Allelic variation of TaABI5-A4 Significantly Affects Seed Dormancy in Bread Wheat

doi:10.21203/rs.3.rs-4710390/v1

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Allelic variation of TaABI5-A4 Significantly Affects Seed Dormancy in Bread Wheat

https://doi.org/10.21203/rs.3.rs-4710390/v1

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ABI5 is a critical transcription factor in regulation of crop seed maturation, dormancy, germination and post-germination. Sixteen copies of homologous sequences of ABI5 were identified in Chinese wheat line Zhou 8425B. Cultivars of two haplotypes TaABI5-A4a and TaABI5-A4bshowed significantly different seed dormancy. Based on two SNPs between the sequences of TaABI5-A4a and TaABI5-A4b, two complementary dominant sequence-tagged site (STS) markers were developed and validated in a natural population of 103 Chinese wheat cultivars and advanced lines and 200 recombinant inbred lines (RILs) derived from the Yangxiaomai/Zhongyou 9507 cross; the STS markers can be used efficiently and reliably to evaluate the dormancy of wheat seeds. The transcription level of TaABI5-A4b was significantly increased in TaABI5-A4a-GFPtransgenic rice lines compared with that in TaABI5-A4b-GFP. The average seed germination index of TaABI5-A4a-GFP transgenic rice lines were significantly lower than those of TaABI5-A4b-GFP. In addition, seeds of TaABI5-A4a-GFP transgenic lines had higher ABA sensitivity and endogenous ABA content, lower endogenous GA content and plant height, and thicker stem internodes than those of TaABI5-A4b-GFP. Allelic variation of TaABI5-A4 affected wheat seed dormancy and the gene function was confirmed in transgenic rice. The transgenic rice lines of TaABI5-A4a and TaABI5-A4b had significantly different sensitivities to ABA and contents of endogenous ABA and GA in mature seeds, thereby influencing the seed dormancy, plant height and stem internode length and diameter.

ABA sensitivity

Plant height

Seed dormancy

TaABI5-A4

Triticum aestivum

We identified a pivotal transcription factor TaABI5-A4 that is significantly associated with pre-harvest sprouting in wheat; its function in regulating seed dormancy was confirmed in transgenic rice.

Pre-harvest sprouting (PHS) is the germination of seeds prior to maturity in the spike when there is excessive moisture before harvest. PHS tolerance of wheat is predominantly attributed to seed dormancy (Li et al. 2004; Gubler et al. 2005; Sun et al. 2012; Yang et al. 2014). The lack of seed dormancy in many wheat cultivars leads to PHS under humid weather conditions (Gubler et al. 2005). Therefore, it is important to understand the genetic mechanism of seed dormancy in wheat.

ABA plays critical roles in plants to respond biotic and abiotic stresses, such as pathogens, low temperatures, drought, salinity, and osmotic stress (Lim et al. 2014; Gonzalez-Guzman et al. 2012; Wei et al. 2022), In the biosynthesis of ABA, many genes are associated with onset and maintenance of seed dormancy (Gubler et al. 2005; Feng et al. 2022; Lynch et al. 2022; Liu et al. 2023; Song et al. 2024). The ABA signaling pathway includes transcription factors of positive and negative regulators. The positive regulatory factors are ABI3, ABI4, and ABI5 (Finkelstein and Lynch 2000), which affect seed development and ABA sensitivity, whereas null mutation of abi3 has weaker seed dormancy and ABA sensitivity than that of abi4 or abi5 (Finkelstein and Lynch 2000; Zhao et al. 2020; Xiao et al. 2021; Lynch et al. 2022). Negative regulators include ABI1, ABI2, and AIP2 E3 ligase for ABI3, RING E3 ligase and ABI5 binding protein (AFP) (Lopez-Molina et al. 2003; Zhang et al. 2005; Stone et al. 2006; Wei et al. 2022).

The basic alkaline leucine zipper transcription factor ABI5 is a critical factor in the regulation of seed maturation, dormancy and germination, and post-germination seedling growth (Finkelstein and Lynch 2000; Lopez-Molina and Chua 2000; Lopez-Molina et al. 2001, 2002; Finkelstein et al. 2002; Dai et al. 2013); it belongs to the ABI5/ABF/AREB/DPBF transcription factor family, binds to the cis-element ABRE, and regulates the expression of many downstream functional genes related to drought resistance and saline-alkali tolerance (Finkelstein and Lynch 2000; Lopez-Molina and Chua 2000). The plants of loss-of-function abi5 mutant are insensitive to ABA, whereas overexpression of ABI5 displays hypersensitive to ABA (Finkelstein and Lynch 2000; Lopez-Molina and Chua 2000; Zhou et al. 2015). The ABI5 protein is activated to be a growth repressor by ABA, resulting in an increase in target promoter occupancy (Lopez-Molina et al. 2002). In addition, degradation of ABI5 protein via the 26S proteasome is inhibited by ABA (Lopez-Molina et al. 2001). The transcription level of ABI5 was highest in late embryonic development and reached a peak during seed drying (Jakoby et al. 2002; Brocard-Gifford et al. 2004). As a transcription factor, ABI5 functions mainly through modulating the expression of target genes (Skubacz et al. 2016), such as ABI3, late embryo genesis abundant (LEA), DELLA, BRI1-EMSSUPPRESSOR1 (BES1), JASMONATEZIM-DOMAIN (JAZ) proteins, TaGATA1, INDUCER OF CBF EXPRESSION1 (ICE1), Pectin methylesterase (PME) and AFP to regulate ABA responses (Hu et al. 2019; Ju et al. 2019; Lim et al. 2013; Pan et al. 2018; Zhao et al. 2019; Xiang et al. 2024; Wei et al. 2022). ABI3 acts together with ABI5 to regulate embryonic gene expression and seed sensitivity to ABA (Lopez-Molina and Chua 2000; Nakamura and Toyama 2001), and ABI3 and ABI5 were degraded during seed germination by the proteasome (Arqyris et al. 2008; Lopez-Molina et al. 2001). ABI5 is insensitive to growth arrest following germination as well as the LEA, induced by ABA, and alters activity of the LEA gene promoter during the later stage of seed development (Lopez-Molina and Chua 2000; Zou et al. 2007). AFP has functions in the development of seedlings, which is a new negative regulator of ABA signaling that facilitates the degradation of ABI5 (Lopez-Molina et al. 2003). During seed development and desiccation, the transcription and translation level of AFP increased, ultimately plateauing in mature seeds (Lopez-Molina et al. 2003; Feng et al. 2019, 2022). HVA1 and HVA22 are ABA-induced genes in barley aleurone cells. HvABI5, a barley bZIP transcription factor, can specifically recognize ABA response complex (ABRC) cis-elements in the promoters of HVA1 and HVA22 (Casaretto and Ho 2003).

Many evidences indicate the functions of ABI5 in seed dormancy formation and releasing in Arabidopsis (Finkelstein and Lynch 2000; Lopez-Molina and Chua 2000), whereas very few proofs are provided in wheat. TaABI5s were isolated in wheat, one of which has the same sequence as TaABF1, a homolog of TaABI5 (Johnson et al. 2008). TaABF1 interacts with PKABA1 (SnRK2-type kinase) to mediate ABA-suppressed and -induced gene expression in aleurone cells. TaABI5s were expressed in developing grains, roots, and leaves (Ohnishi et al. 2008), whereas Zhou et al. (2017) showed that TaABI5 accumulated late in seed development and was expressed in seed only; the cultivar (SHW-L1) with PHS resistance has much higher transcription level of TaABI5 than the PHS-susceptible cultivar Chuanmai 32 (Zhou et al. 2017). In a transient assay in wheat aleurone cells, TaABI5 activated the promoter of Em containing ABRE, an embryogenesis abundant protein gene, indicating that TaABI5 acts as a transcription factor in wheat seeds (Utsugi et al. 2020), and in rice transgenic lines, TaABI5 plays an important role in mature seeds, particularly before seed germination (Utsugi et al. 2020). Our previous studies showed that TaABI5 belongs to a multigene family, and many copies of TaABI5 were identified in wheat, and allelic variations of TaABI5 are potentially associated with seed dormancy in wheat (Wang 2019).

The objectives of the present study were to: (1) identify homologous sequences of ABI5 in bread wheat, (2) evaluate allelic variations of TaABI5 in Chinese wheat cultivars and RILs with different PHS tolerance to develop functional markers associated with PHS tolerance for marker-assisted breeding, (3) characterize the transcript expression level of TaABI5-A4 in wheat, and (4) confirm the function of TaABI5-A4 underlying seed dormancy or PHS tolerance in transgenic rice.

Plant materials

Wheat line Zhou 8425B (germination index (GI%) 56.0) was used to amplify TaABI5 with universal primers. Nine other wheat cultivars were used for PCR amplification of TaABI5 sequences with specific primers, including five PHS-resistant cultivars, Xiaobaiyuhua, Yumai 18, Yangxiaomai, Xiaoyuhua and Xiaoyan 6, with GI values of 4.0, 7.2, 7.5, 9.2 and 13.7 respectively, and four PHS-susceptible cultivars, Jimai 19, Jing 411, Hengshui 7228 and Zhongyou 9507, with GI values of 58.4, 64.2, 68.4 and 70.8, respectively.

One hundred and three Chinese wheat cultivars and advanced lines with different PHS tolerance from the China Autumn-sown Wheat Region (CAWR), representing more than 85% of wheat production areas in China, were used for an association study (Table S1). Among these, 18 cultivars and advanced lines had GI values less than 15, and 85 had GI values between 15–71. GI was determined based on the average data across two cropping seasons at two locations, Anyang in Henan province and Beijing (Yang et al. 2014). Additionally, 200 RILs derived from the Yangxiaomai/Zhongyou 9507 cross also were used to validate the association between allelic variations of TaABI5-A4 and PHS tolerance. Yangxiaomai is a Chinese landrace wheat, with a low GI value (7.5), whereas Zhongyou 9507 had a high GI value (70.8).

Primer design

Based on the sequences of TmABI5 (accession number: AB238933.1), OsABI5 (EF199630.1), and TaABI5 (AB238934.1 and AB238932.1) in GenBank, universal primer sets TaABI5F1/R1 and TaABI5F2/R2 were designed to amplify full-length sequences of TaABI5 in Zhou 8425B. Three primer sets, TaABI5D3F/R, TaABI5A4F/R, and TaABI5D6F/R were used to amplify the sequences of TaABI5-D3, TaABI5-A4, and TaABI5-D6, respectively. Two other primer pairs, TaABI5A4aF/R and TaABI5A4bF/R, were gene-specific markers used for identifying the allelic variants of TaABI5-A4a and TaABI5-A4b, respectively. The primer sets RT-TaABI5F/R and RT-TaABI5A4F/R were designed to analyze mRNA expression level of TaABI5 and TaABI5-A4, respectively. The wheat Actin gene was used as an internal control and included in each reaction to normalize the expression level of TaABI5 and TaABI5-A4 genes; the expected PCR product was 410 bp in length (Table 1).

Table 1

The primer sets used for characterization of *TaABI5* allelic variants and transgenic lines
Primer set	Primer sequence	Annealing temperature (℃)	Fragment size (bp)
TaABI5F1	ATGGCATCGGAGATGAGCA	57.7	948
TaABI5R1	CTCACGGTTCTTGATCATGC	56.5	948
TaABI5F2	CATGATCAAGAACCGTGAGTC	56.4	606
TaABI5R2	TTCACCAGATGCAGCTGC	57.2	606
TaABI5D3F1	ATGGCATCGGAGATGAGCAAGGAC	64.7	1464
TaABI5D3R1	GCTGTAGGAAAAAAAAGATCACGA	57.8	1464
TaABI5A4F1	ATGGCATCGGAGATGAGCA	57.8	823
TaABI5A4R1	CATCGCTCTGTCGCCCATA	58.8	823
TaABI5D6F1	ATGGCATCGGAGATGAGC	55.8	1384
TaABI5D6R1	GATTGTTACAAGCAATTGGC	53.4	1384
TaABI5A4aF	ATGGCATCGGAGATGAGCA	57.8	494
TaABI5A4aR	TGCAACCCCACCACCATTCCTC	64.9	494
TaABI5A4bF	ATGGCATCGGAGATGAGCA	57.8	703
TaABI5A4aR	CGACCATCCCTGCGTACAAGT	59.76	703
RT-TaABI5F	ATGGCATCGGAGATGAGCA	57.0	1174
RT-TaABI5R	TTCACCAGATGCAGCTGC	57.0	1174
RT-TaABI5A4F	ATGGCATCGGAGATGAGCA	57.8	823
RT-TaABI5A4R	CATCGCTCTGTCGCCCATA	58.8	823
Actin F	GTTTCCTGGAATTGCTGATCGCAT	62.0	410
Actin R	CATTATTTCATACAGCAGGCAAGC	59.4	410
Hpt557F	ACACTACATGGCGTGATTTCAT	57.0	557
Hpt557R	TCCACYAYCGGCGAGTACTTCT	57.0	557
Q-TaABI5-A4F	TCCCGTCAAACCACCAAC	60.0	120
Q-TaABI5-A4R	GTCCCCTGCTGTTCGTTC	59.0	120
Q-GFP-F	TGGAGAGGGTGAAGGTGA	56.0	130
Q-GFP-R	TCTTGAAAAGCATTGAAC	50.0	130

PCR amplification and semi-quantitative RT-PCR analysis

Because TaABI5 has multiple copies of gene sequences, semi-quantitative RT-PCR was used for TaABI5 transcript expression analysis. PCR for gene cloning, molecular marker test, and semi-quantitative RT-PCR for gene transcript expression analysis of TaABI5s and TaABI5-A4 were performed in an Applied Biosystems 2720 thermal cycler. Reactions had a total volume of 15 µl, including 1.5 µl of 10×PCR buffer, 1.2 µl of 2.5 µM dNTP each, 4 pmol of each primer, 0.75 U of LaTaq polymerase (TaKaRa), and 30 ng of template DNA (cDNA), and brought to 15 µl with ddH₂O. PCR amplification procedures were 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 56°C–67°C for 45 s, and 72°C for 1 min, with a final extension of 72°C for 10 min. Amplified PCR and semi-quantitative RT-PCR products were separated on 1.5% agarose gels with the nucleic acid dye Gelview.

cDNA was synthesized from 5 µg of total RNA using M-MLV reverse transcriptase (TaKaRa) with random hexamer primer Oligod (T)₁₉ according to the manufacturer’s instructions. Semi-quantitative RT-PCR was performed in an Applied Biosystems 2720 thermal cycler. PCR cycling was performed at 94°C for 5 min, followed by 36 cycles of 94°C for 1 min, 57°C–59°C for 30 s and 72°C for 45 s, with a final extension of 72°C for 10 min. Values were normalized with the amplification rate of the Actin gene as a constitutively expressed internal control. Three replications were carried out for each sample.

Statistical analysis

Statistical analysis was conducted using PROC MIXED (SAS Institute, 8.0). In this study, two types of fragments were amplified using two dominant complementary STS markers TaABI5A4a and TaABI5A4b. These fragments were used to indicate genotype clusters as a categorical variable; mean GI values were derived from each cluster and used in testing the level of significance. GraphPad Prism 8 statistical software was used for data analysis to test significance of differences between clusters.

Construction of transgenic rice materials

Sequences of TaABI5-A4a and TaABI5-A4b were cloned into the pCAMBIA1302-GFP vector by General Biology (Anhui) Co., Ltd (www.generalbiol.com), respectively. The recombinant vectors were transformed into Agrobacterium tumefaciens strain EHA105 to genetically transform embryonic calli of rice (Oryza sativa L. ssp. japonica cv. Nipponbare) to create gene-overexpression rice plants following Feng et al. (2019).

Identification of transgenic plants and phenotyping

All transgenic rice lines were planted at 28/24 ℃ under 16/8 h light/darkness in greenhouse. The genomic DNA was extracted from fresh leaves of 15 plants of each T1 line by the CTAB method. The positive transgenic lines were identified by PCR. The primer sets and amplified fragments are listed in Table 1. At least 10 positive transgenic lines for each genotype were grown in Rice Professional Cooperative in Yiwu city. The positive T2 transgenic rice lines of TaABI5-A4a-GFP and TaABI5-A4b-GFP were analyzed for germination index, ABA sensitivity, exogenous ABA and GA contents, plant height, 100-grain weight, and stem length and diameter.

Germination assays and ABA sensitivity tests

For germination assays, 100 seeds each T2 line were sterilized with 75% ethyl alcohol, and placed in petri dishes covered with damp filter paper at room temperature. Germination for each transgenic line was scored based on radical emergence every 24 h for 7 d for calculating the GI value (Sun et al. 2012). For the ABA sensitivity test, 30 seeds from each T2 line were sterilized with 75% ethyl alcohol and were incubated on filter paper with water or 50 µM ABA solution at room temperature for at least two weeks, then the germination rate was calculated (Sun et al. 2012). Each experiment was performed in three biological replications.

Stress treatments and qPCR analysis

For stress treatments, 100 µM ABA, 150 µM NaCl solution and 300 µM mannitol solution were used to treat transgenic rice leaves at the three-leaf stage for 48 h (Feng et al. 2019; Liang et al. 2022). The samples were treated in greenhouse at 28/24 ℃ under 16/8 h light/darkness. Samples were collected at 0, 6, 12, 24, and 48 h post treatments, then frozen in liquid nitrogen to extract total RNA. Transgenic leaves from empty vector transformants were used as controls. Each experiment was performed in three biological replications.

The expression characteristics of TaABI5-A4 gene were analyzed under different treatments (Table 1), and the primers were designed on Primer Premier 5 (http://www.premierbiosoft.com/primerdesign/index.html). The TB Green Premix Ex Taq™ Ⅱ (TaKaRa, www.takarabiomed.com.cn) was used in PCR. Three biological replications were conducted for each sample and the relative expression level was calculated using the 2^−∆∆ct method (Livak and Schmittgen 2001).

RNA sequencing analysis

Total RNA was extracted from the mature seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines, using TRIzol reagent according to the manufacturer’s instructions. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions, with each sample in three biological replications, and transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Raw reads of fastp format were firstly processed using fastp and the low-quality reads were removed to obtain the clean reads. The clean reads were mapped to the rice reference genome (http://rice.uga.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/) using HISAT2. FPKM of each gene was calculated and the read counts of each gene were obtained by HT Seq-count. Differential expression analysis was performed using the DESeq2. The Q value < 0.05 and fold change > 2 or foldchange < 0.5 were set as the thresholds to determine significantly differentially expressed genes (DEGs). Based on the hypergeometric distribution, GO and KEGG pathway enrichment analysis of DEGs was performed to screen the significantly enriched terms by Bioinformatic analysis using the OECloud tools (https://cloud.oebiotech.cn).

Fluorescent protein expression and Western blotting

Fresh leaves of positive TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice plants at 30 d after germination (DAG) were used to analyze the expression of GFP by qPCR; the primer sets and amplified fragments are shown in Table 1. Each experiment was performed in three biological replications. The total protein of transgenic rice seeds was extracted by Plant Total Protein Extraction Kit (Sangon Biotech, https://www.sangon.com); the proteins were separated by 12% (w/v) SDS-PAGE gel electrophoresis, and transferred to PVDF membrane (Sangon Biotech). Anti-GFP (Shanghai Univ Technology, 1:2000 dilution) was used as a probe to detect the TaABI5-A fusion protein in transgenic rice.

Identification of new copies of TaABI5 in wheat line 8425B

The full-length sequence of TaABI5 was isolated from Zhou 8425B using a universal primer set (Table S1). A total of 16 copies of TaABI5 from Zhou 8425B were detected based on the comparison of ABI5 with the sequences deposited in the IWGSC and the 2005 Supplement of the Wheat Gene Catalogue. These copies were named TaABI5-A1, TaABI5-A2, TaABI5-A3 and TaABI5-A4 on the short arm of chromosome 3A, TaABI5-B1, TaABI5-B2, TaABI5-B3, TaABI5-B4 and TaABI5-B5 on the short arm of chromosome 3B, TaABI5-D1, TaABI5-D2, TaABI5-D3, TaABI5-D4, TaABI5-D5, TaABI5-D6 and TaABI5-D7 on the short arm of chromosome 3D, respectively. The results showed that TaABI5 was present in Triticum aestivum as a multi-copy gene. Sequence analysis indicated that the 16 copies of TaABI5 had 84.43–94.06% similarity with sequence AB238934.1 at the nucleotide level; the length of these copies ranged from 876 to 1556 bp. All sequences were deposited in GenBank under accession numbers MK287847 to MK287860 and MK334234 to MK334236 (https://www.ncbi.nlm.nih.gov/gene/).

Selection of specific primers for identifying the allelic variation of TaABI5 and sequences analysis

Based on the 16 copies of TaABI5, gene specific primers were designed to amplify all copies of TaABI5 sequences. In total, three specific primer pairs, TaABI5D3F/R, TaABI5D6F/R, and TaABI5A4F/R, were identified to specifically amplify the sequences of TaABI5-D3, TaABI5-D6, and TaABI5-A4, respectively. However, no specific primers could be designed for the other 13 copies of TaABI5 because of very high sequence similarities among them.

Ten cultivars (Xiaobaiyuhua, Yumai 18, Yangxiaomai, Xiaoyuhua, Xiaoyan 6, Zhou 8425B, Jimai 19, Jing 411, Hengshui 7228, and Zhongyou 9507) with different levels of seed dormancy were selected to identify allelic variation of TaABI5-D3, TaABI5-D6, and TaABI5-A4. In these ten cultivars, four variants were detected in TaABI5-D3, designated TaABI5-D3a, TaABI5-D3b, TaABI5-D3c, and TaABI5-D3d, respectively. Compared with the sequence of TaABI5-D3a from Zhou 8425B, both TaABI5-D3c and TaABI5-D3d had two SNPs (A to G at position 449 bp changed His into Arg, and A to G at position 705 bp changed Asn into Asp), and TaABI5-D3b had two SNPs (A to G at position 449 bp changed His into Arg, and T to C at position 1270 bp in the intron). In TaABI5-D6, five allelic variants were detected, viz. TaABI5-D6a, TaABI5-D6b, TaABI5-D6c, TaABI5-D6d, and TaABI5-D6e. Compared with the sequence of TaABI5-D6a from Zhou 8425B, InDels and SNPs located in the first exon were identified in TaABI5-D6b, TaABI5-D6c, TaABI5-D6d, and TaABI5-D6e.

There were two allelic variants in TaABI5-A4, i.e. TaABI5-A4a and TaABI5-A4b. Compared with the sequence of TaABI5-A4a, TaABI5-A4b had three SNPs (A to G at positions 269 bp and 473 bp, and C to T at position 686 bp) (Fig. S1). TaABI5-A4a was detected in five PHS-resistant cultivars (Xiaobaiyuhua, Yumai 18, Yangxiaomai, Xiaoyuhua, and Xiaoyan 6), while TaABI5-A4b was identified in five PHS-sensitive cultivars (Zhou 8425B, Jimai 19, Jing 411, Hengshui 7228, and Zhongyou 9507) (Fig. 1).

In addition, the sequence analysis showed that TaABI5-A4a and TaABI5-A4b could not encode full-length proteins, compared with full length of TaABI5 protein, as there is a stop codon at the position of 266th amino acid.

Expression of TaABI5s and TaABI5-A4 from Wanxianbaimaizi and Jing 411 at different seed developmental stages

To evaluate the potential influence of TaABI5 and the specific copy of TaABI5-A4 in Jing 411 (GI = 64.2) and Wanxianbaimaizi (GI = 7.6), the expression patterns of Wanxianbaimaizi (TaABI5-A4a) and Jing 411 (TaABI5-A4b) were determined using semi-quantitative RT-PCR analysis. The transcript expression levels of TaABI5 showed an increasing trend in seeds from 10 to 30 days post-anthesis (DPA) with the highest level at 30 DPA and the lowest at 10 DPA (Fig. 2a). In addition, the transcript expression levels of TaABI5-A4 and TaABI5 had the same trend in Wanxianbaimaizi (TaABI5-A4a) and Jing 411 (TaABI5-A4b) at 10, 20 and 30 DPA (Figs. 2a and 2b), although Wanxianbaimaizi (TaABI5-A4a) had higher transcript levels at all stages than Jing 411 (Figs. 2a and 2b).

Three PHS-resistant (Xiaoyan 6, Wanxianbaimai and Xiaobaiyuhua, TaABI5-A4a) and three PHS-susceptible genotypes (Jing 411, Zhou 8425B and Hengshui 7228, TaABI5-A4b) were chosen to analyze the transcript level by qPCR at mature seeds. Significantly higher transcript levels of TaABI5 and TaABI5-A4 were observed in the genotype TaABI5-A4a than in TaABI5-A4b (Figs. 3a and 3b).

Development and validation of gene-specific markers TaABI5A4a and TaABI5A4b for PHS resistance

Based on the SNPs between the sequences of TaABI5-A4a and TaABI5-A4b, two complementary STS markers were developed, designated TaABI5A4a (primer set TaABI5A4aF/R) and TaABI5A4b (TaABI5A4bF/R), respectively, and used for association analysis with 103 wheat cultivars and advanced lines. It was first to amplify the cultivars with the primer set TaABI5A4F/R, and the PCR product was used as the template in the following PCR system. The primer set TaABI5A4aF/R produced a 494-bp fragment in the TaABI5-A4a genotype and no PCR product in TaABI5-A4b (Table 2 and Fig. 1a), while the primer set TaABI5A4bF/R generated a 703-bp fragment in the TaABI5-A4b genotype (Table 2 and Fig. 1b) and no PCR product in TaABI5-A4a. Among the 103 cultivars and lines tested, 51 had TaABI5-A4a presenting a 494-bp fragment and 52 contained TaABI5-A4b with a 703-bp fragment (Table 2 and Table S1); TaABI5-A4a and TaABI5-A4b accounted for 49.5% and 50.5% with average GI values of 22.2 and 49.6, respectively. The GI values of the 103 lines were consistent across two years (r = 0.966, P < 0.0001), with mean GI values and standard deviations of 36.1 ± 20 in 2006 and 35.9 ± 19 in 2007, respectively. The results indicated that the genotype TaABI5-A4a were more resistant to PHS than TaABI5-A4b (Table 2).

Table 2

Association analysis between *TaABI5-A4* and GI values in 103 wheat cultivars and advanced lines with different PHS tolerance using the GIM model
Genotype	No. of lines	Average GI (%) values	F value	Phenotypic variance explained (%)R²
TaABI5-A4a	51	22.2	114.054*	52.6
TaABI5-A4b	52	49.6	114.054*	52.6
Significant association between TaABI5-A4* and phenotypic values at P < 0.001 level

To further confirm the association, 200 RILs from the cross of Yangxiaomai/Zhongyou 9507 were genotyped using TaABI5A4a and TaABI5A4b specific markers (Fig. 2). Statistical analysis confirmed the significant association (P < 0.001) of allelic variation of TaABI5-A4a/TaABI5-A4b with GI values and PHS tolerance. In this population, TaABI5-A4 explained 42.3%, 60.8%, and 63.7% of the phenotypic variations in Shijiazhuang, Beijing and the averaged GI values of two environments, based on the test of two complimentary STS markers TaABI5A4a and TaABI5A4b (Table 3).

Table 3

Association analysis between *TaABI5-A4* and GI values in 200 RILs using the GIM model
Trait	TaABI5-A4	No. of lines	Average GI% values	F value	Phenotypic variance explained (%) R²
Beijing GI value (%)	TaABI5-A4a	130	20.28	310.001*	60.8
Beijing GI value (%)	TaABI5-A4b	70	56.54	310.001*	60.8
Shijiazhuang GI value (%)	TaABI5-A4a	130	6.40	147.101*	42.3
Shijiazhuang GI value (%)	TaABI5-A4b	70	29.08	147.101*	42.3
Average GI value (%)	TaABI5-A4a	130	13.34	349.687*	63.7
Average GI value (%)	TaABI5-A4b	70	42.81	349.687*	63.7
Significant association between TaABI5-A4* and phenotypic values at P < 0.001 level

Phenotypic evaluation of transgenic rice lines

Fifteen transgenic rice plants (T1) for each of TaABI5-A4a-GFP and TaABI5-A4b-GFP constructs were selected using hygromycin. The primers of hpt557F/R were used to identify transgenic events. The frequencies of positive TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice plants were 73% and 80%, respectively (Fig. S2).

Plant height, germination index, 100-grain weight, stem length and diameter of T2 lines were phenotyped and analyzed (Fig. 4 and Table 4). The plant height of TaABI5-A4b-GFP transgenic lines (84.76 cm) was significantly higher (P < 0.01) than that of the negative control with an empty vector (80.33 cm) and TaABI5-A4a-GFP transgenic lines (79.58 cm). The average GI values of TaABI5-A4a-GFP, TaABI5-A4b-GFP transgenic lines and control plants were 57.7, 67.5, and 62.1, respectively, with significant differences (P < 0.05). The lengths of the first internodes of TaABI5-A4a-GFP, TaABI5-A4b-GFP transgenic lines and control plants were 1.38 cm, 1.57 cm and 1.31 cm, and those of the second internodes were 3.09 cm, 3.65 cm and 3.32 cm, respectively. The data analysis showed that the lengths of the first and second internodes between TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic lines also were significantly different (P < 0.001). The length of the third internode of TaABI5-A4b-GFP transgenic line was 7.98 cm, with significant differences from the control (7.57 cm) and TaABI5-A4a-GFP (7.26 cm) (P < 0.05 and P < 0.01, respectively) (Fig. 4 and Table 4). The diameters of the second and third internodes of TaABI5-A4b-GFP and TaABI5-A4b-GFP transgenic lines were 2.91 mm and 2.76 mm, 2.83 mm and 2.76 mm, respectively, all with significant differences (P < 0.05 and P < 0.01, respectively). Analysis of variance indicated that the TaABI5-A4b-GFP genotype had significantly (P < 0.01) higher average GI values than the TaABI5-A4a-GFP (Fig. 4). These showed that the TaABI5-A4 gene affected not only GI values in transgenic rice lines, but also plant height, internode length and diameters (Fig. 4, Table 4).

Table 4

Phenotypic data of *TaABI5-A4a-GFP* and *TaABI5-A4b-GFP* transgenic rice lines
	Control	TaABI5-A4a-GFP	TaABI5-A4b-GFP
Plant height (cm)	80.33	79.58	84.76^**
GI% value	62.13	57.74^*	67.54^*
100-grain weight (g)	2.18	2.17	2.07
Length of first internode (cm)	1.31	1.38^**	1.51^****
Length of second internode (cm)	3.32	3.09^*	3.65^***
Length of third internode (cm)	7.57	7.26	7.98^*
Diameter of first internode (mm)	3.09	3.14	3.09
Diameter of second internode (mm)	3.09	3.01	2.90^*
Diameter of third internode (mm)	2.88	2.83	2.76^**
, , and ** represent significant differences between transgenic plants and controls at P < 0.05, P < 0.01, P < 0.001, P < 0.0001, respectively. Control stands for transgenic plants with an empty vector as a negative control

Endogenous ABA and GA contents were examined in mature seeds of the control, TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic lines by HPLC. The endogenous ABA in seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP were 0.71 and 0.41 µg/g, respectively, and the endogenous GA contents were 9.82 and 12.14 µg/g, respectively, (Figs. 5a and 5b). These results indicated that the TaABI5-A4a-GFP transgenic rice line had significantly (P < 0.0001) higher endogenous ABA and significantly (P < 0.01) lower endogenous GA contents in mature seeds than TaABI5-A4b-GFP.

ABA sensitivity in transgenic rice lines

In order to determine the ABA responsiveness in the seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP lines, GI values of the seeds were evaluated in water and in 50 µM ABA solution, respectively. The results showed a much lower GI value in seeds of TaABI5-A4a-GFP than that in TaABI5-A4b-GFP lines, indicating that TaABI5-A4a-GFP transgenic rice lines had higher ABA sensitivity than TaABI5-A4b-GFP (Fig. 6).

To understand the expression level of TaABI5-A4 on abiotic stress response, we examined the relative gene expression in TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic lines under different stress conditions. The plants at the three-leaf stage were treated with ABA (100 µM), salt (150 mM NaCl) and drought (300 mM mannitol to mimic dehydration) at 0, 6, 12, 24, 48 h post treatments (Fig. 7). The expression levels of TaABI5-A4 in TaABI5-A4a-GFP transgenic rice were higher than those in TaABI5-A4b-GFP after 6 or 12 h treatment with NaCl, whereas the expression levels of TaABI5-A4 in the TaABI5-A4a-GFP lines were lower than those in the TaABI5-A4b-GFP lines after 12–48 h treatment with manntiol. After 24 h in the ABA treatment, the expression levels of TaABI5-A4 in TaABI5-A4a-GFP lines were higher than those in TaABI5-A4b-GFP, which is consistent with ABA sensitivity tests, indicating that the TaABI5-A4a-GFP transgenic plants were more sensitive to exogenous ABA than the TaABI5-A4b-GFP.

Effect of decreased accumulation of spliced mRNA caused by nonsense-mediated decay (NMD) on phenotypic changes

Although the sequences of TaABI5-A4a and TaABI5-A4b have premature nonsense codons at the position of 266th amino acid, TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines had significantly different GI values, ABA sensitivities, and contents of endogenous ABA and GA. TaABI5-D3 encodes a full-length protein and its DNA sequence has the most similarity (99.07%) with the sequences of TaABI5-A4a and TaABI5-A4b; therefore, TaABI5-D3 was chosen as the positive control to verify whether the premature nonsense codon affects the transcription level and even the translation level. The GFP transcription level and TaABI5-A4-GFP fusion protein level in transgenic rice lines were analyzed with qPCR and Western blotting (WB). In the two-week-old leaves of transgenic rice plants, the transcription expression level of GFP was very much higher in the positive control with the TaABI5-D3-GFP than those in TaABI5-A4a-GFP and TaABI5-A4b-GFP lines (P < 0.0001), and the expression in TaABI5-A4a-GFP lines were significantly (P < 0.01) higher than that in TaABI5-A4b-GFP lines (Fig. 8a). This result indicated that the much lower accumulation of fully spliced RNA in the TaABI5-A4a/b-GFP transgenic lines was caused by nonsense-mediated decay (NMD). Moreover, WB analysis was performed using an Anti-GFP monoclonal antibody to detect the expression level of GFP fusion protein in transgenic rice seeds. The result showed that GFP fusion protein was only detected in the positive control, rather than in transgenic TaABI5-A4a-GFP and TaABI5-A4b-GFP lines (Fig. 8b). These indicated that it was decreased accumulation of spliced mRNA, but fusion protein, that led to phenotypic changes between TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines.

Effect of mRNA accumulation of TaABI5-A4 in transgenic rice on the expression of level ERF13 and GSL-OH associated with seed dormancy

In order to identify the downstream genes regulated by transcription factor TaABI5, we performed RNA sequencing (RNA-seq) using mature seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines. A total of 68 DEGs were identified in TaABI5-A4a-GFP transgenic lines compared with TaABI5-A4b-GFP lines, including 26 up-regulated and 42 down-regulated genes (Fig. 9a). Compared with TaABI5-A4b-GFP transgenic lines, the up-regulated DEGs in TaABI5-A4a-GFP lines belong to the pathways of regulation for response to stress, abiotic stimulus and metabolic process based on GO and KEGG analysis, whereas the down-regulated DEGs in TaABI5-A4a-GFP transgenic lines are involved in the protein metabolic process (Figs. 9b, c and d). Among them, we found ERF113/At5g13330 (LOC_Os08g30100), GSTU16 (LOC_Os10g38360) and GSL-OH (LOC_Os03g08460) in the up-regulated DEGs, and ESD4 (LOC_Os01g16730) in the down-regulated DEGs (Fig. 9e), which may be involved in seed dormancy and germination.

We further examined the transcript expression levels of RAP2.6L, GSTU16 and GSL-OH with qPCR in mature seeds. The results confirmed significantly higher expression levels of these three genes in TaABI5-A4a-GFP transgenic rice lines than those in TaABI5-A4b-GFP (Fig. 9f). However, ESD4 could not be detected by qPCR due to the extremely low expression, which was a significantly down-regulated gene in RNA-seq. These results further verified the up/down-regulated DEGs in the RNA-seq from the mature seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic lines (Fig. 9).

Many genes are known to be involved in ABA biosynthesis (Gubler et al. 2005). Among them, LEC2, ABI3, ABI5, Vp-1, AFP, and FUS3 are related to seed dormancy and PHS resistance, and have dual effects of inhibiting seed germination and promoting embryo maturation (Li et al. 2004). ABI3 is crucial in establishing dormancy and tolerance to desiccation during seed development (Lopez-Molina et al. 2002) and is required for appropriate ABI5 expression (Finkelstein and Lynch 2000), because ABI5 acts in the downstream of ABI3 (Lopez-Molina et al. 2003). In wheat, the TaVp-1 homolog ABI3 is associated with seed dormancy and STS markers Vp1B3 and Vp1A3 have been developed and validated (Yang et al. 2007, 2014). In this study, allelic variants of TaABI5-A4a and TaABI5-A4b were detected and validated, and gene-specific markers of TaABI5 (TaABI5A4a and TaABI5A4b) were significantly (P < 0.001) associated with seed dormancy in wheat lines. The 103 wheat cultivars used in this study also was used to validate the STS marker Vp1A3 associated with seed dormancy (Yang et al. 2014), whereas much higher phenotypic variation was explained by TaABI5A4a and TaABI5A4b in this study (R² = 52.6%). Moreover, the RIL population of Yangxiaomai×Zhongyou 9507 was also used to validate the STS marker Tamyb10D (R² = 22.6%) (Wang et al., 2014), while the TaABI5-A4 gene explained much higher (R² = 63.7%) phenotypic variation in this population than Tamyb10D. These results indicate that TaABI5-A4 is very likely to be a major-effect gene for seed dormancy in ABA signal pathway in wheat. It is very worthwhile to understand the molecular mechanism of TaABI5-A4.

In TaAIB5-A4a-GFP and TaAIB5-A4b-GFP transgenic rice lines, the transcript expression levels of TaABI5-A4 showed different sensitivities to ABA due to different CDS sequences of TaAIB5-A4a and TaAIB5-A4b (Figs. 8 and 9). Compared with the mature seeds of TaAIB5-A4b-GFP, TaAIB5-A4a-GFP showed higher transcript expression level of TaABI5A4 and GFP, more ABA sensitivity, higher endogenous ABA content and lower GI value. But no GFP fusion protein was detected by WB in the mature seeds of TaAIB5-A4a-GFP and TaAIB5-A4b-GFP transgenic lines. These results further indicate that although there is no full-length TaABI5-A4 protein due to the premature termination codon at the position of 266th amino acid, TaABI5-A4 mRNA or some truncated proteins could affect the phenotypes of transgenic lines. Nonsense-mediated decay (NMD) is a mechanism in which abnormal mRNAs containing premature translation termination codons are efficiently eliminated so that production of undesirable truncated proteins is avoided (Culbertson 1999; Hentze and Kulozik 1999), and NMD occurs in rice waxy RNA containing a premature nonsense codon (Isshiki et al. 2001). In this study, transcription of TaABI5-A4a and TaABI5-A4b was obviously observed in transgenic rice lines, but no GFP fusion proteins were detected by WB (Fig. 8b). The NMD for TaABI5-A4a and TaABI5-A4b is also present, thus it must be the transcriptional regulation of TaABI5-A4 that plays a critical function, leading to phenotypic differences between transgenicTaABI5-A4a and TaABI5-A4b rice lines.

The significantly different phenotypes between TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines were not only in ABA sensitivity, endogenous ABA content, and GI value, but also in endogenous GA content, plant height, internode length and diameter (Figs. 5, 6 and 7). The balance of ABA and GA levels and sensitivity is a major regulator of dormancy status. The changes of ABA sensitivity and endogenous ABA and GA contents were observed in seeds of TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines. Both embryo sensitivity to ABA and GA metabolism plays an important role in the expression of dormancy of the developing sorghum grain (Benech-Arnold and Rodríguez 2018). Meanwhile, with a greater embryo sensitivity to ABA and higher expression of SbABA-INSENSITIVE 4 (SbABI4) and SbABA-INSENSITIVE 5 (SbABI5), dormant grains accumulate less active GA4 due to more active GA catabolism (Renata et al. 2013). Moreover, plant height is especially affected by hormones like GA (Eshed and Lippman 2019), and GA is the most important hormone for plant height regulation (Kong et al. 2023). The transgenic lines with higher endogenous ABA content have lower endogenous GA content, leading to shorter plant height, shorter internode length and thicker internode diameter (Figs. 5a and b). Based on the function of GA described above (Renata et al. 2013; Eshed and Lippman 2019; Kong et al. 2023), significantly different phenotypes between TaABI5-A4a-GFP and TaABI5-A4a-GFP transgenic rice lines have a reasonable explanation.

As a transcription factor, TaABI5 probably participates in the regulation of the expression of multiple genes. For instance, the NF-YC9 directly binds to ABI5, which facilitates the function of ABI5 to bind and activate the promoter of a target gene EM6, further positively regulates the response to ABA (Bi et al. 2017). Furthermore, the RNA-seq results revealed that the genes ERF113/At5g13330 (LOC_Os08g30100), GSTU16 (LOC_Os10g38360), GSL-OH (LOC_Os03g08460) and ESD4 (LOC_Os01g16730), which respond to ABA stimulus and may be involved in seed dormancy and germination (Liu et al. 2012; Krishnaswamy et al. 2011; Griffiths et al. 1998; Hansen et al. 2008; Chang et al. 2022; Cui et al. 2022), were differentially expressed between TaABI5-A4a-GFP and TaABI5-A4b-GFP transgenic rice lines. A working model for TaABI5-A4 was predicated according to the data of RNA-seq for mature seeds of TaABI5-A4a/b-GFP transgenic lines (Fig. 10). Overexpression of TaABI5-A4 increased the endogenous ABA content in mature seeds of TaABI5-A4a-GFP transgenic rice. ABA is perceived and bound by ABA receptors PYR/PYL/PCAR; these receptors can form a complex with PP2C, thereby leading to the relief of inhibition of PP2C to the SnRK2s, and then activating SnRK2s (Chen et al. 2020; Cutler et al. 2010; Lin et al. 2021; Soma et al. 2020). Activated SnRK2s in turn activate downstream transcription factors such as ABI5 and its homologs ABRE-binding factors (ABFs), which activate ABA-responsive genes (Fujii et al. 2007; Nakashima et al. 2009), resulting in the change of seed dormancy (Fig. 10). In addition, the ethylene response factor ERF96 increases the content of GSH (Jiang et al. 2020), thus enhances the antioxidative defense function (Van 2013). Therefore, increasing transcript expression level of ERF113 due to the overexpression of TaABI5-A4 (Fig. 10) can lead to the changes of GSH homeostasis in the regulation of ABA signaling regulated by GST (Zhang et al. 2019), and peroxidation of GSH can be catalyzed by GSTs (Wagner et al. 2002), which promotes removal of ROS and leads to seed dormancy (Fig. 10). Transcript expression level of GSL-OH also increased as shown in the RNA-seq data, which could catalyze the 2-hydroxybut-3-enyl glucosinolate, and have biological activities including toxicity to Caenorhabditis elegans, and enhance the seed dormancy (Griffiths et al. 1998; Hansen et al. 2008) (Fig. 10). The ESD4 was significantly decreased based on RNA-seq data; it has negative roles in response to ABA in seed dormancy (Chang et al. 2022), and esd4-3 mutant is ABA-hypersensitive (Cui et al. 2022), and the decreased transcript expression level of ESD4 may also result in much high seed dormancy (Fig. 10).

Author contribution statement Y Han and Z Wang performed the experiments. YJ Zhang and JD Liu constructed the RIL population. Y Yang designed the experiment and assisted in writing the paper.

Acknowledgements

This work was supported by grants from the Key Projects of Inner Mongolia Natural Science Foundation (2023ZD08), Universities directly under the Inner Mongolia Autonomous Region Basal Research Fund (BR231519), and the National Natural Science Foundation of China (31960424).

Compliance with ethical standards

Conﬂict of interest The authors declare no conﬂicts of interest in regard to this manuscript.

Ethical standards We declare that these experiments comply with the ethical standards in China where they were performed.

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Editorial decision: Minor revisions
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Allelic variation of TaABI5-A4 Significantly Affects Seed Dormancy in Bread Wheat

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Abstract

Figures

Key Message

Introduction

Materials and methods

Plant materials

Primer design

PCR amplification and semi-quantitative RT-PCR analysis

Statistical analysis

Construction of transgenic rice materials

Identification of transgenic plants and phenotyping

Germination assays and ABA sensitivity tests

Stress treatments and qPCR analysis

RNA sequencing analysis

Fluorescent protein expression and Western blotting

Results

Development and validation of gene-specific markers TaABI5A4a and TaABI5A4b for PHS resistance

Phenotypic evaluation of transgenic rice lines

ABA sensitivity in transgenic rice lines

Effect of decreased accumulation of spliced mRNA caused by nonsense-mediated decay (NMD) on phenotypic changes

Discussion

Declarations

References

Supplementary Files

Status:

Version 1