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.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

<Emphasis Type="Italic">BcMF11</Emphasis> and its homologous sequences may form a lncRNA family in <Emphasis Type="Italic">Brassica</Emphasis> diploids

BcMF11 is a long non-coding RNA that has been identified in Brassica rapa and shown to be involved in pollen development. Here, when re-cloned the gene sequence, multiple paralogous copies of BcMF11 were identified in B. rapa (A genome). Multiple paralogous copies of BcMF11 were also found in B. nigra (B genome) and Brassica oleracea (C genome), the other two primary diploids of Brassica U triangle. While in the early diverging Brassicaceae lineage including Arabidopsis thaliana, no BcMF11 homolog was found. Phylogenetic analysis showed that the BcMF11 homologous sequences cloned from A genome or C genome could be clustered into a separate branch, respectively. However, there was no distinct cluster defined for BcMF11 homologous sequences cloned from B genome. The expression of BcMF11 in B. rapa was investigated and revealed a different result in the previous study. In addition, 12 expressed sequence tags from B. napus and B. rapa showing high similarities with BcMF11 were identified in the NCBI database, which further verified that rather than the useless repeat fragments in the genome, the BcMF11 homologous genes could transcribe. It is possible that BcMF11 and its homologous sequences may form a large gene family which might be originated in the recent ancestral lineage of Brassica.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Effect of sporophytic <Emphasis Type="Italic">PIRL</Emphasis>9 genotype on post-meiotic expression of the Arabidopsis <Emphasis Type="Italic">pirl1</Emphasis>;<Emphasis Type="Italic">pirl9</Emphasis> mutant pollen phenotype

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander’s staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.     

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) inbred line,Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

A 252-bp insertion in <Emphasis Type="Italic">BoCER1</Emphasis> is responsible for the glossy phenotype in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">capitata</Emphasis>)

Bright green of leaf head is an important characteristic in cabbage breeding. Wax-less cabbage shows glossy phenotype on leaf surface, which facilitates the brilliant green cabbage breeding. In this study, we identified a spontaneous glossy mutant g21-3 in cabbage. Genetic analyses showed that its glossy phenotype is controlled by a single recessive gene. Further analysis indicated that the glossy phenotype of g21-3 and a known glossy cabbage mutant 10Q-961 was controlled by a same locus. According to the fine-mapping of glossy-controlled gene in 10Q-961, BoCER1 was identified as a candidate gene which was found to be closely related to the glossy phenotype in g21-3. Sub-cellular localization showed that BoCER1 protein is localized to the endoplasmic reticulum. Sequence analysis revealed that a 252-bp insertion was included in the fourth intron of BoCER1 in g21-3, but not in the wild-type 21-3. The insertion significantly inhibited the expression of BoCER1. A marker designed to distinguish between the BoCER1 alleles in g21-3 and wild-type cabbage co-segregated perfectly with glossy/waxy phenotypes in backcross population, confirming that the insertion mutation of BoCER1 is responsible for the glossy phenotype. The allele-specific marker is effective for marker-assisted selection of the glossy cabbage.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L^-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L^-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.