DIA-based proteome profiling with PRM verification reveals the involvement of ER-associated protein processing in pollen abortion in Ogura CMS cabbage

Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources.However,only a few cytoplasmic male sterility(CMS)lines of cotton have been produced due to several challenges,like inadequate variation of agronomic traits,incomplete sterility,weak resilience of restorer lines,and difficulty in combining strong dominance.Therefore,the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.Results Two F_(2) segregating populations of cotton were constructed from cytoplasmic male sterile lines(HaA and 01A,maternal)and restorer lines(HaR and 26R,paternal).Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants.Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants.However,sterile plants exhibited smaller floral organs,shortened filament lengths,and anther atrophy on the flowering day in comparison with the fertile plants.When performed scanning electron microscopy(SEM),the two F_(2) populations revealed morphological variations in the anther epidermis.Cellular analysis showed no significant differences in pollen development before pollen maturation.Interestingly,between the pollen maturation and flowering stages,the tapetum layer of sterile plants degenerated prematurely,resulting in abnormal pollen grains and gradual pollen degradation.Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene.Sterile plants exhibit distinctive floral morphology,which is characterized by stamen atrophy and abnormal anthers.Pollen abortion occurs between pollen maturity and flowering,indicating that premature tapetum degradation may be the primary cause of pollen abortion.Overall,our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations,mechanisms of sterility,and the role of sterile and restorer genes.

The essential roles of sugar metabolism for pollen development and male fertility in plants

Sugar metabolism plays an essential role in plant male reproduction. Defects in sugar metabolism during anther and pollen development often result in genic male sterility(GMS). In this review, we summarize the recent progresses of the sugar metabolism-related GMS genes and their roles during plant anther and pollen development, including callose wall and primexine formation, intine development, pollen maturation and starch accumulation, anther dehiscence, and pollen germination and tube growth. We predict 112 putative sugar metabolic GMS genes in maize based on bioinformatics and RNA-seq analyses, and most of them have peak expression patterns during middle or late anther developmental stages.Finally, we outline the potential applications of sugar metabolic GMS genes in crop hybrid breeding and seed production. This review will deepen our understanding on sugar metabolic pathways in controlling pollen development and male fertility in plants.

Organic carbon isotope and pollen evidence for mangrove development and response to human activity in Guangxi(Southwest China) over the last 140 years

Mangrove degradation must reduce carbon sequestration in recent years, thereby aggravating global warming.Thus, short-term impacts of human activity on mangrove ecosystems are cause for concern from local governments and scientists. Mangroves sediments can provide detailed records of mangrove species variation in the last one hundred years, based on detailed 210 Pb data. The study traced the history of mangrove development and its response to environmental change over the last 140 years in two mangrove swamps of Guangxi, Southwest China. Average sedimentation rates were calculated to be 0.48 cm/a and 0.56 cm/a in the Yingluo Bay and the Maowei Sea, respectively. Chemical indicators（δ13Corg and C：N） were utilized to trace the contribution of mangrove-derived organic matter（MOM） using a ternary mixing model. Simultaneous use of mangrove pollen can help to supplement some of these limitations in diagenetic/overlap of isotopic signatures. We found that vertical distribution of MOM was consistent with mangrove pollen, which could provide similar information for tracing mangrove ecosystems. Therefore, mangrove development was reconstructed and divided into three stages： flourishing, degradation and re-flourishing/re-degradation period. The significant degradation, found in the period of 1968–1998 and 1907–2007 in the Yingluo Bay and the Maowei Sea, respectively, corresponding to a rapid increase of reclamation area and seawall length, rather than climate change as recorded in the region.

Molecular Cloning and Characterization of Pollen Development Related Gene RsMF2 from Raphanus sativus L.

In the paper, the full length cDNA of RsMF2 gene, homologous with the BcMF2 gene encoding pollen-specificpolygalacturonase of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) was cloned from Raphanussativus L. cv. Yuanbai by PCR, with a pair of primer designed according to the coding sequence of BcMF2. The largestopening reading frame of RsMF2 gene is 1 266 bp in length and encodes a protein of 421 amino acids with a predictedmolecular mass of 43.9 kDa. Sequence analysis revealed that it has three potential N-glycosylation sites and onepolygalacturonase active position (RVTCGPGHGLSVGS). And the first 32 amino acids of the predicted RsMF2 proteinform a N-terminal hydrophobic domain which displays the properties of a signal peptide. The predicted secondarystructure composition for the protein has 6.9% helix, 42.0% sheet and 51.1% loop. Four domains which are highly conservedin the whole plant and fungal PGs is present in RsMF2. Phylogenetic analysis showed that RsMF2 falls into the categoryof clade-C, which includes PGs related to pollen. These results indicate that RsMF2 may act as polygalacturonase relatedto pollen development.

The miR159a-DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding.Male sterility associated with abnormal pollen development is an important factor in seedlessness.However,our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited.Here,we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus,which further indirectly modulated seed development and fruit size.Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat(Fortunella hindsii)resulted in small and seedless fruit phenotypes.Moreover,pollen was severely aborted in both transgenic lines,with arrested pollen mitotic I and abnormal pollen starch metabolism.Through additional cross-pollination experiments,DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus.Based on DNA affinity purification sequencing(DAP-seq),RNA-seq,and verified interaction assays,YUC2/YUC6,SS4 and STP8 were identified as downstream target genes of DUO1,those were all positively regulated by DUO1.In transgenic F.hindsii lines,the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6,which decreased indoleacetic acid(IAA)levels and modulated auxin signaling to repress pollen mitotic I.The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen.Overall,this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

ZmMS39 encodes a callose synthase essential for male fertility in maize(Zea mays L.)

Callose contributes to many biological processes of higher plants including pollen development,cell plate and vascular tissue formation,as well as regulating the transport function of plasmodesmata.The functions of callose synthase genes in maize have been little studied.We describe a maize male-sterile mutant 39(ms39)characterized by reduced plant height.In this study,we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909(ZmCals12),encoding a callose synthase,is responsible for the male sterility of the ms39 mutant.Compared with male-fertile plants,callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced.Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells,leading to collapse of the anther wall structure.Disordered glucose metabolism in ms39 may have intensified autophagy in anthers.Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

A functional characterization of TaMs1 orthologs in Poaceae plants

TaMs1 encodes a non-specific lipid transfer protein(nsLTP) and is required for pollen development in wheat. Although MS1 is a Poaceae-specific gene, the roles of MS1 genes in other Poaceae plants are unknown, especially in rice and maize. Here, we identified one ortholog in rice(OsLTPg29) and two orthologs in maize(ZmLTPg11 and ZmLTPx2). Similar to TaMs1, both OsLTPg29 and ZmLTPg11 genes are specifically expressed in the microsporocytes, and both OsLTPg29 and ZmLTPg11 proteins showed lipid-binding ability to phosphatidic acid and several phosphoinositides. To determine their roles in pollen development, we created osltpg29 mutants and zmltpg11 zmltpx2 double mutants by CRISPR/Cas9.osltpg29, not zmltpg11 zmltpx2, is defective in pollen development, and only OsLTPg29, not ZmLTPg11,can rescue the male sterility of tams1 mutant. Our results demonstrate that the biological function of MS1 in pollen development differs in the evolution of Poaceae plants.

Signaling in Pollen Tube Growth： Crosstalk, Feedback, and Missing Links

Pollen tubes elongate rapidly at their tips through highly polarized cell growth known as tip growth. Tip growth requires intensive exocytosis at the tip, which is supported by a dynamic cytoskeleton and vesicle trafficking. Several signaling pathways have been demonstrated to coordinate pollen tube growth by regulating cellular activities such as actin dynamics, exocytosis, and endocytosis. These signaling pathways crosstalk to form a signaling network that coordinates the cellular processes required for tip growth. The homeostasis of key signaling molecules is critical for the proper elongation of the pollen tube tip, and is commonly fine-tuned by positive and negative regulations. In addition to the major signaling pathways, emerging evidence implies the roles of other signals in the regulation of pollen tube growth. Here we review and discuss how these signaling networks modulate the rapid growth of pollen tubes.

Control of Cell Wall Extensibility during Pollen Tube Growth

In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underly- ing processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma mem- brane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymeri- zation in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.

Arabidopsis adaptor protein 1G is critical for pollen development

Pollen development is a,pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 （AP1G） mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.

A Genetic Pathway for Tapetum Development and Function in Arabidopsis

In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis （programmed cell death）. The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.

Maize Genic Male-Sterility Genes and Their Applications in Hybrid Breeding: Progress and Perspectives

As one of the most important crops, maize not only has been a source of the food, feed, and industrial feedstock for biofuel and bioproducts, but also became a model plant system for addressing fundamental questions in genetics. Male sterility is a very useful trait for hybrid vigor utilization and hybrid seed production. The identification and characterization of genic male-sterility (GMS) genes in maize and other plants have deepened our understanding of the molecular mechanisms controlling anther and pollen development, and enabled the development and efficient use of many biotechnology-based male-sterility (BMS) systems for crop hybrid breeding. In this review, we summarize main advances on the identification and characterization of GMS genes in maize, and con struct a putative regulatory network controlling maize anther and pollen development by comparative genomic analysis of GMS genes in maize, Arabidopsis, and rice. Furthermore, we discuss and appraise the features of more than a dozen BMS systems for propagating male-sterile lines and producing hybrid seeds in maize and other plants. Finally, we provide our perspectives on the studies of GMS genes and the development of novel BMS systems in maize and other plants. The continuous exploration of GMS genes and BMS systems will enhance our understanding of molecular regulatory networks controlling male fertility and greatly facilitate hybrid vigor utilization in breeding and field production of maize and other crops.

Arabidopsis AT-hook Protein TEK Positively Regulates the Expression of Arabinogalactan Proteins for Nexine Formation

Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK （TEK） gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins （AGPs） as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region （MAR） and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly reg- ulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine laver in the oollen wall.

Expression of a cotton MADS-box gene is regulated in anther development and in response to phytohormone signaling

MADS-box gene family encodes a large number and variety of transcription regulators in plants. In this study, a cDNA, GhMADS9, encoding a typical MADS protein with 230 amino acids was isolated from cotton flower cDNA library. Subsequently, a 1,623 bp genomic DNA fragment of GhMADS9 gene was isolated in cotton by PCR. Compared with its cDNA sequence, six introns were found in GhMADS9 gene. Fluorescent microscopy indicated that GhMADS9 protein localized in the nucleus. Transactivation activity assay in yeast cells revealed that GhMADS9 protein did not show transcriptional activation. Quantitative RT-PCR analysis showed that GhMADS9 was specially expressed in cotton anthers. Further in situ hybridization analysis demonstrated that strong expression of GhMADS9 gene was detected in developing pollens, but no or weak signals were found in the other anther tissues. Furthermore, GhMADS9 expression was dramatically up-regulated in anthers with abscisic acid (ABA) treatment, whereas its activity was down-regulated when treated by gibberellin (GA3). Collectively, our results suggest that GhMADS9 is a transcription factor and might be involved in cotton anther/pollen development and in response to ABA and GA3 signaling.

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis

Photoperiod/temperature-sensitive genic male sterility(P/TGMS)is widely applied for improving crop production.Previous investigations using the reversible male sterile(rvms)mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis.In this work,we isolated a restorer of rvms–2(res3),as the male sterility of rvms–2 was rescued by res3.Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation.Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression,revealing a pathway for tapetum secretory function.Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3.The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3.We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation.A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.