高表达miR396小分子导致拟南芥花柱头弯曲

MicroRNAs（miRNAs）是大小约21个碱基、内源、非编码的小分子RNA。以拟南芥（Arabidopsis thaliana）miR396小分子为研究对象,分别克隆到了miR396小分子的两个前体（MIR396a,MIR396b）,得到了转基因植株。通过转基因植株的遗传学研究发现,高表达miR396小分子导致转基因拟南芥的花柱头弯曲。花柱头的弯曲影响了角果的正常发育。另外,Northern杂交结果表明转基因拟南芥花部位的miR396及其前体的表达量与对照相比显著增加。这些结果表明高表达miR396小分子可以导致拟南芥花柱头弯曲。     

Widespread tissue expression of nepenthesin-like aspartic protease genes in Arabidopsis thaliana.

There are approximately 69 genes encoding aspartyl protease homologues in Arabidopsis thaliana, and most of the gene products constitute a novel subfamily of aspartic proteases. However, their physiological roles are largely unknown. As an initial step to shed light on the roles of these nepenthesin-like aspartic proteases (NAPs), a phylogenetic tree was constructed, which indicated that these proteases are classified into several distinct sub-sub-groups. Based on these results, specific primers were designed for genes selected from several of these groups and their tissue expression was investigated using RT-PCR. The results indicated that these genes are widely expressed in several tissues, such as leaves, stems, seeds and pods, suggesting ubiquitous occurrence and multiple functions of the corresponding proteases in the tissues of A. thaliana.     

Control of sulfate concentration by miR395-targeted APS genes in Arabidopsis thaliana

Sulfur nutrition is crucial for plant growth and development,as well as crop yield and quality.Inorganic sulfate in the soil is the major sulfur source for plants.After uptake,sulfate is activated by ATP sulfurylase,and then gets assimilated into sulfur-containing metabolites.However,the mechanism of regulation of sulfate levels by ATP sulfurylase is unclear.Here,we investigated the control of sulfate levels by miR395-mediated regulation of APS1/3/4.Sulfate was over-accumulated in the shoots of miR395 over-expression plants in which the expression of the APS1,APS3,and APS4 genes was suppressed.Accordingly,reduced expression of miR395 caused a decline of sulfate concentration.In agreement with these results,over-expression of the APS1,APS3,and APS4 genes led to the reduction of sulfate levels.Differential expression of these three APS genes in response to sulfate starvation implied that they have different functions.Further investigation revealed that the regulation of sulfate levels mediated by miR395 depends on the repression of its APS targets.Unlike the APS1,APS3,and APS4 genes,which encode plastid-localized ATP sulfurylases,the APS2 gene encodes a cytosolic version of ATP sulfurylase.Genetic analysis indicated that APS2 has no significant effect on sulfate levels.Our data suggest that miR395-targeted APS genes are key regulators of sulfate concentration in leaves.     

Lipopolysaccharide mobility in leaf tissue of Arabidopsis thaliana

Bacterial lipopolysaccharides (LPS) are triggers of defence responses in plants, and induce local as well as systemic acquired resistance. Arabidopsis thaliana plants pretreated with LPS show an increased resistance to the virulent bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000. To investigate the mobilization and transport of LPS in Arabidopsis leaves, fluorescently labelled LPS (Alexa Fluor® 488 conjugate) from Salmonella minnesota was used. Leaves were pressure infiltrated with fluorescein‐labelled LPS and fluorescence microscopy was used to follow the movement and localization of LPS as a function of time. The observation of leaves 1 h after supplementation with fluorescein‐labelled LPS revealed a fluorescent signal in the intercellular space. Capillary zone electrophoresis was used for the detection and analysis of the labelled LPS in directly treated leaves and systemic leaves. In addition, gel electrophoresis was used to confirm LPS mobilization. The results indicated that LPS mobilization/translocation occurs through the xylem from local, treated leaves to systemic, untreated leaves. Consequently, care should be taken when ascribing the observed biochemical responses and induced resistance from LPS perception as being uniquely local or systemic, as these responses might overlap because of the mobility of LPS in the plant vascular system.     

Hydrogen peroxide-induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.     

Sequence and expression differences underlie functional specialization of Arabidopsis microRNAs miR159 and miR319

Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences.     

An expression atlas of miRNAs in Arabidopsis thaliana

MicroRNAs(miRNAs) are small non-coding RNAs that regulate a variety of biological processes. miRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

Saturating the genetic map of Arabidopsis thaliana with embryonic mutations

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo-defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo-genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty-six of these genes are tagged with T-DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo-defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.     

Protein expression in Arabidopsis thaliana after chronic clinorotation

Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.