Mature Anthers Research Articles

Suppressed expression of RETROGRADE-REGULATED MALE STERILITY restores pollen fertility in cytoplasmic male sterile rice plants

Conflict/reconciliation between mitochondria and nuclei in plants is manifested by the fate of pollen (viable or nonviable) in the cytoplasmic male sterility (CMS)/fertility restoration (Rf) system. Through positional cloning, we identified a nuclear candidate gene, RETROGRADE-REGULATED MALE STERILITY (RMS) for Rf17, a fertility restorer gene for Chinese wild rice (CW)-type CMS in rice (Oryza sativa L.). RNA interference-mediated gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is a candidate gene to be regulated via retrograde signaling. We found that a reduced-expression allele of the RMS gene restored fertility in haploid pollen, whereas a normal-expression allele caused pollen to die in the CW-type CMS. RMS encodes a mitochondrial protein, 178 aa in length, of unknown function, unlike the majority of other Rf genes cloned thus far, which encode pentatricopeptide repeat proteins. The unique features of RMS provide novel insights into retrograde signaling and CMS.

Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis

Magnesium (Mg(2+)) is abundant in plant cells and plays a critical role in many physiological processes. A 10-member gene family AtMGT (also known as AtMRS2) was identified in Arabidopsis, which belongs to a eukaryote subset of the CorA superfamily, functioning as Mg(2+) transporters. Some family members (AtMGT1 and AtMGT10) function as high-affinity Mg(2+) transporter and could complement bacterial mutant or yeast mutant lacking Mg(2+) transport capability. Here we report an AtMGT family member, AtMGT9, that functions as a low-affinity Mg(2+) transporter, and is essential for pollen development. The functional complementation assay in Salmonella mutant strain MM281 showed that AtMGT9 is capable of mediating Mg(2+) uptake in the sub-millimolar range of Mg(2+). The AtMGT9 gene was expressed most strongly in mature anthers and was also detectable in vascular tissues of the leaves, and in young roots. Disruption of AtMGT9 gene expression resulted in abortion of half of the mature pollen grains in heterozygous mutant +/mgt9, and no homozygous mutant plant was obtained in the progeny of selfed +/mgt9 plants. Transgenic plants expressing AtMGT9 in these heterozygous plants can recover the pollen phenotype to the wild type. In addition, AtMGT9 RNAi transgenic plants also showed similar abortive pollen phenotype to mutant +/mgt9. Together, our results demonstrate that AtMGT9 functions as a low-affinity Mg(2+) transporter that plays a crucial role in male gametophyte development and male fertility.

A Novel Fatty Acyl-CoA Synthetase Is Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis

Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein-ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes.

Effects of High Nitrogen Supply on the Susceptibility to Coolness at the Young Microspore Stage in Rice (Oryza sativa L.): Gene Expression Analysis in Mature Anthers

abstractChanges in gene expression patterns by high nitrogen (High-N) and High-N plus cooling at the young microspore stage (High-N-cooling) in rice mature anthers were analyzed by semiquantitative RT-PCR with gene specific primers. Gene expression of alpha-expansin 18 (EXPA18) was repressed under High-N-cooling. Beta-expansin 1 (EXPB1), putative aldehyde dehydrogenase (ALDH) and Fructokinase II (FKII) were upregulated under High-N. EXPB1 and FKII were highly expressed under High-N-cooling. Comprehensive examination of gene expression patterns of 26 alpha-expansins (EXPAs) and 16 beta-expansins (EXPB s) showed that all expansins (EXP s)except EXPA12 were expressed in the anthers. Gene expression of EXP s did not change under High-N except EXPA1, EXPB1 and EXPB5 which were upregulated. In total, 18 EXPAs and 6 EXPB s were repressed under High-N-cooling, and among these, EXPA18, EXPA19 and EXPA20 had high similarities in the amino acid sequences, suggesting that these three genes may constitute a distinct functional gene subfamily related to the decrease in the pollen germination ability.

An anther‐specific dihydroflavonol 4‐reductase‐like gene (DRL1) is essential for male fertility in Arabidopsis

Arabidopsis contains only one functional dihydroflavonol 4-reductase (DFR) gene, but several DFR-like genes encoding proteins with the conserved NAD(P)H binding domain. At4g35420, named DRL1 (Dihydroflavonol 4-reductase-like1), is a closely related homolog of the rice anther-specific gene OsDFR2 reported previously. Two T-DNA mutants (drl1-1 and drl1-2) were found to have impaired pollen formation and seed production. Histological analysis revealed defective microspore development after tetrad release in both mutants. Microspore walls were found to rupture, releasing the protoplasts which eventually degenerated. The DRL1 promoter is anther-specific in closed flower buds. Promoter-GUS analysis in transgenic Arabidopsis revealed expression in tapetum, tetrads, and developing microspores, but not in mature anthers. Enhanced yellow fluorescent protein (EYFP)-localization analysis demonstrated that DRL1 is a soluble cytosolic protein that may also be localized in the nucleus. Restoration of male fertility and seed formation was only achieved by a native promoter-DRL1 construct, but not by a 35S-DRL1 construct, demonstrating the importance of spatial and temporal specificities of DRL1 expression. DRL1 may be involved in a novel metabolic pathway essential for pollen wall development. DRL1 homologs were identified as anther- and floral-specific expressed sequence tags from different species, suggesting that DRL1 may have a conserved functional role in male fertility in flowering plants.

Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis

Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall-associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice.

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA4 accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA4 in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.

Embryology of Swertia (Gentianaceae) relative to taxonomy

The embryological features of three species of Swertia (s.l.) - S. erythrosticta, S. franchetiana, and S. tetraptera were characterized, and the observations were used, together with previously gathered data on other species, to evaluate a recently proposed polyphyly, based on molecular data, of Swertia s.l. Comparisons of species within the genus showed that they have diversified embryologically, and there are significant between-species differences. Notable features that vary between species include the number of cell layers that form the anther locule wall, the construction of the wall of the mature anther, tapetum origin, the cell number in mature pollen grains, the structure of the fused margins of the two carpels, the ovule numbers in placental cross-sections, the shape of the mature embryo sac, the degree of ovule curvature, antipodal variation and the presence of a hypostase, and seed appendages. They share characters that are widely distributed in the tribe Gentianeae, such as a dicotyledonous type of anther wall formation, a glandular tapetum with uninucleate cells, simultaneous cytokinesis following the meiosis of the microsporocytes, tetrahedral microspore tetrads, superior, bicarpellary and unilocular ovaries, unitegmic and tenuinucellar ovules, Polygonum-type megagametophytes, progamous fertilization, nuclear endosperm, and Solanad-type embryogeny. The presence of variation in embryological characters amongst the species of Swertia s.l. strongly supports the view that Swertia s.l. is not a monophyletic group. Frasera is better separated from Swertia s.l. as an independent genus, and is only distantly related to Swertia s. s. judging from the numerous differences in embryology. Swertia tetraptera is very closely related to Halenia, as they show identical embryology. (C) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 383-400.

Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis

Downregulation of the transcription factor AtMYB103 using transgenic technology results in early tapetal degeneration and pollen aberration during anther development in Arabidopsis thaliana. This paper describes the functional analysis of the AtMYB103 gene in three knock-out mutants. Two male sterile mutants, ms188-1 and ms188-2, were generated by ethyl-methane sulfonate (EMS) mutagenesis. A map-based cloning approach was used, and ms188 was mapped to a 95.8-kb region on chromosome 5 containing an AtMYB103 transcription factor. Sequence analysis revealed that ms188-1 had a pre-mature stop codon in the AtMYB103 coding region, whereas ms188-2 had a CCT-->CTT base-pair change in the first exon of AtMYB103, which resulted in the replacement of a proline by a leucine residue in the R2R3 domain. The third mutant, an AtMYB103 transposon-tagging line, also showed a male sterile phenotype. Allelism tests indicated that MS188 and AtMYB103 belong to the same locus. Cytological observation revealed defective tapetum development and altered callose dissolution in ms188 plants. Additionally, most of the microspores in mature anthers were degraded and surviving microspores lacked exine. AtMYB103 encoded an R2R3 MYB protein that is predominantly located in the nucleus. Real-time RT-PCR analysis indicated that the callase-related gene A6 was regulated by AtMYB103. Expression of the exine formation gene MS2 was not detected in mutant anthers. These results implicate that AtMYB103 plays an important role in tapetum development, callose dissolution and exine formation in A. thaliana anthers.

Genome-Wide Gene Expression Profiling Reveals Conserved and Novel Molecular Functions of the Stigma in Rice

In angiosperms, the stigma provides initial nutrients and guidance cues for pollen grain germination and tube growth. However, little is known about the genes that regulate these processes in rice (Oryza sativa). Here, we generate rice stigma-specific or -preferential gene expression profiles through comparing genome-wide expression patterns of hand-dissected, unpollinated stigma at anthesis with seven tissues, including seedling shoot, seedling root, mature anther, ovary at anthesis, seeds 5 d after pollination, 10-d-old embryo, 10-d-old endosperm, and suspension-cultured cells by using both 57 K Affymetrix rice whole-genome array and 10 K rice cDNA microarray. A high reproducibility of the microarray results was detected between the two different technology platforms. In total, we identified 548 genes to be expressed specifically or predominantly in the stigma papillar cells of rice. Real-time quantitative reverse transcription-polymerase chain reaction analysis of 34 selected genes all confirmed their stigma-specific expression. The expression of five selected genes was further validated by RNA in situ hybridization. Gene Ontology analysis shows that several auxin-signaling components, transcription, and stress-related genes are significantly overrepresented in the rice stigma gene set. Interestingly, most of them also share several cis-regulatory elements with known stress-responsive genes, supporting the notion of an overlap of genetic programs regulating pollination and stress/defense responses. We also found that genes involved in cell wall metabolism and cellular communication appear to be conserved in the stigma between rice and Arabidopsis (Arabidopsis thaliana). Our results indicate that the stigmas appear to have conserved and novel molecular functions between rice and Arabidopsis.

Retrograde regulation of nuclear gene expression in CW-CMS of rice

The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS.

Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant

The Arabidopsis male sterility1 mutation results in mature anthers that are devoid of pollen. Meiosis and early development progress normally; however, after microspore release, the microspore cytoplasm and tapetum become abnormally granular and vacuolated, and degeneration occurs. Pollen wall development is seriously affected; primexine formation within the callose wall appears to occur normally, however, once the callose is degraded, abnormal deposits of electrodense material are detected which result in irregular spike-shaped structures, rather than the characteristic rod-like shape of the wild-type bacula. The internal intine wall is also reduced compared with wild type. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) staining and ultrastructural analysis have indicated that programmed cell death (PCD) occurs in the wild-type tapetum after microspore mitosis I. However, no signs of PCD are seen in the ms1 tapetum, where large autophagic vacuoles and mitochondrial swelling suggest that necrotic-based breakdown of the tapetum is occurring in the ms1 mutant rather than the normal, regulated PCD process. After the formation of the large, autophagic vacuole in the tapetum, TUNEL staining is detected in the mutant microspores, indicating that they may go through a PCD-based breakdown as a secondary consequence of the observed tapetal aberrations. Based on these observations, two possible roles for MS1 can be hypothesized; MS1 may function by modifying the transcription of tapetal-specific genes implicated in pollen wall development, which then regulate pollen wall material secretion and in turn wall development and tapetal PCD. Alternatively, the MS1 gene may control tapetal development by directly regulating tapetal PCD and breakdown.

Developmental regulation of K accumulation in pollen, anthers, and papillae: are anther dehiscence, papillae hydration, and pollen swelling leading to pollination and fertilization in barley (Hordeum vulgare L.) regulated by changes in K concentration?

The presence of potassium (K) in pollen, anthers and papillae from barley (Hordeum vulgare L.) flowers with different levels of developmental stages starting from boot stage to fully mature flower, was studied by using the K-sensitive fluorescent dye PBFI (potassium-binding benzofuran isophthalate) and confocal laser scanning microscopy. The presence of heavy K fluorescence was detected only at the aperture area of the mature pollen. Similarly, the presence of K increased with the progression from immature to mature anther and papillae. In addition, a higher concentration of K was observed only at the stomium area (the place of anther dehiscence) of mature anthers. Keeping in view the role of K as an active osmoticum and the consistent and synchronized appearance of K in mature pollen, anthers, and papillae, it was concluded that K may regulate anther dehiscence, pollen imbibition, and papillae hydration leading to pollination and fertilization.

The effect of high temperatures on the expression and activity of sucrose-cleaving enzymes during tomato (Lycopersicon esculentum) anther development

SummaryExposure to high temperatures causes reduced yields in tomato (Lycopersicon esculentum), mainly by affecting male gametophyte development. The effect of heat stress on pollen characteristics is associated with changes in carbohydrate metabolism in the developing anthers. The expression and activities of sucrose-cleaving enzymes and the amounts of starch and soluble sugars were measured in tomato (inbred line ‘NC 8288’) anthers at four stages of development, under ‘normal’ controlled (day/night temperatures of 26°/18°C) and high-temperature (day/night temperatures of 32°/26°C) conditions. Heat stress was found to cause a reduction in cell wall-bound acid invertase activity in the anthers of flower buds 5 d before anthesis (DBA), which correlated with reduced starch accumulation at 3 DBA, and an induction of the activities of sucrose synthase and soluble acid invertase in maturing anthers. The heat stress-induced increases in soluble acid invertase and sucrose synthase activities close to anthesis were found to correlate with a three-fold increase in the hexose:sucrose ratio in mature anthers. The heat-stress conditions applied altered the gene expression profiles of the enzymes tested, decreasing the steady-state level of the mRNA coding for cell wall-bound acid invertase at 5 DBA, increasing expression of the soluble acid invertase gene at 5 DBA, and decreasing expression of the sucrose synthase gene at 5 and 3 DBA. These data indicate that sucrose-cleaving enzymes in tomato anthers respond, at both the mRNA and enzyme activity levels, to high-temperature conditions, that the response is dependent upon the stage of flower development, and that it may involve post-transcriptional control.

Susceptibility to Coolness at the Young Microspore Stage under High Nitrogen Supply in Rice (Oryza Sativa L.). Proteome Analysis of Mature Anthers

In vitro pollen germination experiment using agar plates showed that the growth under high nitrogen conditions enhanced the damage to pollen germination ability caused by the cooling at the young microspore stage. To clarify the physiological factors related to this damage to pollen germination, we performed the comparative proteome analysis of mature anthers and identified proteins that were changed by high nitrogen conditions or high nitrogen plus cooling conditions. Proteins were extracted from mature anther samples and separated by two-dimensional polyacrylamide gel electrophoresis. By comparing anther protein maps of the samples collected from the plants grown under standard nitrogen conditions, high nitrogen conditions and high nitrogen plus cooling conditions, we found 11 protein spots, which varied with the treatment. These protein spots were identified based on the rice proteome database and/or peptide mass fingerprinting (PMF) analysis after digestion with trypsin. Digested samples were analyzed by matrix-assisted laser desorption/ionization-time flight mass spectrometry to produce PMF data. Database searches using these PMF data revealed the identities of 9 proteins. Seven of these proteins were polypeptides involved in cell elongation, stress responses and sugar metabolism. The relation between the fluctuations of these proteins and the decrease in pollen germination are discussed.

Identification and characterization of a heat-inducible ftsH gene from tomato ( Lycopersicon esculentum Mill.)

A heat-inducible filamentation temperature-sensitive H ( ftsH)-like gene was cloned from tomato leaves, and the deduced protein precursor was predicted to be targeted to the chloroplast. Because the protein structure was highly homologous to Arabidopsis AtFtsH6, the cloned tomato ftsH-like cDNA was named LeftsH6. It was characterized as heat-inducible and rapidly responsive to heat stress. However, no LeftsH6 expression was detected after cold, salt, drought or light stress challenges. Heat shock elements (HSEs) were identified in the 5′-flanking sequence of LeftsH6, which specifically bound heat shock factor (HSF) HsfA2 from tomato as determined by electrophoretic mobility shift assay (EMSA) analysis. Subsequently, transgenic tobacco plants carrying the β-glucuronidase (GUS) gene driven by the LeftsH6 promoter were generated. High levels of heat-induced GUS staining were detected in the leaves, roots and flowers of transgenic plants. The ovaries, stigmas, anthers and sepals in the flowers at various developmental stages, as well as the pollen grains of mature anthers, showed strong heat-induced GUS staining. In the leaves of transgenic tobacco, the highest level of fluorometric GUS activity was detected at 40 °C. These results showed that LeftsH6 is a typical heat shock gene and its thermoinducibility is mediated by interactions between HSEs of the LeftsH6 promoter and heat shock factor.

Microarray Analysis of Gene Expression Involved in Anther Development in rice (Oryza sativa L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA3) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA3 and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA3- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA3 or JA treatment, and 24 GA3- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA3. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA3 signals in the control of anther development in rice.

Ultrastructure of Pollen Grains from Forced and Unforced Shrubs of Common Lilac

Forcing makes possible to induce plant flowering independently of the season. In lilac, high temperature is the factor that breaks deep dormancy. The deepest dormancy occurs between the end of October and the end of December. Depending on the depth of dormancy, the starting temperatures required for forcing are 37°C in November, 31°C in December, and 25°C in January–March. Under natural conditions, the temperature inducing the inflorescence bud breaking is 6°C, whereas 9°C and 13°C or more allow inflorescence elongation and flowering, respectively (Kronenberg, 1994). In the present work, the effect of high temperature at the beginning of the forcing cycle on the structure of developing pollen grains of common lilac was investigated. Pollen grains from the outdoor-grown (control) shrubs showed no signs of degeneration. They were spherical, three-colpate to colporoidate, and bicellular, and contained large numbers of lipid bodies. High temperatures at the early forcing cycle (November) resulted in the degeneration of most microspores. The first signs of degeneration (cytoplasm plasmolysis) were observed at the tetrad stage and in mature anthers; the microspores consisted only of the outer and inner sporopolenin layers.

Embryology of Megacodon stylophorus and Veratrilla baillonii (Gentianaceae): descriptions and systematic implications

The embryological characters of Megacodon stylophorus (C. B. Clark) H. Smith and Veratrilla baillonii Franch. are described for the first time and the systematic relationships of Megacodon and Veratrilla are discussed. Both species share the following embryological features. The anthers are tetrasporangiate. The formation of anther walls is of the Dicotyledonous Type. The tapetum is of glandular type with uninucleate cells which have a dual origin. The tapetal cells on the connective side show radial elongation or periclinal division and intrude into the anther locule to form ‘placentoids’. Cytokinesis in the microsporocyte meiosis is of the simultaneous type and the microspore tetrads are nearly always tetrahedral, rarely decussate. The ovary is bicarpellate and unilocular. The ovule is unitegmic and tenuinucellar. The formation of the embryo sac is of the Polygonum Type. Before fertilization, the two polar nuclei fuse into a secondary nucleus. Fertilization is porogamous. The development of the endosperm is of the Nuclear Type. However, the two species show variation in the following features: the number of cell layers which form the anther locule wall; construction of the wall of the mature anther; cell number in mature pollen grains; ovule number in cross sections of placentae; degree of ovule curvature; character of the hypostase and seed shape. In a comparison with the other taxa in the tribe Gentianeae using embryological features, Megacodon is referred to as an independent genus and should be treated as a member of the subtribe Swertiinae; Veratrilla is better separated from Swertia s.l. as an independent genus. Veratrilla is more derived than Swertia s.s. and shows a close relationship with S. tetraptera.

Characterization of SpAPETALA3 and SpPISTILLATA, B class floral identity genes in Spinacia oleracea, and their relationship to sexual dimorphism

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3' and 5' RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.