The R2R3 MYB gene TaMYB305 positively regulates anther and pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

MAIN CONCLUSION: The loss of TaMYB305 function down-regulated the expression of jasmonic acid synthesis pathway genes, which may disturb the jasmonic acid synthesis, resulting in abnormal pollen development and reduced fertility. The MYB family, as one of the largest transcription factor families found in plants, regulates plant development, especially the development of anthers. Therefore, it is important to identify potential MYB transcription factors associated with pollen development and to study its role in pollen development. Here, the transcripts of an R2R3 MYB gene TaMYB305 from KTM3315A, a thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) wheat, was isolated. Quantitative real-time PCR (qRT-PCR) and promoter activity analysis revealed that TaMYB305 was primarily expressed in anthers. The TaMYB305 protein was localized in the nucleus, as determined by subcellular localization analysis. Our data demonstrated that silencing of TaMYB305 was related to abnormal development of stamen, including anther indehiscence and pollen abortion in KAM3315A plants. In addition, TaMYB305-silenced plants exhibited alterations in the transcriptional levels of genes involved in the synthesis of jasmonic acid (JA), indicating that TaMYB305 may regulate the expression of genes related to JA synthesis and play an important role during anther and pollen development of KTM3315A. These results provide novel insight into the function and molecular mechanism of R2R3-MYB genes in pollen development.

TaEXPB5 functions as a gene related to pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

The thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) is completely male sterile under low temperature (< 18 ℃) during Zadoks growth stages 45-52, whereas its fertility can be restored under hot temperature (≥ 20 ℃). The K-TCMS line may facilitate hybrid breeding and hybrid wheat production. Therefore, to elucidate the molecular mechanisms of its male sterility/fertility conversion, we conducted the association analysis of proteins and transcript expression to screen fertility related genes using RNA-seq, iTRAQ, and PRM-based assay. A gene encoding expansin protein in wheat, TaEXPB5, was isolated in K-TCMS line KTM3315A, which upregulated expression in the fertility anthers. Subcellular localization analysis suggested that TaEXPB5 protein localized to nucleus and cell wall. The silencing of TaEXPB5 displayed pollen abortion and the declination of fertility. Further, cytological investigation indicated that the silencing of TaEXPB5 induced the early degradation of tapetum and abnormal development of pollen wall. These results implied that TaEXPB5 may be essential for anther or pollen development and male fertility of KTM3315A. These findings provide a novel insight into molecular mechanism of fertility conversion for thermo-sensitive cytoplasmic male-sterility wheat, and contribute to the molecular breeding of hybrid wheat in the future.

A protein kinase-major sperm protein gene hijacked by a necrotrophic fungal pathogen triggers disease susceptibility in wheat.

Septoria nodorum blotch (SNB), a disease caused by the necrotrophic fungal pathogen Parastagonospora nodorum, is a threat to wheat (Triticum aestivum) production worldwide. Multiple inverse gene-for-gene interactions involving the recognition of necrotrophic effectors (NEs) by wheat sensitivity genes play major roles in causing SNB. One interaction involves the wheat gene Snn3 and the P. nodorum NE SnTox3. Here, we used a map-based strategy to clone the Snn3-D1 gene from Aegilops tauschii, the D-genome progenitor of common wheat. Snn3-D1 contained protein kinase and major sperm protein domains, both of which were essential for function as confirmed by mutagenesis. As opposed to other characterized interactions in this pathosystem, a compatible Snn3-D1-SnTox3 interaction was light-independent, and Snn3-D1 transcriptional expression was downregulated by light and upregulated by darkness. Snn3-D1 likely emerged in Ae. tauschii due to an approximately 218-kb insertion that occurred along the west bank of the Caspian Sea. The identification of this new class of NE sensitivity genes combined with the previously cloned sensitivity genes demonstrates that P. nodorum can take advantage of diverse host targets to trigger SNB susceptibility in wheat.

Comparative transcriptome analysis indicates conversion of stamens into pistil-like structures in male sterile wheat (Triticum aestivum L.) with Aegilops crassa cytoplasm.

BACKGROUND: Aegilops crassa cytoplasm is an important source for investigating cytoplasmic male sterility (CMS). Moreover, the stamens of line C303A exhibit a high degree of pistillody, turning almost white. However, the molecular mechanism that underlies pistillody in C303A remains unclear. Therefore, to obtain a better understanding of pistillody in C303A, the phenotypic and cytological features of C303A were observed to identify the key stage for the homeotic transformation of stamens into pistil-like structures. Transcriptome profiles were determined for stamens using Illumina RNA sequencing. RESULTS: Morphological observations of the CMS wheat line with Aegilops crassa cytoplasm C303A showed that the pistils developed normally, but the stamens were ultimately aborted and they released no pollen when mature. According to paraffin section observations, the stamens began to transform into pistils or pistil-like structures in the binucleate stage (BNS). Therefore, the stamens were collected from line C303A and its maintainer 303B in the BNS for transcriptome sequencing. In total, 20,444 wheat genes were determined as differentially expressed in C303A and 303B stamens, with 10,283 upregulated and 10,161 downregulated genes. Gene Ontology enrichment analyses showed that most of the differentially expressed genes (DEGs) were annotated with GO terms comprising metabolic process, cell, cellular process, catalytic activity, and cell part. Analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that the enriched DEGs were mainly associated with energy metabolism. We also found several essential genes that may contribute to pistillody in C303A. These findings suggest that disrupted energy metabolism and reactive oxygen metabolism induce pistillody and eventually lead to abortion in C303A. CONCLUSION: We determined the complex transcriptome profiles for C303A stamens and demonstrated that disrupted energy metabolism and class B MADS-box genes are related to pistillody. These findings may facilitate future studies of the mechanistic response of the wheat stamen and pollen development in CMS.

Cellular and physiological responses to drought stress in Aegilops tauschii genotypes.

Drought stress is one of the most important limiting factors in crop yield through impact on the cellular and physiological functions of the plant. Therefore, the study of physiological responses of plants can help to better understanding the drought tolerance mechanisms. In this experiment, 125 wild diploid wheat genotypes of Aegilops tauschii were evaluated for the physiological responses under rainfed and supplemental irrigation conditions. The physiological characteristics such as leaf relative water content (RWC), excised leaf water retention (ELWR), relative water loss (RWL), chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, ion leakage, membrane stability index (MSI) and proline content were measured. The results showed that the higher proline content, lower chlorophyll degradation rate and low amount of the membrane stability index (MSI) may inhibit the grain yield reduction under rainfed conditions. It was also found that the lower ion leakage due to the low cell membrane damage may led to the higher yield under rain-fed conditions. The results of regression analysis in both rainfed and supplemental irrigation conditions showed that proline content and total chlorophyll were introduced into the model, and explained the most variation in the grain yield. So, considering the above traits, the genotypes 16, 22, 43, 66 and 106 seems to be more drought tolerant and could be exploited in wheat breeding programs after further assessments.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

Cytological observation of chromosome breakage in wheat male gametophytes caused by gametocidal action of Aegilops triuncialis-derived chromosome 3Ct.

In this study, we investigated the chromosome breakage caused by gametocidal (Gc) chromosome 3Ct and its interaction with the suppressor gene Igc1 (inhibitor of gametocidal gene 1) on wheat chromosome 3B. We demonstrated cytologically that patterns of 3Ct-induced chromosomal fragmentation in microspores differed from patterns observed for other Gc genes. Uninuclear microspores of the monosomic 3Ct addition line had high frequencies of micronuclei, possibly explaining its low fertility. Chromosome fragmentation was observed in prometaphase and metaphase of the first pollen mitosis in the monosomic 3Ct addition line. Patterns of chromosome fragmentation were different from those previously reported for Gc chromosomes 2S of Aegilops speltoides, 4Ssh of Ae. sharonensis and 2Ccy of Ae. cylindrica; many chromosome fragments were observed in prometaphase of the first pollen mitosis in the monosomic 3Ct addition plants. In anthers at the binuclear stage, many microspores at the uninuclear stage coexisted with normally developed microspores.