Anther Tapetum Research Articles

Meiotic and mitotic behaviour of B chromosomes of ryegrass

Mitotic and meiotic analyses using conventional and fluorescent stains were employed in plants (accession ETBAZ 055 - origin: Capão do Leão, Rio Grande do Sul State, Brazil) from the Germplasm Active Bank of Ryegrass (Banco Ativo de Germoplasma de Azevém) of Embrapa, for observing the behaviour of B chromosomes. In several meiotic stages, there were observed up to two B chromosomes, which have presented an unstable behaviour regarding their precocious ascension to metaphase I or delays during anaphase I. At the end of the process, the Bs showed predominantly segregation for the nuclei under formation in order to guarantee their propagation. Concerning the cells that comprise the anther tapetum and the root meristems, the B chromosomes have presented a more stable behaviour.

Embryology of Petrosavia (Petrosaviaceae, Petrosaviales): evidence for the distinctness of the family from other monocots

The affinities of Petrosavia, a rare, leafless, mycoheterotrophic genus composed of two species indigenous to East to Southeast Asia, have long been uncertain. However, recent molecular analyses show that the genus is sister to Japonolirion osense. Japonolirion and Petrosavia comprise the Petrosaviaceae, which are now placed in its own order, Petrosaviales, distinct from other monocots based on molecular analyses. We conducted an embryological study of Petrosavia, comparing it to Japonolirion, as well as to basal monocots (Acorus and Araceae) and more derived monocots (Nartheciaceae, Velloziaceae, and Triuridaceae). Our results showed that Petrosavia is very similar in embryology to Japonolirion, with both genera sharing a glandular anther tapetum, simultaneous cytokinesis in microspore mother cells, anatropous and crassinucellate ovules, T-shaped tetrads of megaspores, ab initio Cellular-type endosperm, and a mature seed coat composed of the exotesta, endotesta, and endotegmen. The two genera of Petrosaviaceae are clearly distinct from Acorus, and all Araceae, Nartheciaceae, Velloziaceae, and Triuridaceae genera in various combinations of characters. Thus, both molecular and embryological evidence support the distinctness of the Petrosaviaceae from other monocots and its placement in its own order, Petrosaviales.

The EMS1 receptor kinase is required for non-meristematic proliferation of the Arabidopsis anther tapetum

The EMS1 receptor kinase is required for non-meristematic proliferation of the Arabidopsis anther tapetum

The EMS1 receptor kinase is required for non-meristematic proliferation of the Arabidopsis anther tapetum

The EMS1 receptor kinase is required for non-meristematic proliferation of the Arabidopsis anther tapetum

A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines

BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070, a member of the BAHD gene family of Arabidopsis thaliana. The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N(1),N(5),N(10)-trihydroxyferuloyl spermidine and N(1),N(5)-dihydroxyferuloyl-N(10)-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene (At1g67990) which co-regulated with SHT during flower development, was shown to be involved in the O-methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the beta-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.

Embryology of Japonolirion (Petrosaviaceae, Petrosaviales): a comparison with other monocots

Japonolirion osense, the sole species of the genus, endemic to Japan, which is placed together with Petrosavia in the Petrosaviaceae and the order Petrosaviales, is still poorly known with respect to systematic characters. Here I present an embryological study of the anther, ovule, and seed of J. osense. Japonolirion is characterized by a glandular anther tapetum, simultaneous cytokinesis in the microspore mother cell, two-celled mature pollen grains, anatropous and crassinucellate ovules, a two-cell-layered nucellar cap formed early in ovule development, antipodal cells hypertrophied in post-fertilization stages, the ab initio cellular mode of endosperm formation, and exotegmic seeds. Comparisons with the basal monocots Acorus (Acorales) and Araceae (Alismatales), and with the more derived monocots Nartheciaceae (Dioscoreales) and Velloziaceae/Triuridaceae (Pandanales), showed that Japonolirion is clearly distinct from those basal and more derived monocots, supporting a distinct position for Petrosaviaceae or Petrosaviales within the monocots. Extensive comparisons further suggest that the two-cell-layered nucellar cap, whose cells are rich in cytoplasm at the time of fertilization in Japonolirion and thus obviously function as the obturator, is likely to be a common characteristic of the basal monocots and may even be a link with the magnoliids.

Arabidopsis INOSITOL TRANSPORTER2 Mediates H+ Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane

Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices. In contrast, AtINT2 codes for a functional transporter. AtINT2 localization in the plasma membrane was demonstrated by transient expression of an AtINT2-GREEN FLUORESCENT PROTEIN fusion in Arabidopsis and tobacco (Nicotiana tabacum) epidermis cells and in Arabidopsis protoplasts. Its functional and kinetic properties were determined by expression in yeast (Saccharomyces cerevisiae) cells and Xenopus laevis oocytes. Expression of AtINT2 in a Deltaitr1 (inositol uptake)/Deltaino1 (inositol biosynthesis) double mutant of bakers' yeast complemented the deficiency of this mutant to grow on low concentrations of myoinositol. In oocytes, AtINT2 mediated the symport of H(+) and several inositol epimers, such as myoinositol, scylloinositol, d-chiroinositol, and mucoinositol. The preference for individual epimers differed from that found for AtINT4. Moreover, AtINT2 has a lower affinity for myoinositol (K(m) = 0.7-1.0 mm) than AtINT4 (K(m) = 0.24 mm), and the K(m) is slightly voltage dependent, which was not observed for AtINT4. Organ and tissue specificity of AtINT2 expression was analyzed in AtINT2 promoter/reporter gene plants and showed weak expression in the anther tapetum, the vasculature, and the leaf mesophyll. A T-DNA insertion line (Atint2.1) and an Atint2.1/Atint4.2 double mutant were analyzed under different growth conditions. The physiological roles of AtINT2 are discussed.

Receptor‐like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.

Arabidopsis male sterility1 regulates programmed cell death in the anther tapetum and pollen wall development

Arabidopsis male sterility1 regulates programmed cell death in the anther tapetum and pollen wall development

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.

Embryology of Eusideroxylon (Cryptocaryeae, Lauraceae) and character evolution in the family

Lauraceae are relatively well-known embryologically and embryological data are available for 23 of about 50 genera. In this paper we present the embryology of Eusideroxylon, an unstudied and key genus within Cryptocaryeae, which are positioned basally in the evolution of Lauraceae, and discuss the evolution of embryological characters in the family. Based on comparisons of over 50 characters, it was found that Eusideroxylon is consistent with Aspidostemon, the core Cryptocaryeae (Beilschmiedia, Cryptocarya, Endiandra and Potameia), Caryodaphnopsis and Cassytha in having a glandular anther tapetum. The core Cryptocaryeae further agrees with both Caryodaphnopsis and Cassytha in having an embryo sac protruding from the nucellus. In light of the phylogenetic trees available, both the glandular tapetum and the embryo sac protruding from the nucellus have evolved as homoplasies in Lauraceae, once each in a clade of Cryptocaryeae, and the Caryodaphnopsis and Cassytha clade, respectively. Such character-state distributions suggest that it is better to place both Caryodapnopsis and Cassytha in the same clade as the core Cryptocaryeae. Embryologically, Eusideroxylon appears to have an intermediate state between Hypodaphnis, a genus positioned basal-most in the family, and the core Cryptocaryeae. Supplementary data on the anther and seed of Hypodaphnis are also provided.

Dominance relationships between self-incompatibility alleles controlled by DNA methylation

In crucifers, the pollen S-determinant gene, SP11, is sporophytically expressed in the anther tapetum, and the pollen self-incompatibility phenotype is determined by the dominance relationships between the two S-haplotypes it carries. We report here that 5' promoter sequences of recessive SP11 alleles are specifically methylated in the tapetum before the initiation of SP11 transcription. These results suggest that tissue-specific monoallelic de novo DNA methylation is involved in determining the dominance interactions that determine the cruciferous self-incompatibility phenotype.

Pollenkitt – its composition, forms and functions

Two types of sticky pollen coat material exist in angiosperms, both produced by the anther tapetum. Pollenkitt is the most common adhesive material present around pollen grains of almost all angiosperms pollinated by animals, whereas tryphine seems to be restricted only to Brassicaceae. Tapetal cell protoplasts have different patterns of development according to the products formed during their development and degeneration. If tryphine is formed, the tapetal cell protoplasts lose their individuality at the microspore stage. If pollenkitt is formed, their contents degenerate at later stages. Cell content is totally reabsorbed, when ripe pollen is not surrounded by any gluing material. Current knowledge of pollenkitt formation, deposition on pollen grains and chemical composition are reviewed and discussed. Methods for detecting this viscous fluid are also presented. The many functions of pollenkitt, deduced from personal observations and the literature, act in the period between anther opening and pollen hydration on the stigma; they are: (1) to hold pollen in the anther until dispersal; (2) to enable secondary pollen presentation; (3) to facilitate pollen dispersal; (4) to protect pollen from water loss; (5) to protect pollen from ultra-violet radiation; (6) to maintain sporophytic proteins responsible for pollen–stigma recognition inside exine cavities; (7) to protect pollen protoplasts from fungi and bacteria; (8) to keep together pollen grains during transport; (9) to protect pollen from hydrolysis and exocellular enzymes; (10) to render pollen attractive to animals; (11) to render pollen visible to animal eyes; (12) to hide pollen from animal eyes; (13) to avoid predation of pollen through smell; (14) to enable adhesion to insect bodies; (15) to enable pollen packaging by bees and to form corbicules; (16) to provide a digestible reward for pollinators; (17) to enable pollen clumps to reach the stigma; (18) to allow self-pollination; (19) to facilitate adhesion to the stigma; (20) to facilitate pollen rehydration. Depending on the developmental program of the species, these functions may act during pollen presentation, in relation to pollinators, during pollen dispersal and when pollen reaches the stigma.

Survey of glycine-rich proteins (GRPs) in the Eucalyptus expressed sequence tag database (ForEST)

The occurrence of quasi-repetitive glycine-rich peptides has been reported in different organisms. Glycine-rich regions are proposed to be involved in protein-protein interactions in some mammalian protein families. In plants, a set of glycine-rich proteins (GRPs) was characterized several years ago, and since then a wealth of new GRPs have been identified. GRPs may have very diverse sub-cellular localization and functions. The only common feature among all different GRPs is the presence of glycine-rich repeat domains. The expression of genes encoding GRPs is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. In addition to the highly modulated expression, several GRPs also show tissue-specific localization. GRPs specifically expressed in xylem, phloem, epidermis, anther tapetum and roots have been described. In this paper, the structural and functional features of these proteins in Eucalyptus are summarized. Since this is the first description of GRPs in this species, particular emphasis has been given to the expression pattern of these genes by analyzing their abundance and prevalence in the different cDNA-libraries of the Eucalyptus Genome Sequencing Project Consortium (ForEST). The comparison of GRPs from Eucalyptus and other species is also discussed.

AtMYB32 is required for normal pollen development in Arabidopsis thaliana

AtMYB32 gene is a member of the R2R3 MYB gene family coding for transcription factors in Arabidopsis thaliana. Its expression pattern was analysed using Northern blotting, in situ hybridization and promoter-GUS fusions. AtMYB32 is expressed in many tissues, but most strongly in the anther tapetum, stigma papillae and lateral root primordia. AtMYB32-GUS was induced in leaves and stems following wounding, and in root primordia by auxin. T-DNA insertion populations were screened and two insertion mutants were identified, both of which were partially male sterile, more than 50% of the pollen grains being distorted in shape and lacking cytoplasm. AtMYB4 is closely related to AtMYB32 and represses the CINNAMATE 4-HYDROXYLASE gene. Distorted pollen grains were produced in both AtMYB4 insertion mutant and overexpression lines. In an AtMYB32 insertion mutant, the transcript levels of the DIHYDROFLAVONOL 4-REDUCTASE and ANTHOCYANIDIN SYNTHASE genes decreased while the level of the CAFFEIC ACID 0-METHYLTRANSFERASE transcript increased. Change in the levels of AtMYB32 and AtMYB4 expression may influence pollen development by changing the flux along the phenylpropanoid pathways, affecting the composition of the pollen wall.

Subcellular heterogeneity of mitochondrial membrane potential in anther tapetum

Studies on animal material have revealed that changes in the mitochondrial permeability transition pore (PTP), which cause a reduction in the mitochondrial transmembrane potential (ΔΨm) followed by release of cytochrome c, belong to the earliest manifestations of some types of apoptosis. We have attempted to monitor the ΔΨm of mitochondria during programmed cell death (PCD) of the secretory tapetum using JC-1, a fluorochrome dye that detects mitochondrial membrane potential and to relate changes in this potential to mitochondrial ultrastructure. Analysis of tapetal cells isolated from Ornithogalum virens anthers revealed that the ΔΨm of mitochondria in the tapetal cells alters during development; the change, however, is not uniform in the mitochondrial population within a single tapetal cell. In young tapetal cells, at the tetrad stage, we detected only the red fluorescence of JC-1 aggregates in all tapetal mitochondria, which indicates highly negative ΔΨm. In an advanced stage of PCD at the late microspore stage, in each tapetal cell we detected both mitochondria with red (as formerly) and mitochondria with green fluorescence. The green fluorescence of JC-1 monomers indicates mitochondria with depolarised membranes. These changes in ΔΨm are related to observed changes in mitochondria ultrastructure. This is the first documentation of intracellular heterogeneity of ΔΨm during anther tapetum development. Alteration in ΔΨm suggests a relationship between mitochondrial function and PCD processes in tapetal cells.

Systematic significance of cell inclusions in Haemodoraceae and allied families: silica bodies and tapetal raphides.

This paper presents the first record of silica deposits in tissues of Haemodoraceae and adds new records of tapetal raphides in this family. Within the order Commelinales, silica is present in leaves of three families (Hanguanacaeae, Haemodoraceae and Commelinaceae), but entirely absent from the other two (Pontederiaceae and Philydraceae). Presence or absence of characteristic cell inclusions may have systematic potential in commelinid monocotyledons, although the existing topology indicates de novo gains and losses in individual families. Silica sand was observed in leaves of five out of nine genera examined of Haemodoraceae, predominantly in vascular bundle sheath cells and epidermal cells. Within Haemodoraceae, silica is limited to subfamily Conostylidoideae. The occurrence of silica in Phlebocarya supports an earlier transfer of this genus from Haemodoroideae to Conostylidoideae. The presence of raphides (calcium oxalate crystals) in the anther tapetum represents a rare character, only reported in a few monocot families of the order Commelinales, and possibly representing a mechanism for regulation of cytoplasmic free calcium levels. Tapetal raphides were observed here in Anigozanthus and Conostylis (both Haemodoraceae), and Tradescantia (Commelinaceae), thus supplementing two earlier records in Haemodoraceae, Philydraceae and Commelinaceae.

Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.