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.

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.

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.

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.

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.

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.

水稻花粉发育过程及其分期

以籼稻品种 IR36为材料 ,采用塑料半薄切片技术、新鲜花药整体观察及游离的雄性细胞观察方法 ,对水稻花粉发育全过程及其药壁组织的情况进行了系统的研究 ,初步将这一过程划分为 8个时期 ,即小孢子母细胞形成期、小孢子母细胞减数分裂期、小孢子早期、小孢子中期、小孢子晚期、二胞花粉早期、二胞花粉晚期和成熟花粉期。并证实了花粉自然发育过程中不存在收缩期。在这一发育过程中 ,绒毡层出现 3种明显不同的发育形态 。

麻竹花粉发育过程观察及其分期

以麻竹为材料,采用醋酸洋红、碘-碘化钾压片以及石蜡切片的观察方法对麻竹花粉发育全过程进行了研究,初步将这一过程划分为8个时期,即小孢子母细胞形成期、小孢子母细胞减数分裂期、小孢子早期、小孢子中期、小孢子晚期、二胞花粉早期、二胞花粉晚期和成熟花粉期,并描述了各个时期的基本特征。麻竹花粉发育过程基本正常,小孢子母细胞减数分裂为连续型,四分体主要为左右对称型,也有四面体形的,成熟的花粉为3-细胞型,具单一萌发孔。

同源四倍体水稻花粉的发育特征

利用激光扫描共聚焦显微技术观察了同源四倍体水稻紫粳-4x花粉的发育过程及其败育时期和方式。在成熟花粉中正常花粉只占45.5%,还有54.5%的花粉在发育过程中发生败育。在小孢子母细胞减数分裂过程中出现了一些异常现象,如染色体不能正常配对、形成异常二分体及二分体分裂不同步等现象。绝大部分花粉败育发生在单核小孢子期和二胞花粉晚期。在败育花粉中典败花粉粒占21.4%,圆败花粉粒占5.0%,染败花粉粒占28.1%。导致紫粳-4x花粉败育的原因可能是由于其染色体数目加倍,在减数分裂过程中有些染色体不能正常配对,从而进行异常分裂造成的。

烟草幼嫩花粉原生质体分离与早期离体发育

建立了两种分离烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）幼嫩花粉原生质体的方法：一为通过花药漂浮培养释放出外壁裂开的花粉，后者转入酶液彻底脱去外壁，内壁降解而分离出原生质体（简称“花药预培养法”）；二为花粉经饥饿处理后，转入酶液释放原生质体（简称“花粉饥饿预处理法”）。对影响分离效果的主要因素作了研究。用花药预培养法分离的幼嫩花粉原生质体，在Ｋ３ 培养基中可再生细胞壁，启动１ 次细胞分裂，或萌发花粉管。表明具有孢子体发育与配子体发育的潜能。观察到花粉原生质体分裂成二细胞后，其中１ 个子细胞又长出花粉管的稀有现象。

牡丹花蕾大小对花药组培诱导率的影响

花蕾大小与花粉母细胞发育时期、以及组培中愈伤组织的诱导率都具有相关性。为确定牡丹花药最佳取样时期,以牡丹"凤丹"大小不同的花蕾为材料,研究了花蕾大小与花药发育情况及愈伤组织诱导的关系。结果表明:花粉处于单核中期是诱导愈伤组织的最佳时期,此时愈伤组织的诱导率最高可达45.8%,相对应的花蕾的大小为20.0～23.0 mm长,瓣尖未张开。

间泽钝绥螨植物花粉食性研究

用19种植物花粉对间泽钝绥螨Amblyseius aizawai Ehara et Bhandliu-falck进行了食性试验,结果表明均能取食,并能使雌螨产卵。以其中9种花粉作发育历期观察,一个生活周期4.25～8.55天。饲育存活率较高的花粉有丝瓜、棕榈、石榴和桦木,存活率达70.00～82.00％;产卵期10.00～40.80天,平均产卵期最长的有玉米、混合花粉和马桑花粉;产卵量10.00～74.00粒,平均产卵量最高的有丝瓜、混合花粉和石榴的花粉;饲育其雌性比最高的是丝瓜花粉;以8种花粉饲养成螨,寿命最长的是马桑、玉米、混合花粉和石榴花粉。

斯氏小盲绥螨取食3种花粉和椭圆食粉螨的实验种群生命表

为了研究斯氏小盲绥螨[Typhlodromips swirskii(Athias-Henriot)]取食3种不同花粉和椭圆食粉螨[Aleuroglyphus ovatus(Troupeau)]的发育和繁殖,为斯氏小盲绥螨人工大量饲养提供理论依据,在光照L∥D=10h∥14h、相对湿度85%,温度(25±1)℃的条件下,研究斯氏小盲绥螨取食向日葵花粉、丝瓜花粉、玉米花粉和椭圆食粉螨的生物学特性,并组建其实验种群生命表。结果表明,斯氏小盲绥螨取食向日葵花粉、丝瓜花粉、玉米花粉和椭圆食粉螨均能从卵发育至成螨并繁殖后代,雌螨发育历期分别为10.42、8.32、9.18、14.43d;平均每雌总产卵量分别为31.20、65.30、40.17、9.62粒;内禀增长率分别为0.134 3、0.150 2、0.144 9、0.089 5;取食丝瓜花粉的净增殖率(R0=45.057 0)、内禀增长率(rm=0.150 2)和周限增长率(λ=1.162 0)都最大;取食玉米花粉的雌雄性比最大(2.27∶1);取食椭圆食粉螨的雌雄性比(0.91∶1)、净增殖率(4.578 8)和内禀增长率(0.089 5)都最小。

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.

萍乡显性核不育水稻幼穗发育时期可溶性蛋白质含量的变化研究

对萍乡显性核不育水稻纯合不育株、杂合不育株与隐性可育株3种不同基因型植株幼穗发育期的叶片、幼穗和花药中的可溶性蛋白质含量变化进行了比较分析。结果表明,显性核不育水稻3种不同基因型植株在幼穗发育时期叶片中的可溶性蛋白质含量差异不大,且变化趋势相同;不育株(纯合与杂合)幼穗中可溶性蛋白质的含量在雌雄蕊形成期与可育株基本相同,而在花粉母细胞形成期和花粉母细胞减数分裂期迅速下降;不育株(纯合与杂合)花药内可溶性蛋白质含量明显低于可育株,这是不育株花粉败育的一个重要生理特征。

6种花粉对卵圆真绥螨实验种群参数的影响

在(25±1)℃、相对湿度(RH)70%±5%、光周期L∶D=13∶11条件下,用小叶龙船花花粉、豇豆花粉、扶桑花粉、台湾相思花粉、棉花花粉、玉米花粉饲喂卵圆真绥螨。结果表明,6种花粉饲喂均能使卵圆真绥螨完成生长发育及繁殖,其中豇豆花粉饲喂卵圆真绥螨时发育历期最短,为(5.07±0.06) d;存活率最高,为93.75%±3.05%;净增殖率最大,为16.78±1.22;单雌总产卵量第二,为(32.33±2.17)粒。综上所述,豇豆花粉较适宜卵圆真绥螨生长发育和繁殖。

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.

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.