 11, 13 and 14 were inhibited. By combining the cis-acting regulatory elements analyzed results, we found that the promoter sequences of TaJAZ2, 4 and 6 lacked the MeJA-responsive elements. Thus, we assumed that TaJAZ1, 3, 5, 7, 8, 9, 10, 12, 13 and 14 were directly involved in JA signaling pathway in wheat, and TaJAZ2, 4 and 6 could also respond to MeJA treatment by other unknown regulative pathways. Likewise, for GA treatment, the relative expression levels of TaJAZ6 and 7 were depressed, and the rest of TaJAZ genes were up-regulated. Based on the composition of cis-acting regulatory elements in promoter sequences, only TaJAZ3, 5, 7 and 12 had GARE element involved in GA response, but the TaJAZ genes lacking GARE element could also respond to GA. For IAA treatment, only TaJAZ5, 7, 8, 12 and 13 had the TGA-element, but all TaJAZ genes could obviously respond to it. For SA treatment, only TaJAZ3, 7, 8, 10 and 13 had the TCA-element, but the response was available in all genes. For ABA treatment only TaJAZ2, 4, 6, 8 and 12 had no ABRE element, but they were still sensitive to the presence of ABA (Fig. 5 and Additional file 2: Table S3). Given this, we found that some TaJAZ genes could respond to various phytohormones, and they might be directly or indirectly involved in the phytohormone crosstalk.

It is clear that the phytohormone crosstalk is universally available in plants, and a very complex signaling regulative network, which constituted by many phytohormone crosstalk, plays a very important role in the regulation of growth and development in plants. There is no doubt that JA signaling pathway plays a leading role in connecting different phytohormone signaling pathways [33]. As a repressor in downstream of JA signaling, JAZ proteins were irreplaceable, and they are the linkers among different crosstalks [33]. By synthesizing the analytic results of the cis-acting regulatory elements composition and the expression patterns under abiotic stresses (Fig. 5 and Additional file 2: Table S3), we speculated that TaJAZ3, 5, 7 and 12 were involved in the JA-GA crosstalk, which is a crosstalk to promote plant growth and defense against pathogens. Moreover, the JA-GA crosstalk can also act synergistically during stamen development, and JAZ proteins appear to play a significant role in this developmental function by interacting with the transcription factors MYB21 and MYB24, which both required for JA- and GA-mediated stamen development and male fertility [54]. Therefore, we thought that TaJAZ3, 5, 7 and 12 had a close contact with the development of stamen in wheat. The mechanism of JA-SA crosstalk in plant remains largely unknown, but the previous evidence has shown the effect of SA on JA signaling through direct or indirect regulation of the stabilization of JAZ proteins [55, 56]. This crosstalk can protect plants from the biotic and abiotic stresses, especially the pathogen infection [57, 58]. Here, we found that TaJAZ3, 7, 8, 10 and 13 had two types of cis-acting regulatory elements CGTCA-motif and TCA-element, and they were sensitive to JA and SA treatments (Fig. 5 and Additional file 2: Table S3). Thus, we thought that the functions of TaJAZ3, 7, 8, 10 and 13 were to enhance or inhibit the antiviral ability of plants. IAA is a very important phytohormone and plays vital roles during the development of plants [59]. The evidences revealed that the function of JA-IAA crosstalk was mainly embodied in regulating the root meristem activity and stem cell maintenance via antagonistic effect in plants [60–62]. Based on the results of cis-acting regulatory element component and the expression patterns (Fig. 5 and Additional file 2: Table S3), we assumed that TaJAZ5, 7, 8, 12 and 13 were directly participated in the JA-IAA crosstalk, and they might be involved in the regulation of primary root growth in wheat. ABA is a “stress hormone” that can regulate growth, stress tolerance, seed germination and senescence in plants. In JA-ABA crosstalk, both synergetic and antagonistic interactions are well known, and it is possible that JAZ proteins play important roles in regulating the JA-ABA crosstalk [33]. Given this, except TaJAZ2, 4, 6, 8 and 12, which lacked ABRE element, we speculated the rest of TaJAZ genes took part in the JA-ABA crosstalk, and played a role in plant stress tolerance, such as high salinity, drought and low temperature stresses (Fig. 5 and Additional file 2: Table S3).

In addition, the analysis of phylogenetic relationship among the JAZ genes can also reveal the putative function of them, because the homologous genes usually have similar biological functions [63]. In this study, we found that TaJAZ2 was clustered with OsJAZ1 into the sub-group G9 (Fig. 4). It is clear that OsJAZ1 protein interacts with a basic helix-loop-helix protein, OsbHLH148, to regulate the drought tolerance in rice [64]. Here, we found 4 drought-inducible cis-acting regulatory elements MBS in the promoter sequence of TaJAZ2, and the expression level of TaJAZ2 was also variational under drought stress. Thus, TaJAZ2 was likely participated in drought tolerance in wheat. Then, we found that TaJAZ4 was clustered with BdJAZ5 into the sub-group G3 (Fig. 4). The expression levels of BdJAZ5 are increased under salt, cold and heat treatments in B. distachyon [45]. There were 2 low temperature related cis-acting regulatory elements LTR in the promoter region of TaJAZ4, and the expression pattern of TaJAZ4 was similar with that of BdJAZ5 under cold stress, so TaJAZ4 might be involved in cold response in wheat. Further, we found that TaJAZ6 shared a high similarity with BdJAZ7 in sub-group G10 (Fig. 4), and there were 2 MBS elements in its promoter sequence. The expression patterns of TaJAZ6 and BdJAZ7 were similar under drought stress (Fig. 5) [45], indicating that TaJAZ6 was also involved in drought tolerance in wheat. Overall, the functions of many TaJAZ genes were overlapping, which needed to be studied minutely in the future.

The evolution analysis of JAZ transcription repressors family

In the ancient terricolous plants, there are 7 and 6 members in the JAZ gene family in P. patens and S. moellendorffii genome, respectively [50]. In neonatal terricolous plants, there are 13 JAZ genes in gymnospermous P. stichensis genome, 12 JAZ genes in dicotyledonous A. thaliana genome, 15, 16 and 15 JAZ genes in gramineous B. distachyon, S. bicolor, and O. sativa genomes, respectively [50]. In the present study, 14 TaJAZ genes were identified from wheat genome (Table 1), and this number was similar to those in gramineous plants, but obviously more than those in older plants P. patens and S. moellendorffii. This result suggested that the number of JAZ genes in higher plants undergone the expansion, and became stable subsequently. Based on the analysis of chromosome localization, we found that the chromosome distribution of TaJAZ genes was tufted. For example, the copies of TaJAZ4, 5, 6 and 7 were densely distributed on the chromosomes 4A, 4B and 4D, respectively (Fig. 2). In addition, both TaJAZ4 and 5 were clustered into sub-group G3 (Fig. 4), while TaJAZ6 and 7 were clustered into sub-groups G10 and G6 (Fig. 4), respectively, indicating that TaJAZ4 and 5 undergone the event of tandem duplication, and maybe the event of divergence happened in the evolutionary process of TaJAZ genes. Moreover, we found that a pair of tandem TaJAZ genes, TaJAZ13-D1 and -D2, shared a high similarity in their protein sequences (Additional file 1: Figure S1), exhibiting that the duplication event of these two genes also happened. Given this, we thought that the gene tandem duplication was the main result leading to the augmentation in the number of TaJAZ gene.

Based on the N-J phylogenetic tree, all JAZ proteins from different plants were clustered into 11 subgroups (Fig. 4). The sub-groups G1, G2, G3, G5, G8 and G9 each had one SmJAZ protein, while none was present in the rest of sub-groups. Thus, we separated these 11 sub-groups into groups I (marked by red) and II (marked by blue) (Fig. 4). In group I, the sub-group G1 had only one JAZ protein, SmJAZ6, indicating that all JAZ proteins in terrestrial plants originated from a common ancestor. In sub-groups G3 and G5, the JAZ proteins from S.moellendorffii and other plants, such as T. aestivum, B. distachyon and A. thaliana, were clustered in these two clades, indicating that the differentiation of these JAZ proteins might have predated the divergence between flowering plants and pteridophyte (Fig. 4). In sub-groups G8 and G9, the JAZ proteins from P. patens and other plants, such as A. thaliana, and O. sativa, were clustered in these two sub-groups, suggesting that the differentiation of those JAZ proteins might have predated the divergence between bryophyte and tracheophyte (Fig. 4). In group II, the JAZ proteins from gymnospermous P. sitchensis and other angiosperm were included in G7, suggesting that those JAZ proteins might have predated the divergence between gymnosperms and angiosperms (Fig. 4). In addition, sub-groups G4, G6 and G10 comprised some JAZ proteins from monogenus and dicotyledonous plants, indicating that these JAZ proteins appeared before the divergence between monogenus and dicotyledonous plants (Fig. 4). The sub-group G11 only included the JAZ proteins from gramineous plants (Fig. 4), suggesting that the differentiation of these JAZ proteins ahead of the formation of gramineous plants.

For TaJAZ protein family, we found that all TaJAZ proteins were directly clustered with the JAZ proteins from Ae. tauschii, B. distachyon, S. bicolor or O. sativa. For example, TaJAZ3 was clustered with BdJAZ10 into sub-group G4; TaJAZ9 was clustered with AetJAZ2 into G6; TaJAZ10 was clustered with BdJAZ14 and SbJAZ11 into G7; and TaJAZ13 was clustered with BdJAZ6 and AetJAZ4, 5, 6, 7 and 8 (Fig. 4). This could be attributed to the fact that these 5 plant species are gramineous. Thus, we speculated that the differentiation of TaJAZ protein family occurred after the divergence between monocotyledon and dicotyledon (90 MYA), and ahead of the formation of gramineous plants (50-80 MYA). In addition, the Ka/Ks ratio revealed that the TaJAZ protein family undergone a process of purifying selection (Table 2), suggesting that the TaJAZ protein family tended to be stable during the long evolutionary process.

TaJAZ7, 8 and 12 were involved in the abnormal anther dehiscence

It is clear that JA, as a kind of important phytohormone, is widely involved in the regulation of anther dehiscence, filaments elongation and pollen fertility in plants [65]. The JA biosynthesis and signaling pathways were important for the development of anther during late developmental stage [66]. JAZ proteins, as repressors in JA signaling pathway, inhibit the transcription of JA response genes [33], and there is no doubt that JAZ genes play an essential role in the JA-mediate regulation pathway of anther dehiscence in plants [66]. The TGMS wheat line BS366 is a temperature dependent variety and its fertility can convert under different environments [48]. Hybrid seed could be produced under sterile condition (TGMS line BS366 as maternal plant), while TGMS line itself could be propagated under fertile condition [41]. The anther dehiscence of conventional wheat line Jing411 is normal under fertile condition (Fig. 6a, a and b), and the pollen can spill out from anthers smoothly. In sterile environment, the anther of TGMS wheat line BS366 is absolutely indehiscent [48], resulting in the male sterility. Interestingly, we found that the anther of BS366 could not fully dehisce, and only the topmost part of anther could crack (Fig. 6a, c and d), leading to the pollen spilling out incompletely. This abnormal phenotype was profitless for the seed multiplication of TGMS wheat line BS366.

In order to explore the potential relationship between the expression patterns of TaJAZ genes and the phenomenon of abnormal anther dehiscence, we divided the heading stage into six periods, and the expression pattern of each TaJAZ gene was checked in the anther tissues of BS366 and Jing411.. As shown in Fig. 6c, the expression patterns of all TaJAZ genes were highly similar from stage 1 to stage 5, and the relative expression levels in stage 2 and stage 4 increased obviously. Given this, we speculated that all 14 TaJAZ genes played important roles in regulation of anther development at stages 1, 2, 3, 4 and 5. Further, we noticed that the relative expression levels of TaJAZ genes at stage 6 were distinguishing in the anther of BS366, and the expression patterns were mainly divided into two types. The first category included TaJAZ1, 2, 3, 7, 8, 11, 13 and 14, and the expression levels of these genes were inhibited (Fig. 6c). The second category included TaJAZ4, 5, 6, 9, 10 and 12, and the expression levels of these genes were induced (Fig. 6c). In the anther of Jing411, the expression patterns of TaJAZ1, 2, 3, 4, 5, 6, 9, 10, 11, 13 and 14 were as same as those in BS366 at the heading stage 6 (Fig. 6c). It was obvious that the expression levels of TaJAZ7, 8 and 12 in the anther of Jing411 were adverse to those in BS366 at stage 6. For example, in the anther of BS366, the expression level of TaJAZ7 was inhibited at stage 6, exhibiting 18 times lower than that at stage 1 (Fig. 6c). In contrast, the transcriptional level of TaJAZ7 increased at the heading stage 6 in the anther of Jing411, displaying 7 to 8 folds higher than that at stage 1 (Fig. 6c). Similar results were also found in the expression patterns of TaJAZ8 and 12. These indicated that not all TaJAZ genes were involved in the regulation of anther dehiscence, and the genes with the same expression patterns in the anthers of Jing411 and BS366 may not be involved in the regulation of the anther dehiscence.

Moreover, the tissue-specific expression assay was performed to check the expression levels of TaJAZ genes in different tissues of BS366 and Jing411.. The relative expression levels of TaJAZ1, 2, 4, 10, 11 and 14 were obviously high in stamen tissues, but they all had high expression levels in other tissues (Fig. 7). Therefore, we thought that the regulatory effect of these genes was constitutive. The relative expression levels of TaJAZ7, 8 and 12 were markedly higher in stamen than that in other tissues, showing a high degree of expression specificity. For most of TaJAZ genes, the tissue-specific expression patterns were consistent in the anther tissues of Jing411 and BS366. In addition, the subcellular localization showed that TaJAZ7, 8 and 12 were all located in nucleus (Additional file 1: Figure S3). Based on the results mentioned above, we thought that TaJAZ7, 8 and 12 were directly participated in JA signaling pathway, and most likely to directly regulate the abnormal anther dehiscence. Thus, TaJAZ7, 8 and 12 were regarded as the candidate genes for the regulation of abnormal anther dehiscence in TGMS wheat line. The functions of TaJAZ7, 8 and 12 will be analyzed in our future works.