Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

Analysis of microspore-specific promoters in transgenic tobacco

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.     

Zm401, a short-open reading-frame mRNA or noncoding RNA, is essential for tapetum and microspore development and can regulate the floret formation in maize

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. Previously, we cloned a pollen specific gene, zm401, from a cDNA library generated from the mature pollen of Zea mays. Expression of partial cDNA of zm401 in maize and ectopic expression of zm401 in tobacco suggested it may play a role in anther development. Here we present the expression and functional characterization of this pollen specific gene in maize. Zm401 is expressed primarily in the anthers (tapetal cells as well as microspores) in a developmentally regulated manner. That is, it is expressed from floret forming stage, increasing in concentration up to mature pollen. Knockdown of zm401 significantly affected the expression of ZmMADS2, MZm3-3, and ZmC5, critical genes for pollen development; led to aberrant development of the microspore and tapetum, and finally male-sterility. Zm401 possesses highly conserved sequences and evolutionary conserved stable RNA secondary structure in monocotyledon. These data show that zm401 could be one of the key growth regulators in anther development, and functions as a short-open reading-frame mRNA (sORF mRNA) and/or noncoding RNA (ncRNA).     

Actin microfilament organization during pollen development ofBrassica napus cv. Topas

Summary The organization of actin microfilaments (MFs) was studied during pollen development ofBrassica napus cv. Topas. Cells were prepared using three techniques and double labelled for fluorescence microscopy with rhodamine-labelled phalloidin for MFs and Hoechst 33258 for DNA. Microfilaments are present at all stages of pollen development with the exception of tricellular pollen just prior to anthesis. Unicellular microspores contain MFs which radiate from the surface of the nuclear envelope into the cytoplasm. During mitosis MFs form a network partially surrounding the mitotic apparatus and extend into the cytoplasm. Both cytoplasmic and phragmoplast-associated MFs are present during cytokinesis. Nuclear associated-, cytoplasmic, and randomly oriented cortical MFs appear in the vegetative cell of the bicellular microspore. Cortical MFs in the vegetative cell organize into parallel MF bundles (MFBs) aligned transverse to the furrows. The MFBs disappear prior to microspore elongation. At anthesis MFs are restricted to the cortical areas subjacent to the furrows of the vegetative cell. The use of cytochalasin D to disrupt MF function resulted in: (1) displacement of the acentric nucleus in the unicellular microspore; (2) displacement of the spindle apparatus in the mitotic cell; (3) symmetrical growth of the bicellular microspore rather than elongation and (4) inhibition of pollen tube germination in the mature pollen grain. This suggests that MFs play an important role in anchoring the nucleus in the unicellular microspore as well as the spindle apparatus during microspore mitosis, in microspore shape determination and in pollen tube germination.Abbreviations MF microfilament - MFB microfilament bundle - rhph rhodamine phalloidin Dedicated to the memory of Professor John G. Torrey     

Development-related changes of protein ubiquitination in pollen from male and female kiwifruit (Actinidia deliciosa)

Kiwifruit (Actinidia deliciosa) is a dioecious vine whose staminate and pistillate flowers nonetheless develop non-functional reproductive structures of the ompposite sex. Ubiquitin is a small, highly conserved protein found in all eucaryotes: a covalent ATP-dependent attachment of ubiquitin marks proteins for degradation. In the present paper, we used immunoblotting to investigate the presence of free ubiquitin and ubiquitin conjugates during pollen development in male (androfertile) and in female (androsterile) genotypes of kiwifruit. In the male, several high molecular mass protein conjugates were present throughout development. On the contrary, such a pattern characterized only early stages of pollen from the female genotype, where conjugates progressively disamppeared, until they were detectable only in trace amounts at anthesis. The highest content of conjugates in the male genotype was observed when microspores were ampproaching the first mitosis. Free ubiquitin increased continuously during development of the male microgametophyte so that mature pollen contained considerable amounts of the ubiquitin monomer at the time of its release from the anther. By contrast, only low levels were detectable in the degenerating microspores in the pistillate flowers. In vitro experiments using labeled ubiquitin indicated that early-uninucleate microspores of the female genotype had a much higher conjugation rate than those of the male genotype at the same stage. However, after feeding α-lactalbumin as exogenous substrate, the rate of ubiquitin conjugation strongly increased and was quite similar in both sexes. Nuclear features of pollen development in both genotypes are also described. The nucleus progressively degenerated in the microspores of the pistillate flowers starting from the early-uninucleate stage, in parallel with the progressive decrease in ubiquitin content and activity. At anthesis, the microspores in the pistillate flowers either had no nucleus or showed only traces of chromatin. Thus, the ubiquitin system seems to play an important role in protein turnover occurring during the normal developmental pathway of the kiwifruit microgametophyte, while it was mainly involved in regressive events related to microspore degeneration in the female genotype.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Isolation of genes abundantly expressed in rice anthers at the microspore stage

A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph