Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

Studies on the Morphology, Ontogeny and Embryology of the Flowers of Hedycarya Arborea J.R. et G. Forst. (Subfamily Monimioideae) and Laurelia Novae-Zelandiae A. Cunn. (Subfamily Atherospermoideae) of the Family Monimiaceae

<p>The inflorescences, flowers and the vascularization of floral parts of Hedycarya arborea and Laurelia novae-zelandiae were described and comparisons made with other members of the family in an attempt to determine the basic types of inflorescences, flowers and floral vascularization in the family. The vegetative, inflorescence and floral meristems of the two genera were compared. It was concluded that the vegetative apices of both had the tunica-corpus configuration typical of many other woody Ranales and other orders. The inflorescence apices were quite similar to the vegetative ones. The young floral apices are in a state of transition from a tunica-corpus to a mantle-core configuration and older floral apices had the mantle-core configuration, which is typical of the floral apices of many woody Ranales. Unusual features of the floral apices of Hedycarya and Laurelia were the lack of a pronounced rib meristem and the occurrence of relatively frequent divisions within vacuolate cells of the core. The ontogeny of the stamens of Hedycarya and Laurelia was described and comparisons were made. In both genera the micro-sporangium developed in a similar fashions: in Hedycarya 5-6 wall layers are formed inside the epidermis; in Laurelia there are 3-5 layers. Both genera had a typically thickened endothecium and a tapetum of the secretory type in which the tapetal cells become binucleate during the first meiotic division of the pollen mother cells. In Hedycarya the meiotic divisions of the pollen mother cells are of the successive type in which walls form by means of centrifugal cell plates Pollen grains remain in permanent tetrads in this genus. In Laurelia wall formation at the end of meiosis is of a modified simultaneous type, which may not have been hitherto described in the literature. Pollen grains are not in permanent tetrads. When the first division occurs in each microspore in Hedycarya, all four cells of a tetrad are at the same stage of division and the generative cell is cut off towards the distal face of the grain. Each microspore is in the two celled condition when shed. It was deduced that the generative cell is cut off against what represents a radial wall of the grain (with reference to the tetrad stage) in Laurelia. Pollen is shed in either the two or three celled condition. Comparisons were made with the development of microsporangia and male gametophytes in other woody Ranales. A study was made of the ontogeny, structure and function of the staminal appendages of Laurelia. It was found that the appendages function as nectaries, the nectar being predominantly sucrose. After a discussion of the various theories as to the morphological nature of the staminal appendages of the Laurales, it was concluded that they are morphologically staminodes. The carpels of Hedycarya and Laurelia have a basically similar ontogeny in which, as in the Lauraceae, the terminal stigmatic region develops from a solid terminal meristem in contrast to many woody Ranales in which the stigma-consists of crests which surround the external part of the cleft of the carpel. The ovules of Hedycarya and Laurelia resemble those of most other woody Ranales in being bitegmic, crassinucellate and anatropous with a monosporic 8-nucleate embryo sac of the Polygonum type. Both linear and T-shaped megaspore tetrads were found in the two genera. Laurelia has pseudocarps which develop after anthesis and enclose plumose achenes, but in Hedycarya the fruits are drupes. It was concluded that Laurelia and Hedycarya belong to two subfamilies which have been separated from each other for a long time and have undergone considerable evolution in different directions. It was also concluded that the Monimiaceae are closely related to the Lauraceae.</p>

Studies on the Morphology, Ontogeny and Embryology of the Flowers of Hedycarya Arborea J.R. et G. Forst. (Subfamily Monimioideae) and Laurelia Novae-Zelandiae A. Cunn. (Subfamily Atherospermoideae) of the Family Monimiaceae

<p>The inflorescences, flowers and the vascularization of floral parts of Hedycarya arborea and Laurelia novae-zelandiae were described and comparisons made with other members of the family in an attempt to determine the basic types of inflorescences, flowers and floral vascularization in the family. The vegetative, inflorescence and floral meristems of the two genera were compared. It was concluded that the vegetative apices of both had the tunica-corpus configuration typical of many other woody Ranales and other orders. The inflorescence apices were quite similar to the vegetative ones. The young floral apices are in a state of transition from a tunica-corpus to a mantle-core configuration and older floral apices had the mantle-core configuration, which is typical of the floral apices of many woody Ranales. Unusual features of the floral apices of Hedycarya and Laurelia were the lack of a pronounced rib meristem and the occurrence of relatively frequent divisions within vacuolate cells of the core. The ontogeny of the stamens of Hedycarya and Laurelia was described and comparisons were made. In both genera the micro-sporangium developed in a similar fashions: in Hedycarya 5-6 wall layers are formed inside the epidermis; in Laurelia there are 3-5 layers. Both genera had a typically thickened endothecium and a tapetum of the secretory type in which the tapetal cells become binucleate during the first meiotic division of the pollen mother cells. In Hedycarya the meiotic divisions of the pollen mother cells are of the successive type in which walls form by means of centrifugal cell plates Pollen grains remain in permanent tetrads in this genus. In Laurelia wall formation at the end of meiosis is of a modified simultaneous type, which may not have been hitherto described in the literature. Pollen grains are not in permanent tetrads. When the first division occurs in each microspore in Hedycarya, all four cells of a tetrad are at the same stage of division and the generative cell is cut off towards the distal face of the grain. Each microspore is in the two celled condition when shed. It was deduced that the generative cell is cut off against what represents a radial wall of the grain (with reference to the tetrad stage) in Laurelia. Pollen is shed in either the two or three celled condition. Comparisons were made with the development of microsporangia and male gametophytes in other woody Ranales. A study was made of the ontogeny, structure and function of the staminal appendages of Laurelia. It was found that the appendages function as nectaries, the nectar being predominantly sucrose. After a discussion of the various theories as to the morphological nature of the staminal appendages of the Laurales, it was concluded that they are morphologically staminodes. The carpels of Hedycarya and Laurelia have a basically similar ontogeny in which, as in the Lauraceae, the terminal stigmatic region develops from a solid terminal meristem in contrast to many woody Ranales in which the stigma-consists of crests which surround the external part of the cleft of the carpel. The ovules of Hedycarya and Laurelia resemble those of most other woody Ranales in being bitegmic, crassinucellate and anatropous with a monosporic 8-nucleate embryo sac of the Polygonum type. Both linear and T-shaped megaspore tetrads were found in the two genera. Laurelia has pseudocarps which develop after anthesis and enclose plumose achenes, but in Hedycarya the fruits are drupes. It was concluded that Laurelia and Hedycarya belong to two subfamilies which have been separated from each other for a long time and have undergone considerable evolution in different directions. It was also concluded that the Monimiaceae are closely related to the Lauraceae.</p>

Dynamic changes in primexine during the tetrad stage of pollen development

Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.

The Role of INAPERTURATE POLLEN1 as a Pollen Aperture Factor Is Conserved in the Basal Eudicot Eschscholzia californica (Papaveraceae)

Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.

Comparative transcriptome analysis reveals key insights into male sterility in Salvia miltiorrhiza Bunge

Background Large-scale heterosis breeding depends upon stable, inherited male sterility lines. We accidentally discovered a male sterility line (SW-S) in the F1progeny of a Salvia miltiorrhiza Bunge from Shandong, China (purple flowers) crossed with a S. miltiorrhiza f. alba from Sichuan, China (white flowers). We sought to provide insights into the pollen development for male sterility in S. miltiorrhiza. Methods The phenotypic and cytological features of the SW-S and fertile control SW-F were observed using scanning electron microscopy and paraffin sections to identify the key stage of male sterility. Transcriptome profiles were recorded for anthers at the tetrad stage of SW-S and SW-F using Illumina RNA-Seq. Results The paraffin sections showed that sterility mainly occurred at the tetrad stage of microspore development, during which the tapetum cells in the anther compartment completely fell off and gradually degraded in the sterile line. There was little-to-no callose deposited around the microspore cells. The tetrad microspore was shriveled and had abnormal morphology. Therefore, anthers at the tetrad stage of SW-S and fertile control SW-F were selected for comparative transcriptome analysis. In total, 266,722,270 clean reads were obtained from SW-S and SW-F, which contained 36,534 genes. There were 2,571 differentially expressed genes (DEGs) in SW-S and SW-F, of which 63.5% were downregulated. Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes were enriched in 56 functional groups (GO terms); of these, all DEGs involved in microgametogenesis and developmental maturation were downregulated in SW-S. These results were confirmed by quantitative RT-PCR. The two GO terms contained 18 DEGs, among which eight DEGs (namely: GPAT3, RHF1A, phosphatidylinositol, PFAS, MYB96, MYB78, Cals5, and LAT52) were related to gamete development. There were 10 DEGs related to development and maturation, among which three genes were directly related to pollen development (namely: ACT3, RPK2, and DRP1C). Therefore, we believe that these genes are directly or indirectly involved in the pollen abortion of SW-S. Our study provides insight into key genes related to sterility traits in S. miltiorrhiza, and the results can be further exploited in functional and mechanism studies.

Characterization and transcriptome analysis of a dominant genic male sterile cotton mutant

Background : Male sterility is an efficient trait for hybrid seed production and germplasm innovation. Until now, most studies on male sterility were on cytoplasmic and recessive genic sterility, with few on dominant genic male sterility, especially in cotton, due to lack of such mutant.Results : We discovered a natural male sterile (MS) Sea Island cotton ( G. barbadense ) mutant, Genetic analysis showed the mutation was caused by a dominant mutation in a single nuclear gene. Comparative cytological observation of anther sections from MS and WT uncovered cellular differences in anther at and after the tetrad stage of pollen mother cells (PMC). In the MS anthers, the outer wall of pollen grains was free of spinules, the tapetum was vacuolated and showed delayed degradation, consequently, no functional pollen grains. Comparison of transcriptomes from meiosis, tetrad, mononuclear and binuclear pollen, and pollen maturation stages identified 13,783 non-redundant differentially expressed genes (DEGs) between MS and WT. Based on the number of DEGs, analyses of enriched GO terms and KEGG pathways, it was evident that significant transcriptomic changes occurred at and after the tetrad stage, consistent with cytological observation, and that the major differences were on metabolism of starch, sucrose, ascorbate, aldarate, alanine, aspartate and glutamate, and biosynthesis of cutin, suberine and wax. WGCNA analysis identified five modules containing 920 genes highly related to anther development, especially the greenyellow module with 54 genes that was highly associated with PMC meiosis and tetrad formation. A NAC transcription factor ( Gh_D11G2469 ) was identified as a hub gene for this module, which warrants further functional characterization.Conclusions : We demonstrated that the MS trait was controlled by a single dominant nuclear gene and caused by delayed tapetum degradation at the tetrad stage. Comparative transcriptome analysis and gene network construction identified DEGs, enriched GO terms and metabolic pathways, and hub genes potentially associated with anther development and the MS trait, which will contribute to important ideas and basis of the experimental data related to the molecular mechanism of DGMS and the innovation of cotton germplasm resources.

Temporal Distinction between Male and Female Floral Organ Development in Nicotiana tabacum cv. Xanthi (Solanaceae)

Early floral developmental investigations provide crucial evidence for phylogenetic and molecular studies of plants. The developmental and evolutionary mechanisms underlying the variations in floral organs are critical for a thorough understanding of the diversification of flowers. Ontogenetic comparisons between anthers and pistil within single flowers were characterized over time in Nicotiana tabacum cv. Xanthi. The ages of 42 tobacco flower or flower primordia were estimated using corolla growth analysis. Results showed that the protodermal layer in carpel primordia contributes to carpel development by both anticlinal and periclinal divisions. Periclinal divisions in the hypodermal layer of the placenta were observed around 4.8 ± 1.3 days after the formation of early carpel primordia (ECP) and ovule initiation occurred 10.0 ± 0.5 days after ECP. Meiosis in anthers and ovules began about 8.9 ± 1.1 days and 14.4 ± 1.3 days after ECP, respectively. Results showed an evident temporal distinction between megasporogenesis and microsporogenesis. Flower ages spanned a 17-day interval, starting with flower primordia containing the ECP and anther primordia to the tetrad stage of meiosis in megasporocytes and the bicellular stage in pollen grains. These results establish a solid foundation for future studies in order to identify the developmental and molecular mechanisms responsible for the mating system in tobacco.

Integrative Analysis of the lncRNA and mRNA Transcriptome Revealed Genes and Pathways Potentially Involved in the Anther Abortion of Cotton (Gossypium hirsutum L.)

Cotton plays an important role in the economy of many countries. Many studies have revealed that numerous genes and various metabolic pathways are involved in anther development. In this research, we studied the differently expressed mRNA and lncRNA during the anther development of cotton between the cytoplasmic male sterility (CMS) line, C2P5A, and the maintainer line, C2P5B, using RNA-seq analysis. We identified 17,897 known differentially expressed (DE) mRNAs, and 865 DE long noncoding RNAs (lncRNAs) that corresponded to 1172 cis-target genes at three stages of anther development using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DE mRNAs; and cis-target genes of DE lncRNAs probably involved in the degradation of tapetum cells, microspore development, pollen development, and in the differentiation, proliferation, and apoptosis of the anther cell wall in cotton. Of these DE genes, LTCONS_00105434, LTCONS_00004262, LTCONS_00126105, LTCONS_00085561, and LTCONS_00085561, correspond to cis-target genes Ghir_A09G011050.1, Ghir_A01G005150.1, Ghir_D05G003710.2, Ghir_A03G016640.1, and Ghir_A12G005100.1, respectively. They participate in oxidative phosphorylation, flavonoid biosynthesis, pentose and glucuronate interconversions, fatty acid biosynthesis, and MAPK signaling pathway in plants, respectively. In summary, the transcriptomic data indicated that DE lncRNAs and DE mRNAs were related to the anther development of cotton at the pollen mother cell stage, tetrad stage, and microspore stage, and abnormal expression could lead to anther abortion, resulting in male sterility of cotton.