Arabidopsis ecotype variability in camalexin production and reaction to infection by Alternaria brassicicola

Camalexin production was compared in 24 ecotypes of Arabidopsis thaliana. Detached Arabidopsis leaves were inoculated with Cochliobolus carbonum, an incompatible pathogen of Arabidopsis, to test the ability of each ecotype to produce camalexin. Whole plants were inoculated with Alternaria brassicicola, a crucifer pathogen, to determine if there was a correlation between the ability of an ecotype to produce camalexin and its resistance to A. brassicicola. All ecotypes were capable of producing camalexin, but the amounts produced relative to the Columbia ecotype (used as a standard) varied within and among ecotypes, and among experiments. Different degrees of resistance to A. brassicicola were observed among ecotypes, both macroscopically and microscopically. Extraction of A. brassicicola-inoculated leaves revealed that only four ecotypes (two resistant and two susceptible) produced easily detectable amounts of camalexin in response to this pathogen. TLC plate bioassays suggested that A. brassicicola was relatively insensitive to camalexin, thus casting some doubt on the importance of this compound in defense. These studies suggest that the role of camalexin in disease resistance varies among different Arabidopsis populations in nature, and they provide some clues to other possible determinants of resistance to A. brassicicola.     

基于植物p53保守序列构建RNAi的DNA片段方法

构建p53基因RNA干涉DNA片段的目的,是将其应用于植物悬浮培养中的细胞周期调节．依据拟南芥p53基因与其他高等植物具有高度保守区域的特点,合成其两个区域的核苷酸片段（Ⅰ和Ⅱ）及其相应的反向片段（Ⅰ＇和Ⅱ＇）,并在Ⅱ和Ⅱ＇端部分别引入内含子两个端部核苷酸序列．在相互连接后进行PCR选择扩增,其产物再与克隆裁体连接并经过蓝白斑筛选获得重组DNA;在电泳和核苷酸测序鉴定后表明,最终得到了Ⅰ-5＇-内含子-Ⅱ＇-Ⅱ-内含子-3＇-Ⅰ＇序列的重组DNA片段该片段两端含多克隆位点,通过插入植物的表达载体进入细胞基因组,在细胞中其转录产物将形成发夹结构,经胞内酶切后可以形成短的双链RNA片段,将具有干涉p53基因表达的功能     

Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean.     

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

GABA transaminase (GABA‐T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA‐T from Saccharomyces cerevisiae (ScGABA‐TKG) is an α‐ketoglutarate‐dependent enzyme encoded by the UGA1 gene, while higher plant GABA‐T is a pyruvate/glyoxylate‐dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA‐T). The GABA‐T from A. thaliana is localized in mitochondria and mediated by an 18‐amino acid N‐terminal mitochondrial targeting peptide predicated by both web‐based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA‐TKG and AtGABA‐T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA‐TKG and AtGABA‐T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA‐TKG deletion mutant Δuga1 with AtGABA‐T or ScGABA‐TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat‐induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA‐T is functionally interchangeable with ScGABA‐TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA‐T is about half as efficient in doing so as ScGABA‐TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate‐limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.     

Arabidopsis serine decarboxylase mutants implicate the roles of ethanolamine in plant growth and development

Ethanolamine is important for synthesis of choline, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in plants. The latter two phospholipids are the major phospholipids in eukaryotic membranes. In plants, ethanolamine is mainly synthesized directly from serine by serine decarboxylase. Serine decarboxylase is unique to plants and was previously shown to have highly specific activity to l-serine. While serine decarboxylase was biochemically characterized, its functions and importance in plants were not biologically elucidated due to the lack of serine decarboxylase mutants. Here we characterized an Arabidopsis mutant defective in serine decarboxylase, named atsdc-1 (Arabidopsis thaliana serine decarboxylase-1). The atsdc-1 mutants showed necrotic lesions in leaves, multiple inflorescences, sterility in flower, and early flowering in short day conditions. These defects were rescued by ethanolamine application to atsdc-1, suggesting the roles of ethanolamine as well as serine decarboxylase in plant development. In addition, molecular analysis of serine decarboxylase suggests that Arabidopsis serine decarboxylase is cytosol-localized and expressed in all tissue.     

Chemoproteomic Profiling of O-GlcNAcylation in Arabidopsis Thaliana by Using Metabolic Glycan Labeling

Protein O-GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O-GlcNAcylated proteins have been identified in animals, the plant O-GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O-GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both N-azidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz) can metabolically label O-GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP-galactose 4-epimerases were found to interconvert UDP-GalNAz and UDP-GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide-incorporated O-GlcNAc with alkyne-biotin via click chemistry, the O-GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O-GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O-GlcNAcylated proteins were enriched in various plant-specific processes such as hormone responses. By co-expression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O-GlcNAc transferase, we validated that the MGL-identified proteins were O-GlcNAc-modified. Our work establishes a powerful tool for profiling plant O-GlcNAylation and provides an invaluable resource for investigating the functional role of O-GlcNAc in Arabidopsis.     

Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two-photon laser scanning microscopy

Two-photon laser scanning microscopy (TPLSM) was used to directly measure glutathione (GSH) as its fluorescent glutathione S-bimane conjugate (GSB) in developing root hair cells (trichoblasts) and non-root hair cells (atrichoblasts) of intact Arabidopsis roots. In comparison to confocal microscopy, TPLSM showed more detail deep within the tissue with less signal attenuation. The total level of GSB labelling reached a plateau after 60 min in both trichoblasts and atrichoblasts, reflecting depletion of GSH. GSB was formed initially in the cytoplasm and was subsequently transported into the vacuole. The volume ratio of vacuole to cytoplasm was determined using the Cavalieri estimator of volume and used to calculate the amount of GSB per volume of cytoplasm in each cell type. At the end of the time-course the cytoplasmic concentration of GSB was 2.7 ± 0.5 m m ( n  = 5) in trichoblasts and 5.5 ± 0.8 m m ( n  = 5) in atrichoblasts. In trichoblasts this value represents the initial concentration of GSH in the cytoplasm. Labelling of roots with monochlorobimane (MCB) on ice led to the formation of GSB in the cytoplasm, but prevented vacuolar sequestration. After washing prelabelled roots and transfer to room temperature, vacuolar transport resumed. Although no free MCB was present the total amount of GSB in atrichoblasts increased further, indicating that the higher values recorded in the atrichoblasts might reflect additional symplastic transport and sequestration of GSB from neighbouring cells.