Overexpression of phyA and appA Genes Improves Soil Organic Phosphorus Utilisation and Seed Phytase Activity in Brassica napus

Phytate is the major storage form of organic phosphorus in soils and plant seeds, and phosphorus (P) in this form is unavailable to plants or monogastric animals. In the present study, the phytase genes phyA and appA were introduced into Brassica napus cv Westar with a signal peptide sequence and CaMV 35S promoter, respectively. Three independent transgenic lines, P3 and P11 from phyA and a18 from appA, were selected. The three transgenic lines exhibited significantly higher exuded phytase activity when compared to wild-type (WT) controls. A quartz sand culture experiment demonstrated that transgenic Brassica napus had significantly improved P uptake and plant biomass. A soil culture experiment revealed that seed yields of transgenic lines P11 and a18 increased by 20.9% and 59.9%, respectively, when compared to WT. When phytate was used as the sole P source, P accumulation in seeds increased by 20.6% and 46.9% with respect to WT in P11 and a18, respectively. The P3 line accumulated markedly more P in seeds than WT, while no significant difference was observed in seed yields when phytate was used as the sole P source. Phytase activities in transgenic canola seeds ranged from 1,138 to 1,605 U kg^–1 seeds, while no phytase activity was detected in WT seeds. Moreover, phytic acid content in P11 and a18 seeds was significantly lower than in WT. These results introduce an opportunity for improvement of soil and seed phytate-P bioavailability through genetic manipulation of oilseed rape, thereby increasing plant production and P nutrition for monogastric animals.     

Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus

Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.     

Seed Coat Microsculpturing Is Related to Genomic Components in Wild Brassica juncea and Sinapis arvensis

It has been reported that wild Brassica and related species are widely distributed across Xinjiang, China, and there has been an argument for species identification. Seed coat microsculpturing (SCM) is known to be an excellent character for taxonomic and evolutionary studies. By identifying collections from Xinjiang, China, and combining SCM pattern, flow cytometry, and genome-specific DNA markers as well as sexual compatibility with known species, this study aimed to detect potential relationships between SCM and genomic types in wild Brassica and related species. Three wild collections were found to be tetraploid with a SCM reticulate pattern similar to B. juncea, and containing A and B genome-specific loci, indicating relatively high sexual compatibility with B. juncea. The others were diploid, carrying S-genome-specific DNA markers, and having relatively high sexual compatibility with Sinapis arvensis. Moreover, their SCM was in a rugose pattern similar to that of S. arvensis. It was suggested that SCM, as a morphological characteristic, can reflect genomic type, and be used to distinguish B-genome species such as B. juncea from the related S. arvensis. The relationship between SCM and genomic type can support taxonomic studies of the wild Brassica species and related species.     

Genome-Wide Delineation of Natural Variation for Pod Shatter Resistance in Brassica napus

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.     

BraLTP1, a Lipid Transfer Protein Gene Involved in Epicuticular Wax Deposition,Cell Proliferation and Flower Development in Brassica napus

Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17–80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.     

Dissecting Quantitative Trait Loci for Boron Efficiency across Multiple Environments in Brassica napus

High yield is the most important goal in crop breeding, and boron (B) is an essential micronutrient for plants. However, B deficiency, leading to yield decreases, is an agricultural problem worldwide. Brassica napus is one of the most sensitive crops to B deficiency, and considerable genotypic variation exists among different cultivars in response to B deficiency. To dissect the genetic basis of tolerance to B deficiency in B. napus, we carried out QTL analysis for seed yield and yield-related traits under low and normal B conditions using the double haploid population (TNDH) by two-year and the BQDH population by three-year field trials. In total, 80 putative QTLs and 42 epistatic interactions for seed yield, plant height, branch number, pod number, seed number, seed weight and B efficiency coefficient (BEC) were identified under low and normal B conditions, singly explaining 4.15–23.16% and 0.53–14.38% of the phenotypic variation. An additive effect of putative QTLs was a more important controlling factor than the additive-additive effect of epistatic interactions. Four QTL-by-environment interactions and 7 interactions between epistatic interactions and the environment contributed to 1.27–4.95% and 1.17–3.68% of the phenotypic variation, respectively. The chromosome region on A2 of SYLB-A2 for seed yield under low B condition and BEC-A2 for BEC in the two populations was equivalent to the region of a reported major QTL, BE1. The B. napus homologous genes of Bra020592 and Bra020595 mapped to the A2 region and were speculated to be candidate genes for B efficiency. These findings reveal the complex genetic basis of B efficiency in B. napus. They provide a basis for the fine mapping and cloning of the B efficiency genes and for breeding B-efficient cultivars by marker-assisted selection (MAS).     

Dynamic Metabolic Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of Brassica napus

Canola (Brassica napus) is one of several important oil-producing crops, and the physiological processes, enzymes, and genes involved in oil synthesis in canola seeds have been well characterized. However, relatively little is known about the dynamic metabolic changes that occur during oil accumulation in seeds, as well as the mechanistic origins of metabolic changes. To explore the metabolic changes that occur during oil accumulation, we isolated metabolites from both seed and silique wall and identified and characterized them by using gas chromatography coupled with mass spectrometry (GC-MS). The results showed that a total of 443 metabolites were identified from four developmental stages. Dozens of these metabolites were differentially expressed during seed ripening, including 20 known to be involved in seed development. To investigate the contribution of tissue-specific carbon sources to the biosynthesis of these metabolites, we examined the metabolic changes of silique walls and seeds under three treatments: leaf-detachment (Ld), phloem-peeling (Pe), and selective silique darkening (Sd). Our study demonstrated that the oil content was independent of leaf photosynthesis and phloem transport during oil accumulation, but required the metabolic influx from the silique wall. Notably, Sd treatment resulted in seed senescence, which eventually led to a severe reduction of the oil content. Sd treatment also caused a significant accumulation of fatty acids (FA), organic acids and amino acids. Furthermore, an unexpected accumulation of sugar derivatives and organic acid was observed in the Pe- and Sd-treated seeds. Consistent with this, the expression of a subset of genes involved in FA metabolism, sugar and oil storage was significantly altered in Pe and Sd treated seeds. Taken together, our studies suggest the metabolite profiles of canola seeds dynamically varied during the course of oil accumulation, which may provide a new insight into the mechanisms of the oil accumulation at the metabolite level.     

Coat Color Variation and Pigmentation Gene Expression in Rhesus Macaques (Macaca mulatta)

Light-dark coat color variation is a common aspect of color diversity within and across mammalian taxa. This variation in pelage brightness is associated with aspects of evolutionary ecology, particularly for primates, but little is known about the genetic mechanisms underlying light-dark differences in pelage pigmentation. Previous work, focusing particularly on macaques (Genus Macaca), has found no clear relationship between color variation and coding sequences of key pigmentation genes. This suggests that other loci and/or gene regulatory differences underlie this variation and raises the question of how patterns of gene expression differ in light verses dark hair follicles. Here, we examine relative expression levels of pigmentation genes in hair follicles from free-ranging rhesus macaques (Macaca mulatta) showing stark light-dark coat color variation. We quantified the brightness (reflectance) of plucked hair tufts using a spectrophotometer. We extracted RNA from the follicles and used quantitative RT-PCR to measure the relative amounts of gene product (mRNA) for seven candidate pigmentation genes (MITF, MC1R, MGRN1, ATRN, SLC24A5, TYRP1, and DCT). Expression values were normalized with the house-keeping gene ACTB. All candidate genes were expressed at similar levels in dark, intermediate, and light hair, and thus, light-dark variation in macaque coat color is unlikely to be due to differences in the expression of these key pigmentation genes. This study represents the first examination of gene expression and natural color variation in a non-human primate population. Our results indicate that even in a system, like pigmentation, where a candidate-gene approach is promising, identifying important intra-specific gene regulatory differences remains challenging.     

Characterization of the PMT Gene Family in Cryptococcus neoformans

Background

Protein-O-mannosyltransferases (Pmt''s) catalyze the initial step of protein-O-glycosylation, the addition of mannose residues to serine or threonine residues of target proteins.

Methodology/Principal Findings

Based on protein similarities, this highly conserved protein family can be divided into three subfamilies: the Pmt1 sub-family, the Pmt2 sub-family and the Pmt4 sub-family. In contrast to Saccharomyces cerevisiae and Candida albicans, but similar to filamentous fungi, three putative PMT genes (PMT1, PMT2, and PMT4) were identified in the genome of the human fungal pathogen Cryptococcus neoformans. Similar to Schizosaccharomyces pombe and C. albicans, C. neoformans PMT2 is an essential gene. In contrast, the pmt1 and pmt4 single mutants are viable; however, the pmt1/pmt4 deletions are synthetically lethal. Mutation of PMT1 and PMT4 resulted in distinct defects in cell morphology and cell integrity. The pmt1 mutant was more susceptible to SDS medium than wild-type strains and the mutant cells were enlarged. The pmt4 mutant grew poorly on high salt medium and demonstrated abnormal septum formation and defects in cell separation. Interestingly, the pmt1 and pmt4 mutants demonstrated variety-specific differences in the levels of susceptibility to osmotic and cell wall stress. Delayed melanin production in the pmt4 mutant was the only alteration of classical virulence-associated phenotypes. However, the pmt1 and pmt4 mutants showed attenuated virulence in a murine inhalation model of cryptococcosis.

Conclusion/Significance

These findings suggest that C. neoformans protein-O-mannosyltransferases play a crucial role in maintaining cell morphology, and that reduced protein-O-glycosylation leads to alterations in stress resistance, cell wall composition, cell integrity, and survival within the host.     

Genome-Wide Identification of the Invertase Gene Family in Populus

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.     

Characterization of the Pichia pastoris Protein-O-mannosyltransferase Gene Family

The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P . pastoris , thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P . pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P . pastoris PMT family. Like Candida albicans, P . pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P . pastoris platform as a suitable system for the production of therapeutic glycoproteins.