Maize MS2 encodes an ATP-binding cassette transporter that is essential for anther development

The anther cuticle and pollen exine play a critical role in male gametophyte development. The sporopollenin precursors and cuticular lipid monomers are transported to the surface of the microspores and the epidermis by lipid transport proteins(LTPs) and ATP-binding cassette G(ABCG) transporters for the formation of the pollen wall and anther cuticle, respectively. However, the function of ABCG transporters in maize anther development is unclear. Here, we cloned the MS2 gene from the maize male sterile2 mutant using map-based cloning and determined that it encodes an ABCG transporter. MS2 protein was experimentally confirmed to be located on the cell membrane. The quantitative real-time PCR(qRT-PCR)results showed that MS2 was ubiquitously expressed in all vegetative and reproductive tissues, whereas a high transcriptional level of MS2 was observed in anthers, especially at the young microspore stage. Gas chromatography-mass spectrometry(GC–MS) analysis showed decreased accumulation of cutin and wax components in ms2 anthers, indicating that MS2 plays a role in the transport of lipid molecules to anther cuticle and pollen exine. To our knowledge, MS2 is the first reported ABCG transporter gene that participates in anther development in maize.

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development

The tapetum,a crucial innermost layer encompassing male reproductive cells within the anther wall,plays a pivotal role in normal pollen development.The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1,a gatekeeper TF in the tapetum.Nevertheless,the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown.In this study,we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089,probably through their K259 site.Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089.Intriguingly,elevated CCoAOMT1 expression resulted in defective pollen development,mirroring the phenotype observed in bhlh010 bhlh089 mutants.Moreover,our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1.Importantly,transgenic plants with the probHLH089::bHLH089^(K259A)construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants.Collectively,these findings emphasize the need to maintain balanced TF homeostasis for male fertility.They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.

GAMYB transcription factor LoMYB65 from lily plays a vital role in pollen development

Lily(Lilium spp.)is an important horticultural crop,but its use is limited due to serious pollen contamination problems.There are many studies on pollen development in model plants,but few on flower crops such as lilies.Gibberellin(GA)is a large class of hormones and plays an important role in plant vegetative growth and reproductive development.GAMYB is a group of the R2R3-MYB family upregulated by gibberellin,and plays an important role in anther development.Here,we isolated a novel GAMYB,named LoMYB65,from lily,which was closely related to the AtMYB65 and AtMYB33 in Arabidopsis.Fluorescence quantitative PCR results showed that LoMYB65 was mainly expressed in lily anthers.LoMYB65 could be activated by 288μmol·L^(-1)GA3treatment and the LoMYB65 protein was located in the nucleus and cytoplasm,and had transactivation in yeast and tobacco leaf cells.The conserved motif within 226 amino acids of the C-terminal of LoMYB65 contributed to its transactivation.Overexpression of LoMYB65 caused dwarf phenotype,unnormal tapetum development,less seeds of siliques in transgenic Arabidopsis plants,the transgenic plants showed partly male sterile.Simultaneously,silencing of LoMYB65 with VIGS(Virus Induced Gene Silencing)in lily anthers caused unnormal pollen development and reduced the pollen amount.Overexpression of LoMYB65 in Arabidopsis and silencing of LoMYB65 in lily resulted in decreased pollen counts,so we speculate that LoMYB65 may be dose-dependent.Overall,these findings suggest that LoMYB65 may play an important role in anther development and pollen formation in lily.LoMYB65 may provide a useful candidate gene for pollenless breeding of lily.

Normal Structure and Function of Endothecium Chloroplasts Maintained by ZmMs33-Mediated Lipid Biosynthesis in Tapetal Cells Are Critical for Anther Development in Maize

Genic male sterility(GMS)is critical for heterosis utilization and hybrid seed production.Although GMS mutants and genes have been studied extensively in plants,it has remained unclear whether chloroplast-associated photosynthetic and metabolic activities are involved in the regulation of anther development.In this study,we characterized the function of ZmMs33/ZmGPAT6,which encodes a member of the glycerol-3-phosphate acyltransferase(GPAT)family that catalyzes the first step of the glycerolipid synthetic pathway.We found that normal structure and function of endothecium(En)chloroplasts maintained by ZmMs33-mediated lipid biosynthesis in tapetal cells are crucial for maize anther development.ZmMs33 is expressed mainly in the tapetum at early anther developmental stages and critical for cell proliferation and expansion at late stages.Chloroplasts in En cells of wild-type anthers function as starch storage sites before stage 10 but as photosynthetic factories since stage 10 to enable starch metabolism and carbohydrate supply.Loss of ZmMs33 function inhibits the biosynthesis of glycolipids and phospholipids,which are major components of En chloroplast membranes,and disrupts the development and function of En chloroplasts,resulting in the formation of abnormal En chloroplasts containing numerous starch granules.Further analyses reveal that starch synthesis during the day and starch degradation at night are greatly suppressed in the mutant anthers,leading to carbon starvation and low energy status,as evidenced by low trehalose-6-phosphate content and a reduced ATP/AMP ratio.The energy sensor and inducer of autophagy,SnRK1,was activated to induce early and excessive autophagy,premature PCD,and metabolic reprogramming in tapetal cells,finally arresting the elongation and development of mutant anthers.Taken together,our results not only show that ZmMs33 is required for normal structure and function of En chloroplasts but also reveal that starch metabolism and photosynthetic activities of En chloroplasts at different developmental stages are essential for normal anther development.These findings provide novel insights for understanding how lipid biosynthesis in the tapetum,the structure and function of En chloroplasts,and energy and substance metabolism are coordinated to maintain maize anther development.

Regulation of Arabidopsis Early Anther Development by Putative Cell-Cell Signaling Molecules and Transcriptional Regulators

Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS （also called NOZZLE）, to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 （DYT1） gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

Exploring key developmental phases and phase-specific genes across the entirety of anther development in maize

Anther development from stamen primordium to pollen dispersal is complex and essential to sexual reproduction.How this highly dynamic and complex developmental process is controlled genetically is not well understood,especially for genes involved in specific key developmental phases.Here we generated RNA sequencing libraries spanning 10 key stages across the entirety of anther development in maize(Zea mays).Global transcriptome analyses revealed distinct phases of cell division and expansion,meiosis,pollen maturation,and mature pollen,for which we detected 50,245,42,and 414 phase-specific marker genes,respectively.Phase-specific transcription factor genes were significantly enriched in the phase of meiosis.The phase-specific expression of these marker genes was highly conserved among the maize lines Chang7-2 and W23,indicating they might have important roles in anther development.We explored a desiccationrelated protein gene,ZmDRP1,which was exclusively expressed in the tapetum from the tetrad to the uninucleate microspore stage,by generating knockout mutants.Notably,mutants in ZmDRP1 were completely male-sterile,with abnormal Ubisch bodies and defective pollen exine.Our work provides a glimpse into the gene expression dynamics and a valuable resource for exploring the roles of key phase-specific genes that regulate anther development.

ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development

Arabidopsis abcbl abcb19 double mutants defective in the auxin transporters ABCB1/PGP1 and ABCB19/PGP19 are altered in stamen elongation, anther dehiscence and pollen maturation. To assess the contribution of these transporters to stamen development we performed phenotypic, histological analyses, and in situ hybridizations on abcbl and abcbl9 single mutant flowers. We found that pollen maturation and anther dehiscence are precocious in the abcbl but not in the abcb19 mutant. Accordingly, endothecium ligniflcation is altered only in abcbl anthers. Both abcbl and abcb1 abcb19 stamens also show altered early development, with asynchronous anther Iocules and a multilayer tapetum. DAPI staining showed that the timing of meiosis is asynchronous in abcbl abcb19 anther Iocules, while only a small percentage of pollen grains are non- viable according to Alexander＇s staining. In agreement, TAM （TARDY ASYNCHRONOUS MEIOSIS）, as well as BAM2 （BARELY ANY MERISTEM）involved in tapetal cell development--areoverexpressed in abcbl abcb19 young flower buds. Corre- spondingly, ABCB1 and ABCB19 mRNA localization supports the observed phenotypes of abcbl and abcbl abcb19 mutant anthers. In conclusion, we provide evidence that auxin transport plays a significant role both in early and late stamen development： ABCB1 plays a major role during anther development, while ABCB19 has a synergistic role.

Cytological analysis and genetic control of rice anther development

Microsporogenesis and male gametogenesis are essential for the alternating life cycle of flowering plants between diploid sporophyte and haploid gametophyte generations. Rice （Oryza sativa） is the world＇s major staple food, and manipulation of pollen fertility is particularly important for the demands to increase rice grain yield. Towards a better understanding of the mechanisms controlling rice male reproductive development, we describe here the cytological changes of anther development through 14 stages, including cell division, differentiation and degeneration of somatic tissues consisting of four concentric cell layers surrounding and supporting reproductive cells as they form mature pollen grains through meiosis and mitosis. Furthermore, we compare the morphological difference of anthers and pollen grains in both monocot rice and eudicot Arabidopsis thaliana. Additionally, we describe the key genes identified to date critical for rice anther development and pollen formation.

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.

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.

橡胶花药体细胞植株的优良性状

花药体细胞植株,集芽接树和实生树的优点于一体,是一种幼态自根无性系,表现出许多优良性状: 1.生长快。比母体芽接树快9—20％,大致可提早一年开割投产。 2.产量高。海垦1和海垦2花药体细胞植株头两割年平均单株产量分别为母体无性系的131％和143％。 3.发育阶段从老态回复到幼态。表现在:和实生苗一样有一条粗壮的主根及一轮侧根;茎干圆锥度大,酷似实生树;取1.5年生植株的芽片芽接,抽生的嫩枝与砧木夹角小。这和老态型(抽生嫩枝与砧木夹角大)有明显差别,植株性状整齐一致;抗逆性强,经济寿命长,可排除砧木对接穗的不良影响。因此花药体细胞植株是一种很有发展前途的新一代种植材料。

牡丹花蕾与花粉发育关系的研究

以牡丹品种洛阳红为材料，取直径为14．18-43．68mm的花蕾，用1％的醋酸洋红压片法制片，在光学显微镜下观察花粉的发育状况。结果表明：花蕾直径在14．18-24．40mm时，花粉的发育基本处于单核早期，占总数的89．15％；直径在25．08-34．02mm时，花粉的发育主要处于单核早期到有丝分裂前期，二者占总数的62．40％；直径在35．00～43．68mm时，花粉大部分已成熟，占总数的52．75％。通过观察牡丹花蕾外观形态，可以直观确定花药发育时期，为花药单倍体培养奠定基础。

水稻不育系和保持系不同发育时期花药mRNA差异展示

应用DDRT－PCR技术分析了粤泰不育系及保持系单核、二核及三核期的mRNA。不育系和保持系本身不同发育时期花药CDNA扩增带型相似，不育系和保持系同时期花药的CDNA扩增带型也很相似，说明大多数基因都是组成型表达的。在实验中共观察到24条差异带，通过与总RNA斑点杂交对部分差异带初步筛选，表明AGCK－20仅与不育系总RNA有杂交信号。

利用花药相关基因及启动子创制雄性不育种质研究进展

杂种优势利用是大幅度提高作物产量、改良作物品质的有效途径,而作物雄性不育及优良的育性恢复种质是利用杂种优势的关键因素。为此,综述了近年来有关花药发育重要基因克隆和功能验证研究方面的重要成果,以及运用植物遗传转化工程创制植物雄性不育种质的进展。同时讨论了利用转基因技术创制作物雄性不育系、恢复系和保持系的技术策略,并对利用植物遗传转化技术创制植物杂种优势利用中"三系"种质的现状进行了分析和展望。

Genome-wide analyses on transcription factors and their potential microRNA regulators involved in maize male fertility

Anther development is a programmed biological process crucial to plant male reproduction. Genomewide analyses on the functions of transcriptional factor(TF) genes and their microRNA(miRNA) regulators contributing to anther development have not been comprehensively performed in maize. Here, using published RNA-Seq and small RNA-Seq(sRNA-Seq) data from maize anthers at ten developmental stages in three genic male-sterility(GMS) mutants(ocl4, mac1, and ms23) and wild type W23, as well as newly sequenced maize anther transcriptomes of ms7-6007 and lob30 GMS mutants and their WT lines, we analyzed and found 1079 stage-differentially expressed(stage-DE) TF genes that can be grouped into six(premeiotic, meiotic, postmeiotic, premeiotic-meiotic, premeiotic-postmeiotic, and meiotic-postmeiotic clusters) expression clusters. Functional enrichment combined with cytological and physiological analyses revealed specific functions of genes in each expression cluster. In addition, 118 stage-DE miRNAs and99 miRNA-TF gene pairs were identified in maize anthers. Further analyses revealed the regulatory roles of zma-miR319 and zma-miR159 as well as ZmMs7 and ZmLOB30 on ZmGAMYB expression. Moreover,ZmGAMYB and its paralog ZmGAMYB-2 were demonstrated as novel maize GMS genes by CRISPR/Cas9 knockout analysis. These results extend our understanding on the functions of miRNA-TF gene regulatory pairs and GMS TF genes contributing to male fertility in plants.

Male Sterile Lines of Zinnia elegans and Their Cytological Observations

In order to find out a new pathway for utilizing heterosis of Zinnia elegans and accelerate breeding process, the mechanism of anther development of a male sterile line was explored. Backcross, sibmating, selling of fertile plants and testcross with inbred lines were analyzed and identified in the field, and cytology was observed. Recessive nucleus male sterile line AH209AB capable of being a maintainer was obtained by successive backcrosses with male sterile plants and fertile F1 plants as male parents. Cytological and anatomical studies indicated that： （1） The wall of normal anther was constituted of four layers of cells such as epidermis, powder chamber wall, middle level and tapetum cells. The process in meiosis of pollen mother cell in Zinnia elegans was normal and cytoplasm divided simultanously. Mature pollen grain was tricellular type. （2） The petal of male sterile plant degraded as a thread-like structure, the stamens were villiform in appearance and no pollens were formed. The result showed that the anther of male sterile plant no longer proceed to differentiate spore mother cell and the pollen sac after the formation of the tissue of sporogenous cells, there was no evident boundary between tapetum cell, middle lamella and inner wall of PMC, tapetal cells did not develop from the very beginning. So the abortion type was completely structural male sterility. The male sterile line belongs to non-sporange male sterile type and is of great use in F1 seeds production.

Floret-specific differences in gene expression and support for the hypothesis that tapetal degeneration of Zea mays L. occurs via programmed cell death

The maize （Zea mays） spikelet consists of two florets, each of which contains three developmentally synchronized anthers. Morphologically, the anthers in the upper and lower florets proceed through apparently similar developmental programs. To test for global differences in gene expression and to identify genes that are coordinately regulated during maize anther development, RNA samples isolated from upper and lower floret anthers at six developmental stages were hybridized to cDNA microarrays. Approximately 9% of the tested genes exhibited statistically significant differences in expression between anthers in the upper and lower florets. This finding indicates that several basic biological processes are differentially regulated between upper and lower floret anthers, including metabolism, protein synthesis and signal transduction. Genes that are coordinately regulated across anther development were identified via cluster analysis. Analysis of these results identified stage-specific, early in development, late in development and bi-phasic expression profiles. Quantitative RT-PCR analysis revealed that four genes whose homologs in other plant species are involved in programmed cell death are up-regulated just prior to the time the tapetum begins to visibly degenerate （i.e., the mid-microspore stage）. This finding supports the hypothesis that developmentally normal tapetal degeneration occurs via programmed cell death.

Cloning and Characterization of a Somatic Embryogenesis Receptor-Like Kinase Gene in Cotton (Gossypium hirsutum)

A novel gene, GhSERK1, was identified in cotton. It encoded a protein belonging to the somatic embryogenesis receptor- like kinase （SERK） family. The genomic sequence of GhSERK1 was 6 920 bp in length, containing a predicted transcriptional start site （TSS）. Its full-length cDNA was 2 502 bp, encoding a protein of 627 amino acids. Sequence analysis of GhSERK1 revealed high levels of similarity to other reported SERKs, as well as a conserved intron/exon structure that was unique to members of the SERK family. Expression analysis showed that GhSERK1 mRNA was present in all organs of cotton plants and at different developmental stages, but its transcripts were most abundant in reproductive organs. Compared with that of the male-fertile line, the level of GhSERK1 mRNA was lower in the anther of the male-sterile cotton line, in which the pollen development was defected. Taken together, these findings illustrated that the GhSERK1 play a critical role during the anther formation, and may also have a broad role in other aspects of plant development.

几个甘蓝型油菜细胞质雄性不育系与保持系雌、雄蕊发育动态的比较

在初花期对几个甘蓝型油菜 (BrassicanapusL .)细胞质雄性不育系与相应保持系的雌、雄蕊及花药发育动态进行了观察。结果表明 ,不育系与保持系间在雌蕊发育过程中差异相对较小 ,仅在花蕾发育后期表现出一定的差异。到开花前 ,不育系的雌蕊略长于保持系。而雄蕊则在蕾长 3～ 4mm时期开始表现出较明显的差异 ,花药则在蕾长 1mm后开始表现出差异。临花前 ,不育系雄蕊及花药明显短于保持系 ,一般仅为保持系的 1/ 2左右。在不育系的花蕾中 ,雌蕊与雄蕊长度间在蕾长 3～ 4mm时开始表现出明显的差异 ,这时 ,雌、雄蕊长度差异可达 0 5～ 1mm ,可以用肉眼直接剥蕾观察出来。

低温预处理对刺梨花药培养小孢子发育的影响

为明确低温预处理对刺梨花药离体培养过程中小孢子发育及愈伤组织诱导率的影响,进行低温预处理试验,结果表明:离体培养条件下,刺梨雄核存在3条发育途径,即A-V途径、A-G途径和B途径,以A途径为主;随着培养时间的增加,小孢子逐渐退化,低温预处理3d可在一定程度上延缓刺梨小孢子退化,提早雄核发育,并增加参与雄核发育小孢子数量,提高花药愈伤组织诱导率。