Development Of Pollen Grains Research Articles

Pollen grains as a target for introduction of foreign genes into plants: an assessment.

Introduction of foreign genes and development of transgenic plants have become an integral part of crop improvement programmes in the last decade. However, most of the present day plant transformation protocols require long periods for development of transgenic plants and need skilled personnel. Development of alternate, simple and rapid transformation protocols for development of transgenic plants can overcome the constraints of in vitro culture, regeneration and associated problems. Pollen grains, due to their abundance and ease with which they can be handled are ideal targets for introduction of foreign genes into the germ line. However, progress in introduction of transgenes into pollen grains and their subsequent use in fertilization leading to development of transgenic plants are limited. With the recent progress made in understanding of pollen development along with reports of successful pollen-mediated transformation in important crop plants, it should be possible to extend this simple method of transformation to other crop plants. The review deals with development of pollen grains as a target for introduction of genes with special emphasis on recent developments.

Pollen morphology of Polish native species of the Rosa genus (Rosaceae) and its relation to systematics

The morphology of pollen grains of 16 species from the <em>Rosa </em>L. genus were studied (i.e. <em>R. agrestis</em>, <em>R. canina</em>, <em>R. dumalis</em>, <em>R. gallica</em>, <em>R. inodora</em>, <em>R. jundzillii</em>, <em>R. kostrakiewiczii</em>, <em>R. majalis</em>, <em>R. micrantha</em>, <em>R. mollis</em>, <em>R. pendulina</em>, <em>R. rubiginosa</em>, <em>R. sherardii</em>, <em>R. tomentosa</em>, <em>R. villosa</em>, and <em>R. zalana</em>). The material came from 16 native localities of those species in Poland. The measurements are based on at least 30-50 randomly selected, fully developed pollen grains per specimen. In total, 500 pollen grains were examined. They were analysed for 13 quantitative features of pollen grains and exine sculpturing and the following qualitative traits: outline, shape, "operculum" structure. The diagnostic features of pollen grains of studied species were: length of polar and equatorial axes and length of ectocolpi. The above-mentioned pollen grain morphological features make isolation of one species possible: <em>R. gallica</em>. <em>R. gallica </em>is distinguished for its highest values of the length of polar and equatorial axes, and the length of ectocolpi. The obtained analytical results of operculum and exine sculpture features, considered as diagnostic, corroborated only slightly their priority significance for the isolation of the examined species and sections. The collected data failed to confirm fully the current taxonomical division of the <em>Rosa </em>genus into sections (only section <em>Gallicanae</em> from <em>R. gallica </em>is isolated) as well as consanguinity relationships between the examined species from the <em>Caninae </em>section. On the dendrogram, both species closely related with each other as well as those from other developmental lines were found in the same group. These equivocal results are by no means surprising because the <em>Caninae </em>section is the most polymorphic group in the <em>Rosa </em>genus, and contemporary <em>Caninae </em>are of the nature of a swarm of <em>R. canina </em>hybrids as a link combining all taxons of the section.

Effect of the water-soluble fraction of diesel exhaust particles on the development and protein patterns of pollen grains in bean plants (Phaseolus vulgaris L.)

Excessive air pollution may adversely affect plant growth and development. The overall objective of this research was to elucidate some microscopic effects of diesel exhaust particles (DEP) on the formation, development, and structure of pollen grains and its proteins in Phaseolus vulgaris L. Bean plants were grown in experimental pots and treated with different concentrations of the water-soluble fraction of DEP. Flowers and young buds were removed, fixed in FAA70 (formalin : acetic acid : ethanol, 2 : 1 : 17), and subjected to developmental studies. Our results show that DEP cause some abnormalities during pollen development. Pollen grains in normal plants are spherical in equatorial view and triangular in polar view; in DEP-treated plants, they were changed into irregular shapes in equatorial view and asteroidal in polar view. Delay in degeneration of the tapetum layer of the anther, and formation of giant, irregular, and non-fertile pollen grains are other results of DEP treatment. Gel electrophoretic studies revealed that a new protein with a molecular mass of 80 kD was formed in the DEP-treated group. Some pollen proteins may act as allergens, considering the fact that pollen allergy frequency is increased in polluted areas; the possibility arises that DEP could be an effective agent entail formation of detoxifying proteins which, on the other hand, can also act as allergens.

Emergence and patterning of the five cell types of the Zea mays anther locule

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize ( Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2′-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.

Biological features of flowering of some medicinal plants introduced in the vicinity of Tomsk

Data on the seasonal rhythms of development of the representatives of Lamiaceae (Ballota nigra L., Dracocephalum ruyschiana L., Hyssopus officinalis L., Scutellaria baicalensis Georgi) and Asteraceae (Eupatorium cannabinum L.) are presented. The cytoembryological study of these species was carried out, focused on the phases of flower development, morphological features, and fertility of pollen grains.

Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in beta-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway.

Microsporogenesis and pollen formation in Scotch pine (Pinus sylvestris L.) under modern climatic conditions of Siberia

The prolongation of the warm season during autumn, which has become typical for Siberia in the past 5–6 years, has caused changes in the terms of reproductive processes in microsporangia of Scotch pine in the Krasnoyarsk region. Meiosis has started to begin in the autumn of the year preceding pollination, and then come to an end the next spring, whereas usually this process has occurred in the spring of the year of pollination. Under such conditions the division of microsporocytes is accompanied by different abnormalities, causing various anomalies and the incomplete development of pollen grains.

The bouquet of grapevine (Vitis viniferaL. cv. Cabernet Sauvignon) flowers arises from the biosynthesis of sesquiterpene volatiles in pollen grains

Terpenoid volatiles are important information molecules that enable pollinators to locate flowers and may protect reproductive tissues against pathogens or herbivores. Inflorescences of grapevine (Vitis vinifera L.) are composed of tiny green flowers that produce an abundance of sesquiterpenoid volatiles. We demonstrate that male flower parts of grapevines are responsible for sesquiterpenoid floral scent formation. We describe temporal and spatial patterns of biosynthesis and release of floral volatiles throughout the blooming of V. vinifera L. cv. Cabernet Sauvignon. The biosynthesis of sesquiterpene volatiles, which are emitted with a light-dependent diurnal pattern early in the morning at prebloom and bloom, is localized to anthers and, more specifically, within the developing pollen grains. Valencene synthase (VvValCS) enzyme activity, which produces the major sesquiterpene volatiles of grapevine flowers, is present in anthers. VvValCS transcripts are most abundant in flowers at prebloom stages. Western blot analysis identified VvValCS protein in anthers, and in situ immunolabeling located VvValCS protein in pollen grains during bloom. Histochemical staining, as well as immunolabeling analysis by fluorescent microscopy and transmission electron microscopy, indicated that VvValCS localizes close to lipid bodies within the maturing microspore.

NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development

Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a Km of 2.20 mg ml−1 and Vmax of 11.07 μg ml−1 min−1. The recombinant NtCP56 retained more than 50% of its maximum enzymatic activity from 20 °C to 60 °C with an optimum Tm range of 30–50 °C. The enzyme had a maximum activity at approximately pH 6.5. Suppression of the NtCP56 gene in anti-sense transgenic tobaccos resulted in the sterility of pollen grains. Our data indicated that, as a cysteine protease, NtCP56 might play an important role in pollen development.

Meiosis and pollen formation in haploid Thuja plicata gracilis Oud.

Male strobili development and the course of microsporogenesis were studied on a tree growing in the Botanical Garden at Stockholm as well as on cuttings from a haploid and a diploid plant of Thuja plicata. The plants from cuttings were kept in a phytotron experiment at different photoperiods and treated with gibberellin in order to get rich flowering. The development of the strobili and the course of microsporogenesis were very variable under different ecological conditions. The best result (similar to that of the tree in Botanical Garden) was obtained when the plants from cuttings were kept for 17 weeks in long day during which period the plants were sprayed with gibberellin, twice a week. A rich initiation of buds was obtained but the development remained arrested at this initiation stage (corresponding to preprophase-pachytene). After transferring the plants to short day the strobili development and microsporogenesis continued and were completed after 14 weeks. Thus, the photoperiodical sequence necessary for complete development of pollen grains was long day — short day. The pollen grains in diploid plants were normally developed but those in haploid ones showed irregularities caused by meiotic disturbances.

Characterization of a Lolium multiflorum accessory chromosome

AbstractThe meiotic behavior and the male and female transmission rates of a Lolium multiflorum accessory chromosome were investigated. Typically, accessory chromosomes display an accumulative behavior termed non‐disjunction that allows them to increase their numbers in a population. Root‐tip counts, utilizing individuals with known numbers of accessory chromosomes were evaluated to determine male and female transmission rates and cytogenetic examinations of microspores and developing pollen grains were performed at the metaphase and anaphase stages where such accumulative non‐disjunction events occur. Results of the investigation identified that the accessory chromosome of this Lolium cv. did not exhibit a strong accumulation mechanism and were relatively normal during meiosis and pollen mitosis. Accessory chromosome transmission through the egg was normal without accumulation and frequent non‐disjunction events did not occur during the microspore or pollen development stages. The pattern of chromosome movement was more similar to that of the A‐chromosome set than that observed for the characterized accessory chromosome of Festuca pratensis. Because the Lolium accessory chromosome meiotic and pollen mitotic behavior are relatively normal with a low rate of accumulation, such a chromosome may serve as a mini‐chromosome carrier platform for directed biolistic‐mediated gene transformation or the generation of accessory chromosome‐Lolium and accessory chromosome‐Festuca translocations.

Influence of UV-B radiation on the reproductive function of Hordeum vulgare L. plants

It has been shown that ultraviolet-B radiation induced acceleration of flowering and differentiation of sexual spike elements in barley. Increasing of pollen asynchronous development and variability of pollen grains with growing of pollen grain sterility were observed as well. The productivity of plants irradiated with UV-B radiation did not change. Considerable doses of irradiation resulted in decreasing of pollen sterility by intensification of haplontic cell selection. The influence of UV-B radiation on the plants can be considered as a genotoxical effects via formation of initial cell DNA damages and photoinduction.

The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis

We have isolated the LLA23 gene in the pollen of Lilium longiflorum. The LLA23 gene encodes an ASR (named after abscisic acid, stress and ripening) protein that has a nuclear localization sequence at the C terminus. The gene is interrupted by one single intron and possesses a long 5'-untranslated region. Southern blots of lily genomic DNA indicated that LLA23 is a member of a small gene family. We examined the link between LLA23 location and the desiccation that naturally occurs in developing anthers using immunogold labeling. When pollen reached maturity, a significant increase in LLA23 labeling was observed in the nuclei of both vegetative and generative cells from 10- to 12-cm buds and thereafter. This clearly demonstrates that a marked increase in LLA23 translocation from the cytoplasm to both nuclei of pollen grains occurs in 12-cm buds, a stage shortly before the commencement of desiccation during anther development. In addition, microarray analysis showed that 410 (206 up-regulated and 204 down-regulated) genes have altered expression in LLA23-overexpressing plants. Quantitative PCR analysis confirmed the changes in mRNA levels observed in our microarray analysis. This genome-wide overview of gene expression supports the theory that LLA23 acts as a regulator.

Anatomical differences on development of fertile and sterile pollen grains of Prunus salicina Lindl.

Anatomical changes occurring during the microsporogenic development of P. salicina Lindl. were studied in male fertile and male sterile genotypes. Male fertile pollen grains showed three well determined pore regions, without ektexine. Intine was thick and surrounded the vegetative cell. Vegetative cells enclosed the generative cells; their cytoplasm was rich in plastids, abundant RER and active mitochondria. Development of sterile pollen was different from the meiosis step. Microspores did not show germination pores and ektexine was continuous around the whole grain. Pollen grains showed an atypical shape. The tapetum persisted after the tetrad stage and showed hypertrophy and vacuole development, resulting in abnormal microspore development. Only a few pollen grains and rudiments of collapsed microspores close to the anther wall were formed at anthesis.

Cytological Observation of Microsporogenesis in Male-Sterile Lines of Chinese Pink ( Dianthus chinensis L.)

This study addressed the differences in microsporogenesis between male sterile and fertile lines of Chinese pink. The microsporogenesis processes of male sterile and fertile lines were histologically examined in squashed pollen grains and in paraffin embedded sections. A stable male-sterile line (H-37B) was obtained following six generations of inbreeding in a self-fertile line, followed by two generations of backcrossing. In the corresponding fertile line, development of the mature pollen grains was followed through the initiation of the sporogenous cell, microsporocyte formation, and the tetrad developmental period. In the male-sterile line, abortion of the developing pollen grains was observed to take place at various stages, namely, sporogenous cell growth, mother cell meiosis, and tetrad transformation to the uninuclear state. The pollen grains of the fertile line were spheroid, turgid, and viable. By contrast, the male-sterile line produced pollen that was irregular in shape, empty, and nonviable. The abortion of the microspore in the male-sterile line appeared to relate to abnormal growth of the tapetum layer.

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus)

Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cell's organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.

Isolation and characterization of 4 gametophytic male sterile mutants in Arabidopsis thaliana

In flowering plants, male fertility depends on the formation and development of normal male gametophytes or pollen grains. However, little is known about the molecular mechanisms that regulate the processes. Here, we report the identification of four novel independent Arabidopsis gametophytic male sterile mutants, apam1, apam2, apam3 and apam4 (Arabidopsis pollen abortion mutant). The four mutants that were generated by the insertions of gene-and enhancer-trap Ds transposon elements were defective in pollen development. Genetic analysis results showed that all four mutations resulted in the loss of male gametophytic function, but did not affect female gametophytic function, and the Ds elements were linked to the mutations tightly in all four mutants. Localization of the Ds insertion sites by thermal asymmetric interlaced PCR (TAIL-PCR) showed that the Ds elements were inserted in four different loci distributed on three chromosomes, chromosomes II, III and V. In summary, the apam4 is allelic to AHA3, while the other three were located in places where there are no genes that have been known to be involved in pollen development, suggesting that they are novel mutations involved in pollen development.

Overexpression of Zm401, an mRNA-like RNA, has distinct effects on pollen development in maize

We previously identified a 0.7 Kb cDNA fragment of Zm401, a novel pollen-specific gene in maize (Zea mays). However, little information is known about the function of Zm401 in pollen development. The full-length of Zm401 cDNA was amplified by 5′ RACE and 3′ RACE and both sequence analysis and in vitro translation of Zm401 showed that it belonged to an mRNA-like non-coding gene. To analyze its possible biological roles in pollen development, the Zm401 cDNA was overexpressed in transgenic maize under the control of a pollen specific promoter Zm13 or a CaMV 35S promoter. RT-PCR and RNA gel blot analysis indicated that the expression level of Zm401 in leaves and anthers of transgenic plants was much higher than that of non-transformants. Compared with the non-transformed maize, transgenic maize showed distinct phenotypes, such as abnormal tassels and degenerate anthers. The histological observation showed that the development of pollen grains and anthers in transgenic plants were abnormal. These abnormalities include delayed degradation of tapetum, asynchronous fusion of pollen sacs, and aborted pollen grain development. Furthermore, the pollen viability in six transgenic plants ranged from 1.24% to 6.63%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. These results suggest that Zm401 is involved in the regulation of pollen development. This article demonstrated Zm401, as a non-coding RNA, plays an essential role in pollen development.

Inheritance of the ps mutation in sugar beet

Genetic analysis of the inheritance of mutation ps in sugar beet was conducted. This mutation causes the meiotic abnormalities leading to the development of diploid pollen grains and influences several other morphological traits, namely, annual or biennial habit, stem color, and aggregation of pollen grains into tetrads, which are controlled by the genes B, Stc, and ap, respectively. The literature data on the linkage of genes B and Stc were confirmed; the obtained recombination coefficient between these genes amounts to 15.0 ± 3.6%. It was demonstrated that gene ap was inherited independently of genes B and Stc. Statistical analysis of the data shows that the mutation ps is recessive and is inherited independently of the mutation ap but in a linked manner with the traits development habit and stem color. The conclusion is made that a gene with a strong phenotypic effect that determines the development of the phenotype characteristic of mutation ps is located in the first linkage group near genes B and Stc.