Stages Of Pollen Development Research Articles

Cytological investigation of male sterility in sorghum caused by a dominant mutation (Ms tc ) derived from tissue culture

Male sterility mutations are an important tool in the investigation of anther and pollen development and for obtaining hybrid seeds in plant breeding. Cytological analysis of microsporo- and microgameto-genesis in sorghum plants with dominant mutation of male sterility (Ms tc ) derived from tissue culture has been carried out. Using substitution backcrosses, this mutation was introduced first into the nuclear background of the fertile sorghum line SK-723 and from this line into Volzhskoe-4w (V-4w). The mechanism of Ms tc action on anther and pollen development differed in different nuclear backgrounds. In SK-723, phenotypic expression of Ms tc began before the beginning of meiosis, which resulted in degeneration of sporogenous tissue in some anthers and in significant disturbances of anther morphology. In microsporocytes that did not degenerate, the frequency of non-specific meiotic abnormalities characteristic of the fertile line SK-723 significantly increased. In addition, in the mutant plants, a number of specific meiotic abnormalities—almost complete desynapsis, and formation of syncytial structures—were observed, apparently the consequence of Ms tc action. In mono- or bi-nucleate microspores, degenerative processes resulting in formation of empty or anomalously coloured pollen grains led to almost complete male sterility. In the V-4w nuclear background, changes in anther structure and meiotic disturbances were infrequent. The degenerative processes began at the uni- or binucleate microspore stage and resulted in formation of empty or abnormally coloured pollen grains, and in partial pollen sterility. Thus, the same nuclear male sterility-inducing mutation in different nuclear backgrounds affects different stages of pollen development.

Reproductive Studies in Ipecac (Cephaelis ipecacuanha (Brot.) A. Rich; Rubiaceae): Flower Bud and Anther Size Associated to Male Gamete Development Stages

Reproductive studies were carried out on Brazilian accessions of Cephaelis ipecacuanha, which is considered a threatened species. Meiotic behavior was studied using squashing technique with 1% acetic carmine and floral buds and anther were measured for the following male gamete development stages: meiosis I, meiosis II, tetrads, microspores and pollen. Bud and anther length showed significant differences (P<0.01) among the stages of pollen development. There was high significant correlation between bud and anther length by Pearson correlation. Tukey test results indicated that each analyzed stage could be associated to bud length mean but it was not found the same association for anther length.

Proteomic analysis reveals developmentally expressed rice homologues of grass group II pollen allergens.

Three isoallergens of Ory s 2, homologues of grass group II pollen allergens, were identified from rice and characterised by proteome and immunochemical analyses. The N-terminal amino acid sequence profiles of three proteins on a 2-dimensional electrophoresis (2-DE) gel of rice pollen proteins matched 100% to the protein sequences encoded by three rice expressed sequence tags (ESTs). The deduced protein sequences from these ESTs share sequence identities of 41-43% with the protein sequences of the group II pollen allergens of different grasses, and sequence identity of 39% with the C-terminal portion of rice group I pollen allergens. Signal peptide sequences, which are similar to the leader peptides of other major pollen allergens, are also present in the deduced amino acid sequences. Polyclonal antibodies, produced in rabbits using Ory s 2 proteins purified by 2-DE, were used to investigate the developmental-stage- and tissue-specific expression of Ory s 2 by immunochemical analysis. Results of immunochemical experiments show that Ory s 2 proteins are expressed only at the late stage of pollen development and they do not have cross-reactivity with group II pollen allergens from some other common grasses.

Functional analysis of a β-1,3-glucanase gene ( Tag 1) 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.

Male gametophyte development in bread wheat (Triticum aestivum L.): molecular, cellular, and biochemical analyses of a sporophytic contribution to pollen wall ontogeny

Bread wheat (hexaploid AABBDD genome; 16 billion basepairs) is a genetically complex, self-pollinating plant with bisexual flowers that produce short-lived pollen. Very little is known about the molecular biology of its gametophyte development despite a longstanding interest in hybrid seeds. We present here a comprehensive characterization of three apparently homeologous genes (TAA1a, TAA1b and TAA1c) and demonstrate their anther-specific biochemical function. These eight-exon genes, found at only one copy per haploid complement in this large genome, express specifically within the sporophytic tapetum cells. The presence of TAA1 mRNA and protein was evident only at specific stages of pollen development as the microspore wall thickened during the progression of free microspores into vacuolated-microspores. This temporal regulation matched the assembly of wall-impregnated sporopollenin, a phenylpropanoid-lipid polymer containing very long chain fatty alcohols (VLCFAlc), described in the literature. Our results establish that sporophytic genes contribute to the production of fatty alcohols: Transgenic expression of TAA1 afforded production of long/VLCFAlc in tobacco seeds (18 : 1; 20 : 1; 22 : 1; 24 : 0; 26 : 0) and in Escherichia coli (14 : 0; 16 : 0; 18 : 1), suggesting biochemical versatility of TAA1 with respect to cellular milieu and substrate spectrum. Pollen walls additionally contain fatty alcohols in the form of wax esters and other lipids, and some of these lipids are known to play a role in the highly specific sexual interactions at the pollen-pistil interface. This study provides a handle to study these and to manipulate pollen traits, and, furthermore, to understand the molecular biology of fatty alcohol metabolism in general.

Erratum to: Transformation of wheat (Triticum aestivum L.) microspore-derived callus and microspores by particle bombardment

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4◦C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at −20◦C or −80◦C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4◦C for a couple of weeks and then at −80◦C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).

Gene-expression analysis of sucrose-starch metabolism during pollen maturation in cytoplasmic male-sterile and fertile lines of sorghum

During pollen maturation, single-celled microspores undergo a rapid phase of starch biosynthesis, presumably from sucrose stored in the vacuoles. Very little is known about the genes and the intracellular controls that regulate the sucrose → starch pathway in such microspores. We show here RNA profiles in sorghum microspores during the transition from vacuolated microspore to immature pollen at two specific stages of pollen development: early, showing little or no detectable starch, and late, when pollen are active in starch-filling. We have also examined two near-isogenic cytoplasmic male sterile lines that retain fully turgid immature pollen until the starch-filling stage, but remain starch-deficient, and ultimately form inviable pollen. Using maize cDNA clones, both temporal and genotypic differences were observed in the expression profiles of four metabolic genes (soluble or vacuolar invertase, Ivr2; sucrose synthase, Sus1; phosphoglucomutase, Pgm; and a subunit of ADPG pyrophosphorylase, Bt2), two similar metabolic regulatory genes (Grf1 and Grf2, encoding 14-3-3 proteins) and a transcription factor, ZmMADS1. Western blot analyses were conducted to visualize sucrose synthase, SS2; AGPase, BT2 and the GRF (14-3-3) proteins. Temporal differences of both a qualitative and quantitative nature were observed in all three proteins in both male-fertile and male-sterile lines. We suggest that these changes in gene expression may result from increased and decreased sink strengths and associated changes in carbohydrate metabolism in starch-filled (fertile) and starch-deficient (sterile) pollen, respectively.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

Analyses of the Genomic Sequence and Promoter Activity of a Gene for a Protein Similar to Tat Binding Protein Isolated from Brassica rapa.

We have previously isolated a cDNA clone, which is similar in sequence to the Tat binding protein (TBP) of human immunodeficiency virus 1, from the anthers of Brassica rapa and designated it BrTBP. In the present study we isolated a genomic clone of BrTBP from B. rapa and found several sequence motifs conserved in various pollen-expressed genes. The 1.7-kb promoter region of BrTBP was fused to the GUS gene and introduced into tobacco and Arabidopsis thaliana. GUS expression was observed exclusively in mature pollen after anthesis in transgenic tobacco, while it was observed in tricellular pollen starting two days before anthesis in A. thaliana. Such expression patterns at the late stage of pollen development have not been reported in other pollen-specific promoters. These results indicate that the BrTBP promoter region used in this study has a unique activation pattern in tobacco and A. thaliana.

Stress, as assessed by the appearance of sHsp transcripts, is required but not sufficient to initiate androgenesis

A number of abiotic stresses can elicit young pollen grains of Brassica napus to irreversibly alter their development to androgenic (embryo formation) pathway. In our experiments, temperature and colchicine were used as abiotic stresses capable of inducing androgenesis. The most effective temperature was 32–33°C. This absolute temperature requirement, but only in combination with a temperature differential of at least 5–10°C between the donor plant and in vitro culture conditions, had a major stimulatory effect to elicit androgenesis in young pollen grains. Colchicine teatment also resulted in induction of androgenesis but at lower levels than the temperature treatment. In both instances, application of abiotic stresses resulted in the elicitation of a general stress response in young pollen grains as indicated by the appearance of sHsp transcripts. The appearance of sHsp transcripts can be used as a marker to estimate the level of stress necessary to change the differentiation pathway of young pollen grains that leads to induction of androgenesis since androgenesis never took place without the sHsps gene transcription. The need for the presence of sHsp transcripts is supported by observation that a mutant form of B. napus cv. Topas, producing approximately 50 times fewer embryos, had a substantially reduced expression of sHsps in young pollen grains subjected to the androgenesis inductive heat stress. It remains to be determined to what extent sHsps are directly involved in induction of androgenesis as, under stress, sHsps gene products were also detected in microspores and pollen grains developmentally too young or old to undergo the process. Nevertheless, it can be concluded that application of stress and the subsequent stress response, which includes the transcription of sHsps, are some of the major events associated with changes in the pollen differentiation process that can lead to androgenesis. Other factors, such as the pollen developmental stage and the phase of the cell cycle at the time of stress imposition, play an important role as well.

The MADS box gene ZmMADS2 is specifically expressed in maize pollen and during maize pollen tube growth

We screened a cDNA library of mature maize pollen for clones encoding transcription factors. Two novel cDNAs (ZmMADS1 and ZmMADS2) encoding MADS box proteins were isolated. To our knowledge, ZmMADS1 and ZmMADS2 represent the first transcription factors isolated from pollen of a monocot plant species. ZmMADS1 can be classified as a member of the TM3 subfamily of MADS box proteins whereas ZmMADS2, which is described here in more detail, belongs to the AGL17 subfamily. Temporal and spatial expression analyses showed that ZmMADS2 expression is restricted to pollen and roots. During pollen development ZmMADS2 is detectable at all stages of pollen development analyzed by RT-PCR but is most abundant in mature pollen after dehiscence. The establishment of a whole-mount RNA in situ hybridization protocol with in-vitro germinated pollen enabled the localization of ZmMADS2 transcripts in pollen tubes. ZmMADS2 transcripts are translocated into pollen tubes immediately upon germination and display a gradient with increasing concentration towards the tube tip. The vegetative nucleus and the sperm cells, putative targets of ZmMADS2, enter the pollen tube about 2 h later.

Ubiquitin and ubiquitin-conjugated proteins in the olive ( olea europaea l.) pollen

A study of free ubiquitin and ubiquitinated proteins has been carried out in olive (Olea europaea L.) during the late stages of pollen development and during in-vitro pollen hydration. As described in relatively few plant species, immunoblot experiments carried out using crude protein extracts have shown a reduction in the free ubiquitin level taking place during the mature pollen stage. This reduction does not coincide with a parallel reduction in the level of the corresponding transcripts, as detected by RT-PCR analysis, suggesting the existence of a post-transcriptional regulatory mechanism. Relatively high levels of free ubiquitin are quickly restored upon in-vitro pollen hydration. TEM immunocytochemical localisation of ubiquitin-conjugated compounds in the mature pollen grain showed the antibodies bound to the cytoplasm, the chromatin and the nucleolus of the vegetative and generative cells. Within the cytoplasm, labelling was concentrated in the cytosol, the endoplasmic reticulum membranes and minuscule vesicles associated to the Golgi apparatus. The fibrillar material adhering to the exine, and probably derived from the tapetum, also showed gold particles. Lipid bodies, aperture regions, mitochondria, the intine and the cell wall were devoid of labelling. The changes observed throughout pollen development, as well as the specific localisation, reflect the involvement of the ubiquitin-mediated degradative pathway in regulation of pollen metabolism.

Hermodactylus tuberosus L. (Iridaceae) pollen organisation before and after anther dehiscence

ABSTRACT The morphology, cytology and viability of Hermodactylus tuberosus L. (Iridaceae) pollen were examined from the first mitosis until maturation and after anther opening. During maturation, the pollen coat becomes modified, and the vegetative cell cytoplasm accumulates several types of reserve substances. In the vegetative cell cytoplasm, starch is quickly utilised whereas lipid inclusions of different dimensions, shape and composition occur during pollen maturation. Pollen from opened anthers have a thin pollen coat; the cytoplasm has mostly lipid reserves, and many small vesicles and vacuoles. It is similar in size or larger than pollen located inside the anther, and its viability does not decrease until one day after anther dehiscence. Large osmiophilic bodies, different from those of the vegetative cell cytoplasm, are present in the generative cell cytoplasm starting from the first stage of pollen development. The poorly developed pollen coat in pollen from opened anthers suggests that it plays a minor role in attracting insects for pollination. The size and structural and ultrastructural features of mature pollen indicate that it does not undergo dehydration and possesses sufficient vigour for immediate germination.

Determination of Optimum Pollen Developmental Stage for Inducing Callus in Anther Culture of Rice

Determination of Optimum Pollen Developmental Stage for Inducing Callus in Anther Culture of Rice

Pleiotropic effects of a nuclear restorer-of-fertility locus on mitochondrial transcripts in male-fertile and S male-sterile maize.

Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

N-glycoproteins specific for different stages of microspore and pollen development in tobacco

Glycoproteins (GPs) specifically linked to stages of microspore and pollen development were identified and characterized by: 1D- and 2D-electrophoresis, treatment with glycosidases, affinity blotting with lectins ConA, GNA, AAA, WGA, DSA, PNA and RCA, and according to cell location. Most GPs reacted with ConA and GNA and were hydrolyzed with PNGase F and endoglycosidase H, indicating N-linkage of high-mannose and/or hybrid type glycans. Early microspores were unique by the occurrence of ConA and AAA binding 50- and 52-kD GPs. Specific for mitotic microspores were cytoplasmic neutral GPs 39 and 92 kD, and membrane GP 98 kD. Maturing pollen was characterized by a new set of ConA-binding GPs: acidic, membrane GP 38 kD, neutral GPs 51, 66 and 75 kD, GP 53 kD separated in IEF into spots with p I 6.8–7.5 and 5.9, and IEF variants of GP 55 kD with p I 6.5–7.5. GPs 48, 59, 70, 83 and 114 kD showed developmental changes in reactivity with various lectins. In contrast to GPs, no clear qualitative changes were observed in profiles of Coomassie blue stained proteins during pollen development. The oligosaccharide structures appeared as important determinants of specificities of GPs associated with phases of microspore and pollen development.

Transient expression of the green fluorescent protein in pollen

ZM13 is a pollen-specific maize gene which is expressed in the late stages of pollen development. We wished to utilize the ZM13 promoter to examine the expression of a synthetic green fluorescent protein (SGFP) in germinating pollen. The usefulness of the SGFP expression product is that its appearance and distribution can be monitored non-destructively in vivo. A plasmid containing the SGFP coding region under the control of the ZM13 promoter was constructed and then transiently transformed into pollen of Tradescantia paludosa and Nicotiana tabacum by the use of microprojectile bombardment. The expression of the green fluorescent protein was analyzed by fluorescence microscopy using a fluorescein filter. Expression began about 3 h post-bombardment, and all parts of the pollen grain and tube fluoresced. High levels of fluorescence were observed for several days following treatment.

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants.

Seven new male-sterile mutants (ms7-ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4',6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified.

Detection and quantification of rRNA by high-resolutionin situ hybridization in pollen grains

We examined changes in the localization of cytoplasmic rRNA during pollen development inNicotiana tabacum SR-1. The rRNA was visualized byin situ hybridization, and the signal intensity of rRNA in microspore, vegetative and generative cell was quantified by microphotometry. The amount of rRNA per microspore or pollen section increased about 5 times from microspore to mature pollen grain and kept increasing even in the late stage of pollen development after PMI. The increase of rRNA occur in both vegetative and generative cells. The results suggest that synthesis of rRNA occur even after PM I in both vegetative and generative cells.

Production of microspore-derived plants by anther culture of an interspecific F1 hybrid between Cyclamen persicum and C. purpurascens

Anthers of a F1 hybrid (2n=41) between Cyclamen persicum (2n=2x=48) and C. purpurascens (2n=2x=34) were cultured to produce microspore-derived plants. Embryoids were produced when anthers, containing microspores at the early uninucleate stage of pollen development, were cultured in B5 medium containing sucrose (90 g l-1) and NAA (0.1, 1 mg l-1) or 2,4-D (0.1 mg l-1) in the dark at 5 °C for 4 days, then at 25 °C for 60 days. The embryoids usually developed into plantlets when cultured in B5 medium containing sucrose (30 g l-1) in the dark at 25 °C. At meiosis, the F1 hybrid, used as source for anther culture, formed some cells with restitution nuclei at telophase and dyads at the tetrad stage, which resulted in the production of viable pollen grains as unreduced gametes. Plants produced by anther culture were grouped into sterile plants with 2n=41 chromosomes and fertile plants with 2n=82 chromosomes. The present findings suggested that the sterile plants were polyhaploids originating from unreduced microspores (n=41) of the F1 hybrid and that the fertile plants were amphidiploids induced by a spontaneous doubling during culture of chromosomes of such unreduced microspores.