Anther Development Research Articles

OsMS188 Is a Key Regulator of Tapetum Development and Sporopollenin Synthesis in Rice

BackgroundDuring anther development, the tapetum provides essential nutrients and materials for pollen development. In rice, multiple transcription factors and enzymes essential for tapetum development and pollen wall formation have been cloned from male-sterile lines.ResultsIn this study, we obtained several lines in which the MYB transcription factor OsMS188 was knocked out through the CRISPR-Cas9 approach. The osms188 lines exhibited a male-sterile phenotype with aberrant development and degeneration of tapetal cells, absence of the sexine layer and defective anther cuticles. CYP703A3, CYP704B2, OsPKS1, OsPKS2, DPW and ABCG15 are sporopollenin synthesis and transport-related genes in rice. Plants with mutations in these genes are male sterile, with a defective sexine layer and anther cuticle. Further biochemical assays demonstrated that OsMS188 binds directly to the promoters of these genes to regulate their expression. UDT1, OsTDF1, TDR, bHLH142 and EAT1 are upstream regulators of rice tapetum development. Electrophoretic mobility shift assays (EMSAs) and activation assays revealed that TDR directly regulates OsMS188 expression. Additionally, protein interaction assays indicated that TDR interacts with OsMS188 to regulate downstream gene expression.ConclusionOverall, OsMS188 is a key regulator of tapetum development and pollen wall formation. The gene regulatory network established in this work may facilitate future investigations of fertility regulation in rice and in other crop species.

Differential expression of transport and signalling genes in leaves and panicle regulates the development of pollen-free anthers in TGMS red rice

Thermosensitive genic male sterile (TGMS) plants are male sterile above a critical sterility temperature (CST) and become male fertile below CST during a critical thermosensitive stage. It is essential to analyse the gene expression pattern under fertility- and sterility-inducing conditions to understand the mechanisms of male sterility since it is regulated by a single recessive nuclear gene sensitive to environment during a specific stage of panicle development. Hence, this study aims at understanding the molecular mechanism associated with pollen sterility in TGMS system. The newly developed TGMS line on red rice background exhibited male sterility with pollen-free anthers and hence can be recommended as a stable female parent for the development of suitable red rice hybrids to the state of Kerala. Microarray gene expression analysis of TGMS leaf and young panicle revealed that occurrence of pollen-free anthers in the TGMS line during sterility-inducing condition is mainly due to the down-regulation of genes encoding ABC transporter proteins, lipid transfer protein and strictosidine synthase, glucose-methanol-choline oxidoreductase, male sterility protein 2, wax synthase and β-1,3-glucanase. Moreover, pathways involved in the carbohydrate synthesis and transport were also down-regulated during sterility condition. The differential expression of transport and signalling genes regulates the development of pollen-free anthers in TGMS rice.

Uncovering ceRNA integrated networks that associate with fertility in a photoperiod and temperature sensitive male sterile wheat line

The pollen fertility of photoperiod/temperature sensitive genic male sterile (P/TGMS) wheat is controlled by light and/or temperature. Circular RNA (circRNA) and long non-coding RNA (lncRNA) are known to participate in the development of anthers in plants, but their impact on male sterility in the P/TGMS line is not well understood. In this study, we carried out high-throughput sequencing to investigate the differential expression of lncRNAs and circRNAs and their biological functions in anthers of photo-thermosensitive genic male sterile (PTGMS) wheat line BS366-42L during the transition phase of male fertility under four different photoperiod and temperature treatments. Eight lncRNAs, 40 mRNAs and three circRNAs were screened out and thought as essential candidates that closely related to male sterility. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential functions of differentially expressed RNAs. The results indicated that carbohydrate-related metabolism was important for male sterility in the wheat PTGMS line BS366-42L. lncRNA/circRNA-mRNA-miRNA (ceRNA) integrate networks were constructed to reflect their complex inner association with male sterility. Our study provides a systematic perspective on the potential function of RNAs in male fertility in PTGMS lines of wheat.

Morphological characterization of floral traits to predict ideal stage for haploid production in bell pepper

Double haploid (DH) breeding not only helps in shortening the breeding cycle, also in fastening the release of homozygous inbred lines with superior and desirable traits. In vitro microspore culture is the fastest approach for the production of haploid plantlets. The key parameter for success of the haploid production in any crop species is the stage of microspore or pollens at the time of culture as it decides the fate of cultured anthers/pollens. Therefore an easy, precise, fast, and reliable criterion to identify flower buds carrying microspores or pollen at particular stages are essential. In the present investigation, we developed easiest and accurate criterion to correlate visible, measurable traits of bud and anther development with individual stages of microsporogenesis in two sweet pepper F1 hybrids (Orobelle and Bomby). Anthers containing microspores at late uninucleate to early binucleate stage are optimal for induction of androgenesis in wide range of crop species. Anther containing uninucleate pollens were observed in flower buds with an average size of 5.39 mm in Orobelle and 4.8mm in Bomby, average anther length of 2.75 mm in Orobelle and 2.71mm in Bomby and corolla was slightly longer than that of the calyx in both the hybrids. Anthers were without any purple pigmentation in Orobelle and slight/no pigmentation at the top of the anther sac end in Bomby.

A Novel Method to Map Small RNAs with High Resolution.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

Embryology and fertility of the natural tetraploid Lessingianthus plantaginoides (Asteraceae, Vernonieae): taxonomic implications

Abstract Lessingianthus plantaginoides (Vernonieae, Asteraceae) is a small natural tetraploid shrub that inhabits rocky highlands from South America. The population studied inhabits and covers an extensive region of a private reserve with high local biodiversity and animal and plant endemisms. With the purpose of providing insights into the cyto-embryology of this tetraploid species, the aims of this study were: to perform an ontogenetic study of the male and female gametophytes of L. plantaginoides; to carry out detailed meiotic analysis and evaluate the fertility of this species; to document and provide highlights on taxonomic implications of their reproductive aspects. Lessingianthus plantaginoides presented the following male and female gametophyte traits: dicotyledonous type of anther wall development, tetrahedral tetrads, 3-celled mature pollen grains; development of the chalazal megaspore, monosporic embryo sac and Polygonum type of megagametophyte development. The meiotic behavior was regular, the spores were tetrads of equal size and the pollen grains were highly stainable. Lessingianthus plantaginoides is a highly diplodized autotetraploid that reproduces sexually and has high meiotic regularity; which is apparently responsible for its colonization potential. It now seems certain that polyploid speciation plays a significant role in the establishment and diversification of the genus.

Advances in the role of auxin for transcriptional regulation of lignin biosynthesis.

Lignin is a natural polymer interlaced with cellulose and hemicellulose in secondary cell walls (SCWs). Auxin acts via its signalling transduction to regulate most of plant physiological processes. Lignification responds to auxin signals likewise and affects the development of anther and secondary xylem in plants. In this review, the research advances of AUXIN RESPONSE FACTOR (ARF)-dependent signalling pathways regulating lignin formation are discussed in detail. In an effort to facilitate the understanding of several key regulators in this process, we present a regulatory framework that comprises protein-protein interactions at the top and protein-gene regulation divided into five tiers. This characterises the regulatory roles of auxin in lignin biosynthesis and links auxin signalling transduction to transcriptional cascade of lignin biosynthesis. Our works further point to several of significant problems that need to be resolved in the future to gain a better understanding of the underlying mechanisms through which auxin regulates lignin biosynthesis.

Classification and Computational Analysis of Arabidopsis thaliana Sperm Cell-Specific F-Box Protein Gene 3p.AtFBP113.

In plants, F-box proteins (FBPs) constitute one of the largest superfamilies of regulatory proteins. Most F-box proteins are shown to be an integral part of SCF complexes, which carry out the degradation of proteins and regulate diverse important biological processes. Anthers and pollen development have a huge importance in crop breeding. Despite the vast diversity of FBPs in Arabidopsis male reproductive organs, their role in anther and pollen development is not much explored. Moreover, a standard nomenclature for naming FBPs is also lacking. Here, we propose a standard nomenclature for naming the FBPs of Arabidopsis thaliana uniformly and carry out a systematic analysis of sperm cell-specific FBP gene, i.e., 3p.AtFBP113 due to its reported high and preferential expression, for detailed functional annotation. The results revealed that 3p.AtFBP113 is located on the small arm of chromosome and encodes 397 amino acid long soluble, stable, and hydrophilic protein with the possibility of localization in various cellular compartments. The presence of the C-terminal F-box associated domain (FBA) with immunoglobulin-like fold anticipated its role in protein binding. Gene ontology based functional annotation and tissue-specific gene co-expression analysis further strengthened its role in protein binding and ubiquitination. Moreover, various potential post/co-translational modifications were anticipated and the predicted tertiary structure also showed the presence of characteristic domains and fold. Thus, the outcomes of the study will be useful in developing a better understating of the function of 3p.AtFBP113 during the process of pollen development, which will be helpful for targeting the gene for manipulation of male fertility that has immense importance in hybrid breeding.

Regulation of plant MYB transcription factors in anther development

MYB transcription factor is one of the largest transcription families and involved in plant growth and development, stress response, product metabolism and other processes. It regulates the development of plant flowers, especially anther development, a key role in the reproduction of plant progeny. Here, we discuss the regulatory effects of MYB transcription factors on the development of anther, including tapetum development, anther dehiscence, pollen development, carbohydrates and hormone pathways. We provide a reference for the further study of the regulation mechanism and network of plant anther development.

Silencing of the Ortholog of DEFECTIVE IN ANTHER DEHISCENCE 1 Gene in the Woody Perennial Jatropha curcas Alters Flower and Fruit Development.

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.

Identification of ceRNA and candidate genes related to fertility conversion of TCMS line YS3038 in wheat

Previous studies have indicated that noncoding RNAs are important factors in gene functions. To explore the mechanism of male sterility of YS3038, the sterile genes were mapped, and based on previous work, the expression of long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and their target genes was studied. Weighted gene coexpression network analysis (WGCNA) and competitive endogenous RNA (ceRNA) analysis were further performed for differentially expressed noncoding RNAs and target genes. At last, the candidate genes were silenced by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) to prove their function. The sterile genes were mapped on chromosomes 1B and 6B based on chip mix pool analysis, and one major effect QTL (27.3190% variation) was found based on SSR primers. The WGCNA analysis revealed that the dark turquoise and steel blue modules were highly correlated with anther development and fertility conversion, respectively. The ceRNA analysis showed that a total of 184 RNAs interacted with each other, including 115 mRNAs, 55 microRNAs (miRNAs), eight circRNAs, and six lncRNAs. Finally, the seed setting rate of the plant was significantly decreased after fatty acyl-CoA reductase 5 silencing. This study provides breeders with a new option for the development of thermosensitive cytoplasmic male-sterile (TCMS) wheat lines, which will favor the sustainable development of two-line hybrid wheat.

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

Retraction Note: Cloning and characterization of TaPUB4, a U-box gene from common wheat (Triticum aestivum L.) involved in regulation of pollen development by influencing sucrose-starch pathway in anther

Development of functional pollen grains in plants is regulated through complex genetic networks and environmental clues. The ubiquitin-26S proteasome system is important in the post-translational modification of intracellular proteins, and the system includes the ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes. Here, a novel plant U-box-containing cDNA was isolated in anther from common wheat (Triticum aestivum) and designed as TaPUB4. The cDNA encoding comprised 2598 bp, with an open reading frame of 865 amino acids. The deduced amino acid sequence of TaPUB4 showed 41.2–92.3% identity with other plant U-box homologous genes. Self-ubiquitination assay indicated that the bacterially expressed TaPUB4 protein had E3 ligase activity, and subcellular localization results showed that TaPUB4 was located in the nucleus and cytoplasm in onion epidermal cells. Overexpression of TaPUB4 gene in Arabidopsis atpub4 mutant restored partially the fertility of the mutant, increased the number of pollens in anthers, enhanced pollen viability, and displayed normal degradation of tapetum as anther development. The expression levels of genes related to starch deposition also changed greatly in the anthers of transgenic lines. These results support the notion that as a homolog of AtPUB4, TaPUB4 might control male fertility by regulating sucrose-starch metabolism in pollen grains, and this process may be accomplished through ubiquitin-proteasome system. The development and utilization of TaPUB4 as a functional molecular marker may be helpful for improving its fertility in wheat breeding programs in the future.

Anther and ovule development of Clematis serratifolia (Ranunculaceae)-with new formation types in megaspore and nucellus.

Morphological indices of vegetative organs or reproductive organs, which are often used to analyze the evolution and classify Clematis, indicate that Clematis serratifolia and C. glauca could be related members at similar evolutionary levels. However, this assumption differs with phylogenetic studies based on genetics. Embryonic characteristics, which are more stable, are commonly used to estimate the phylogeny and evolution of angiosperms. We studied the microsporogenesis, microgametogenesis, megasporogenesis and macrogametogenesis development of C. serratifolia, and compared the early embryological characteristics among C. serratifolia, C. serratifolia and other Clematis species reported to provide a reference for the taxonomy of the genus Clematis. Our results showed that C. serratifolia and C. glauca differ in megaspore formation and nucellus types suggesting that they have originated from different ancestors. The differences among Clematis were mainly found in the type of the anther wall development, tapetum, pollen grains, megaspore formation and nucellus types.

Tightly controlled expression of OsbHLH35 is critical for anther development in rice

Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72 % on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (pOsbHLH35::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

Comprehensive Atlas of Wheat (Triticum aestivum L.) AUXIN RESPONSE FACTOR Expression During Male Reproductive Development and Abiotic Stress.

AUXIN RESPONSE FACTOR (ARF) proteins regulate a wide range of signaling pathways, from general plant growth to abiotic stress responses. Here, we performed a genome-wide survey in wheat (Triticum aestivum) and identified 69 TaARF members that formed 24 homoeologous groups. Phylogenetic analysis clustered TaARF genes into three clades, similar to ARF genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Structural characterization suggested that ARF gene structure and domain composition are well conserved between plant species. Expression profiling revealed diverse patterns of TaARF transcript levels across a range of developmental stages, tissues, and abiotic stresses. A number of TaARF genes shared similar expression patterns and were preferentially expressed in anthers. Moreover, our systematic analysis identified three anther-specific TaARF genes (TaARF8, TaARF9, and TaARF21) whose expression was significantly altered by low temperature in thermosensitive genic male-sterile (TGMS) wheat; these TaARF genes are candidates to participate in the cold-induced male sterility pathway, and offer potential applications in TGMS wheat breeding and hybrid seed production. Moreover, we identified putative functions for a set of TaARFs involved in responses to abscisic acid and abiotic stress. Overall, this study characterized the wheat ARF gene family and generated several hypotheses for future investigation of ARF function during anther development and abiotic stress.

Identification of fertility-related genes for maize CMS-S via Bulked Segregant RNA-Seq

Cytoplasmic male sterility (CMS) is extensively used in maize hybrid production, and identification of genes related to fertility restoration for CMS is important for hybrid breeding. The fertility restoration of S type CMS is governed by several loci with major and minor effects, while the mechanism of fertility restoration for CMS-S is still unknown. In this study, BSR-Seq was conducted with two backcrossing populations with the fertility restoration genes, Rf3 and Rf10, respectively. Genetic mapping via BSR-Seq verified the positions of the two loci. A total of 353 and 176 differentially expressed genes (DEGs) between the male fertility and male sterile pools were identified in the populations with Rf3 and Rf10, respectively. In total, 265 DEGs were co-expressed in the two populations, which were up-regulated in the fertile plants, and they might be related to male fertility involving in anther or pollen development. Moreover, 35 and seven DEGs were specifically up-regulated in the fertile plants of the population with Rf3 and Rf10, respectively. Function analysis of these DEGs revealed that jasmonic acid (JA) signal pathway might be involved in the Rf3 mediated fertility restoration for CMS-S, while the small ubiquitin-related modifier system could play a role in the fertility restoration of Rf10.

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.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady\u2019s slipper orchid endemic to East Asia

BackgroundThe genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List.ResultsWe investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage.ConclusionOur overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.