Morphological development of anthers induced by the dimorphic smut fungus<Emphasis Type="Italic"> Microbotryum violaceum</Emphasis> in female flowers of the dioecious plant<Emphasis Type="Italic"> Silene latifolia</Emphasis>

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.Abbreviations DAPI 4,6-diamidino-2-phenylidole - i infected - PMC pollen mother cell     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Characterization of <Emphasis Type="Italic">SpAPETALA3</Emphasis> and <Emphasis Type="Italic">SpPISTILLATA</Emphasis>, B class floral identity genes in <Emphasis Type="Italic">Spinacia oleracea</Emphasis>, 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.Electronic Supplementary Material Supplementary material is available for this article at Edited by G. Jürgens     

<Emphasis Type="Italic">odd-skipped</Emphasis> homologs function during gut development in<Emphasis Type="Italic"> C. elegans</Emphasis>

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family.Edited by C. Desplan     

<Emphasis Type="Italic">AOM3</Emphasis> and<Emphasis Type="Italic"> AOM4</Emphasis>: two MADS box genes expressed in reproductive structures of<Emphasis Type="Italic"> Asparagus officinalis</Emphasis>

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Microarray Analysis of Gene Expression Involved in Anther Development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> 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.     

Induction of male sterile cabbage using a tapetum-specific promoter from<Emphasis Type="Italic"> Brassica campestris</Emphasis> L. ssp.<Emphasis Type="Italic"> pekinensis</Emphasis>

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5 noncoding (promoter) region were different, except for the sequence from –281 to –89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.Abbreviations At Arabidopsis thaliana - Bc Brassica camepstris - Bn Brassica napus - DTx-A Diphtheria toxin A-chain gene - hpt Hygromycin phosphotransferase - PCR Polymerase chain reaction - SDS Sodium dodecyl sulfate - SSC Sodium chloride-sodium citrate bufferThis revised version was published online September 2003 with corrections to Figure 6.Communicated by I.S. Chung     

Meiotic abnormality in dominant genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis>

Rs1046AB is a dominant genic male sterile (DGMS) Brassica napus line derived from Yi-3A. Until now the molecular mechanism of its male sterility is still unknown. In this paper, cytological observations demonstrated that all cells in sterile plants contained condensed nuclei at the beginning stage of meiosis; this implied that meiotic cells were degenerating. Although 31% (93/300) cells escaped from the state of nuclei condensation in buds about 3 mm in length (in such length, normal plants are at tetrade stage), no cells could pass the pachytene stage. Then pachytene-or zygotene-like chromatin/chromosomes sometimes congregated into two or more groups with different size, which resulted in the formation of micronuclei. A nucleoplasmic bridge could also be found in some meiotic cells. Even when the “microspore’s analogue” appeared in sterile buds about 4 mm in length (in such length, mature pollens could be detected in normal buds), the nuclei condensation and escaped cells with a pachytene-like chromosome still could be found in the sterile anthers. So it could be concluded that male sterility was caused by meiotic abnormality. According to our previous research, four genes related to cell cycle/DNA processing were identified in fertile plants. RT-PCR further confirmed that three DNA repair genes were partially or completely repressed in the sterile plants and were only expressed in the early stage fertile flower buds, i.e., the buds <3 mm in length. Therefore, DGMS of rapeseed was probably caused by the abnormality in the DNA damage repair system during meiosis. According to these results, some possible mechanisms of fertility control were discussed.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

Gene expression pattern at desiccation in the anther of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Although gene expression profile of pollen has been described, there is limited information regarding a particular phase during anther/pollen development. This work characterizes gene expression pattern at desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) anthers. We have applied a suppression-subtractive hybridization (SSH) strategy, through which 90 clones were identified and sequenced. These clones resulted in the identification of 42 individual cDNAs among which 33 genes were specifically expressed at the desiccation phase of anthers of >150-mm buds. Fourteen cDNAs were chosen for further examination. Six genes were both dehydration- and abscisic acid (ABA)-inducible whereas the other eight genes were apparently dehydration-irrelevant. The group of dehydration- and ABA-induced genes was also induced by desiccation that developmentally occurs in the anther. The application of fluridone has a significant effect of inhibition on mRNA accumulation of these genes in maturing anthers during which desiccation occurs. Pollen germination analysis indicated that, of those dehydration-irrelevant genes, three were ABA-responsive and the other five were not. Thus, three separate signal pathways that function in the activation of late genes at desiccation during anther development are established. The first is the ABA-dependent pathway induced by environmental stress of dehydration. The other two pathways of signaling triggered by developmental cues, through which one is ABA-dependent and another is ABA-independent. The 14 gene proteins showed spatial and temporal expression patterns and may participate in membrane/cell wall synthesis, cytoskeletal organization, signaling, RNA binding, ubiquitin-mediated degradation and transportation during germination and tube growth. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The pea<Emphasis Type="Italic"> END1</Emphasis> promoter drives anther-specific gene expression in different plant species

END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the –330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity.Abbreviations GUS -Glucuronidase - uidA -Glucuronidase gene - Nos Nopaline synthase gene - nptII Neomycin phosphotransferase II gene - SEM Scanning electron microscopy GenBank accession numbers for the END1 cDNA and the END1 promoter: AY 091466 and AY 324651, respectively