Chemical agents that inhibit pollen development: tools for research

Summary Several unrelated compounds are known to selectively inhibit the development of the male gametophyte. When applied at suitable dosages to plants at the appropriate stages of anther development, these substances block the formation of fertile pollen. The affected stage of pollen development is characteristic of the specific chemical structure of the compound, ranging from effects on microspore meiosis to the formation of pollen defective in the ability to germinate or fertilize. The range of effects mediated by these substances, and by known male-sterile mutants, indicates that microspore development has several critical phases that are particularly sensitive to fatal inhibition. We propose that chemical inhibitors of pollen development deserve attention as tools for elucidating the regulation of pollen development.     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

Analysis of gene expression profile in pollen development of recessive genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. line S45A

Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

A lectin from Bryonia dioica root stocks

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.Abbreviations BDA Bryonia dioica agglutinin - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

Structural, histochemical, and protein analysis of male reproductive development in willow

Information on the development of the male reproductive structures in willow will help advance our understanding of its reproductive behavior and contribute to our ability to work towards its improvement. Willow also offers the opportunity to study male sterility, a subject matter which is not typically dealt with in woody plants. As compared to the three willow species examined (Salix eriocephala, S. exigua, and S. purpurea), pollen development in S. discolor S365 showed several abnormalities starting with the delay in meiosis. This lasted for about 10 days and meiosis eventually occurred as manifested by the formation of microspores. However, most of the resulting microspores collapsed, while only a few developed into pollen grains. The large number of undeveloped and disintegrated microspores appeared to make the few pollen grains sticky, preventing them from being dispersed. Histochemical analysis showed that meiosis in most species of willow was associated with the presence of large amounts of insoluble polysaccharides in the anther wall layers, but only very few of these were observed in S. discolor. Also, a 32-kDa protein which is the most abundant protein in the reproductive structures of willow, was absent in S. discolor S365. Proteomic analysis showed that this is similar to the storage proteins in Populus x canadensis and P. deltoides. Therefore, male sterility in S. discolor may be due to some genetic defects affecting the accumulation of essential reserves in its reproductive structures. The mechanism behind this is unknown, but this study has established the nature of sterility in S. discolor S365.     

Ragweed pollen: The aeroallergen is spreading in Italy

Presently in Europe, ragweed pollen as an aeroallergen is not as important as Poaceae,Parietaria or Betulaceae, even if in some countries the plant is beginning to influence the local composition of the airborne pollen spectra. In northern Italy, the presence of ragweed airborne pollen has only been reported since the beginning of the 1980’s and it is increasingly spreading from year to year. Given this situation, the allergologists have begun to regard the potential risk of sensitisation to ragweed pollen with much attention. Up to now, such pollen has not been included in the routine allergological tests. In 1995 in some sites of northern Italy (Turin, Milan, Trieste), the concentration values of ragweed pollen were remarkable (∼ 20–30 p/m³) and on the increase with respect to the previous years. This investigation aims at focusing the atmospheric concentration trend on this new aeroallergen (Ambrosia sp.) in Italy from 1991 throughout 1995.     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric pointTo whom correspondence should be addressed.     

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. Data deposition: The sequence reported in this paper have been deposited in the GeneBank database (Accession No. U12171)     

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear “chimeric” at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.Shelley R. Hepworth and Jennifer E. Klenz contributed equally to this work.