Tapetal Cells Research Articles

BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus

Here, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function.

Flowering Research in Taiwan

Flowering is an important and highly regulated process. Taiwanese researchers have contributed significantly to understanding flowering biology, including cell biology, cytogenetics and functional genomics, in rice, lily and especially orchids. In recognition of this effort, the PCP Editorial Board is publishing a special issue on flowering research in Taiwan. This issue contains seven articles on cutting-edge topics or reviews of flowering research carried out in Taiwan, focusing on research into orchids and lily. An anther consists of sporophytic tissues of three outer cell layers and an innermost layer, the tapetum. The tapetum has attracted the most attention. Tapetum cells are important in nurturing the maturing microspores, and their ablation leads to microspore death and thus male sterility (Hesse et al. 1993). In a review paper, M.-D. Huang et al. (pp. 1459–1466) provide information about current studies on the transcriptome of the anther sporophyte, including microarray and next-generation sequencing data. The authors illustrate a proposed gene regulation network of anther development in Arabidopsis using the most updated references. In addition, they point out potential studies worthwhile pursuing, including those of ‘secretomes’, antisense transcripts and small RNAs. Finally, they propose a model for exine precursor biosynthesis in tapetum cells and transport of these precursors from the tapetum cell to the microspore surface. With an estimated >25,000 species, orchids are the most species-rich of all angiosperm families (Leitch et al. 2009). They show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. The expansion of the orchid family probably took place in a comparatively short period as compared with that of most flowering plant families, which suggests exceptionally high speciation rates (Gill 1989). The production of orchids in the world has increased 12–18% every year since 2000. Taiwan is one of the world-renowned countries exporting orchids, because the climate is congenial to the growth of orchids and because of the rich resource of native species and advances in breeding techniques to produce more beautiful and elegant new varieties. Taiwan’s orchid industry has been recognized by the international community as prosperous in terms of sales volume, orchid plantation, orchid-growing technologies and financial resources. The most popular potted flowering plants are Phalaenopsis hybrids, and Oncidium are the top-traded cut flowers. Most, if not all, orchid flowers on the market are hybrids, such as Phalaenopsis spp., although most researchers use inbred lines such as Phalaenopsis equestris as materials for detailed studies. Fig. 1 illustrates several examples of orchid varieties, their mutants, disease and insect infection, as well as orchid production. In the second review paper of this issue, Hsiao et al. (pp. 1467–1486) provide an overview of the research activities in orchid biology and biotechnology, including the status of genomics, transformation technology, flowering regulation, molecular regulatory mechanisms of floral development, scent production and color presentation. The authors also point out the pros and cons of using orchids for genome research. Oncidium is a valuable floral genus of orchid. The yellow color is most often seen in the flower market. The largest flower structure is the labellum, also known as the lip (fig. 1E, p. 1533). Chang et al. (pp. 1532–1545) used 454 sequencing to perform deep sequencing of the Oncidium transcriptome using six organs. A value-added expressed sequence tag (EST) database, OncidiumOrchidGenomeBase (http://predictor.nchu. edu.tw/oogb/), was constructed and provides a foundation for molecular genetics and functional genomics for modifying the flowering quality and desirable agronomic traits of Oncidium. The authors also identified flowering time-associated genes, including photoreceptors and floral pathway integrators. Su et al. (pp. 1501–1514) performed transcriptome analysis of mixed vegetative and reproductive tissues of the moth orchid Phalaenopsis aphrodite and constructed the Orchidstra genome database (http://orchidstra.abrc.sinica.edu.tw) for searching and linking to corresponding Pfam, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Fu et al. (2011) prepared an EST database from 11 tissues collected from three orchid species, i.e. P. equestris, P. aphrodite and P. bellina, using both traditional and next-generation sequencing. This OrchidBase is freely available at http://lab.fhes.tn.edu.tw/est. Altogether, these three transcriptome databases consist of about 100,000 contigs from four orchid species and thus provide tremendous resources for orchid functional genomics studies as well as breeding application. Although the floral ABCDE model is generally thought to be ubiquitous in the core eudicots, studies of basal angiosperms,

Microspore Wall Morphogenesis of Isoetes piedmontana (Pfeiffer) Reed

Microspore wall morphogenesis of Isoetes piedmontana was studied with SEM and TEM. The microspore wall consists of four layers: perispore, paraexospore, exospore, and endospore. Immediately after meiosis, the paraexospore forms around the microspore. The exospore forms next between the cell membrane and the paraexospore. Finally, the perispore is deposited on the paraexospore, and the endospore is formed within the exospore. The paraexospore, exospore, and endospore are largely derived from the cytoplasm of the microspore. The sporopollenin materials of the perispore are derived from the secretory tapetal cells along the inner sporangial wall.

Characterization of a caleosin expressed during olive (Olea europaeaL.) pollen ontogeny

BackgroundThe olive tree is an oil-storing species, with pollen being the second most active site in storage lipid biosynthesis. Caleosins are proteins involved in storage lipid mobilization during seed germination. Despite the existence of different lipidic structures in the anther, there are no data regarding the presence of caleosins in this organ to date. The purpose of the present work was to characterize a caleosin expressed in the olive anther over different key stages of pollen ontogeny, as a first approach to unravel its biological function in reproduction.ResultsA 30 kDa caleosin was identified in the anther tissues by Western blot analysis. Using fluorescence and transmission electron microscopic immunolocalization methods, the protein was first localized in the tapetal cells at the free microspore stage. Caleosins were released to the anther locule and further deposited onto the sculptures of the pollen exine. As anthers developed, tapetal cells showed the presence of structures constituted by caleosin-containing lipid droplets closely packed and enclosed by ER-derived cisternae and vesicles. After tapetal cells lost their integrity, the caleosin-containing remnants of the tapetum filled the cavities of the mature pollen exine, forming the pollen coat. In developing microspores, this caleosin was initially detected on the exine sculptures. During pollen maturation, caleosin levels progressively increased in the vegetative cell, concurrently with the number of oil bodies. The olive pollen caleosin was able to bind calcium in vitro. Moreover, PEGylation experiments supported the structural conformation model suggested for caleosins from seed oil bodies.ConclusionsIn the olive anther, a caleosin is expressed in both the tapetal and germ line cells, with its synthesis independently regulated. The pollen oil body-associated caleosin is synthesized by the vegetative cell, whereas the protein located on the pollen exine and its coating has a sporophytic origin. The biological significance of the caleosin in the reproductive process in species possessing lipid-storing pollen might depend on its subcellular emplacement. The pollen inner caleosin may be involved in OB biogenesis during pollen maturation. The protein located on the outside might rather play a function in pollen-stigma interaction during pollen hydration and germination.

Small heat shock protein LimHSP16.45 protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily

Plant meiotic prophase I is a complicated process involving the late zygotene and pachytene stages, both crucial for completing synapsis and recombination. Using David Lily (Lilium davidii var. Willmottiae) as our research material, we performed suppression subtractive hybridization to construct EST library of anthers at various stages of development by the pollen mother cells. From this library, we identified small heat shock protein LimHSP16.45 was highly expressed during the late zygotene to pachytene stages. Our results also showed that LimHSP16.45 was almost specifically expressed in the anther compared with the root, stem, or leaf, and in situ expression of LimHSP16.45 mRNAs showed strong signals in the pollen mother cells and tapetal cells. LimHSP16.45 could be induced by heat and cold in lily anthers, and its ectopic expression enhanced the viability of E. coli cells under both high and low temperatures. In vitro, it acted as molecular chaperone and could help luciferase refolding after heat shock stress. All of these data suggest that LimHSP16.45, working as molecular chaperone, possibly protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily.

Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

Immunolocalization of Lipoxygenase in the Anther Wall Cells of Lathyrus undulatus Boiss. during Programmed Cell Death

Lipoxygenase catalyzes oxygenation of long chain fatty acids to hydroperoxides and is involved in the degradation of membranes occuring in some types of programmed cell death (PCD). The localization of lipoxygenase in the anther wall layers of L. undulatus during cellular degradation was analyzed by immunogold labeling technique at young and vacuolated pollen stage, due to the close relation between lipoxygenase activity and membrane degradation in programmed cell death. Immunoreaction to lipoxygenase was monitored slightly at young pollen stage in the anther wall cells. As programmed cell death signals progress, lipoxygenase revealed in anther wall cells intensely. At vacuolated pollen stage tapetal cells came forward with ultrastructural changes such as cell, organelle and membrane disintegration. At the indicated stage immunogold particles indicating sites of LOX PAb-binding epitopes were located in the nucleus (chromatin was condensed and lined at the periphery), cytoplasm and close to long dilated rough endoplasmic reticulum (RER) cisterna. In conclusion lipoxygenase increase which has a role in the membrane degeneration, possibly induced the collapse of tonoplast, nuclear and plasma membrane and triggered programmed cell death in the tapetal cells of L. undulatus as well as the other wall cells.

Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522

The present work was aimed at developing vector construct(s) suitable for restoring fertility in transgenic male sterile tobacco plants expressing male-sterility-inducing ORFH522 in tapetal cell layer (Nizampatnam et al. Planta 229:987-1001, 2009). PTGS vectors that could produce either intron spliced hairpin RNA against the orfH522 or induce silencing of orfH522 by heterologous 3'UTR region were developed using the selected 316bp (orf316) fragment of orfH522. The constructs were independently mobilized into Agrobacterium and used for transforming tobacco. The T(1) generation plants carrying the restorer gene cassettes in homozygous condition were identified and crossed with the male sterile transgenic tobacco plants to obtain the hybrid seeds. PCR analysis of hybrid plants indicated segregation for the sterility inducing cassette while all the plants carried the restorer cassette. Hybrid plants produced fertile pollen grains and formed normal capsules upon selfing. Further molecular analyses of these hybrid plants with RT-PCR, Northern blotting and siRNA detection, revealed that intron interrupted hairpin RNA (ihp-RNA) mediated gene silencing was more effective compared to silencing by heterologous 3'UTR (SHUTR) as indicated by the complete degradation of orfH522 transcripts and formation of higher levels of orf316 specific siRNA molecules in plants carrying ihp-RNA restorer construct. Segregation analyses of F(2) (selfed hybrid) plants confirmed the co-segregation of gene cassettes and the traits in Mendelian di-hybrid ratio (9:3:3:1). Taken together, the results established that intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restored fertility in transgenic male sterile tobacco plants expressing orfH522 and ihp-RNA was more efficient in silencing orfH522 transcripts.

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having 'two-celled' groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of 'non-invasive' tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.

PERSISTENT TAPETAL CELL1Encodes a PHD-Finger Protein That Is Required for Tapetal Cell Death and Pollen Development in Rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

Tapetum development in transgenic tobacco (Nicotiana tabacum L.) plants with modlfied level of histone H1 variants

The phenomenon of male sterility has often been observed in investigations on the role of histone H1 in regulation of morphogenetic and cytological processes in transgenic tobacco plants. These changes were accumulated by disturbances in flower development, consisting in lengthening of the pistil style in relation to stamen heads. This prevented pollination and production of seeds. As similar abnormalities occurred also in the present investigations (depending on combination, the sterility% was 84.4 to 19.9, at only 8.1 in the control), the main problem of our investigations was an attempt to explain their reasons. It is commonly known that one of the conditions for formation of fertile pollen is the properly functioning tapetum. Here, we carried out observations of ultrastructure of anther tapetum control cells in respect of abnormalities which occurred during microsporogenesis of transgenic plants with inactivated expression of two major (A, B) and two minor (C, D) histone H1 variants. The investigations were carried out on the following groups of plants: (1) control group with a full set of histone variants (K), (2) with inactivated A and B variants (-AB); (3) with inactivated A, B, C and D variants (-ABCD), (4) with inactivated C and D variants (-CD). It was found that tapetal development was normal in all the investigated groups of plants, and the sequence of changes was similar as in the control. However, certain ultrastructural differences appeared when tapetum functioned as secretory tissue, and in the degeneration phase. In tapetal cell cytoplasm, with participation of rER, lipid bodies were formed, which, having penetrated to the cell surface and to locules, took part in formation of pollen grain sporoderm. Both in the control and in the remaining combination, excluding -ABCD, these bodies looked similar: they were grey, homogenous and surrounded by black jagged deposits. In -ABCD plants, these bodies were more translucent, slightly rarefied, and not surrounded by the deposits. Moreover, in -CD plants, large lipid deposits were frequently observed between remainders of degraded tapetal cells. They did not occur in the control and the remaining combinations.

Sweets – The Missing Sugar Efflux Carriers

One of the last mysteries in plant physiology is the efflux of sugars from mesophyll cells into the apoplasmic space. Depending on the strategy of long-distance assimilate transport, text books divide plants into symplasmic and apoplasmic phloem loaders. In both cases large concentration gradients are built up between mesophyll cells and the sieve element–companion cell complex (SE–CCC) of the phloem. In case of apoplasmic loaders this gradient is achieved by active transport of sucrose from the apoplasm into the SE–CCC. This uptake is mediated by well characterized plasma membrane sucrose–H+ symporters (Kuhn and Grof, 2010). As a consequence of this model, sucrose synthesized within mesophyll cells must be exported into the apoplasmic space, but the efflux mechanism is as yet unknown (Lemoine, 2000; Williams et al., 2000). In addition, sugar efflux must play a crucial role in fuelling pollen and embryo development as well as nectar production and secretion of carbohydrates to the rhizosphere. Furthermore, phytopathogenic bacteria usually colonize the apoplasmic space and are dependent on nutrient uptake from the host. Hence, it is very likely that efflux carriers from the host are required for bacterial growth. Despite these important functions, attempts to isolate and characterize sugar efflux carriers failed so far, probably because of unsuitable screening systems. Using a new and elegant screening system, Chen et al. (2010) were able to isolate and characterize the first sugar efflux carrier from plants and later on could demonstrate that these efflux carriers belong to a novel transporter family with homologs in Caenorhabditis elegans and humans. The screening system was based on a mammalian cell line (HEK293T) co-expressing a genetically encoded glucose sensor (Frommer et al., 2009) and a library of polytopic A. thaliana membrane proteins of unknown function as predicted by the membrane protein database Aramemnon (Schwacke et al., 2003). This system allowed the sensitive detection of glucose uptake and let to the identification of AtSWEET1. In Arabidopsis thaliana, AtSWEET1 is a member of a gene family comprising 17 members. Mutant phenotypes of some members of the SWEET family have already been described in A. thaliana and rice. Mutation of AtSWEET8 (ruptured pollen grain 1, RPG1) for example, prevents microsporocyte development by inhibiting glucose efflux from tapetum cells (Guan et al., 2008). Interestingly, several members of the AtSWEET gene family are inducible by pathogens. This let to the hypothesis that some pathogens might hijack host efflux systems to support their own growth in the apoplastic space of infected host plants. Support for this hypothesis comes from rice. Mutations in OsSWEET11 (Xa13) results in resistance against late blight (Chu et al., 2006). Moreover, in wild-type rice plants, Xanthomonas oryzae, the causative agent of late blight, activates OsSWEET11 expression by binding of TAL effectors to promoter elements of the OsSWEET11 gene (Romer et al., 2010). Mutations in these binding sites lead to the failure of gene induction and hence prevent colonization of the host. Interestingly, mutations in the same gene also result in reduced fertility of pollen. Thus pathogens seem to be able to reprogram host cells to support their nutrient supply by interfering with the host transport system. Taken together, assigning a function to the SWEET gene family opens up exiting perspectives for both plant physiology and plant–pathogen interaction. It could clearly be shown that SWEETS are in involved in glucose efflux and play a crucial role during pollen development and plant–pathogen interaction. Since the SWEETS transport glucose, this still leaves the question which proteins are responsible for sucrose efflux around the phloem or from the seed coat. However the base has been laid – one could use the FRET sucrose sensors (Lager et al., 2006) to use the same principal approach as was used for identifying the SWEETS, namely to screen a library of unknowns for novel sucrose transporters that might be responsible for sugar efflux around the phloem. But it seems to be clear that on the basis of the important role of members of the SWEET family during plant–pathogen interaction, novel strategies in plant protection become feasible. Therefore, the article by Chen et al. (2010) represents an exiting milestone in furthering our understanding of transport processes in plants and their relevance in plant–microbe interactions (for illustration see Figure ​Figure11). Figure 1 Schematic representation of sugar transport processes during plant–bacterium interaction and phloem loading. S, sucrose; H+, proton.

Global transcriptome analysis of two ameiotic1 alleles in maize anthers: defining steps in meiotic entry and progression through prophase I

BackgroundDevelopmental cues to start meiosis occur late in plants. Ameiotic1 (Am1) encodes a plant-specific nuclear protein (AM1) required for meiotic entry and progression through early prophase I. Pollen mother cells (PMCs) remain mitotic in most am1 mutants including am1-489, while am1-praI permits meiotic entry but PMCs arrest at the leptotene/zygotene (L/Z) transition, defining the roles of AM1 protein in two distinct steps of meiosis. To gain more insights into the roles of AM1 in the transcriptional pre-meiotic and meiotic programs, we report here an in depth analysis of gene expression alterations in carefully staged anthers at 1 mm (meiotic entry) and 1.5 mm (L/Z) caused by each of these am1 alleles.Results1.0 mm and 1.5 mm anthers of am1-489 and am1-praI were profiled in comparison to fertile siblings on Agilent® 4 × 44 K microarrays. Both am1-489 and am1-praI anthers are cytologically normal at 1.0 mm and show moderate transcriptome alterations. At the 1.5-mm stage both mutants are aberrant cytologically, and show more drastic transcriptome changes. There are substantially more absolute On/Off and twice as many differentially expressed genes (sterile versus fertile) in am1-489 than in am1-praI. At 1.5 mm a total of 4,418 genes are up- or down-regulated in either am1-489 or am1-praI anthers. These are predominantly stage-specific transcripts. Many putative meiosis-related genes were found among them including a small subset of allele-specific, mis-regulated genes specific to the PMCs. Nearly 60% of transcriptome changes in the set of transcripts mis-regulated in both mutants (N = 530) are enriched in PMCs, and only 1% are enriched in the tapetal cell transcriptome. All array data reported herein will be deposited and accessible at MaizeGDB http://www.maizegdb.org/.ConclusionsOur analysis of anther transcriptome modulations by two distinct am1 alleles, am1-489 and am1-praI, redefines the role of AM1 as a modulator of expression of a subset of meiotic genes, important for meiotic progression and provided stage-specific insights into the genetic networks associated with meiotic entry and early prophase I progression.

Cytochemical and ultrastructural observations of anthers and pollen grains in Lathyrus undulatus Boiss

Cytochemical and ultrastructural observations of anthers and pollen grains inLathyrus undulatusBoissInLathyrus undulatusBoiss. (Fabaceae), the young microspore stage of anther development was characterized by the enlarged secretory tapetal cells, which presented an intense reaction with regard to protein, insoluble polysaccharides and lipids. At bicellular pollen stage, the middle layer and the tapetum degenerated. After degradation of the tapetum, epidermis and single row U-shaped endothecium existed in the mature anther wall, and pollen grains remained in the locus. Young microspores had a spherical and centrally located nucleus with one or two nucleoli, many spherical lipid bodies and starchy plastids. A mature pollen grain contains insoluble polysaccharides, proteins, lipids and calcium. The mature pollen had the following morphological characteristics: 3-zonocolporate, prolate, tectate (imperforate) type of exine and perforate type of structure. The intine formed an important constituent portion of the wall, and consisted two sublayers: an outer intine (exintine) and an inner intine (endintine). The well-defined exine was made up of lipoidal substances and protein, but the intine composed of insoluble polysaccharides and protein. The bicellular state of the pollen grains persisted to anthesis.

Reproductive Meristem22 is a unique marker for the early stages of stamen development

Stamens undergo a very elaborate development program that gives rise not only to many specific tissue types, but also to the male gametes. The specification of stamen identity is coordinated by a group of homeotic genes such as APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA (SEP1-4) genes. Genome-wide transcriptomic comparisons between floral buds of wild-type and ap3 mutants led to the identification of the REM22 gene, which is expressed in the early stages of stamen development. This gene is member of the plant-specific B3 DNA-binding superfamily. In this work, we dissect the spatio-temporal expression pattern of REM22 during the early stages of stamen development. To this end, both in situ hybridization analyses as well as in vivo fluorescence strategies were employed. At stage 4 of flower development, REM22 is expressed exclusively in those undifferentiated cells of the floral meristem that will give rise to the stamen primordia. At stage 5, REM22 expression is restricted to the epidermal and the subepidermal layers of anther primordia. Later, this expression is confined to the middle layer and the differentiating tapetal cells. After stage 10 when all the tissues of the anther have differentiated, REM22 expression is no longer detectable. Furthermore, we examined the pREM22::GUS-GFP marker line in an inducible system where the ectopic AG function is used to promote microsporogenesis. The data support the idea that REM22 expression is a useful marker to study the early stages of stamen development.

LAP6/POLYKETIDE SYNTHASE AandLAP5/POLYKETIDE SYNTHASE BEncode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis thaliana

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.

Characteristics and abortion cytological pathways of male sterility of Bupleurum chinense

To study the characteristics of male sterility of Bupleurum chinense and further explore the developmental period and reason of abortion for the male sterile plants. The morphological characteristics of B. chinense male sterile and normal plants were investigated and compared. The anther development process and pollen viability of two types of plants were examined by microscopic assay. The shapes and sizes of anther and filament were different between the male sterile and the normal plants. For the male sterile plant's, the filament size was no more than 1/2 of that of normal plants and the anthers were shriveled, failed to dehisce and pollinate naturally, and the pollen grains in the anthers had no vitality. Other morphological characteristics were similar between two types of plants. The main reason leading to male sterility of B. chinense was the abnormal development of tapetum cells with two circumstances. The one is that the tapetum cells degraded early during the period of microsporocyte phase to tetrad phase and the other is that the tapetum cells proliferated with delayed degradation in the tetrad to uninucleate phase.

An ABCG/WBC‐type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.

A cis regulatory element in the TAPNAC promoter directs tapetal gene expression

The tapetum is a single cell layer surrounding the anther locule and its major function is to provide nutrients for pollen development. The ablation of tapetal cells interferes with pollen production and results in plant male sterility. In spite of the importance of this tissue in the quality and production of pollen grains, studies on promoter gene regulation of tapetal expressed genes are very few and there are no reports on specific cis regulatory sequences that control tapetal gene expression. We have identified a NAC gene, TAPNAC (At1g61110), specifically expressed in the Arabidopsis tapetum via transcriptional profiling. The TAPNAC promoter was studied in detail to identify cis regulatory sequences that confer tapetal specific expression. For this purpose, TAPNAC promoter elements were fused to the β-glucuronidase (GUS) reporter gene, and spatial and temporal GUS expression was monitored. The results showed that TAPNAC promoter-driven GUS expression emulates the expression of TAPNAC mRNA in anthers. A conserved TCGTGT motif was identified in the TAPNAC promoter and other tapetal expressed promoters. The TCGTGT motif enhances GUS expression in anthers of transgenic plants but only in the context of the TAPNAC promoter proximal region.