Mago nashi Interacts with a Taiwania (Taiwania cryptomerioides) Pectin Methylesterase-like Protein

Mago nashi proteins are highly conserved among eukaryotes. They are involved in oogenesis, embryogenesis and germ-line determination during animal development, and play important roles in pollen tube growth, root development and spermatogenesis during plant development. In this study, we used yeast two-hybrid screening to show that the TcMago protein can interact with a Taiwania (Taiwania cryptomerioides) pectin methylesterase-like protein (TcPME1) which consists of a transmembrane domain, a pectin methylesterase inhibitor (PMEI) domain and a pectin methylesterase (PME) domain. The PME domain of TcPME1 was necessary for binding with the TcMago protein. The PME domain was highly conserved in all the plants assayed and had five well conserved active site residues. The predicted protein tertiary structures revealed that the PMEI domain and PME domain of TcPME1 are similar to kiwi (Actinidia deliciosa) PMEI and carrot (Daucus carota) PME, respectively. TcPME1 was expressed abundantly in the early stage of root elongation and accumulated at root tip. Moreover, TcPME1 expression was inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Thus, TcPME1 might be involved in root elongation, shoot development and auxin transport during Taiwania development.     

Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system

A new CMS system designated as ‘msH1’ has been reported in bread wheat using the cytoplasm of H. chilense. While testing this system in different wheat backgrounds, a highly fertile line with chromosome number 42 plus an extra acrocentric chromosome was obtained. The extra chromosome did not pair with any wheat chromosome at meiosis, and progeny from this line which lack the acrocentric chromosome showed pollen abortion and male sterility. In order to establish the origin of this chromosome, FISH using H. chilense genomic DNA as probe was used and showed that it had originated from H. chilense chromosome(s). The novel chromosome did not possess sequences similar to wheat rDNA; however, the probe pSc119.2 from S. cereale containing the 120 bp family was found to occur at the end of its long arm. Data obtained from FISH and EST molecular markers confirm that the long arm of the acrocentric chromosome is indeed, the short arm of chromosome 1H^ch from H. chilense. We suggest that the novel chromosome originated from a deletion of the distal part of the long arm of chromosome 1H^ch. Neither the 1H^chS short arm, nor the whole chromosome 1H^ch restores pollen fertility of the alloplasmic wheat. Therefore, the restorer gene on the acrocentric chromosome must be located on the retained segment from the hypothetical 1H^chL, while some pollen fertility inhibitor could be present on the deleted 1H^chL distal segment. Disomic addition of the acrocentric chromosome was obtained and this line resulted fully stable and fertile.     

Characterization and Expression Pattern of a Putative Pectin Methylesterase Gene, <Emphasis Type="Italic">BcMF27</Emphasis>, in <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Pectin methylesterases (PMEs) play an important role in modifying cell wall. PMEs catalyze the de-esterification of pectin, an important compound of cell wall, to affect fertility in plant reproduction. However, little especially molecular mechanism about pectin methylesterase is studied in recent years despite its importance to reproductive development in flower plant. Here the bioinformatics analysis of BcMF27 (Brassica campestris Male Fertility 27) (BRAD: Bra000541 GenBank: KT600012) sequence isolated from Brassica campestris L. ssp. chinensis showed its highly and characteristically conserved structure as a pectin methylesterase. Transient expression analysis in the onion epidermal cells revealed the product of BcMF27 was a transmembrane protein. Real-time RT-PCR and in situ hybridization suggested that BcMF27 was expressed in pollen grain and pollen tube. This study demonstrates that BcMF27 encodes a transmembrane pollen- and pollen tube-specific PME gene, and is also considered to help further understand the biological function of pectin methylesterases and the molecular mechanism of pollen development, pollen tube growth as a genic tool.     

Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation

Summary. Antisense transgenesis of tobacco (Nicotiana tabacum) with a partial flax (Linum usitatissimum L.) pectin methylesterase (Lupme3) cDNA sequence yielded plants with altered pollen content. Moreover, the characteristically sculptured cell wall surrounding the pollen grains was modified in transgenic tobacco plants: the wavy ornamentation was dramatically reduced, suggesting the involvement of the demethylation of pectin in the pollen cell wall-specific structure. Germination of pollen was decreased and the pollen tube surface aspect was also different in transgenic plants.Correspondence and reprints: Laboratoire de Biotechnologies et Physiologie Végétales, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France.     

Isolation and Characterization of an Anther-Specific Polygalacturonase Gene, BcMF16, in Brassica campestris ssp. chinensis

Many genes in the genic male sterile A/B line (Bajh97-01A/B) of Chinese cabbage pak choi (Brassica campestris L. subsp. chinensis Makino) are expressed differentially, and some play critical roles in the formation of pollen walls. In this study, one of these genes, Brassica campestris Male Fertility 16 (BcMF16), has been isolated and characterized. The BcMF16 gene shares approximately 85% nucleotide sequence homology with two exopolygalacturonase (EC3.2.1.67) genes of Arabidopsis thaliana. Cluster analysis of polygalacturonase peptides indicate that BcMF16 belongs to the pollen polygalacturonase clade. Quantitative real-time PCR analysis has revealed that BcMF16 is specifically expressed in reproductive tissues of the fertile line of genic male sterile A/B line of Chinese cabbage pak choi, and that expression levels dramatically increased during later stages of pollen development. In situ hybridization has demonstrated that BcMF16 is specifically and transiently expressed in both tapetum and pollen following microspore separation at the tetrad stage.     

BoPMEI1, a pollen-specific pectin methylesterase inhibitor, has an essential role in pollen tube growth

Pectin methylesterase (PME) is known to have important roles in pollen development and pollen tube growth. As pivotal regulatory factors in PME activity modulation, PME inhibitors (PMEIs) are thought to be key regulators of cell wall stability at the tip of the pollen tube. We report on the cloning and characterization of a novel B. oleracea PMEI gene, BoPMEI1. Heterologously expressed BoPMEI1 showed PMEI activity. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoPMEI1 was specifically expressed in mature pollen grains and pollen tubes. Based on in vivo transient assays, we found that BoPMEI1 appears to be largely localized to the plasma membrane. Transgenic Arabidopsis plants expressing antisense BoPMEI1 under the control of the CaMV 35S promoter suppressed the expression of the orthologous gene At1g10770, which led to partial male sterility and decreased seed set by inhibition of pollen tube growth. Our study demonstrates the involvement of BoPMEI1 in pollen tube growth.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

调控拟南芥花瓣脱落的果胶甲酯酶基因表达和功能分析

在基因序列和表达信息分析的基础上,重点研究初选出的两个拟南芥果胶甲酯酶基因At8g47550和At4g02330的表达和功能。半定量RT-PCR和转基因植株检测表明,At2g47550主要在花粉粒中大量表达,其次在微管组织中有明显表达。然而,At4g02330在拟南芥花和荚果整个发育时期都有不同程度的表达。主要在花的离层组织、柱头、微管组织和花粉粒中表达。结果说明,At2g47550可能参与花粉粒和花粉管的发育,而At4g02330则可能通过参与细胞壁中果胶代谢,从而达到调控细胞分离和花瓣脱落的目的。     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Chromosomal location of a pollen fertility-restoring gene, Rf, for CMS in Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization

In a previous study, we developed cytoplasmic male sterile lines of Allium fistulosum possessing the cytoplasm of A. galanthum, a wild species, by continuous backcrossing. Furthermore, we reported the presence of a pollen fertility-restoring gene (Rf) for cytoplasmic male sterility (CMS) in A. fistulosum from segregation of pollen fertility of backcross progenies. In the present study, genomic in situ hybridization (GISH), using genomic DNA of A. galanthum as the probe DNA and that of A. fistulosum as the blocking DNA, was applied to F₁ hybrids between both species and backcross progenies to determine the chromosomal location of the Rf locus. By means of GISH, eight chromosomes from A. galanthum were clearly discriminated from those of A. fistulosum in the F₁ hybrids, and chromosome substitution process through continuous backcrossing was visualized. Interestingly, the chromosome region from A. galanthum, specific to male fertile plants, was detected in one chromosome of BC₄ to BC₇ generations. Based on the karyotype analysis of the male fertile plants, the chromosome was identified as the 5F chromosome. Our results confirm that the Rf locus is located on the 5F chromosome of the male fertile plants. This is the first report that identified the chromosomal location of the pollen fertility-restoring gene in A. fistulosum.