| Pre-harvest sprouting (PHS) of grain while it is still in the ear, usually in response to damp conditions, is due to an early disruption of seed dormancy. As a worldwide natural disaster, PHS reduces the quality of wheat and the value of the grain, seriously affects the yield of wheat. In particular, the synthesis ofα-amylase results in starch breakdown and the production of breads with a lower volume and a compact, sticky crumb structure. Producers suffer great losses when wheat damaged by sprouting is sold at a discount; millers are faced with reduced flour yields and bakers encounter problems in processing and quality due to starch damage. In China, accounting for about 83% of the cultivating area of wheat was threatened by Sprouting. Moreover, since the over emphasis on the yield ability of wheat, the quality was often more or less overlooked in the past two decades. This directly leads to a number of wheat varieties which are sensitive to PHS, and great threat to food security.PHS is, generally speaking, a multi-factor induced phenomenon. It has a strong relationship with the environmental conditions, which makes things more complex. At present, it has been reported that PHS was mainly controlled by hereditary factors. But due to the huge hexaploid wheat genome and multi-location of the PHS-related genes (all seven chromosome groups except Group 1), the reason why some species possess resistance ability to PHS is still not fully explained. Thus, the problem of finding out a number of genes involved in PHS tolerance and clarifying the mechanism of pre-harvest sprouting resistance is not only a world wide important topic in wheat variety improvement, but also one of the most important traits in Chinese wheat breeding.Abscisic acid (ABA) has broad influence on many complex reactions during plant development, including seed maturation, dormancy and germination. ABA sensitivity in embryos is one of the key factors affecting seed dormancy. Maize Vp-1(Viviparous-1) and Arabidopsis ABI3 (Abscisic acid-insensitive 3) are orthologous transcription factors that regulate key aspects of plant seed development and ABA signal transduction. Vp-1 gene encodes a seed-specific transcription factor and plays an important role in wheat PHS resistance. Three orthologues of Vp-1 genes are present in bread wheat, which are located on the long arms of chromosomes 3A, 3B and 3D, respectively. Semi-quantitative RT-PCR analysis showed that there were difference in the splicing patterns or abundances of Vp-1B between PHS-tolerant and susceptible cultivars. This result indicated that the expression of the Vp-1B gene may affect its sensitivity to ABA, and thus its resistance to PHS. ABI3 is a central regulator in ABA sigaling, but little is known of how this factor is regulated. AIP2 (ABI3-interacting protein 2), which encodes a E3 ligase, can polyubiquitinate ABI3 and negatively regulate ABA signaling by targeting ABI3 in seed of Arabidopsis. This result indicates that AIP2 gene may affect embryo's sensitivity to ABA, and thus its tolerance to PHS, indirectly.In this study, three typical genotypes with different levels of PHS tolerance were selected and used for PCR amplification. Zhongyou 9507, a PHS susceptible cultivar, its GI (Germinated index) value was 0.84; Xinong 979, a PHS tolerant cultivar, its GI value was 0.50; Yongchuan white wheat, a typical PHS tolerant Chinese landrace, its GI value was 0.14. Moreover, two alleles were located in wheat genotypes with distinct PHS tolerance and ABA responsiveness and designated as Vp-1Bb (Yongchuan white wheat, YC) and Vp-1Bc (Xinong 979, XN). Compared with Vp-1Ba (Zhongyou 9507, ZY), these alleles had an insertion of 193-bp and a deletion of 83-bp in the third intron in the region of B3 domain, respectively. Further analysis indicated that different Vp-1B alleles showed different expression and responsiveness to ABA exposure at transcriptional level. We inferred that the insertion or deletion in the third intron region may affect the structure of pre-mRNA and thus the resistance to PHS. Therefore, in this experiment, we analyzed the characters and functions of Vp-1B and AIP2 gene with three typical materials of different levels of PHS tolerant wheat.Different cDNA clones of Vp-1B alleles were isolated from seeds of three typical wheats and named them as YCCVp-1B (GenBank accession no. FJ640558), XNCVp-1B and ZYCVp-1B (FJ640559), respectively. The sequence analysis revealed that the sequence homology between YCCVp-1B and XNCVp-1B is 100%. YCCVp-1B and XNCVp-1B comprised a 2,064 bp complete open reading frame (ORF), encoding a polypeptide of 72.66 kDa, consist of 687 amino acid residues, with its pI 8.23. The sequence homology of ZYCVP-1B with them is 99.85%. The cDNA sequence of ZYCVP-1B was 2,067 bp in length, encoding a protein of 688 amino acids with a predicted molecular mass of 72.79 kDa and a predicted pI of 8.46. The deduced amino acid sequence of YCCVp-1B contains a highly conserved B3 domain. Phylogenetic tree analysis showed that YCCVp-1B shares greater similarity with the other plant ABI3/Vp-1(25%-87%). Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that YCCVp-1B was a typical cytoplasmic protein.To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5'- and 3'-ends of the YCGVp-1B cDNA and named them as YCGVp-1B(FJ643534), XNGVp-1B(FJ517258) and ZYGVp-1B(FJ643535), respectively. The sequence analysis revealed that the DNA sequence of YCGVp-1B ,XNGVp-1B and ZYCVP-1B were 4244 bp, 3965 bp , 4051 bp in length, respectively.To investigate the biological activity of the YCCVp-1B protein, the full-length cDNAs of YCCVp-1B was fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into onion epidermal cells for analysis of their subcellular localizations. Fluorescence of YCCVp-1B:hGFP occurred predominantly in the cell nucleus and also distributed a little in the plasma membrane.In order to define the expression patterns of the three Vp-1B alleles and their relationship with PHS tolerance, semi-quantitative RT-PCR analysis was carried out to determine the expression levels of Vp-1B alleles at different seed developmental stages, Under H2O treatment and upon ABA treatment conditions in the three wheat cultivars differing in PHS tolerance. The result of semi-quantitative RT-PCR shown that during seed developmental stages, especially in 55 days after pollination (DAP) embryos, the Vp-1Bb and Vp-1Bc alleles had a higher abundances of transcripts than Vp-1Ba, with the level being highest in the Vp-1Bb allele and lowest in Vp-1Ba. Under H2O treatment,the transcript levels of Vp-1Ba ,Vp-1Bb and Vp-1Bc reached a maximum at 24 h after treatment then decreased, at 96 h, no Vp-1Ba allele was detected but the transcript levels of Vp-1Bb and Vp-1Bc were still maintained highly. The influences of exogenous ABA on transcription of three Vp-1B alleles in embryos were also investigated. Vp-1Bb was rapidly induced following treatment with 50μM ABA solution, the transcript levels of Vp-1Bb reached a maximum level at 12 h. However, Vp-1Ba and Vp-1Bc mRNA levels were upregulated in a time-dependent manner, reaching peak levels after 24 h. These results suggested that the three alleles differed in their response to ABA exposure. In conclusion, the analysis revealed that the expression level of three Vp-1B alleles have a positive correlation between embryo's sensitivity to ABA and seed dormancy, thus resistance to PHS.To further investigate the function of VP-1B gene, Using Gateway cloning technology, four plant ovexpression vectors of Vp-1B gene including ZYGVp-1B, XNGVp-1B, YCGVp-1B and YCCVp-1B sequence which were driven by the double 35S promoter were successfully constructed and induced into abi3-1, a deletion mutant allele of abi3, via agrobacterium. The results of experiments showed that expression of Vp-1B can partially complement abi3-1. ABI5, a positive regulator of ABA signaling, was activated by Vp-1B. ABI1 and ABI2, negative regulators of ABA signaling were strongly inhibited. These results indicated that Vp-1B strongly modified ABA signaling through feed forward regulation of ABI1 / ABI5-related genes. Furthermore, the wheat VP-1B fully restores ABA sensitivity of abi3-1 during seed germination and suppresses the early flowering phenotype of abi3-1. The expression of YCCVp-1B could perfectly compensate the inability of mutated genes in abi3-1.The complement ability of YCGVp-1B was the highest among three genomic sequences. This result showed that the sequence insertion and deletion in B3 domain affected the expression level of the Vp-1B and thus PHS tolerance..To further investigate the function of VP-1B gene, according to YCVp-1B sequence, the overexpression vector of YCVp-1B gene named pAHC-YCCVp-1B was successfully constructed and transformed into wheat variety Xinchun 9. Finally, three transgenic lines both with Bar and YCVp-1B gene were identified, with the transformation frequency of 0.17%. GI analysis showed that the value in transgenic seeds was significantly lower compared with control. It was suggested that overexpression of YCVp-1B gene could inhibit seed germination.To further confirm the function of VP-1 gene, according to the Vp-1 sequence from wheat, the RNAi expression vector of Vp-1 gene named pAHC-WVpRi was successfully constructed and transformed into wheat variety Xinchun 9. In total, 1825 embryos were bombarded. 34 regenerated plants with resistance to Bialaphos were obtained. Among them, four transgenic lines both with the Bar gene and interference fragment were identified, with the transformation frequency of 0.16%. The transgenic plants showed a higher GI value due to Vp-1 interference phenomena.In conclusion, our results suggest that Vp-1B proteins are important transcription factors that influence resistance to PHS by regulating embryo development and repressing germination of seed.A full-length cDNA sequence of AIP2-1 was isolated using RACE and DNA fragment assembly method from three typical wheats and named them as ZYCAIP2-1 (GenBank accession no. FJ640556), XNCAIP2-1 and YCCAIP2-1, respectively. The sequence analysis revealed that the sequence homology between them is 100%.The cDNA sequence of ZYCAIP2-1 was 1,111 bp in length, including a complete open reading frame of 972 bp with a 5'-UTR of 69 bp and a 3'-UTR of 70 bp, encoding a protein of 323 amino acids with a predicted molecular mass of 36.16 kDa and a predicted pI of 4.83. The deduced amino acid sequence of ZYCAIP2-1 contains a highly conserved Ring motif.Another cDNA copy of AIP2 gene were isolated from seeds of three typical wheat and named them as ZYCAIP2-2 (GenBank accession no. FJ640557), XNCAIP2-2 and YCCAIP2-2, respectively. The sequence analysis revealed that the sequence homology between them is 100%. ZYCAIP2-2 comprised a 972 bp complete open reading frame (ORF), encoding a polypeptide of 36.10 kDa, consist of 323 amino acid residues, with its pI 4.83. The cDNA sequence homology between ZYCAIP2-1 and ZYCAIP2-2 is 98.56%. Although there were 14 single-base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was almost completely conserved (99.69%, V76A).Studies on transmembrane domain indicated that there was no transmembrane domain in the predicted amino acid residues suggested that ZYCAIP2-1 was a typical cytoplasmic protein. Homology alignment analysis showed that ZYCAIP2-1 and ZYCAIP2-2 shares greater similarity with the other plant AIP2 (48%-82%).To further investigate the gene structures, we isolated the genomic sequences using PCR primers corresponding to the 5'- and 3'-ends of the AIP2 cDNA and named them as ZYGAIP2-1(3298 bp, FJ643532),ZYGAIP2-2 (3597 bp, FJ643533), respectively. Sequence alignment of the cDNA with their genomic counterparts revealed that both AIP2-1 and AIP2-2 were composed of 5 exons and 4 introns.AIP2 protein does not have a recognizable NLS. Fluorescence of AIP2-1:hGFP and AIP2-2:hGFP uniformly distributed throughout the onion epidermal cell just like control hGFP. These results suggest that AIP2-1 and AIP2-2 can't localize into the nuclei, they may depends on its complexation with other proteins into the nucleus. AIP2-1 and AIP2-2 genes were assigned respectively to chromosomes 5A and 5B by PCR using nullisomic–tetrasomic series of T. aestivum L.cv. Chinese Spring as materials with specific primers. Using three diploid relative-species (AA: Triticum urartu; BB: Aegilops speltoides; DD: Aegilops tauschii) as materials, further analysis suggested that AIP2-1 gene may be derived from the evolution of future generations but AIP2-2 gene may be derived from ancestral species- Aegilops speltoides.The result of semi-quantitative RT-PCR shown that during seed developmental stages, the expression of AIP2-1 and AIP2-2 genes had a gradually downward trend which reach the minimum at 55 DAP in three varieties. But the two transcripts indicated different expression pattern. The expression of AIP2-1 gene had a higher abundance of transcripts in tolerant cultivars than susceptible ones. Under H2O treatment,the transcript levels of AIP2-1 and AIP2-2 genes reached a maximum at 24 h after treatment, then decreased, at 96 h, the transcript levels of AIP2-1 and AIP2-2 genes were no significant difference among three varieties. But at 24 h, the expression of AIP2-1 and AIP2-2 genes had a higher abundance of transcripts in the susceptible cultivars than tolerant ones. The influences of exogenous ABA on transcription of AIP2-1 and AIP2-2 genes in embryos of three varieties were also investigated. The susceptible and tolerant cultivars had a greater sensitivity to ABA than resistant ones. From these results we conclude that the expression levels of AIP2-2 gene has a positive correlation with the resistance to PHS during seed developmental stages. Furthermore, the AIP2-1 and AIP2-2 genes were induced by exogenous ABA.Two overexpression vectors of ZYAIP2-1 and ZYAIP2-2 were constructed from the plant expression vector pLeela and transformed into the Arabidopsis mutant aip2-1 by Agrobacterium-mediated method. Seeds of T2 generation plants were tested by ABA. The results showed that transformed plants tended to have a higher germination rate and earlier flowering time compared with control. This indicated that the introduced wheat gene AIP2 was able to activate the endogenous genes ABI1, ABI2 in Arabidopsis thaliana and led to less sensitivity to ABA. Comparing two different copies of AIP2, we observed stronger activation ability of ZYAIP2-2 than ZYAIP2-1. Based on the conclusion above, another two vectors were constructed for further function analysis in wheat. pAHC-ZYAIP2-2 was built for overexpression of ZYAIP2-2 and pAHC- Vp-AIPRi was a RNAi vector with a Vp-1 promoter. Two plasmids were transformed separately into wheat by microprojectile bombardment. Ten transformed wheat lines were finally obtained in total: 7 lines in the overexpression population while 3 lines in the RNAi population. Lines with alteration of ear type only accounted for a small part of these two populations. These lines had a GI value as we expected before.Combining results of the above two functional analysis experiment in model plant Arabidopsis thaliana and common wheat, we believe that wheat AIP2 gene plays an important role in seed germination acceleration and embryo dormancy inhibition.
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