Gene atlas of iron-containing proteins in Arabidopsis thaliana

Iron (Fe) is an essential element for the development and physiology of plants, owing to its presence in numerous proteins involved in central biological processes. Here, we established an exhaustive, manually curated inventory of genes encoding Fe-containing proteins in Arabidopsis thaliana, and summarized their subcellular localization, spatiotemporal expression and evolutionary age. We have currently identified 1068 genes encoding potential Fe-containing proteins, including 204 iron-sulfur (Fe-S) proteins, 446 haem proteins and 330 non-Fe-S/non-haem Fe proteins (updates of this atlas are available at https://conf.arabidopsis.org/display/COM/Atlas+of+Fe+containing+proteins ). A fourth class, containing 88 genes for which iron binding is uncertain, is indexed as ‘unclear’. The proteins are distributed in diverse subcellular compartments with strong differences per category. Interestingly, analysis of the gene age index showed that most genes were acquired early in plant evolutionary history and have progressively gained regulatory elements, to support the complex organ-specific and development-specific functions necessitated by the emergence of terrestrial plants. With this gene atlas, we provide a valuable and updateable tool for the research community that supports the characterization of the molecular actors and mechanisms important for Fe metabolism in plants. This will also help in selecting relevant targets for breeding or biotechnological approaches aiming at Fe biofortification in crops.     

杂交构树UDP-葡萄糖脱氢酶基因编码蛋白的亚细胞定位及其启动子5'端缺失片段的功能分析

为了研究杂交构树UDP-葡萄糖脱氢酶基因（DDBJ,BpUGDH基因登录号为LC457701）启动子不同区域的表达活性,利用5'端缺失及同源重组实验技术,将5个不同长度的BpUGDH启动子5'端缺失片段与GUS基因连接,并通过农杆菌介导法瞬时转化烟草;同时,为了定位BpUGDH基因编码的蛋白在细胞中表达的具体位置,利用GFP报告基因融合目的基因进行蛋白质的亚细胞定位。结果显示：BpUGDH基因启动子-244 bp以内的序列均能介导GUS基因的诱导表达,并且-973、-465、-355、-281和-244 bp之间的区域可能对BpUGDH基因启动子的活性发挥着至关重要的作用。另外,BpUGDH基因编码蛋白的亚细胞定位结果显示：BpUGDH位于叶绿体中。     

Septin14, a gene specifically expressed in the testis and seminal vesicle of the Banna mini-pig inbred line (BMI)

Septin14 is an important spermatogenesis related gene involved in the pathogenesis of male infertility that has not been well studied. Here, full-length Septin14 cDNA of the Banna mini-pig inbred line (BMI) was cloned using the RACE method and expressed in pig kidney epithelial cells (PK15) and E. coli Rosetta (DE3) cells. Septin14 expression was identified in somatic tissues and testis in different developmental stages. The pig Septin14 CDS is 1,299 bp long, and encodes a peptide (or protein) of 432 amino acids (MW=50.4 kDa). Phylogenetic analysis indicated that pig Septin14 was highly evolutionarily conserved. Subcellular localization of GFP-tagged Septin14 fusion protein revealed that Septin14 was distributed throughout the testicular cells. Among 34 pig tissues, Septin14 mRNA was found specifically in testis and seminal vesicle. In six different postnatal developmental stages, the testicular level of Septin14 mRNA was barely detectable on day 2, while the highest level occurred on day 75. The spatiotemporal expression profile of Septin14, reported herein for the first time in pig, indicated that Septin14 might be involved in the division, development and apoptosis of germ cells. Furthermore, using a pET prokaryotic expression system, we expressed and isolated recombinant 67.9 kDa Septin14 protein.     

Perception of double-stranded RNA in plant antiviral immunity

RNA silencing and antiviral pattern-triggered immunity (PTI) both rely on recognition of double-stranded (ds)RNAs as defence-inducing signals. While dsRNA recognition by dicer-like proteins during antiviral RNA silencing is thoroughly investigated, the molecular mechanisms involved in dsRNA perception leading to antiviral PTI are just about to be untangled. Parallels to antimicrobial PTI thereby only partially facilitate our view on antiviral PTI. PTI against microbial pathogens involves plasma membrane bound receptors; however, dsRNAs produced during virus infection occur intracellularly. Hence, how dsRNA may be perceived during this immune response is still an open question. In this short review, we describe recent discoveries in PTI signalling upon sensing of microbial patterns and endogenous ‘danger’ molecules with emphasis on immune signalling-associated subcellular trafficking processes in plants. Based on these studies, we develop different scenarios how dsRNAs could be sensed during antiviral PTI.     

Trichloroacetic acid of different origin in Norway spruce needles and chloroplasts

Trichloroacetic acid (TCA), a secondary atmospheric pollutant, is also formed in forest soil and thus ranked among natural organohalogens. The observed biooxidation of atmospheric tetrachloroethene (PER) to TCA in chloroplasts has led to the investigation of the mode of action of TCA in spruce needles, since TCA is also accumulated in the needles after its rapid uptake from soil by roots. Being phytotoxic, TCA considerably influences conifers by affecting their photosynthetic apparatus. We examined the transport of TCA from soil into chloroplasts in order to compare the effects of TCA on conifers from both sources, i.e. endogenously produced within chloroplasts or taken up by roots. The influence of TCA formed in chloroplasts was found to be much more adverse than that of “soil” TCA.     

Proteomics of total membranes and subcellular membranes

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.     

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD₅₀ light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.     

Localization of fatty acid hydroperoxide cleavage activity in membranes of cucumber fruit

The subcellular localization of the fatty acid hydroperoxide cleavage enzyme from cucumber fruit has been studied. Activity from the flesh tissue has been located in 3 fractions; plasma and Golgi membranes and endoplasmic reticulum, at equilibrium densities on sucrose gradients of 1.17, 1.15 and 1.12 g/cm³ respectively. Enzymatic activity and electron microscopy studies were carried out to identify plasma and Golgi membranes. Little activity was associated with microbodies (1.23), plastids (1.21) and mitochondria (1:19 g/cm³). However, chloroplasts isolated from the peel of the cucumber fruit contained a large amount of hydroperoxide cleavage activity.     

Quantification and its Applications in Fluorescent Microscopy Imaging

Fluorescent microscope imaging technologies have developed at a rapid pace in recent years. High-throughput 2D fluorescent imaging platforms are now in wide use and are being applied on a proteome wide scale. Multiple fluorophore 3D imaging of live cells is being used to give detailed localization and subcellular structure information. Further, 2D and 3D video microscopy are giving important insights into the dynamics of protein localization and transport. In parallel with these developments, significant research has gone into developing new methodologies for quantifying and extracting meaning from the imaging data. Here we outline and give entry points to the literature on approaches to quantification such as segmentation, tracking, automated classification and data visualization. Particular attention is paid to the distinction between and application of concrete quantification measures such as number of objects in a cell, and abstract measures such as texture.     

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation

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