Effects of Low-Oxygen Atmosphere on the Growth and Development of Arabidopsis Thaliana (L.) Heynh.

Interest in the improvement of crop productivity through repression of photorespiration led to experimental growth of plants in low O2 and the surprising finding that reproduction is inhibited under these conditions. Using Arabidopsis thaliana (L.) Heynh. cv. Columbia, studies were undertaken to understand the mechanism behind oxygen control of plant development. The hypothesis that developmental changes observed in low O 2 are due to repression of the plant hormones ethylene and brassinolide (which require O2 for biosynthesis) was tested. Arabidopsis was grown for 35 days in Sun bags in one of five altered O 2 atmospheres (210, 160, 100, 50, 25 mmol/mol) with 0.35 mmol/mol CO 2 in N2. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding ACC synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. To understand if low O2 inhibition of brassinolide biosynthesis was responsible for developmental changes in low O2, brassinolide-replacement experiments were performed. Arabidopsis was grown for 10 days on nutrient agar or nutrient agar + 10-7 M brassinolide in magenta vessels coupled to gas mixtures of 210 or 25 mmol/mol O2, 0.35 mmol/mol CO2 in N2. When grown in 25 mmol/mol O2, plants exhibited dwarf morphology resembling the brassinolide-deficient mutant det2, and this line was incorporated into the brassinolide studies at low O2 for comparison. Leaf development under low O2 resulted in changes in leaf size, stomatal density and stomatal patterning. Stomatal density increased in all low O2 treatments but was not linked to changes in ethylene or brassinolide. Low O2 changes in leaf size, however, were mediated through depression of brassinolide biosynthesis. There was a loss of ethylene biosynthesis in siliques from plants grown at 50 and 25 mmol/mol O2, and silique ACC oxidase mRNA increased as O2 was lowered. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. Loss of ethylene in the silique was tightly correlated with loss of the embryo in seeds produced in low O2. These data show that changes in development under low oxygen can be explained by repression of O2-sensitive plant hormones

ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst

Arabidopsis Toxicos en Levadura 12 Modulates Salt Stress and ABA Responses in Arabidopsis thaliana

Salt is one of the most common abiotic stresses, causing ionic and osmotic pressure changes that affect plant growth and development. In this work, we present molecular and genetic evidence that Arabidopsis Toxicos en Levadura 12 (ATL12) is involved in both salt stress and in the abscisic acid response to this stress. We demonstrate that ATL12 is highly induced in response to salt stress and that atl12 mutants have a lower germination rate, decreased root length, and lower survival rate compared to the Col-0 wild-type in response to salt stress. Overexpression of ATL12 increases expression of the salt stress-associated genes SOS1/2, and ABA-responsive gene RD29B. Additionally, higher levels of reactive oxygen species are detected when ATL12 is overexpressed, and qRT-PCR showed that ATL12 is involved in the AtRBOHD/F-mediated signaling. ATL12 expression is also highly induced by ABA treatment. Mutants of atl12 are hypersensitive to ABA and have a shorter root length. A decrease in water loss and reduced stomatal aperture were also observed in atl12 mutants in response to ABA. ABA-responsive genes RD29B and RAB18 were downregulated in atl12 mutants but were upregulated in the overexpression line of ATL12 in response to ABA. Taken together our results suggest that ATL12 modulates the response to salt stress and is involved in the ABA signaling pathway in Arabidopsis thaliana

Short-Chain Chitin Oligomers: Promoters of Plant Growth

Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.Funding was provided in part by grants to Shauna Somerville from the Carnegie Institution of Science and the National Science Foundation, USA (#0114783). Alexander J. Winkler was funded by the Erasmus Program at Universidad Politécnica de Madrid (UPM). We acknowledge the kind contributions, at different stages of this project, of Fernando García Arenal, Pablo González Melendi and Mark Wilkinson from CBGP (UPM-INIA), Luis Díaz Balteiro and Carlos Calderón from MONTES, Carmen Muñoz from E.T.S.I. Forestales (UPM), Norma García from UPM and Elisabeth Magel from Hamburg UniversityWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Developmental and Reproductive Effects of Iron Oxide Nanoparticles in Arabidopsis thaliana

Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%–6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed

Loss-of-Function Mutations in Chitin Responsive Genes Show Increased Susceptibility to the Powdery Mildew Pathogen Erysiphe cichoracearum

Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways

ATL9, a RING zinc finger protein with E3 ubiquitin ligase activity implicated in chitin- and NADPH oxidase-mediated defense responses.

Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst

Microarray analysis of chitin elicitation in Arabidopsis thaliana

Chitin oligomers, released from fungal cell walls by endochitinase, induce defence and related cellular responses in many plants. However, little is known about chitin responses in the model plant Arabidopsis. We describe here a large-scale characterization of gene expression patterns in Arabidopsis in response to chitin treatment using an Arabidopsis microarray consisting of 2375 EST clones representing putative defence-related and regulatory genes. Transcript levels for 71 ESTs, representing 61 genes, were altered three-fold or more in chitin-treated seedlings relative to control seedlings. A number of transcripts exhibited altered accumulation as early as 10 min after exposure to chitin, representing some of the earliest changes in gene expression observed in chitin-treated plants. Included among the 61 genes were those that have been reported to be elicited by various pathogen-related stimuli in other plants. Additional genes, including genes of unknown function, were also identified, broadening our understanding of chitin-elicited responses. Among transcripts with enhanced accumulation, one cluster was enriched in genes with both the W-box promoter element and a novel regulatory element. In addition, a number of transcripts had decreased abundance, encoding several proteins involved in cell wall strengthening and wall deposition. The chalcone synthase promoter element was identified in the upstream regions of these genes, suggesting that pathogen signals may suppress the expression of some genes. These data indicate that Arabidopsis should be an excellent model to elucidate the mechanisms of chitin elicitation in plant defence

Expression and regulation of ATL9, an E3 ubiquitin ligase involved in plant defense.

Plants are continually exposed to a variety of pathogenic organisms, including bacteria, fungi and viruses. In response to these assaults, plants have developed various defense pathways to protect themselves from pathogen invasion. An understanding of the expression and regulation of genes involved in defense signaling is essential to controlling plant disease. ATL9, an Arabidopsis RING zinc finger protein, is an E3 ubiquitin ligase that is induced by chitin and involved in basal resistance to the biotrophic fungal pathogen, Golovinomyces cichoracearum (G. cichoracearum). To better understand the expression and regulation of ATL9, we studied its expression pattern and the functions of its different protein domains. Using pATL9:GUS transgenic Arabidopsis lines we found that ATL9 is expressed in numerous tissues at various developmental stages and that GUS activity was induced rapidly upon wounding. Using a GFP control protein, we showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Using a series of deletion mutants, we found that the PEST domain and the RING domain have effects on ATL9 degradation. Further infection assays with G. cichoracearum showed that both the RING domain and the TM domains are important for ATL9's resistance phenotype. Interestingly, the PEST domain was also shown to be significant for resistance to fungal pathogens. This study demonstrates that the PEST domain is directly coupled to plant defense regulation and the importance of protein degradation in plant immunity