Abnormal Pollen Development Research Articles

Overexpression of the CmACS-3 gene in melon causes abnormal pollen development

Sexual diversity expressed by the Curcurbitaceae family is a primary example of developmental plasticity in plants. Most melon genotypes are andromonoecious, where an initial phase of male flowers is followed by a mixture of bisexual and male flowers. Over-expression of the CmACS-3 gene in melon plants showed an increased number of flower buds, and increased femaleness as demonstrated by a larger number bisexual buds. Transformation of CmACS-3 in melons showed earlier development of and an increased number of bisexual buds that matured to anthesis but also increased the rate of development of the bisexual buds to maturity. Field studies showed that CmACS-3-overexpressing melons had earlier mature bisexual flowers, earlier fruit set, and an increased number of fruits set on closely spaced nodes on the main stem.

Comparative cytological and transcriptomic analysis of pollen development in autotetraploid and diploid rice

Autotetraploid rice has greater genetic variation and higher vigor than diploid rice, but low pollen fertility is one of the major reasons for low yield of autotetraploid rice. Very little is known about the molecular mechanisms of low pollen fertility of autotetraploid rice. In this study, cytological observations and microarray analysis were used to assess the genetic variation during pollen development in autotetraploid and diploid rice. Many abnormal chromosome behaviors, such as mutivalents, lagged chromosomes, asynchronous cell division, and so on, were found during meiosis in autotetraploid. Microsporogenesis and microgametogenesis in autotetraploid rice was similar to diploid rice, but many different kinds of abnormalities, including microspores degeneration, multi-aperture, and abnormal cell walls, were found in autotetraploid rice. Compared with diploid rice, a total of 1,251 genes were differentially expressed in autotetraploid rice in pollen transcriptome, among them 1,011 and 240 genes were up-regulated and down-regulated, respectively. 124 and 6 genes were co-up-regulated and co-down-regulated during three pollen development stages, respectively. These results suggest that polyploidy induced up-regulation for most of the genes during pollen development. Quantitative RT-PCR was done to validate 12 differentially expressed genes selected from functional categories based on the gene ontology analysis. These stably expressed genes not only related to the pollen development genes, but also involved in cell metabolism, cell physiology, binding, catalytic activity, molecular transducer activity, and transcription regulator activity. The present study suggests that differential expression of some key genes may lead to complex gene regulation and abnormal pollen development in autotetraploid rice.

Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.

The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.

ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) has attracted great interest because of its application in crop breeding. Despite increasing knowledge of CMS, not much is understood about its molecular mechanisms. Previously, orfH79 was cloned and identified as the CMS gene in Honglian rice, but how the ORFH79 protein causes pollen abortion is still unknown. Through bacterial two-hybrid library screening, P61, a subunit of the mitochondrial electron transport chain (ETC) complex III, was selected as a candidate that interacts with ORFH79. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (coIP) assays verified their interaction inside mitochondria. Blue native polyacrylamide gel electrophoresis (BN-PAGE) and western blotting showed ORF79 and P61 colocalized in mitochondrial ETC complex III of CMS lines. Compared with the maintainer line, Yuetai B (YB), a significant decrease of enzyme activity was detected in mitochondrial complex III of the CMS line, Yuetai A (YA), which resulted in decreased ATP concentrations and an increase in the reactive oxygen species (ROS) content. We propose that the CMS protein, ORFH79, can bind to complex III and decrease its enzyme activity through interaction with P61. This defect results in energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals that lead to abnormal pollen development.

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility

KEY MESSAGE : BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology. We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes' extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.

Over-expression of Arginine Decarboxylase Gene in Tapetal Tissue Results in Male Sterility in Tomato Plants

Homeostasis of cellular Polyamines (PAs) is very important for proper growth and development of living organisms. Development of male sex organ and gametophyte in plants is a complex process involving many known and unknown pathways. PAs are actively involved in the development of anther and pollen. In the present study, oat arginine decarboxylase (ADC) gene has been over-expressed in tapetum of tomato using tapetum-specific TA29 promoter to perturb the PA homeostasis in order to see its implication on male fertility. Tapetum-specific ADC overexpression resulted in PA accumulation, mainly in free and bound fractions in the anther of transgenics. Transgenic lines displayed abnormal pollen and tapetum development. Pollen grains were aborted and distorted in morphology and tapetum showed premature degeneration in ADC transgenics. These transgenics failed to set seeds, but cross pollination with normal pollen from wild type plants restored the seed setting indicating that s ADC over-expression did not affect female fertility. ADC over-expression also did not affect the growth and morphology of transgenics. The present study suggests that the excess of PAs in anther is lethal for pollen and tapetum development and overexpression of ADC gene can be an effective approach for the engineering of male sterility in plants.

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.

Identification of Mutants for Abnormal Pollen Development in Rice

To gain a comprehensive understanding of the genetic regulation of male gametogenesis, we screened and identified mutants for abnormal pollen development in rice (Oryza sativa L.). We gamma-irradiated spikelets of a japonica rice cultivar, Taichung 65, just before and after anthesis. Among 1,000 M2 lines, 24 lines were identified as mutants for pollen sterility. Progeny tests in M3 demonstrated that pollen sterility in 12 lines was controlled by single recessive genes, designated sps1–sps12 (sporophytic pollen sterility). In the remaining 12 lines, pollen semi-sterility was under the control of gametophytic genes, designated gps1–gps12 (gametophytic pollen sterility). Linkage analysis revealed that sps6, sps9, and sps12 were mapped on chromosomes 3, 9, and 7, respectively.

Abnormal pollen development of bread wheat-Leymus mollis partial amphiploid

Octoploid Tritileymus was the partial amphiploid obtained from Triticum aestivum × Leymus mollis, its chromosome constitution was 14 Leymus chromosomes and 42 wheat chromosomes. In the pollen development of Tritileymus, some abnormalities were observed, such as the absence of tetrad formation, cytomixis of microspores, irregular shape of some microspores and multi-germination pores, symmetrical first mitosis, chromosomes or chromatids randomly dispersed in the cytoplasm of microspore.

Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley

A chimaeric gene was constructed comprising a wheat high molecular weight glutenin subunit gene promoter, a 304-bp sucrose non-fermenting-1-related (SnRK1) protein kinase sequence in the antisense orientation, and the cauliflower mosaic virus 35S RNA gene terminator. Transgenic barley plants containing the antisense SnRK1 chimaeric gene were produced by particle bombardment of barley immature embryos with the aim of obtaining plants expressing the antisense SnRK1 sequence in the seeds. Despite the fact that the promoter was expected to be active only in seeds, two independent transgenic lines were found to fail to transmit the transgene to the T1 generation. These T0 plants had matured and died before this was discovered, but subsequently four other independent transgenic lines were found to be affected in the same way. Cytological analysis of the pollen grains in these lines showed that about 50% were normal but the rest had arrested at the binucleate stage of development, were small, pear-shaped, contained little or no starch and were non-functional. The presence of antisense SnRK1 transcripts was detected in the anthers of the four lines analyzed and a ubiquitin promoter/UidA (Gus) gene, one of the marker genes codelivered with the antisense gene, was found to be expressed only in the abnormal pollen. Expression analyses confirmed that SnRK1 is expressed in barley anthers and that expression of one class of SnRK1 transcripts (SnRK1b) was reduced in the abnormal lines. All of the abnormal lines showed approximately 50% seed set, and none of the transgenes were detected in the T1 generation.

Effects of high‐temperature stress on microsporogenesis in heat‐sensitive and heat‐tolerant genotypes of Phaseolus vulgaris

AbstractHigh ambient temperature (32/27 °C, day/night, 12 h photoperiod) applied prior to anthesis to Phaseolus vulgaris plants results in abnormal pollen and anther development during microsporogenesis. Scanning and transmission electron microscopy were used to examine anther and pollen morphology and pollen wall architecture after heat stress was applied to two genotypes that differ with respect to yield potential under high‐temperature field conditions: one, a heat‐sensitive, Mesoamerican genotype, A55, the second, a heat‐tolerant, Andean genotype, G122. High‐temperature treatment of both genotypes was applied 1–13 d before anthesis. Under heat stress, the heat‐tolerant genotype showed anther and pollen characteristics that were generally similar to the low temperature controls. In contrast, after 9 d of heat treatment before anthesis, the anthers of the heat‐sensitive genotype were indehiscent and contained abnormal pollen. Pollen wall architecture was also affected in the 12 and 13 d treatments. In addition to the morphological changes, the heat‐sensitive genotype also experienced reduced pollen viability and reduced yield in high‐temperature experiments conducted in both the greenhouse and field.

Cytological and Genetic Investigation of Abnormal Pollen Development in Sugarbeet

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The expression of alternative oxidase and alternative respiratory capacity in cytoplasmic male sterile common bean

The alternative respiratory pathway is present in all plant species investigated to date. Yet, the role of the alternative pathway is not clear. Some evidence suggests an important role in pollen development. We undertook this study to investigate the expression of alternative oxidase, in comparison with expression of a component of cytochrome oxidase, during pollen formation in common bean (Phaseolus vulgaris L.). Expression was evaluated immunohistochemically. In addition, we compared both the alternative oxidase capacity in young seedling tissues and alternative oxidase expression in developing flower buds of isonuclear cytoplasmic male sterile and male fertile bean lines. We observed no evidence of an association between the abnormal pollen development of CMS bean and changes in alternative oxidase expression or capacity. We did observe a tissueand stage-specific pattern of expression of alternative oxidase, differing from the expression pattern of cytochrome oxidase subunit II, during anther development in normal bean lines. Although no association was evident between the cytoplasmic male sterility phenotype and differential expression of alternative oxidase, the regulated pattern of alternative oxidase expression in developing anthers does suggest that the alternative pathway may play a role in microgametogenesis and microsporogenesis.

A Mitochondrial DNA Sequence Is Associated with Abnormal Pollen Development in Cytoplasmic Male Sterile Bean Plants

A Mitochondrial DNA Sequence Is Associated with Abnormal Pollen Development in Cytoplasmic Male Sterile Bean Plants

A mitochondrial DNA sequence is associated with abnormal pollen development in cytoplasmic male sterile bean plants.

Cytoplasmic male sterility (CMS) in common bean is associated with the presence of a 3-kb unique mitochondrial sequence designated pvs. The pvs sequence encodes at least two open reading frames (297 and 720 bp in length) with portions derived from the chloroplast genome. Fertility restoration by the nuclear restorer gene Fr results in the loss of this transcriptionally active unique region. We examined the effect of CMS (pvs present) and fertility restoration by Fr (pvs absent) on the pattern of pollen development in bean. In the CMS line, pollen aborted in the tetrad stage late in microgametogenesis. Microspores maintained cytoplasmic connections throughout pollen development, indicating aberrant or incomplete cytokinesis. Pollen-specific events associated with pollen abortion and fertility restoration imply that a gametophytic factor or event may be involved in CMS. In situ hybridization experiments suggested that significant reduction or complete loss of the mitochondrial sterility-associated sequence occurred in fertile pollen of F2 populations segregating for fertility. These observations support a model of fertility restoration by the loss of a mitochondrial DNA sequence prior to or during microsporogenesis/gametogenesis.

Transformation of an Australian Cotton Cultivar: Prospects for Cotton Improvement Through Genetic Engineering

Somatic embryogenesis and regeneration of whole plants is a highly genotype-dependent process in cotton. We have identified at least one highly regenerable Australian cultivar, Siokra 1-3, which is a sister line to the current major variety being grown in Australia. A number of plants have been regenerated and although some are showing abnormal pollen development, most can produce fertile seed when selfed or crossed with a normal pollen donor. Agrobacterium tumefaciens has been used to efficiently produce fertile transgenic Siokra 1-3 plants expressing novel genes such as the bacterial neomycin phosphotransferase or the β-glucuronidase. This is the first example of the transformation of an elite commercial cultivar. Critical factors in the transformation are the use of a supervirulent disarmed Ti-plasmid with a binary transformation vector, and a highly regenerable genotype of cotton. Bacterial concentration at the time of infection, tissue age, kanamycin selection regime, and co-cultivation support and media composition all have an influence on transformation efficiency and were optimised in our protocol. The ability to transform an elite Australian cultivar of cotton paves the way for agronomic improvements through genetic engineering. We have concentrated on increasing the tolerance of Australian cotton to the herbicide 2,4-D (to protect it from spray drift damage from adjacent cereal crops), and increasing its tolerance to insect pests, such as Helicoverpa armigera, using BT-toxin genes, protease inhibitors and other novel insect resistance genes.

Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant.

Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development.

Reproductive responses of cowpea (Vigna unguiculata (L.) Walp.) to heat stress. II. Responses to night air temperature

The influence of high night temperature and drought on the abscission of reproductive organs were studied with cowpea (Vigna unguiculata (L.) Walp. cv. ‘California Blackeye No. 5’) in controlled environments. Floral development of cowpeas at 33/22°C day/night temperature appeared to be normal. However, at 33/30°C day/night temperature all flowers abscissed within 48 h after anthesis due to male sterility resulting from abnormal pollen development and anther indehlscience. By moving plants between growth chambers with high and moderate night temperatures, it was demonstrated that the stage of floral development most sensitive to high night temperature occurred 5–7 days before anthesis. This corresponds with the stage just after the release of the tetrads from the microscope mother cell sac. Plants subjected to drought were smaller, and at the lower night temperature drought resulted in less seed production but this was due to fewer reproductive nodes and the percent abscission of reproductive organs was not influenced by drought. Observations were made on the effects of high temperatures on reproductive growth of six cowpea strains grown under field conditions. Complete abscission of flowers was observed during hot weather under field conditions followed by pod set approximately 6 days after decrease in night temperature. These data are consistent with the observations on cowpeas in growth chambers which demonstrated that high night temperatures can cause male sterility and complete abscission of flowers.

Genetic Male Sterility in Barley. III. Pollen and Anther Characteristics1

Pollen staining, pollen and anther appearance, and anther size were observed in 18 genetic male sterile mutants in barley, Hordeum vulgare L. The stains used were 2,3,5‐triphenyltetrazolium chloride (TTC), acetocarmine, and iodine potassium iodide. Stain uptake with the vital stain TTC was not a good indication of selfing ability, but nonstainability indicated sterility. To facilitate allelism studies four groups of mutants were delimited: A. Selfing ability‐ms9; B. Near‐normal pollen and anther development but no selfing‐ms6, ms8, ms16; C. Extremely abnormal pollen and anther development‐ ms3, ms7; and D. Variable pollen and anther development‐ms1, ms2, ms4, ms5, ms10, ms11, ms11az, ms13, ms14, ms17, ms18, ms19. The two allelic mutants, ms11 and ms11az, behaved differently for pollen stainability and appearance and for anther size.