Jane Geisler-Lee

A Predicted Interactome for Arabidopsis

The complex cellular functions of an organism frequently rely on physical interactions between proteins. A map of all protein-protein interactions, an interactome, is thus an invaluable tool. We present an interactome for Arabidopsis (Arabidopsis thaliana) predicted from interacting orthologs in yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila melanogaster), and human (Homo sapiens). As an internal quality control, a confidence value was generated based on the amount of supporting evidence for each interaction. A total of 1,159 high confidence, 5,913 medium confidence, and 12,907 low confidence interactions were identified for 3,617 conserved Arabidopsis proteins. There was significant coexpression of genes whose proteins were predicted to interact, even among low confidence interactions. Interacting proteins were also significantly more likely to be found within the same subcellular location, and significantly less likely to be found in conflicting localizations than randomly paired proteins. A notable exception was that proteins located in the Golgi were more likely to interact with Golgi, vacuolar, or endoplasmic reticulum sorted proteins, indicating possible docking or trafficking interactions. These predictions can aid researchers by extending known complexes and pathways with candidate proteins. In addition we have predicted interactions for many previously unknown proteins in known pathways and complexes. We present this interactome, and an online Web interface the Arabidopsis Interactions Viewer, as a first step toward understanding global signaling in Arabidopsis, and to whet the appetite for those who are awaiting results from high-throughput experimental approaches.

Poplar Carbohydrate-Active Enzymes. Gene Identification and Expression Analyses

Over 1,600 genes encoding carbohydrate-active enzymes (CAZymes) in the Populus trichocarpa (Torr. & Gray) genome were identified based on sequence homology, annotated, and grouped into families of glycosyltransferases, glycoside hydrolases, carbohydrate esterases, polysaccharide lyases, and expansins. Poplar (Populus spp.) had approximately 1.6 times more CAZyme genes than Arabidopsis (Arabidopsis thaliana). Whereas most families were proportionally increased, xylan and pectin-related families were underrepresented and the GT1 family of secondary metabolite-glycosylating enzymes was overrepresented in poplar. CAZyme gene expression in poplar was analyzed using a collection of 100,000 expressed sequence tags from 17 different tissues and compared to microarray data for poplar and Arabidopsis. Expression of genes involved in pectin and hemicellulose metabolism was detected in all tissues, indicating a constant maintenance of transcripts encoding enzymes remodeling the cell wall matrix. The most abundant transcripts encoded sucrose synthases that were specifically expressed in wood-forming tissues along with cellulose synthase and homologs of KORRIGAN and ELP1. Woody tissues were the richest source of various other CAZyme transcripts, demonstrating the importance of this group of enzymes for xylogenesis. In contrast, there was little expression of genes related to starch metabolism during wood formation, consistent with the preferential flux of carbon to cell wall biosynthesis. Seasonally dormant meristems of poplar showed a high prevalence of transcripts related to starch metabolism and surprisingly retained transcripts of some cell wall synthesis enzymes. The data showed profound changes in CAZyme transcriptomes in different poplar tissues and pointed to some key differences in CAZyme genes and their regulation between herbaceous and woody plants.

Phytotoxicity, accumulation and transport of silver nanoparticles by Arabidopsis thaliana

Abstract The widespread availability of nano-enabled products in the global market may lead to the release of a substantial amount of engineered nanoparticles in the environment, which frequently display drastically different physiochemical properties than their bulk counterparts. The purpose of the study was to evaluate the impact of citrate-stabilised silver nanoparticles (AgNPs) on the plant Arabidopsis thaliana at three levels, physiological phytotoxicity, cellular accumulation and subcellular transport of AgNPs. The monodisperse AgNPs of three different sizes (20, 40 and 80 nm) aggregated into much larger sizes after mixing with quarter-strength Hoagland solution and became polydisperse. Immersion in AgNP suspension inhibited seedling root elongation and demonstrated a linear dose–response relationship within the tested concentration range. The phytotoxic effect of AgNPs could not be fully explained by the released silver ions. Plants exposed to AgNP suspensions bioaccumulated higher silver content than plants exposed to AgNO3 solutions (Ag+ representative), indicating AgNP uptake by plants. AgNP toxicity was size and concentration dependent. AgNPs accumulated progressively in this sequence: border cells, root cap, columella and columella initials. AgNPs were apoplastically transported in the cell wall and found aggregated at plasmodesmata. In all the three levels studied, AgNP impacts differed from equivalent dosages of AgNO3.

IMMUTANS Does Not Act as a Stress-Induced Safety Valve in the Protection of the Photosynthetic Apparatus of Arabidopsis during Steady-State Photosynthesis

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O2 to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P700 to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6×) and 16 (OE-16×) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1). Similar results were observed either after 3-d cold stress at 5°C or after full-leaf expansion at 5°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1. Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25°C or 5°C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P700+ during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Expression of the ethylene biosynthetic machinery in maize roots is regulated in response to hypoxia.

Ethylene regulates plant growth in response to many adverse environmental conditions, including the induction of aerenchyma, i.e. the formation of air spaces, in flooded roots in an effort to maintain oxygen levels. In this work, quantitative RT-PCR and in situ RNA hybridization were used to determine how the expression of the ethylene biosynthetic machinery in maize roots is spatially and temporally regulated following exposure to 4% oxygen (i.e. hypoxia) for up to 24 h, conditions that induced aerenchyma formation in the fully-expanded region of the root and reduced cytoplasmic density throughout the root. Expression of ACC oxidase, the ethylene forming enzyme, was observed in the root cap, protophloem sieve elements, and companion cells associated with metaphloem sieve elements. Exposure to 4% oxygen induced ACC oxidase expression in these cell types as well as in the root cortex. ACC synthase, which generates the ethylene precursor, was expressed in the root cap and the cortex and its expression was induced in cortical cells following low oxygen treatment. The induction of expression of the ethylene biosynthetic machinery was accompanied by an induction of ethylene evolution and a reduced rate of root growth. These results suggest that maize roots respond to conditions of hypoxia by inducing the spatially restricted expression of the ethylene biosynthetic machinery, resulting in increased ethylene production.

Tissue-specific expression of the ethylene biosynthetic machinery regulates root growth in maize.

Although the hormonal control of root growth and development has been extensively studied, relatively little is known about the role that ethylene plays in cereal root development. In this work, we have investigated how the ethylene biosynthetic machinery is spatially regulated in maize roots and how changes in its expression alter root growth. ACC synthase (ZmACS) expression was observed in the root cap and in cortical cells whereas ACC oxidase (ZmACO) expression was detected in the root cap, protophloem sieve elements, and the companion cells associated with metaphloem sieve elements. Roots from Zmacs6 mutants exhibited significantly reduced ethylene production, a smaller root cap of increased cell number but smaller cell size, accelerated elongation of metaxylem, cortical, and epidermal cells, and increased vacuolation of cells in the calyptrogen of the root cap, phenotypes that were complemented by exogenous ACC. Zmacs6 mutant roots exhibited increased growth when largely unimpeded, a phenotype complemented by exogenous ACC, whereas loss of ZmACS2 expression had less of an effect. In contrast, Zmacs6 plants exhibited reduced root growth in soil. These results suggest that expression of ZmACS6 is important in regulating growth of maize roots in response to physical resistance.

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family

BACKGROUND AND AIMS Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene. METHODS Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4.1b10). KEY RESULTS Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides. CONCLUSIONS This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.

Reproductive Toxicity and Life History Study of Silver Nanoparticle Effect, Uptake and Transport in Arabidopsis thaliana

Concerns about nanotechnology have prompted studies on how the release of these engineered nanoparticles impact our environment. Herein, the impact of 20 nm silver nanoparticles (AgNPs) on the life history traits of Arabidopsis thaliana was studied in both above- and below-ground parts, at macroscopic and microscopic scales. Both gross phenotypes (in contrast to microscopic phenotypes) and routes of transport and accumulation were investigated from roots to shoots. Wild type Arabidopsis growing in soil, regularly irrigated with 75 μg/L of AgNPs, did not show any obvious morphological change. However, their vegetative development was prolonged by two to three days and their reproductive growth shortened by three to four days. In addition, the germination rates of offspring decreased drastically over three generations. These findings confirmed that AgNPs induce abiotic stress and cause reproductive toxicity in Arabidopsis. To trace transport of AgNPs, this study also included an Arabidopsis reporter line genetically transformed with a green fluorescent protein and grown in an optical transparent medium with 75 μg/L AgNPs. AgNPs followed three routes: (1) At seven days after planting (DAP) at S1.0 (stages defined by Boyes et al. 2001 [41]), AgNPs attached to the surface of primary roots and then entered their root tips; (2) At 14 DAP at S1.04, as primary roots grew longer, AgNPs gradually moved into roots and entered new lateral root primordia and root hairs; (3) At 17 DAP at S1.06 when the Arabidopsis root system had developed multiple lateral roots, AgNPs were present in vascular tissue and throughout the whole plant from root to shoot. In some cases, if cotyledons of the Arabidopsis seedlings were immersed in melted transparent medium, then AgNPs were taken up by and accumulated in stomatal guard cells. These findings in Arabidopsis are the first to document specific routes and rates of AgNP uptake in vivo and in situ.

Aleurone cell identity is suppressed following connation in maize kernels.

Expression of the cytokinin-synthesizing isopentenyl transferase enzyme under the control of the Arabidopsis (Arabidopsis thaliana) SAG12 senescence-inducible promoter reverses the normal abortion of the lower floret from a maize (Zea mays) spikelet. Following pollination, the upper and lower floret pistils fuse, producing a connated kernel with two genetically distinct embryos and the endosperms fused along their abgerminal face. Therefore, ectopic synthesis of cytokinin was used to position two independent endosperms within a connated kernel to determine how the fused endosperm would affect the development of the two aleurone layers along the fusion plane. Examination of the connated kernel revealed that aleurone cells were present for only a short distance along the fusion plane whereas starchy endosperm cells were present along most of the remainder of the fusion plane, suggesting that aleurone development is suppressed when positioned between independent starchy endosperms. Sporadic aleurone cells along the fusion plane were observed and may have arisen from late or imperfect fusion of the endosperms of the connated kernel, supporting the observation that a peripheral position at the surface of the endosperm and not proximity to maternal tissues such as the testa and pericarp are important for aleurone development. Aleurone mosaicism was observed in the crown region of nonconnated SAG12-isopentenyl transferase kernels, suggesting that cytokinin can also affect aleurone development.

Gene Expression, Protein Function and Pathways of Arabidopsis thaliana Responding to Silver Nanoparticles in Comparison to Silver Ions, Cold, Salt, Drought, and Heat

Silver nanoparticles (AgNPs) have been widely used in industry due to their unique physical and chemical properties. However, AgNPs have caused environmental concerns. To understand the risks of AgNPs, Arabidopsis microarray data for AgNP, Ag+, cold, salt, heat and drought stresses were analyzed. Up- and down-regulated genes of more than two-fold expression change were compared, while the encoded proteins of shared and unique genes between stresses were subjected to differential enrichment analyses. AgNPs affected the fewest genes (575) in the Arabidopsis genome, followed by Ag+ (1010), heat (1374), drought (1435), salt (4133) and cold (6536). More genes were up-regulated than down-regulated in AgNPs and Ag+ (438 and 780, respectively) while cold down-regulated the most genes (4022). Responses to AgNPs were more similar to those of Ag+ (464 shared genes), cold (202), and salt (163) than to drought (50) or heat (30); the genes in the first four stresses were enriched with 32 PFAM domains and 44 InterPro protein classes. Moreover, 111 genes were unique in AgNPs and they were enriched in three biological functions: response to fungal infection, anion transport, and cell wall/plasma membrane related. Despite shared similarity to Ag+, cold and salt stresses, AgNPs are a new stressor to Arabidopsis.