Plant Reproduction最新文献

Hybridization plays an indispensable role in creating the diversity associated with plant evolution and genetic improvement of crops. Production of hybrids requires control of pollination and avoidance of self-pollination for species that are predominantly autogamous. Hand emasculation, male sterility genes or male gametocides have been used in several plant species to induce pollen sterility. However, in cowpea (Vigna unguiculata (L.) Walp), a self-pollinated cleistogamous dryland crop, only hand emasculation is used, but it is tedious and time-consuming. In this study, male sterility was effectively induced in cowpea and two dicotyledonous model species (Arabidopsis thaliana (L.) Heynh. and Nicotiana benthamiana Domin) using trifluoromethanesulfonamide (TFMSA). Pollen viability assays using Alexander staining showed that 30 ml of 1000 mg/l TFMSA with two-time treatments of one-week interval at the early stage of the reproductive phase under field or greenhouse conditions induced 99% pollen sterility in cowpea. TFMSA treatment induced non-functional pollen in diploid A. thaliana at two-time treatment of 10 ml of 125-250 mg/l per plant and N. benthamiana at two-time treatment of 10 ml of 250-1000 mg/l per plant. TFMSA-treated cowpea plants produced hybrid seeds when used as the female parent in crosses with non-treated plants used as male parents, suggesting that TFMSA had no effect on female functionality in cowpea. The ease of TFMSA treatment and its effectiveness to induce pollen sterility in a wide range of cowpea genotypes, and in the two model plant species tested in this study, may expand the scope of techniques for rapid pollination control in self-pollinated species, with potential applications in plant breeding and plant reproduction science.

During angiosperm sexual reproduction, pollen tubes must penetrate through multiple cell types in the pistil to mediate successful fertilization. Although this process is highly choreographed and requires complex chemical and mechanical signaling to guide the pollen tube to its destination, aspects of our understanding of pollen tube penetration through the pistil are incomplete. Our previous work demonstrated that disruption of the Arabidopsis thaliana O-FUCOSYLTRANSFERASE1 (OFT1) gene resulted in decreased pollen tube penetration through the stigma-style interface. Here, we demonstrate that second site mutations of Arabidopsis GALACTURONOSYLTRANSFERASE 14 (GAUT14) effectively suppress the phenotype of oft1 mutants, partially restoring silique length, seed set, pollen transmission, and pollen tube penetration deficiencies in navigating the female reproductive tract. These results suggest that disruption of pectic homogalacturonan (HG) synthesis can alleviate the penetrative defects associated with the oft1 mutant and may implicate pectic HG deposition in the process of pollen tube penetration through the stigma-style interface in Arabidopsis. These results also support a model in which OFT1 function directly or indirectly modifies structural features associated with the cell wall, with the loss of oft1 leading to an imbalance in the wall composition that can be compensated for by a reduction in pectic HG deposition.

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

Key message: S²⁹ haplotype does not require the MLPK function for self-incompatibility in Brassica rapa. Self-incompatibility (SI) in Brassicaceae is regulated by the self-recognition mechanism, which is based on the S-haplotype-specific direct interaction of the pollen-derived ligand, SP11/SCR, and the stigma-side receptor, SRK. M locus protein kinase (MLPK) is known to be one of the positive effectors of the SI response. MLPK directly interacts with SRK, and is phosphorylated by SRK in Brassica rapa. In Brassicaceae, MLPK was demonstrated to be essential for SI in B. rapa and Brassica napus, whereas it is not essential for SI in Arabidopsis thaliana (with introduced SRK and SP11/SCR from related SI species). Little is known about what determines the need for MLPK in SI of Brassicaceae. In this study, we investigated the relationship between S-haplotype diversity and MLPK function by analyzing the SI phenotypes of different S haplotypes in a mlpk/mlpk mutant background. The results have clarified that in B. rapa, all the S haplotypes except the S²⁹ we tested need the MLPK function, but the S²⁹ haplotype does not require MLPK for the SI. Comparative analysis of MLPK-dependent and MLPK-independent S haplotype might provide new insight into the evolution of S-haplotype diversity and the molecular mechanism of SI in Brassicaceae.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Callose, a β-1,3-glucan, lines the pollen tube cell wall except for the apical growing region, and it constitutes the main polysaccharide in pollen tube plugs. These regularly deposited plugs separate the active portion of the pollen tube cytoplasm from the degenerating cell segments. They have been hypothesized to reduce the total amount of cell volume requiring turgor regulation, thus aiding the invasive growth mechanism. To test this, we characterized the growth pattern of Arabidopsis callose synthase mutants with altered callose deposition patterns. Mutant pollen tubes without callose wall lining or plugs had a wider diameter but grew slower compared to their respective wildtype. To probe the pollen tube's ability to perform durotropism in the absence of callose, we performed mechanical assays such as growth in stiffened media and assessed turgor through incipient plasmolysis. We found that mutants lacking plugs had lower invading capacity and higher turgor pressure when faced with a mechanically challenging substrate. To explain this unexpected elevation in turgor pressure in the callose synthase mutants we suspected that it is enabled by feedback-driven increased levels of de-esterified pectin and/or cellulose in the tube cell wall. Through immunolabeling we tested this hypothesis and found that the content and spatial distribution of these cell wall polysaccharides was altered in callose-deficient mutant pollen tubes. Combined, the results reveal how callose contributes to the pollen tube's invasive capacity and thus plays an important role in fertilization. In order to understand, how the pollen tube deposits callose, we examined the involvement of the actin cytoskeleton in the spatial targeting of callose synthases to the cell surface. The spatial proximity of actin with locations of callose deposition and the dramatic effect of pharmacological interference with actin polymerization suggest a potential role for the cytoskeleton in the spatial control of the characteristic wall assembly process in pollen tubes.

Impaired activity of centromeric histone CENH3 causes inaccurate chromosome segregation and in crosses between the Arabidopsis recombinant CENH3 mutant GFP-tailswap and CENH3^G83E with wild-type pollen it results in chromosome loss with the formation of haploids. This genome elimination in the zygote and embryo is not absolute as also aneuploid and diploid progeny is formed. Here, we report that a temporal and moderate heat stress during fertilization and early embryogenesis shifts the ratio in favour of haploid progeny in CENH3 mutant lines. Micronuclei formation, a proxy for genome elimination, was similar in control and heat-treated flowers, indicating that heat-induced seed abortion occurred at a late stage during the development of the seed. In the seeds derived from heat-treated crosses, the endosperm did not cellularize and many seeds aborted. Haploid seeds were formed, however, resulting in increased frequencies of haploids in CENH3-mediated genome elimination crosses performed under heat stress. Therefore, heat stress application is a selective force during genome elimination that promotes haploid formation and may be used to improve the development and efficacy of in vivo haploid induction systems.

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial-temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future.

The presence of a pollinium is a distinct character in Apocynaceae which is important for phylogenetic analysis. The pollinium of Hoya has an outer sporopollenin wall and a pellucid margin which are adaptive features. However, their ontogeny and related evolutionary implications are not entirely understood. Therefore, a representative species Hoya carnosa was selected to investigate the pollinium development using light and electron microscopy and cytochemical tests. In contrast to the microsporogenesis in most angiosperms, which is associated with callose, the non-callosic intersporal walls in Hoya carnosa, together with the successive cytokinesis and linear form of the tetrad, represent an alternative pattern of microsporogenesis. This pattern has specific implication for the early stages of pollen morphogenesis. The absence of exine and apertures in the pollen grains in the pollinium could result from a combination of factors including the absence of callose in the early stages and the modifications in later developmental pathways, e.g., the sporopollenin accumulation pathway. The pollinium wall is an exine without stratification, its surface lacks sculptures, and it provides structural support and protection. The pollen tubes germinate through the pellucid margin and germinating ridge which are specialized features. The pellucid margin originates from aborted microspores. The germinating ridge that lies on the outer side of the pellucid margin develops in the same way as a classic pollen exine. The pollen grains are aggregated by intine fusion which is favorable for tube germination and growth. Comparing Asclepiadoideae with the other two subfamilies of Apocynaceae that develop a pollinium, the pollinium of Asclepiadoideae has reduced deposition of sporopollenin in the inner walls but an increase in the outer pollinium wall, thus making the inner walls more reduced and simplified, and the outer walls more solid. The adaptive characters of the pollen wall structure and the cohesion mechanism suggest that the pollinium of Hoya carnosa is a derived form of pollen aggregation.