The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.)

doi:10.21203/rs.3.rs-3558175/v1

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Research Article

The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.)

https://doi.org/10.21203/rs.3.rs-3558175/v1

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published 05 Aug, 2024

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Background

The male sterile lines are an important foundation for heterosis utilization in wheat (Triticum aestivum L.). Thereinto, pollen development is one of the indispensable processes of wheat reproductive development, and its fertility plays an important role in wheat heterosis utilization, and are usually influencing by genes. However, these key genes and their regulatory networks during pollen abortion are poorly understood in wheat.

Results

The rice gene TDF1, a member of the R2R3-MYB family, is crucial for early tapetum development and pollen grain fertility. TaTDF1, an ortholog of rice gene TDF1, was identified in wheat, with an average bolting time of 33 d in TaTDF1-OE Arabidopsis plants. The anthers of TaTDF1-OE plants appeared red. TaTDF1-EAR plants were shrunken and small, showing a blue-green color after Alexander's staining. The TaTDF1 interacting protein (TaMAP65) was confirmed using Yeast Two-Hybrid Assay and Bimolecular-Fluorescence Complementation experiments. The results showed that downregulated expression of TaTDF1 and TaMAP65 could cause anthers to be smaller and shrunken, leading to pollen abortion in TaTDF1 wheat plants induced by virus-induced gene-silencing technology. The expression pattern of TaTDF1 was influenced by TaMAP65.

Conclusions

Thus, systematically revealing the regulatory mechanism of wheat TaTDF1 during anther and pollen grain development may provide new information on the molecular mechanism of pollen abortion in wheat.

wheat

male sterility

Tapetum

Transcription factor

TaTDF1

Wheat is an important global food crop, and its yield plays an important role in stabilizing social and economic development and ensuring human health [1]. Therefore, it is important to use molecular biological technology to create excellent male-sterile lines and realize heterosis use to significantly increase the yield of wheat [2]. Male sterility is a complex physiological process involving the specific expression and silencing of many genes that can occur during differentiation of stamen into mature pollen grains [3]. It manifests as stamen degeneration or morphological abnormalities, meiosis abnormalities, microspore degeneration, tapetoid function defects, and formation of fertile pollen without anther cracking [4]. In recent years, progress has been made in the study of key genes related to anther development and pollen formation in higher plants and their regulatory mechanisms in their networks. Several key genes involved in stamen development, pollen sac cell differentiation, pollen mother cell meiosis regulation, and the promotion of pollen wall formation and anther cracking have been identified in Arabidopsis, rice (Oryza sativa L.), and other plants [5].

R2R3-MYB belongs to one of the largest transcription factor (TF) families in the plant genome [6]. The N-terminus of R2R3-MYB comprises two homologous MYB domains (R2 and R3) that bind to specific target sequences through the synergistic effect of R2 and R3. Its C-terminus is a transcriptional regulatory region, and its sequence is rich in acidic amino acids that have transcriptional activation functions and participate in regulating the entire plant growth and development [7]. These include regulation of secondary metabolism, environmental factor response, development, and cell differentiation, and are especially for anther development and tapetum in higher plants [8, 9]. AtMYB33 and AtMYB65 are homologous to barley GAMYB. The tapetum cells of the myb33 and myb65 mutants increased abnormally during meiosis, leading to stagnation of microspore development in the prophase of meiosis [10]. AtMYB21, AtMYB24, and AtMYB57 control the synthesis of lignin by interacting with hormones such as methyl jasmonate or by regulating the metabolic pathway of phenylpropane, thus regulating the dehiscence of anther [11–13]. OsMYB103 positively regulates tapetum cell degradation by modulating the TFs ETERNAL TAPETUM 1 (EAT1) and PERSISTENT TAPETAL CELL 1 (PTC1), resulting in delayed tapetum cell degradation and pollen grain defects [14]. The mutant pksb of ZmMYB84 is characterized by thinning of the pollen outer wall, thickening and densification of the anther cuticle, and delayed degeneration of the tapetum cells. A study of its internal mechanism revealed that the ZmMYB84-ZmPKSB regulatory module could alter the expression of a series of genes related to the biosynthesis and transportation of sporopollen, keratin, and wax. Furthermore, regulation of the formation of the anther cuticle and pollen outer wall helps maintain the normal development of pollen grains [15].

DYT1-TDF1-AMS-MS188-MS1 TF synergistically regulates the development of tapetum cells and plays an important role in the normal degradation of anther tapetum cells. Anormal expression of TFs can cause pollen abortion [16]. TDF1 belongs to the R2R3-MYB TF family, and its abnormal function significantly affects the degradation of tapetum cells. AtTDF1 was found to be a key regulator in the early stages of the Arabidopsis anther tapetum, which can directly regulate AtAMS, thus regulating the development of tapetum cells and promoting pollen wall formation [17]. Cai et al. showed that overexpression of OsTDF1 in Arabidopsis tdf1 mutants can restore pollen fertility. The tapetum cells of ostdf1 gene knockout mutant showed a vacuolation phenotype similar to Arabidopsis tdf1 mutant, which caused male sterility [18]. AtTDF1 is a negative regulator of ascorbic acid (vitamin C) accumulation in Arabidopsis anthers, which prevents tapetum cells from excessive division. It affects ROS homeostasis in anthers by mediating antioxidant L-ascorbic acid content, thus promoting tapetum cell division and differentiation [19]. Moriyama et al. successfully transformed the allele of the male sterility gene lfltdf1 into Easter lilies using a hybridization test and observed the pollen sterility phenotype [20]. However, there are few reports on genes related to pollen abortion during anther development in wheat. Wu et al. found that both TaTDRL-EAR and TaMYB103-EAR transgenic Arabidopsis plants showed abnormal pollen phenotypes, abortions, and low seed-setting rates. The homolog of the TaTDRL gene in Arabidopsis thaliana is AtAMS, which is the downstream TF of TaTDF1 [21].

In this study, the tapetal development and function 1 homologous gene (TaTDF1) of rice OsTDF1 was identified and analyzed. Gene structure, protein structure, protein sequence, physical and chemical properties of proteins, evolution and development, gene expression levels, and subcellular localization were studied. Furthermore, the interacting proteins were screened by the yeast two-hybrid test. Moreover, TaTDF1 transgenic Arabidopsis plants and TaTDF1 silences in wheat were generated to clarify the molecular mechanism of TaTDF1 in the regulation of the wheat pollen abortion process. This study helps build a solid theoretical foundation for the subsequent creation of new excellent wheat male-sterile lines.

Cloning and sequence analysis of TaTDF1

The cDNA sequence of TaTDF1 was obtained by comparison with the Ensemble Plant database according to the gene number TraesCS4D02G192400.1. Using MR-90-110 meiosis anthesis cDNA as a template, the target band was amplified to 924 bp, encoding 308 amino acid residues. The molecular weight of the protein was approximately 32.9 kDa, pI = 5.46 (Table S3). TaTDF1 contains three exons, two introns and the conserved domain spans amino acids 6-116aa (Fig. 1, Fig. 2). Cluster X multisequence alignment showed that the similarity of protein sequences between TaTDF1, OsTDF1, and AtTDF1 was 75.24% and 33.02%, respectively. Phylogenetic tree analysis of TaTDF1 using MEGA showed that TaTDF1 was closely related to barley (Hordeum vulgare L., XP.044979101.1). It is relatively conserved on homologous chromosomes A, B, and D (Fig. 3). The prediction of the secondary structure of TaTDF1 in the protein showed that the helix accounted for 38.31%, the random curl for 46.10%, extended chain accounted for 7.79%, and the turn for 7.79% (Fig. S1).

The expression pattern analysis of TaTDF1

TaTDF1 expression levels in different tissues and anthers of MS-90-110 and MR-90-110 cells at different developmental stages were analyzed using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The results showed that the expression level of TaTDF1 in anthers was significantly higher than that in other tissues. Expression levels in the leaf sheath, ovary, and bran were relatively low (Fig. 4A). TaTDF1-4A, TaTDF1-4B, and TaTDF1-4D were decreased from meiosis to early uninucleate stage, and then increased sharply at late uninucleate stage, whereafter, their expression levels were persisting declination in MS-90-110; however, their expression levels in the anthers of MR-90-110 during meiosis were considerably higher than those in MS-90-110 (Fig. 4B, Fig. 4C, Fig. 4D). TaTDF1-4D has been speculated to be involved in anther development and pollen fertility.

35S:EGFP and 35S:TaTDF1-EGFP were transformed into Agrobacterium tumefaciens and injected into tobacco epidermal cells. The transient expression results showed that 35S:EGFP produced signals of green fluorescent proteins in the cytoplasm and nucleus. However, 35S: TaTDF1-EGFP exhibited only green fluorescence in the nucleus (Fig. 5).

Yeast two hybridization (Y2H) screening of interacting proteins that bind to the TaTDF1 protein

To verify whether TaTDF1 has toxic and transcriptional activation functions, we tested its transcriptional activation activity by constructing the yeast expression vector BD-TaTDF1. PGBKT7 (BD) and BD-TaTDF1 were transformed into yeast-competent cells Y2H, respectively. The colonies of BD and BD-TaTDF1 were white and there was no obvious difference in growth (Fig. 6A). At the same time, BD-TaTDF1 of OD₆₀₀ = 0.85. These results showed that BD-TaTDF1 was not toxic to yeast cells. Negative control BD and target protein BD-TaTDF1 can be grown only on the SD/-Trp medium, not SD/-Trp/-Ade/-His medium. The positive control P53 was grown on SD/-Trp and SD/-Trp/-Ade/-His media. These results indicate that BD-TaTDF1 does not have transcriptional activation activity and can be used for verification of yeast interactions and library screening (Fig. 6B).

The wheat anther cDNA library plasmids were transformed into yeast cells containing the bait vector BD-TaTDF1 and the products were coated onto SD/-Ade/-His/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp/X-α-gal media for screening. Therefore, 25 well-growing plaques with a bluish color were obtained (Fig. 6C). Twenty-five positive clones were amplified, plasmids were extracted, and sent to Shanghai Biotech for sequencing. The sequencing results were compared with those from the Ensembl Plants and NCBI databases. Seventeen non-redundant proteins were obtained, and their functions were further annotated (Table S3). These 17 proteins are involved in multiple metabolic pathways. TaMAP65, a protein screened for microtubule kinetic arrays associated with the microspore nuclear division process, was used as a candidate interaction protein.

Cloning and sequence analysis of TaMAP65

A candidate interacting protein of TaMAP65 was screened by the yeast two hybrid with an annotation of the MAP65_ASE1 superfamily (TraesCS1A02G256800.1). The full-length CDS of the TaMAP65 gene is 2034 bp, encoding 677 amino acids. The molecular weight of the protein is approximately 76.15 kDa, and the pI = 6.07 (Table S3). The 5' and 3' untranslated regions (UTR) of the TaMAP65 gene are 161 bp and 306 bp, respectively (Fig. 7). Cluster X multisequence alignment showed that the sequence similarities of the TaMAP65, OsMAP65, and AtMAP65 proteins were 70.58% and 49.09% (Fig. 8), respectively. Using MEGA.X, we studied the evolutionary relationship of TaMAP65 and found that TaMAP65 is closely related to the evolutionary relationship of Aegilops tauschii Coss (XP.040246772.1) and is relatively conserved on the homologous ABD chromosome (Fig. 9). The secondary structure of TaMAP65 in the protein showed that helix accounted for 59.38%, random curl accounted for 34.12%, extended chain accounted for 3.84% and turn accounted for 2.66% (Fig. S2).

Analysis of TaMAP65 expression pattern

The expression levels of TaMAP65 in different tissues and anthers of MS-90-110 and MR-90-110 in different developmental stages were analyzed using qRT-PCR. The expression level of TaMAP65 in the anther was significantly higher than the other tissues; however, the expression levels in the ovary and bran were relatively low (Fig. 10A). TaMAP65-1A, TaMAP65-1B, and TaMAP65-1D were detected in MS-90-110. Meanwhile, the expression levels of TaMAP65-1A and TaMAP65-1B were decreased from meiosis to Trinucleate stage. However, the TaMAP65-1D were decreased from meiosis to late uninucleate stage, and then increased sharply at binucleate stage. Whereafter, the expression levels of TaMAP65-1D was persisting declination. Furthermore, their expression levels in the anthers of MR-90-110 during meiosis and early uninucleate stage were significantly higher than those in MS-90-110 except TaMAP65-1D (Fig. 10B, Fig. 10C, Fig. 10D). TaMAP65 has been speculated to be involved in the development of anther and pollen fertility. 35S:EGFP and 35S:TaMAP65-EGFP were transformed into A. tumefaciens and injected into tobacco epidermal cells. The results of transient expression showed that a signal of green fluorescent protein was observed in the cytoplasm and nucleus of 35S:EGFP. However, 35S:TaMAP65-EGFP exhibited only a green fluorescence signal in the nucleus (Fig. 11).

Verification of the interaction between the TaTDF1 and TaMAP65 protein

To further confirm the interaction between TaMAP65 and TaTDF1, recombinant vectors AD-TaMAP65 and BD-TaTDF1 were co-transformed into yeast-competent Y2H cells. AD-T + BD-Lam and AD-T + BD-53 cells were used as negative and positive controls, respectively. The results showed that the combination of the positive controls AD-T + BD-53 and BD-TaTDF1 + AD-TaMAP65 grew on SD/-Trp/-Ade/-Leu/-His medium, and the plaques were blue. The negative control, AD-T + BD-Lam, did not grow on the SD/-Trp/-Ade/-Leu/-His medium (Fig. 12A, Fig. 12B). These results show that there is an interaction between the TaTDF1 and TaMAP65 proteins.

pUN-SPYNE, pUC-SPYCE, pUN-TaTDF1, and pUC-TaMAP65 were transformed into the A. tumefaciens strain GV3101 (pSoup-19) and transiently expressed in Nicotiana tabacum leaves. The interaction between TaTDF1 and TaMAP65 was observed under a laser confocal microscope, in which TaTDF1-SPYNE + pUC-SPYCE and pUN-SPYNE + TaMAP6-SPYCE were used as negative controls. The results showed that there was no green fluorescent protein (GFP) fluorescence signal in negative control tobacco cells; however, there was a green fluorescence signal in the nucleus of the pUN-TaTDF1 + pUC-TaMAP65 experimental group, indicating that TaTDF1 and TaMAP65 proteins could interact with the nucleus (Fig. 13).

Functional analysis of TaTDF1 in Arabidopsis thaliana

Analysis of the function of TaTDF1 TFs in the development of wheat anthers and pollen. In this study, TaTDF1 overexpression and dominant chimeric suppression silencing vectors were constructed and transfected into A. thaliana. TaTDF1-OE overexpression lines and TaTDF1-EAR dominant chimeric suppression silencing lines were generated. By observing the phenotype, it was found that compared with wild A. thaliana, TaTDF1-OE overexpression lines and TaTDF1-EAR dominant chimeric suppression silent lines also bloomed normally (Fig. 14A). The average bolting time was 33 d in TaTDF1 overexpression (TaTDF1-OE) Arabidopsis plants, which was significantly earlier than in wild-type Arabidopsis plants by approximately 7 d (Fig. 14B, Fig. 14C). The anthers of wild A. thaliana and TaTDF1-OE plants appeared red after staining with Alexander’s stain (Fig. 14D, Fig. 14E). The average bolting period in dominant chimerism inhibition-silenced Arabidopsis plants (TaTDF1-EAR) was approximately 4 d later than that in wild-type Arabidopsis plants (Fig. 14B, Fig. 14C). The anthers from TaTDF1-EAR plants were shrunken and small and showed a blue-green color upon staining with Alexander’s stain (Fig. 14F). These results indicate that the TF TaTDF1 is important for the development of reproductive organs in Arabidopsis plants and is an important positive regulatory protein factor that maintains the normal development of pollen grains.

Functional analysis of TaTDF1 and TaMAP65 in wheat

In this study, γ-PDS, γ-TaTDF1 and γ-TaMAP65 recombinant plasmids were constructed, the MR-90-110 wheat leaves were inoculated with virus-induced gene-silencing technology. The phenotype of the leaves was observed 10 d after inoculation, anther morphology and pollen fertility were observed in the flowering stage, and the expression levels of TaTDF1 and TaMAP65 were analyzed. The leaves of wheat inoculated with barley stripe mosaic virus (BSMV):PDS showed photobleaching, whereas those inoculated with BSMV:00, BSMV:TaTDF1, or BSMV:TaMAP65 showed striped lesions (Fig. 15A). Thus, the technical system for barley stripe mosaic virus-induced wheat TaTDF1 and TaMAP65 gene silencing was reliable. The silencing efficiency of tatdf1 in TaTDF1 silencing strain was 0.095 and that of TaMAP65 was 0.96 (Fig. 15B). The silencing efficiency of tamap65 in the TaTDF1 silencing strain was 0.108 and that of TaMAP65 was 0.151 (Fig. 15C). Compared with the BSMV:00 negative control plants, the expression levels of TaTDF1 and the TaMAP65 genes decreased significantly (Fig. 15B, Fig. 15C). Investigation of the panicle seed-setting rate in BSMV:TaTDF1 and BSMV: TaMAP65 silenced plants, which revealed that the seed-setting rate of BSMV:TaTDF1 was the lowest (Fig. 15D). Downregulation of TaTDF1 and TaMAP65 significantly reduced grain fertility, suggesting that the expression of TaTDF1 and TaMAP65 could be closely related to pollen fertility.

Karyotypes and fertility of the anthers and pollen grains of MOCK, BSMV:00, BSMV:PDS, BSMV:TaTDF1, and BSMV:TaMAP65 plants in the pollen-scattering stage were observed (Fig. 16A1-A5). Compared with MOCK, BSMV:PDS, and BSMV:00, the anthers of BSMV:TaTDF1 and BSMV:TaMAP65 were shriveled, thin, and dull during the pollen-scattering stage (Fig. 16B1-B5, Fig. 16C1-C5). Using 10 g/mL of DAPI dye solution to detect the karyotype of pollen grains in the loose pollen stage, the pollen grains of the MOCK, BSMV:PDS, and BSMV:00 plants developed into fish-like sperm nuclei (Fig. 17A1-A3). However, the pollen grains of BSMV:TaTDF1 and BSMV:TaMAP65 were abnormal, and most were seedless, and few developed into fish-like sperm nuclei (Fig. 17A4,17A5). The pollen grains of MOCK, BSMV:00, and BSMV:PDS plants were stained with 1% I₂-KI solution and blue-black (Fig. 17B1-B3). However, most pollen grains of the BSMV:TaTDF1 and BSMV:TaMAP65 were stained with 1% I₂-KI solution, although some pollen grains were still stained blue-black (Fig. 17B4, Fig. 17B5). Therefore, TaMAP65 could be located upstream of TaTDF1, and the downregulated expression of TaTDF1 and TaMAP65 could affect the development of the anther, causing starch in pollen grains to accumulate with difficulty, thus causing pollen grains to abort.

Pollen grains are important to plant male reproductive organs. The growth and development of pollen grains are closely related to the differentiation of the microspore mother cells, the degradation of the tapetum cells, and the formation of the callose and pollen walls [22]. Mutations in genes related to pollen grain development or environmental changes can also cause abnormal pollen development, resulting in male sterility. Previous studies have found that the DYT1-TDF1-AMS-MYB80 gene pathway strictly controls tapetum development and plays an important role in maintaining normal degradation of tapetum cells and ensuring pollen fertility. This has been confirmed in A. thaliana, rice, tomatoes, and asparagus [23–26].

The TDF1 gene belongs to the DYT1-TDF1-AMS-MYB80 pathway. Mutation or deletion of its base sequence leads to abnormal degradation of tapetum cells, leading to an imbalance in nutrient metabolism and, eventually, to pollen abortion. Zhu et al. created a mutant of A. thaliana attdf1 using EMS mutagenesis technology. Compared to the tapetum cells of wild A. thaliana, the tapetum cells of the stamen anthers were swollen, vacuolated, and excessively divided, preventing them from being unable to transform into the secretory tapetum. Simultaneously, callose around the tetrad was deposited for a long time and the microspores could not be released in time, leading to the failure to form pollen grains, resulting in pollen abortion [27]. The tapetum cell development of ogu-CMS in black cabbage was studied, and the tapetum cell development characteristics were like those of the rice mutant ostdf1. Tapetum vacuoles rapidly form after microspore meiosis, and they cannot secrete enough callose enzyme, so most microspores cannot be released in time. Even if a few microspores are released, the tapetum cells squeeze them until they degenerate and disappear, indicating male sterility. Furthermore, overexpression of the rapeseed BcTDF1 gene in the A. thaliana attdf1 mutant restored the pollen fertility of the A. thaliana attdf1 mutant to some extent and produced some mature and fertile pollen grains [28]. The ability of the tdf1 mutant to induce plant pollen abortion was confirmed in this study. In this study, TaTDF1 was overexpressed in A. thaliana and dominant chimerism was used to suppress the silencing of the TDF1 gene. It was found that the TaTDF1-OE and TDF1-EAR transgenic lines bloomed normally, and the average bolting time of the TaTDF1-OE overexpression lines was 7 d earlier. The results of Alexander staining of the anthers are shown in red. However, the silencing lines of the TDF1-EAR dominant chimera suppression delayed the bolting period by an average of 4 d. Its anthers are shriveled and thin, and Alexander staining of the anthers is blue-green. Virus-induced gene-silencing technology for TaTDF1 silencing in wheat showed that the anthers of the wheat-silent strain tatdf1 were shriveled, thin, and dull in the loose pollen stage; the seed-setting rate decreased significantly. DAPI staining of pollen grains revealed abnormal nuclear development, 82.05% were seedless or unable to form normal fish-like sperm nuclei. However, 17.95% of the pollen grains developed into mature and fertile fish-like sperm nuclei (Fig. 18). After 1% I₂-KI staining, most pollen grains were yellow-brown, and a few pollen grains remained blue-black. The results showed that downregulation of TaTDF1 could induce pollen abortion in wheat. Tapetum cells from the ostdf1 gene knockout mutant showed a vacuolated phenotype similar to the Arabidopsis attdf1 mutant, and TUNEL stained nuclei were not observed in anthers. PCD defects were confirmed in the tapetum cells of ostdf1 anthers, leading to pollen abortion [18].

In summary, these findings suggest that MYB family transcription factor TDF1 may play a key role in regulating tapetal development and late function during wheat anther development. Similar to AtTDF1 of Arabidopsis thaliana, TDF1 affects pollen fertility by regulating tapetal production and degradation. Thus, the process of tapetum development in wheat may be similar to that of Arabidopsis thaliana, which is controlled by the conserved genetic pathway DYT1-TDF1-AMS-MYB80-MS1. In this study, the protein TaMAP65 interacting with TaTDF1 was screened by yeast two-hybrid test. Further qRT-PCR was performed on tatdf1 and tamap65 silent strains, and the results preliminarily indicated that TaTDF1 was located in the upstream of TaMP65, which was a supplement to the above genetic pathway (Fig. 19). Unfortunately, this study could not further study the regulatory relationship between DYT1 and MAP65 to further improve this genetic pathway. The process of pollen abortion in tatdf1 silenced strains could not be studied during the process of tapetum cell development. And their relations could not be illustrated. Compared with MF-90-110, the expression level of TaTDF1 in MS-90-110 was lower, suggesting that TaTDF1 may be related to pollen abortion in wheat K-type male sterile lines. This results was confirmed in previous study. Wu et al. showed that TaTDRL ( AtAMS homolog gene) is down-regulated during the development of anther in MS-90-110 [21]. Meanwhile, AtTDF1 is the upstream gene of AtAMS in Arabidopsis thaliana, indicating TaTDRL is possibly located at downstream of TaTDF1. It can be inferred that TaTDF1 and TaTDRL are also key genes involved in pollen abortion during the development of anther in MS-90-110. But this hypothesis needs further study.

Jasmonic acid (JA) is an important hormone that is widely involved in plant growth and development, metabolic regulation, stress, and defense responses [29]. Among these, MYB transcription factors promote anther dehiscence by interacting with methyl jasmonate to ensure normal anther development and pollen fertility. Qi et al. found that the development of JA-mediated stamen and grain setting were regulated by the bHLH-MYB complex. Among them, the bHLH transcription factor MYC5, the target gene of the JAZ repressor, plays an important regulatory role in stamen development and grain setting, together with the other bHLH factors MYC2, MYC3, and MYC4. Furthermore, the bHLH transcription factor MYC5 interacts with the MYB transcription factors MYB21 and MYB24 to form a bHLH-MYB transcription complex that synergistically regulates stamen development [30]. The R2R3-MYB transcription factors MYB21, MYB24, and MYB108 have been shown to affect stamen development by regulating the JA content. Its abnormal expression can lead to filament elongation, delayed anther dehiscence, and decreased pollen viability, eventually leading to the abortion of male pollen [31–33]. MYB21 and MYB24 are direct targets of JAZs, and their functions are affected by their content. Once the JA signal was detected, COI1 recruited JAZs into the SCF(COI1) complex and used the 26S proteasome for ubiquitination and degradation. MYB21 and MYB24 are released to activate the expression of various genes for JA-regulated anther development and filament elongation [33]. Both LoMYB21 in silent lilies and overexpression of LoMYB21 in Arabidopsis caused abnormal JA content and signal transduction, delayed anther dehiscence, reduced Jasmonic acid accumulation, and pollen abortion [34]. On top of that, Abscisic acid is also an important plant hormone that regulates plant resistance to various biotic and abiotic stresses. These include drought, cold, and salt stress, seed dormancy, and pollen fertility [35]. The pollen activity of plants (L4) overexpressing AtNCED3 decreased. The results of semi-thin sections and electron microscope observations of anthers showed that tapetum cells in L4 degraded too fast, microspores could not absorb nutrients, and their pollen fertility decreased. However, after exogenous spraying of ABA, the surface of the wax content on the anther surface increased and nutrients in the tapetum cells were continuously supplied, thus improving the fertility of the pollen grains [36]. The atu2af65b mutant showed an early flowering phenotype under both long- and short-d conditions, and the transcript of the flowering inhibition gene FLC decreased in the shoot tip of atu2af65b. This was mainly because of abnormal splicing of ABI5 and a decrease in transcript abundance. Furthermore, ABA promotes the expression of AtU2AF65b during ABA-induced flowering. However, the transition and splicing of FLC and ABI5 in the atu2af65b mutant were impaired; therefore, they could not normally induce male germ cell development [37]. Plants overexpressing the ABA-dependent SnRK2 kinase members SAPK8 and ABF1 showed delayed flowering. It is more sensitive to exogenous spraying of ABA-mediated flowering inhibition, whereas simultaneously knocking out ABF1 and its homologous gene bZIP40 can promote flowering [38]. These results indicate that JA and ABA hormones interact with MYB and bZIP transcription factors, respectively, by recognizing the CGTCA, TGACG-motif, and ABRE (ACGTG/CACGTG) motifs. This interferes with gene expression, affecting pollen fertility. This result is discussed preliminarily in this study.

In this study, the regulatory elements of the TaTDF1 promoter region were analyzed using PlantCARE and NewPLACE. Two cis-acting regulators, the CGTCA motif and the TGACG-motif, related to the methyl jasmonate reaction; one cis-acting regulator, ABRE (ACGTG/CACGTG), related to the abscisic acid reaction; four photoperiod response elements, GT1-motif, ATCT motif, G-box, and GA-motif; and three cis-acting regulators, ARE, GC-motif, and MBS, related to adversity stress, were identified (Fig. 20; Table S4). Among these, MeJA and ABRE cis-acting elements may interact with the upstream MYB and bZIP transcription factors to regulate the development of anthers or pollen grains. Therefore, Yeast One-Hybrid (Y1H) and Electrophoretic Mobility Shift Assay (EMSA) experiments can be used to further study the degradation of transcription factors interacting with MeJA and ABRE elements in TaTDF1 promoter region in wheat tapetum cells and the regulatory mechanism during anther or pollen grain development. This study provides new insights into the extraction of key genes that regulate the development of male reproductive organs in wheat.

Materials

In this study, the wheat K-type cytoplasmic male-sterile line MS (Kots) -90-110 (MS) and its fertile control line MR (Kots) -90-110 (MR) were used. When microspore development reached the meiosis, early uninucleate (Euns), late uninucleate (Luns), binucleate (Bns), and trinucleate (Tns) stages, samples were taken from the field, placed on ice, and brought back to the laboratory for microscopic examination of pollen kernel development. After confirmation, another 30 ears of anther were mixed and placed into 2.0 mL EP tube, and frozen in liquid nitrogen and stored in the refrigerator at -80 ℃ for future use. The wheat variety material used in BSMV-VIGS test was field. The above wheat variety materials Provided by the Key Laboratory of Crop Heterosis Research and Utilization at the Northwest A&F University.

RNA extraction and qRT-PCR analysis

Total RNA was extracted using the Total RNA Extraction Reagent RNA Isolator (Vazyme, Nanjing, China) and was purified according to the manufacturer’s instructions. The first strand of cDNA was synthesized using the HiScript® II Reverse Transcriptase kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. Real-time quantitative PCR (qRT-PCR) was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on Biosystems 7500 Fast Real-Time PCR System. Using an internal reference gene (GenBank accession No. AB181991.1), the relative expression of TaActin was calculated according to 2^−∆∆Ct method with three biological repeats. Data were analyzed using Duncan’s method in SPASS 12.0.

Cloning and sequence analysis of TaTDF1

The OsTDF1 protein sequence was obtained from GenBank (accession number: XP.015630216.1). The nucleotide sequence of TaTDF1 and its encoded amino acid sequence were retrieved from the NCBI for Biotechnology Information and Ensembl Plant databases. The full-length coding sequence CDS (Table S1) of MR-90-110 during meiosis was amplified with TaTDF1 gene-specific primers. The PCR product was purified using the EasyPure® rapid gel extraction kit (Transgen, China, Beijing). This was connected to the pEASY-T1 cloning vector (Transgen, China, Beijing) to construct a recombinant plasmid, which was then inserted into competent cells of E. coli DH5α (Protein Interaction, Wuhan, China). Positive clones were verified by PCR and sequencing (Shenggong, China, Shanghai).

Bioinformatic analysis of TaTDF1

TaTDF1 protein sequence. The TaTDF1 protein domain was analyzed using the online tool SMART (https://smart.embl.de/). The online software programs PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/) and SWISS-MOLD (https://swissmodel.expasy.org/interactive) were used to predict the secondary and tertiary structures of TaTDF1. The physical and chemical properties of TaTDF1 were analyzed using EXPASY (https://web.expasy.org/protparam/) software. The TMHMM Server V.2.0 (http://www.cbs.dtu.dk/services/TMHM) and SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/0) predicted the transmembrane domain and the signal peptide of the TaTDF1 protein, respectively. MEGA software was used to study the evolutionary relationship of TDF1 in different species. The conserved domain of TaTDF1 was identified by multisequence alignment using DNAMAN software.

Subcellular localization of TaTDF1

The open reading frame of TaTDF1 was amplified using specific primers (without a stop codon). The PCR product was ligated into the BstBI site of the pCAMBIA1302-GFP (CaMV 35S promoter) vector. The primers used are listed in Table S1. The fusion and empty control vectors were transferred to A. tumefaciens strain (GV3101), and the bacteria were introduced to Nicotiana Benthamian leaves for transient expression. GFP signals were observed using a laser confocal fluorescence microscope (Olympus, Tokyo, Japan).

Yeast two-hybrid screening (Y2H) assay

The full-length TaTDF1 cDNA was cloned using specific primers and attached to the pGBKT7 vector as bait (Table S1). The BD-TaTDF1 recombinant plasmid was transfected into Y2H gold-receptive yeast cells (Protein Interaction, Wuhan, China) using the lithium acetate conversion method [39]. After the non-toxic and self-activating effects were confirmed using SD/-Trp and SD/-Trp/-Ade/-His media, the successfully transformed positive monoclonal solution was mixed with the wheat anther cDNA library solution and coated onto SD/-Leu/-Trp/-His/-Ade media. It was then combined with X-α-gal chromogenic solution to screen for blue and white spots. Blue-positive clones were amplified by PCR and verified by sequencing (Shenggong, China, Shanghai).

Rotation verification assay of TaTDF1 and TaMAP65

Specific primers were designed to clone the full-length cDNA of TaMAP65 and inserted into the pGADT7 vector (Table S1). BD-TaTDF1, AD-TaMAP65, and recombinant plasmids were co-transformed into Y2HGold yeast receptive cells (Protein Interaction, Wuhan, China) using the lithium acetate conversion method. A positive control (co-transformed AD-T and BD-53 plasmids) and a negative control (co-transformed AD-T and BD-lam plasmids) were used. The interaction between SD/-Trp/-Ade/-Leu/-His medium and X-α-gal color reaction was verified.

Bimolecular-fluorescence complementation (BiFC) assay

The full-length cDNA of TaTDF1 was cloned into the pUN-SPYNE vector. The full-length cDNA of TaMAP65 was cloned into the pUC-SPYCE vector (see Table S1 for primer information). PUN-SPYNE, pUC-SPYCE, pUN-TaTDF1, and pUC-TaMAP65 were transformed into A. tumefaciens strain GV3101 (pSoup-19), which was then introduced into Nicotiana Benthamian leaves for transient expression. GFP signals were observed using confocal laser fluorescence microscopy (Olympus, Tokyo, Japan).

Genetic transformation of Arabidopsis thaliana

The CDS of TaTDF1 was cloned and inserted between the NcoI and SpeI cleavage sites of the pCAMBIA1302 vector to construct the TaTDF1 overexpression vector, and p35S::TaTDF1 (TATDF1-OE) was obtained. To construct a chimeric suppressor of TaTDF1, we fused TaTDF1 CDSs with coding ear motif (LDLDLELRLGFA) suppressor domain (RD) DNA. Subsequently, it was inserted into the downstream 35S promoter of the pCAMBIA1302 vector to construct the p35S::TaTDF1 (TATDF1-EAR) fusion vector. The primers used are listed in Supplementary Table S1. A. tumefaciens with the recombinant plasmids TaTDF1-OE and TaTDF1-EAR were used to infect Arabidopsis floss using the flower impregnation method to obtain Arabidopsis-positive plants.

Gene silencing of TaTDF1, TaMAP65 in wheat

The BSMV comprises three righteous RNA strands, α, β, and γ. Specific primers were designed to clone conserved cDNA fragments of TaTDF1 (+ 95–+188) and TaMAP65 (+ 483–+589), and were inserted into γ vector by the restriction endonucleases PacI and NotI (see Table S1 for primer information). α, β, γ, γ-PDS, γ-TaTDF1, and γ-TaMAP65 recombinant plasmids were digested by a single enzyme (Table S2). In vitro transcription was performed using the T7 High-Yield RNA Synthesis Kit (Shenggong, China, Shanghai).

α, β, γ, γ-PDS, γ-TaTDF1, and γ-TaMAP65 transcribed in vitro were mixed on ice according to the volume of α:β:γ (γ-PDS/γ-TaTDF1/γ-TaMAP65):GPK buffer = 0.5:0.5:9 (10.5 µL). The flag leaf and inverted wheat leaf were infected with recombinant virus, and then cultured in greenhouse under the conditions of 25°C, 16 h light, 20 ℃ and 8 h darkness. After 7–10 d of inoculation, symptoms were observed, and photos were taken. When the pollen of infected plants developed into the binucleate and trinucleate stages, the silencing efficiency of TaTDF1 and TaMAP65 in Mock and silenced plants was detected by qRT-PCR.

Acknowledgements

Wheat materials used in this study were provided by Shaanxi Key Laboratory of Crop Heterosis. We would like to appreciate the Innovation Center for Crop Biology staff for technical assistance.

Authors contributions

S.S., and P.T. contributed equally to this work. Y.S. managed the project. Y.S. and N.N. conceived and designed the experiments. S.S., and P.T. carried out the experiments and analyzed datum. S.S., and Y.S. wrote and finalized the manuscript, with the advice from G.Z. R.W., Y.R., and N.Y. involved in anther collection, RNA extraction, and phenotype investigation. B.L. carried out field experiment and gave some important suggestions for improving the design of the experiments. All authors have read and approved the manuscript.

Funding

The design of the study, sample collection, genes identification and analysis, gene quantification, interpretation of data and the writing of the manuscript was financially supported by National Natural Science Foundation of China (No.31701500); Shaanxi Province Key Research and Development Program General Project (No.2022NY-176); Fundamental Research Funds of Northwest A&F University (No.1090322148); Ph.D. Start-up Fund of Northwest A&F University (No.2452015263); the Fundamental Research Funds for the Central Universities (2452022109); Yangling Seed Industry Innovation Key Research and Development(Ylzy-xm-03).

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the NCBI for Biotechnology Information and Ensembl Plant databases , https://www.ncbi.nlm.nih.gov/ or https://plants.ensembl.org/index.html.

Ethics approval and consent to participate

Experimental research and feld studies on plants (either cultivated or wild), including the collection of plant material, must comply with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Authors and Affiliations

College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, P.R.China

Sicong Shan, Peng Tang, Rui Wang, Yihang Ren, Baolin Wu, Nuo Yan, Gaisheng Zhang, Na Niu, Yulong Song

Corresponding author

Correspondence to Yulong Song.

Conflict of interest

The authors declare that they have no competing interests.

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The characteristic analysis of TaTDF1 reveals its function related to male sterility in wheat (Triticum aestivum L.)

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Cloning and sequence analysis of TaTDF1

The expression pattern analysis of TaTDF1

Yeast two hybridization (Y2H) screening of interacting proteins that bind to the TaTDF1 protein

Cloning and sequence analysis of TaMAP65

Analysis of TaMAP65 expression pattern

Verification of the interaction between the TaTDF1 and TaMAP65 protein

Functional analysis of TaTDF1 in Arabidopsis thaliana

Functional analysis of TaTDF1 and TaMAP65 in wheat

Discussion

Materials and methods

Materials

RNA extraction and qRT-PCR analysis

Cloning and sequence analysis of TaTDF1

Bioinformatic analysis of TaTDF1

Subcellular localization of TaTDF1

Yeast two-hybrid screening (Y2H) assay

Rotation verification assay of TaTDF1 and TaMAP65

Bimolecular-fluorescence complementation (BiFC) assay

Genetic transformation of Arabidopsis thaliana

Gene silencing of TaTDF1, TaMAP65 in wheat

Declarations

References

Additional Declarations

Supplementary Files

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