Shoucai Ma

Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.)

Highlight A mitochondrial protein–protein interaction network in wheat explains why microspores suffered from severe oxidative stress during pollen development, triggered premature tapetal PCD, and consequently resulted in pollen abortion.

Cytochemical investigation at different microsporogenesis phases of male sterility in wheat, as induced by the chemical hybridising agent SQ-1

Abstract. This study used semi-thin sectioning and cytochemistry to investigate the relationship between pollen nutrient metabolism and pollen abortion in male sterile lines of wheat induced by SQ-1 (a chemical hybridising agent). Anthers were collected from the tetrad to trinucleate stages, and 4′ ,6-diamidino-2-phenylindole staining was used to visualise nuclei and confirm the development stage. Sudan Black B, periodic acid–Schiff, Coomassie Brilliant Blue, and toluidine blue were used to detect lipids, starch, proteins, and acidic polyanions, respectively. Semi-thin sectioning indicated that nutrient accumulation was much higher in the fertile line 1376 than in the sterile line 1376-PHYMS. Further, no lipids were found in the free microspore stage in the sterile line; however, at the late microspore stage, more proteins and acidic polyanions were found in the sterile line 1376-PHYMS pollen than in the fertile line 1376 pollen. From the binucleate to trinucleate pollen stages, the starch content was low and the intine considerably thinner in the pollen of the 1376-PHYMS line. SQ-1 probably hampered nutrient metabolism in the anthers, leading to decreased nutrient supply and abnormal intine formation, ultimately resulting in pollen abortion. A new mechanism for nutrient absorption, i.e. endocytosis of Ubisch bodies or orbicules by the intine through the germinal aperture, was revealed.

De Novo Assembly and Transcriptome Analysis of Wheat with Male Sterility Induced by the Chemical Hybridizing Agent SQ-1

Wheat (Triticum aestivum L.), one of the world’s most important food crops, is a strictly autogamous (self-pollinating) species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1–induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1–induced male sterile wheat and is a valuable source of data for future research in SQ-1–induced wheat male sterility.

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

Differential proteomic analysis of polyubiquitin-related proteins in chemical hybridization agent-induced wheat (Triticum aestivum L.) male sterility

Abstract Protein polyubiquitination is a significant regulator of diverse physiological functions, including sexual reproduction, in plants. Chemical hybridizing agents (CHA) SQ-1 has been shown to induce male sterility in wheat (Triticum aestivum L.) through inhibition of pollen development. This mechanism by which CHA induces male sterility in wheat is unclear. In this study, differential proteomic analysis of polyubiquitinated proteins associated with wheat male sterility was investigated. Wheat plants of the same genetic background were treated with or without CHA. Ubiquitinated proteins were then extracted and enriched for proteomic analysis. Differentially expressed polyubiquitinated proteins in trinuclear stage anther were identified by nanospray liquid chromatography/tandem mass spectrometry. A total of 127 and 131 differentially expressed polyubiquitinated proteins, including heat shock protein 70, ATPase subunit, glycosyltransferase, ubiquitin-related enzyme, and 20S proteasome subunit, were successfully identified by searching against wheat protein database and NCBInr database, respectively. Most of these proteins are related to photosynthesis, carbohydrate and energy metabolism, and multiple metabolic processes. These findings show that alteration of polyubiquitinated proteins is associated with male sterility in wheat.

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.

Special heterogeneous cytoplasm suppresses the expression of the gene producing multi-ovary in common wheat

Generally, there is only one seed in each floret of the common wheat (Triticum aestivum L.). However, DUOII is a multi-ovary variety that has 2–3 pistils and 3 stamens. The genetics of DUOII are very stable; and it can set 2–3 seeds similar to mono-ovary wheat. Previously, we found that special heterogeneous cytoplasm influenced the expression of the multi-ovary gene. To study the genetics governing DUOII under the influence of special heterogeneous cytoplasm, we made a reciprocal cross between DUOII and TeZhiI, which has the nucleus of common wheat with the cytoplasm of Aegilops. By investigating the multi-ovary trait in the reciprocal cross and the F2, F3, BC1, and BC1F1 offspring, we found that the DUOII multi-ovary trait was controlled by a dominant gene, and that the special heterogeneous cytoplasm of TeZhiI suppressed the expression of this gene. In addition, the special heterogeneous cytoplasm could only suppress the expression of the heterozygous, but not homozygous, dominant multi-ovary gene, which resulted in the heterozygous dominant multi-ovary plant producing a mono-ovary phenotype, and the homozygous dominant plant producing the multi-ovary trait. The multi-ovary trait of DUOII was determined by nuclear-cytoplasm interactions and that the special heterogeneous cytoplasm of TZI could suppress the expression of the multi-ovary gene in a new particular way. Hence, the DUOII is an ideal model for further research into nuclear-cytoplasm interactions, and the genetic basis of DUOII provides a theoretical foundation for the practical application of the multi-ovary trait in hybrid wheat.

Changes in DNA methylation are associated with heterogeneous cytoplasm suppression of the multi-ovary gene in wheat (Triticum aestivum)

Abstract. Variety DUOII is a multi-ovary line of common wheat (Triticum aestivum L.) that has two or three pistils and three stamens. The multi-ovary trait is controlled by a dominant gene, the expression of which can be suppressed by the special heterogeneous cytoplasm of line TeZhiI (TZI). TZI has the nucleus of common wheat and the cytoplasm of Aegilops. DUOII (♀) × TZI (♂) shows the multi-ovary trait, whereas TZI (♀) × DUOII (♂) shows the mono-ovary trait. DNA methylation affects gene expression and plays a crucial role in organ and tissue differentiation. In order to study the relationship between DNA methylation and the suppression of the multi-ovary gene, we used methylation-sensitive amplification polymorphisms (MSAP) to assess the DNA methylation status of the reciprocal crosses. Genome-wide, 14 584 CCGG sites were detected and the overall methylation levels were 31.10% and 30.76% in the respective crosses DUOII × TZI and TZI × DUOII. Compared with DUOII × TZI, TZI × DUOII showed 672 sites (4.61%) in which methylation–demethylation processes occurred. The results showed that the special heterogeneous cytoplasm significantly changed DNA methylation, and this might have suppressed the multi-ovary gene. The results provide insight into the changing patterns of DNA methylation in the suppression of the multi-ovary gene, and provide essential background for further studies on the underlying mechanisms of heterogeneous cytoplasm suppression of the expression of the multi-ovary gene in wheat.

Chloroplast structure and DNA methylation polymorphisms in an albino mutant of wheat (Triticum aestivum) cv. Xinong 1376

Abstract. DNA methylation plays an important role in regulating plant development, including organ and tissue differentiation, which may determine variations in agronomic traits. However, no reports exist for the regulation of leaf colour in wheat. The present study investigated the chloroplast structure and epigenetic mechanisms regulating leaf colour in an albino mutant of wheat (Triticum aestivum L.) cv. Xinong 1376. Structural analysis was performed by scanning and transmission electron microscopy, and epigenetic modifications were detected by methylation-sensitive amplification polymorphism (MSAP) analysis. Mesophyll cells of green leaves showed a well-ordered arrangement and they were filled with chloroplasts with intact lamellar structures and thylakoid membranes. By contrast, mesophyll cells of red and white leaves were disorganised and contained only a few plastids or chloroplasts with no lamellar structures or thylakoid membranes. Comparison of MSAP profiles revealed that white or red leaves had higher levels of cytosine methylation and showed changes in polymorphic loci compared with green leaves (4.35% and 4.10%, respectively). We sequenced 150 DNA fragments that were differentially displayed in MSAP patterns of white or red and green leaves of the Xinong 1376 albino mutant. A further BLAST search of 77 cloned sequences located them in coding regions. Most of these sequences were found to be involved in processes such as signal transduction, transcription regulation, post-transcriptional processing, DNA modification and repair, transport, biosynthesis of cellulose, photosynthesis, protein ubiquitination, stress responses, and retroposition. Expression analysis demonstrated a decrease in the transcription of two methylated genes, psaA and psbD, which are involved in the photosystem. Although the DNA methylation changes and leaf colour changes were not directly associated, these results may indicate that methylation of specific genes is an active and rapid epigenetic response to variation of leaf colour in the Xinong 1376 albino mutant, further elucidating the mechanism of variation in leaf colour.

Chemical hybridizing agent SQ-1-induced male sterility in Triticum aestivum L.: a comparative analysis of the anther proteome

BackgroundHeterosis is widely used to increase the yield of many crops. However, as wheat is a self-pollinating crop, hybrid breeding is not so successful in this organism. Even though male sterility induced by chemical hybridizing agents is an important aspect of crossbreeding, the mechanisms by which these agents induce male sterility in wheat is not well understood.ResultsWe performed proteomic analyses using the wheat Triticum aestivum L.to identify those proteins involved in physiological male sterility (PHYMS) induced by the chemical hybridizing agent CHA SQ-1. A total of 103 differentially expressed proteins were found by 2D–PAGE and subsequently identified by MALDI-TOF/TOF MS/MS. In general, these proteins had obvious functional tendencies implicated in carbohydrate metabolism, oxidative stress and resistance, protein metabolism, photosynthesis, and cytoskeleton and cell structure. In combination with phenotypic, tissue section, and bioinformatics analyses, the identified differentially expressed proteins revealed a complex network behind the regulation of PHYMS and pollen development. Accordingly, we constructed a protein network of male sterility in wheat, drawing relationships between the 103 differentially expressed proteins and their annotated biological pathways. To further validate our proposed protein network, we determined relevant physiological values and performed real-time PCR assays.ConclusionsOur proteomics based approach has enabled us to identify certain tendencies in PHYMS anthers. Anomalies in carbohydrate metabolism and oxidative stress, together with premature tapetum degradation, may be the cause behind carbohydrate starvation and male sterility in CHA SQ-1 treated plants. Here, we provide important insight into the mechanisms underlying CHA SQ-1-induced male sterility. Our findings have practical implications for the application of hybrid breeding in wheat.