Characterization of TaDREB1 in wheat genotypes with different seed germination under osmotic stress

Background The cis-acting element DRE/CRT plays an important role in activating gene expression responsive to osmotic stress, low temperature and high-salinity. DREB1/CBF genes encode DRE-binding proteins with the function as transcript activators. TaDREB1 was also found to be induced by osmotic stress. Methods The dates of osmotic stress was assessed by seed germination drought resistance index; the full-length cDNA sequences of TaDREB1 gene were downloaded from NCBI datebase; identification of allelic variation and transcript expression were assessed by PCR and semi-quantitive RT-PCR analysis, respectively. Results Total 13 new allele variations of TaDREB1 were identified in the germplasms tested in the paper, including 5 TaDREB1-A on chromosome 3AL, 4 TaDREB1-B on chromosome 3BL and 4 TaDREB1-D on chromosome 3DL. In each variety, there existed two loci of TaDREB1-D genes, named TaDREB1-D1 and TaDREB1-D2, both of which had the similar nucleotide sequence except an 11 bp insertion in the former. In wheat seeds under osmotic stress, we did not detect the transcript expression level of TaDREB1-A and TaDREB1-B, but that of TaDREB1-D. Conclusions The capacity of osmotic stress tolerance was closely correlated with the expression level and tendency of TaDREB1-D. Electronic supplementary material The online version of this article (10.1186/s41065-018-0064-6) contains supplementary material, which is available to authorized users.


Background
Drought is already widespread in many regions and one of the primary reason causing plant loss worldwide. [1]. Even in many irrigated regions, shortage of water supply allows only limited irrigation. Therefore, it is imperative to strengthen the study on osmotic stress resistance of wheat.
Dehydration-responsive element binding (DREB) proteins constitute a large family of transcription factors regulating some functional genes closely related to drought, high-salinity and low temperature [10,11]. Arabidopsis genome has six DREB1/CBF genes, namely DREB1A/CBF3, DREB1B/CBF1, DREB1C/CBF2, DREB1D/CBF4, DDF1/DREB1F and DDF2/DREB1E [12]. DREB genes feature three conserved regions, an EREBP/AP2 DNA binding domain [13,14], an N-terminal nuclear localization signal, and a conserved Ser/Thr-rich region adjacent to the EREBP/AP2 domain [15,16]. The characteristics of above three regions determine the DREB characteristics [17]. Most of the functional studies on DREB transcription factors were focused on Arabidopsis thaliana before 2002, and then in wheat, rice, soybean, cotton and other plants [18][19][20][21][22][23][24]. DREB factors have been extensively characterized and grouped into several molecular classes based on similarities in the function or the amino acid sequence of the proteins they encode [24]. DREB1 and DREB2 are two main subgroups of the DREB subfamily, involved in two different signal transduction pathways under cold and dehydration, respectively [25]. DREB1 genes include DREB1-A, DREB1-B, DREB1-C,DREB1-D,DREB1-E and DREB1F [26]. TaDREB1 genes were located on chromosomes 3A, 3B and 3D [11], and isolated from a drought-induced cDNA library of wheat, which was found to be induced by low temperature, abscisic acid (ABA), salinity and drought [27]. The cDNA length of TaDREB1 is 1292 bp, including 837 bp open reading frame, 251 bp 5'-UTR and 204 bp 3'-UTR [18]. The study results showed that the heterogeneity of TaDREB1 gene haplotypes is inconsistent in drought-resistant materials or in drought-sensitive wheat materials, indicating the complexity of drought resistance. In addition, the nucleic acid polymorphisms of TaDREB1 gene are richer in wheat [26].
The present research aimed at identifying the seed germination resistance varieties under osmotic stress for wheat breeding program, searching the new allelic variation of TaDREB1, looking for SNPs, InDels and transcript expression level associated with seed germination tolerance under osmotic stress, and charactering the transcript expression level of TaDREB1-A, TaDREB1-B and TaDREB1-D in wheat varieties or lines with different values of GDRI (germination drought resistance index).

Plant materials
Six spring wheat genotypes were used for cloning of TaDREB1-A, TaDREB1-B and TaDREB1-D genes, including three osmotic stress-resistant genotypes (08-1783, 08-1826 and 07-6239 with relative germination rate values of 55.98, 47.74 and 47.11%, respectively) and three osmotic stress-susceptible ones (08dongzhong 275, Zhangye 1 and 05 cm 178 with relative germination rate values of 1.27, 0.51 and 0%, respectively). Eighty Chinese spring wheat genotypes, with different osmotic stress tolerance from China Spring-sown Wheat Region (CSWR) were used for identifying the osmotic stress tolerance (Table 1). Mature seeds of 08-1783 and Zhangye 1 were used to extract RNA for analysis of mRNA transcription.

Assay for seed germination drought tolerance
Osmotic stress was assessed based on seed germination drought resistance index (GDRI). Ears were harvested at dough-yellow ripening stage, hand-threshed and sterilized with HgCl 2 , and then placed in plastic petri dishes on filter paper with 6 mL of treatment solution (− 1.00 MPa) at room temperature, and distilled water was used as a control. Germinated seeds were counted and removed daily. The number of germinated seeds was investigated on the 2nd, 4th, 6th and 8th days (seeds with over 1 mm length of radicle were counted). The GDRI values was calculated according to the following formula: GDRI-germination drought resistance index of seeds per reparation. GIs-germination index of seeds under stress. GIc-germination index of seeds as control. nd2, nd4, nd6, nd8 -represents the number of seeds germinated on day 2, 4, 6 and 8, respectively. N-represents the total number of seeds. Then using the membership function method in fuzzy mathematic, the average germination index of wheat under osmotic stress was calculated to evaluate the difference of osmotic stress resistance among the varieties [28,29].
X j ----the jth measured value of osmotic stress resistance index; X j max-----the maximum value of the jth osmotic stress resistance index. X j min------the minimum value of the jth osmotic stress resistance index. U(x j )--the membership function value of the jth index. Rj-----The correlation coefficient between the jth index and the comprehensive 106 osmotic stress resistance coefficient.
R j / ∑ nj = 1rj ------As the indicator weight, indicating the importance of the jth index in all indicators. D---The evaluated value of each osmotic stress resistance indicator for each species under mannitol stress. If the measured index is positively correlated with the osmotic stress resistance of the genotypes, then formula (1) is used to calculate the membership function value, otherwise (2). Using D values to evaluate the osmotic stress resistance of the species, that is, D > 0.80 is for Level 1 (strong resistance), 0.50 < D < 0.80 is for Level 2 (resistance), 0.30 < D < 0.5 is for Level 3 (weak resistance), D < 0.30 is for Level 4 (sensitive). The entire osmotic stress resistance index was treated with Excel system.

Primer design
Based on the full-length cDNA sequence of TaDREB1 gene(AF303376), the primers were designed using DNA-MAN software and were synthesized in Takara Biotechnology (Dalian) Co. Ltd. in China (Table 2).

PCR amplification and semi-quantitive RT-PCR analysis
For each varieties, 3~5 seeds were selected and cultured in a clean culture dish at 25°C for 5~7 d. 1~2 g leaves were frozen by liquid nitrogen. Then genomic DNA was extracted by method of CTAB. The TaDREB1-A, TaDREB1-B and TaDREB1-D were amplified by specific primers (Table 1) in genotypes with different osmotic stress tolerance.
PCR reaction was performed in a PTC-100 TMProgrammable Thermal Controller in a total volume of 15 μL, including 30 ng genomic DNA, 10 × PCR reaction Mix buffer 1.5 μL, 0.3 μM of each primer, 0.5 U of Taq DNA polymerase (TaKaRaCo., China). It was carried out using the following programs: initial denaturation at 94°C for 5 min; 35 cycles of 72°C for 30-90s, annealing at 58°C~64°C for 30~90s, 72°C for 30~90s and a final extension at 72°C for 10 min. The PCR products recovered by agarose gel were cloned into the cloning vector PMD19-T (TaKaRaCo., China). The sequencing was performed by Nanjing Kingsley Sequencing Co., Ltd. Sequencing data analysis was performed using DNAMAN software (https://www.lynnon.com/pc/framepc.html).
Total RNA was extracted from the mature dry seeds treated with − 1.00 MPa mannitol for 0 h, 12 h, 24 h and 36 h, using TaKaRa Mini BEST plant RNA extraction kit (TaKaRa Co., China). The concentration and purity of the total RNA extracted was determined using a NanoDropND-2000C spectrophotometer. According to the manuscript, cDNA was synthesized with the reverse transcriptase kit RTaseM-MLV (TaKaRa Co., China). RT-PCR reaction was performed in a PTC-100

Identification of allelic variation of TaDREB1 gene
The specific primers of DREB1-A1, DREB1-A2, DREB1-B, DREB1-D1 and DREB1-D2 were used to amplify the TaDREB1-A1, TaDREB1-B1 and TaDREB1-D1, respectively, in six genotypes (including three osmotic stress-resistant materials and three osmotic stress-sensitive ones). Total 13 new allele variations of TaDREB1-A1, TaDREB1-B1 and TaDREB1 Figure S1). 33SNPs were found in the full sequence of TaDREB1-B1, the frequency of SNPs was 4.78%. Among them, 6 SNPs were found in 5'UTR, twenty-seven SNPs were located in exons of TaDREB1-B11 and TaDREB1-B12. One T is inserted at 485 bp site of coding region. T-A, E-D, G-R, T-I, A-P, K-N, E-R, V-I, S-E, N-Q changes were found in the protein sequences. Most of the amino acid mutations were detected in TaDREB1-A13 genotypes, and the hydrophilicity is enhanced after the amino acid mutation. Lysine (k) is missed. The changes were not consistent with the osmotic stress resistance of the wheat genotypes (Additional file 2: Figure S2).
Expression characterization of TaDREB1-A, TaDREB1-B, and TaDREB1-D in two genotypes with different seed germination osmotic stress tolerances In order to define the expression patterns of the three TaDREB1 homologues and their relationship with osmotic stress tolerance, semi-quantitative RT-PCR analysis was carried out to determine the expression levels of TaDREB1-A, TaDREB1-B, and TaDREB1-D in two wheat genotypes differing in seed germination osmotic stress tolerance, using the ACTIN gene as an internal control. The results showed that the transcriptions of TaDREB1-A and TaDREB1-B genes were not detected in dry seeds and seeds treated with − 1.00 MPa mannitol for 12, 24 and 36 h via agarose gel electrophoresis. However, the expression level of TaDREB1-D had the tendency to increase gradually and then decrease when treated with − 1.00 MPa mannitol for 0 h, 12 h, 24 h and 36 h. Higher transcript expression level was detected in seeds treated by mannitol than in dry seeds. The highest transcript expression level came from osmotic stress-resistant line 08-1783 for 24 h treatment and from osmotic stress-sensitive variety Zhangye 1 after 12 h treatment (Fig. 3).

Discussion
osmotic stress is the most significant environmental stress in agriculture worldwide, so the improvement of grain yield under water limitation is increasingly targeted in plant breeding program. Seed germination osmotic stress resistance is important for wheat cultivation, especially in seeding stage. TaDREB play an important role in response to drought stress [2]. Compared with the result studied by Wei et al. [11], much rich allelic variations were founded in our study. From the 6 genotypes tested, a total of 13 gene variations (TaDREB1-A11, TaDREB1-A12, TaDREB1-A13, TaDREB1 -A14, TaDREB1-A15, TaDREB1-B11, TaDREB1-B12, TaDREB1-B13, TaDREB1-B14, TaDREB1-D11, TaDREB1-D12, TaDREB1-D21 and TaDREB1-D22) were identified, indicating that much richer allelic TaDREB1variations existed in common wheat. However, no relationship was found between the SNPs and GDRI for the time being.
In addition, two TaDREB1-D genes was detected in each of the 6 wheat genotypes tested, named TaDREB1-D1 and TaDREB1-D2; And two alleles of TaDREB1-D1 (TaDREB1-D11 and TaDREB1-D12) and two alleles of TaDREB1-D2 (TaDREB1-D21 and TaDREB1-D22) were identified. It was interesting to find that TaDREB1-D21 had the same sequence with TaDREB1-D11 (DQ195068.1) except a 11 bp deletion (CCCATGCGGCG) at position of 481 bp-491 bp in intron; And TaDREB1-D22 had the same sequence with TaDREB1-D12 except a 11 bp deletion (CCCATGCGGCG) at position of 481 bp-491 bp in intron and a 3 bp (ATT) insertion at positon 1645-1647 bp in 3 'UTR (Additional file 3: Figure S3). However, several SNPs were found between TaDREB1-D21 and TaDREB1-D22, the same as between TaDREB1-D11 and TaDREB1-D12. The base similarity between TaDREB1-D11 and TaDREB1-D21 (or between TaDREB1-D12 and TaDREB1-D22) was higher than between the two alleles of TaDREB1-D1 (or between the two alleles of TaDREB1-D2). Besides, TaDREB1-D11 and TaDREB1-D21 always appeared together in the same genotype, so as for TaDREB1-D12 and TaDREB1-D22. The results indicated that the duplication of TaDREB1-D gene occurred after the SNP mutations, and the copy of TaDREB1-D11 and TaDREB1-D12 (TaDREB1-D21 and TaDREB1-D22) was independent in different genotypes or environments. Higher transcript expression level of TaDREB1-D presented in seeds treated with mannitol, but no transcript product detected in TaDREB1-A and TaDREB1-B, the results showed that TaDREB1-D was more important to seed germination under osmotic stress than TaDREB1-A and TaDREB1-B. There were some reports that some genes homologies to DREB1 were weakly induced by osmotic stress [10,19,31], so same resulit were founded in TaDREB1-A and TaDREB1-B because of no transcript product detected in agrose gel. In addition, according to the complicated function of DREB in Arabidopsis thaliana, rice, soybean, cotton and other plants [18][19][20][21][22][23] Bohnert et al. 1995 and the transcript expression character of TaDREB1-D, more work needs to clarify the function of TaDREB1-D in further experiment.
When seeds were treated with − 1.00 MPa mannitol, the time of the highest transcript expression level of TaDREB1-D presented at 24 h in the line with osmotic stress resistance, but the highest transcript expression level of TaDREB1-D presented at 12 h in the variety with osmotic stress sensitivity. If the time that the highest transcript expression level of TaDREB1-D appeare in more genotypes with different osmotic stress tolerance and relate with seed germonation tolerance under osmotic stress, it could be used as a potential marker to identify the genotypes with more seed germination drought resistance in breeding program, and then to breed the stable and high yield varieties in dry farming conditions. It is necessary to characterize genetic resources based on osmotic stress adaptation, determine suitable genotypes, and use them to improve the wheat resistance. In the germplasms tested, 7 genotypes belong to level 1 osmotic stress tolerance were identified, (08-1783, 07-5866, 08-1826, 07-6239, Bayou 2, 34-206 and Linyou 2), which can be used as the parents in wheat breeding program for seed germination osmotic stress resistance.

Conclusions
In summy, our study was intended to identify the seed germination resistance varieties, search the new allelic variation of TaDREB1, and character the transcript expression level of TaDREB1-A, TaDREB1-B and TaDREB1-D in wheat varieties or lines with different values of GDRI.
The results indicated that seven genotypes belong to the level 1 osmotic stress tolerance, which might be used in wheat breeding for drought resistence; thirteen new allele variations of TaDREB1 were identified in the germplasms tested, in addition, TaDREB1-D1 and TaDREB1-D2 existed in each variety; In wheat seeds under osmotic stress, no transcript expression level of TaDREB1-A and TaDREB1-B were detected, but that of TaDREB1-D, and the capacity of osmotic stress tolerance was closely correlated with the expression level and tendency of TaDREB1-D.