A novel plastid localization of chalcone synthase in developing grape berry

Chalcone synthase (CHS, E.C. 2.3.1.74) is an entrance enzyme of flavonoid metabolism and a critical point to regulation of biosynthesis of different flavonoid compounds that directly contribute to color and monthfeel of grape and wine. In the present experiment, subcellular localization of CHS in developing grape berry was performed via immunogold electron microscopy technique. The result showed that CHS was localized in rough endoplasmic reticulum (ER) and cytoplasm of the skin cells, while few gold particles representing CHS were found on the cell wall. Besides, two novel localized sites of CHS were observed within a cell of developing grape berry, one being the plastid-distributed throughout developmental stages and the other being vacuole-distributed at late developmental stage. It is speculated that these novel localized patterns may relate to abundant and multi-branch flavonoid metabolism in grape berry. This work will provide new insight for the regulation of different branch pathways leading to diverse flavonoid compounds.     

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.     

Global patterns of gene regulation associated with the development of ectomycorrhiza between birch (Betula pendula Roth.) and Paxillus involutus (Batsch) Fr

The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct morphological and developmental stages, such as preinfection and adhesion, mantle, and Hartig net formation. The global pattern of gene expression during these stages in the birch (Betula pendula)-Paxillus involutus ECM association was analyzed using cDNA microarrays. In comparison with nonsymbiotic conditions, 251 fungal (from a total of 1,075) and 138 plant (1,074 in total) genes were found to be differentially regulated during the ECM development. For instance, during mantle and Hartig net development, there were several plant genes upregulated that are normally involved in defense responses during pathogenic fungal challenges. These responses were, at later stages of ECM development, found to be repressed. Other birch genes that showed differential regulation involved several homologs that usually are implicated in water permeability (aquaporins) and water stress tolerance (dehydrins). Among fungal genes differentially upregulated during stages of mantle and Hartig net formation were homologs putatively involved in mitochondrial respiration. In fully developed ECM tissue, there was an upregulation of fungal genes related to protein synthesis and the cytoskeleton assembly machinery. This study highlights complex molecular interactions between two symbionts during the development of an ECM association.     

A novel cell ablation strategy blocks tobacco anther dehiscence.

We utilized a new cell ablation strategy to ablate specific anther cell types involved in the dehiscence process. The tobacco TA56 gene promoter is active within the circular cell cluster, stomium, and connective regions of the anther at different developmental stages. We introduced a cytotoxic TA56/barnase gene into tobacco plants together with three different anticytotoxic barstar genes. The anticytotoxic barstar genes were used to protect subsets of anther cell types from the cytotoxic effects of the TA56/barnase gene. The chimeric barstar genes were fused with (1) the tobacco TP12 gene promoter that is active at high levels in most anther cell types; (2) the soybean lectin gene promoter that is active earlier in the connective, and at lower levels in the circular cell cluster and stomium, than is the TA56 promoter; and (3) the tobacco TA20 gene promoter that is active at high levels in most anther cell types but has a different developmental profile than does the TP12 promoter. Normal anther development and dehiscence occurred in plants containing the TA56/barnase and TP12/barstar genes, indicating that barstar protects diverse anther cell types from the cytotoxic effects of barnase. Anthers containing the TA56/barnase and lectin/barstar genes also developed normally but failed to dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous connective regions. Anthers containing the TA56/barnase and TA20/barstar genes failed to dehisce as well. However, only the stomium region was ablated in these anthers. The connective, circular cell cluster, and adjacent wall regions were protected from ablation by the formation of barnase/barstar complexes. We conclude that anther dehiscence at flower opening depends on the presence of a functional stomium region and that chimeric barnase and barstar genes containing promoters that are active in several overlapping cell types can be used for targeted cell ablation experiments.     

Phenotypic characterization of Streptococcus pneumoniae biofilm development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.     

Expression analysis of irisin during different development stages of skeletal muscle in mice

BackgroundAs a newly discovered muscle factor secreted by skeletal muscle cells, irisin is a polypeptide fragment formed from hydrolysis of fibronectin type Ⅲ domain-containing protein 5 (FNDC5). Irisin can promote beigeing of white adipose tissue (WAT) and regulate glucose and lipid metabolisms. However, the functions of irisin in skeletal muscle development remain largely unknown. In order to characterize the expression of irisin, this study investigated the expression of irisin precursor FNDC5 in myoblasts and skeletal muscles during different developmental stages of SPF mice.ResultsThe Western blot, quantitative real-time PCR (qRT-PCR), and immunofluorescence assay results showed that FNDC5 was expressed in all the developmental stages of myoblasts and gastrocnemius, but its expression differed at different stages. FNDC5 protein exhibited the highest expression in gastrocnemius of sexually mature mice, followed by elderly mice and adolescent mice, and it displayed the lowest expression in pups. Additionally, FNDC5 protein was mainly expressed in cytoplasm, and it had the highest expression in primary myoblasts, followed by the myotubes with the lowest expression in C2C12 myogenic cells.ConclusionsOverall, FNDC5 was mainly expressed in cytoplasm and extracellular matrix with different expression levels at different developmental stages of skeletal muscle cells and tissues in mice. This study will provide new strategies for promoting skeletal muscle development and treating muscle- and metabolism-related disease by using irisin.     

Dynamics of vegetative cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana

Summary Ultrastructural changes of pollen cytoplasm during generative cell formation and pollen maturation inArabidopsis thaliana were studied. The pollen cytoplasm develops a complicated ultra-structure and changes dramatically during these stages. Lipid droplets increase after generative cell formation and their organization and distribution change with the developmental stage. Starch grains in amyloplasts increase in number and size during generative and sperm cell formation and decrease at pollen maturity. The shape and membrane system of mitochondria change only slightly. Dictyo-somes become very prominent, and numerous associated vesicles are observed during and after sperm cell formation. Endoplasmic reticulum appears extensively as stacks during sperm cell formation. Free and polyribosomes are abundant in the cytoplasm at all developmental stages although they appear denser at certain stages and in some areas. In mature pollen, all organelles are randomly distributed throughout the vegetative cytoplasm and numerous small particles appear. Organization and distribution of storage substances and appearance of these small particles during generative and sperm cell formation and pollen maturation are discussed.     

Analysis of cell-type-specific gene expression during mouse spermatogenesis

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of specific cells, or changes in the cell-type composition. This reflects the cellularity of the tissue. Here we have combined techniques that assess the expression profiles of genes at the whole-tissue level, differential display and DNA array, and, at the level of cellularity, in situ hybridization. Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII-XI). This pachytene cluster was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination of these cell-type-specific expression patterns.     

The dynamic gene expression from different genetic systems for protein and lysine contents of indica rice

The dynamic expression of genes for protein and lysine contents of rice grain under different environments was carried out with time-dependent measures by using the developmental genetic models for quantitative traits of triploid endosperm in cereal crops. The results showed that the genetic effects, especially genotype × environment (GE) interaction effects from the genes expression of different genetic systems including triploid endosperm, cytoplasm and diploid maternal plant were important for the performance of both nutrient quality traits at all developmental times/stages of rice grain. The conditional genetic variance analysis found that the activation of quantitative genes especially from endosperm and maternal plant genetic systems for protein and lysine contents was gradually carried through the developmental process of rice grain. The net genetic effects showed that the new expression of quantitative genes for protein and lysine contents was more active at late filling stage (15–21 days after flowering) and maturity stage (22–28 days) of rice grain. Also the sequential expression of cytoplasmic genes cannot be ignorable for the development of nutrient quality traits. The phenomena that some genes could continuously express for several developmental stages or the genes expression could be interrupted among developmental stages of rice grain was detected especially for net endosperm additive main effects or maternal additive main effects. The differences of genetic relationships from different genetic systems were found for protein and lysine contents among developmental times of rice grain.     

Developmental behavior of gene expression for brown rice thickness under different environments

The dynamic changes of genetic effects, including main effects, and genotype x environment (GE) interaction effects on brown rice thickness (BRT) across environments were investigated by using the developmental genetic models. Seven cytoplasmic male sterile lines of indica rice (Oryza sativa L.) as females and five restoring lines as males were used in a factorial design to produce grains of F(1)s and F(2)s in two environments (years) for developmental genetic analysis. The results indicate that genetic effects, especially GE interaction effects of triploid endosperm genes, cytoplasm genes, and diploid maternal plant genes were important to the performance of BRT at various filling stages of rice. The BRT was genetically controlled by the net genetic effects of genes expressed at the early and late filling stages (1-7 days and 15-21 days after flowering, respectively). The differences in net genetic effects under different environments for endosperm, cytoplasm, and maternal plant genes were found, and the net GE interaction effects were more important to BRT at the early filling and mature stages of rice. Some net genetic effects, especially for net cytoplasm effects spasmodically expressed, were detected among filling stages. Higher additive and cytoplasm main effects, along with their interaction effects, were found, which would be useful for selection for BRT in breeding programs. The predicated genetic effects at different filling stages show that the parents of V20 and Xieqingzao were better than others for improving BRT of progenies.     

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.     

葡萄SAUR基因家族鉴定与生物信息学分析

为了研究葡萄早期应答生长素基因SAUR(Small auxin-up RNA)家族,本研究利用全基因组信息鉴定了葡萄64个SAUR家族成员,并对SAUR家族成员的基因结构、氨基酸特性、染色体定位、基因进化、基因功能以及组织表达进行分析。结果表明,葡萄全基因组上64个SAUR家族成员在19条染色体中的8条染色体上呈现簇状分布,主要分布在3、4号染色体上,其中3号染色体上数量最多为37个;葡萄SAUR家族基因长度较短,有59个基因是无内含子基因;蛋白理化特征分析显示,多数SAUR蛋白呈碱性,结构稳定性较差,蛋白脂溶指数高,呈亲水性;基因功能预测结果表明,葡萄SAUR基因主要担当生长因子、结构蛋白、转录、转录调控以及响应胁迫应答和免疫应答6种功能,其中更多参与生长调节功能;根据系统进化分析将其分为10个分支,另外不同组织表达谱的分析结果表明SAUR基因家族成员具有不同的组织表达模式,对于非生物胁迫具有一定的调节作用。这些信息为葡萄SAUR基因家族功能分析奠定了一定的工作基础。     

Helminthosporic acid functions as an agonist for gibberellin receptor

Helminthosporol was isolated from a fungus, Helminthosporium sativum, as a natural plant growth regulator in 1963. It showed gibberellin-like bioactivity that stimulated the growth of the second leaf sheath of rice. After studying the structure–activity relationship between the compound and some synthesized analogs, it was found that helminthosporic acid (H-acid) has higher gibberellin-like activity and chemical stability than helminthosporol. In this study, we showed that (1) H-acid displays gibberellin-like activities not only in rice but also in Arabidopsis, (2) it regulates the expression of gibberellin-related genes, (3) it induces DELLA degradation through binding with a gibberellin receptor (GID1), and (4) it forms the GID1-(H-acid)-DELLA complex to transduce the gibberellin signal in the same manner as gibberellin. This work shows that the H-acid mode of action acts as an agonist for gibberellin receptor.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.