Project description:Peptidases are known to play key roles in multiple biological processes in all living organisms. In a model plant Arabidopsis, the vast majority of many putative aminopeptidases remain uncharacterized. We therefore aim to explore physiological function of uncharacterized aminopeptidase in higher plants using Arabidopsis as a model to study.We performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression, and transcriptomic and metabolomic analyses of the mutants. We found that LAP2, one of the 28 aminopeptidases in Arabidopsis, regulates plant growth, leaf longevity and stress response by controlling intracellular protein turnover. Loss-of-function alleles of LAP2 reduced in vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the quiescent center cells in the root meristem, cotyledons and leaf veins. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular protein turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis, however, this influenced the flux of glutamate strikingly. As a result, gamma-aminobutyric acid, a metabolite which is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant.Our results provide molecular and biochemical evidence that LAP2 is indeed an enzymatically active aminopeptidase. LAP2 plays key roles in senescence, stress response and protein turnover. Regulated proteolysis is an important mechanism in all stages of the plant life cycle. The present study would contribute to further understanding of the aminopeptidases which have several implications in higher plants.
Project description:Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis thaliana (Arabidopsis), but the underlying mechanisms remain largely unknown. Here we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR 5 (ERF5) and ERF6 as master regulators which adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving GA and DELLA signaling. Using an ERF6 inducible overexpression line, we demonstrate that the GA-degrading enzyme GA2-OX6 is transcriptionally induced by ERF6 and that consequently DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while growth of erf5erf6 loss-of-function mutants is less affected by stress. Next to its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51 and WRKY33. Interestingly, the activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth-inhibition.
Project description:Background: Polycyclic aromatic hydrocarbons (PAHs) are toxic, widely-distributed, environmentally persistent, and carcinogenic byproducts of carbon-based fuel combustion. Previously, plant studies have shown that PAHs induce oxidative stress, reduce growth, and cause leaf deformation as well as tissue necrosis. To understand the transcriptional changes that occur during these processes, we performed microarray experiments on Arabidopsis thaliana L. under phenanthrene treatment, and compared the results to published Arabidopsis microarray data representing a variety of stress and hormone treatments. In addition, to probe hormonal aspects of PAH stress, we assayed transgenic ethylene-inducible reporter plants as well as ethylene pathway mutants under phenanthrene treatment. Results: Microarray results revealed numerous perturbations in signaling and metabolic pathways that regulate reactive oxygen species (ROS) and responses related to pathogen defense. A number of glutathione S-transferases that may tag xenobiotics for transport to the vacuole were upregulated. Comparative microarray analyses indicated that the phenanthrene response was closely related to other ROS conditions, including pathogen defense conditions. The ethylene-inducible transgenic reporters were activated by phenanthrene. Mutant experiments showed that PAH inhibits growth through an ethylene-independent pathway, as PAH-treated ethylene-insensitive etr1-4 mutants exhibited a greater growth reduction than WT. Further, phenanthrene-treated, constitutive ethylene signaling mutants had longer roots than the untreated control plants, indicating that the PAH inhibits parts of the ethylene signaling pathway. Conclusions: This study identified major physiological systems that participate in the PAH-induced stress response in Arabidopsis. At the transcriptional level, the results identify specific gene targets that will be valuable in finding lead compounds and engineering increased tolerance. Collectively, the results open a number of new avenues for researching and improving plant resilience and PAH phytoremediation.
Project description:Background: Polycyclic aromatic hydrocarbons (PAHs) are toxic, widely-distributed, environmentally persistent, and carcinogenic byproducts of carbon-based fuel combustion. Previously, plant studies have shown that PAHs induce oxidative stress, reduce growth, and cause leaf deformation as well as tissue necrosis. To understand the transcriptional changes that occur during these processes, we performed microarray experiments on Arabidopsis thaliana L. under phenanthrene treatment, and compared the results to published Arabidopsis microarray data representing a variety of stress and hormone treatments. In addition, to probe hormonal aspects of PAH stress, we assayed transgenic ethylene-inducible reporter plants as well as ethylene pathway mutants under phenanthrene treatment. Results: Microarray results revealed numerous perturbations in signaling and metabolic pathways that regulate reactive oxygen species (ROS) and responses related to pathogen defense. A number of glutathione S-transferases that may tag xenobiotics for transport to the vacuole were upregulated. Comparative microarray analyses indicated that the phenanthrene response was closely related to other ROS conditions, including pathogen defense conditions. The ethylene-inducible transgenic reporters were activated by phenanthrene. Mutant experiments showed that PAH inhibits growth through an ethylene-independent pathway, as PAH-treated ethylene-insensitive etr1-4 mutants exhibited a greater growth reduction than WT. Further, phenanthrene-treated, constitutive ethylene signaling mutants had longer roots than the untreated control plants, indicating that the PAH inhibits parts of the ethylene signaling pathway. Conclusions: This study identified major physiological systems that participate in the PAH-induced stress response in Arabidopsis. At the transcriptional level, the results identify specific gene targets that will be valuable in finding lead compounds and engineering increased tolerance. Collectively, the results open a number of new avenues for researching and improving plant resilience and PAH phytoremediation. Arabidopsis thaliana (ecotype Columbia) plants were long-day grown with +/- 0.25 mM phenanthrene in sterile plates at 23C for 21d before harvest. At least 20 plants were pooled prior to each mRNA extraction.
Project description:UV radiation is a ubiquitous component of solar radiation that affects plant growth and development. Analysis of natural variation in response to UV radiation revealed significant differences among natural accessions of Arabidopsis thaliana. However, the genetic basis of this is to a large extent unknown. Here, we analyzed the response of Arabidopsis accessions to UV radiation stress by performing RNA-sequencing of three UV sensitive and three UV resistant accessions. The genome-wide transcriptional analysis revealed a large number of genes significantly up- or down-regulated only in sensitive or only in resistant accessions, respectively. Mutant analysis of few selected candidate genes suggested by the RNA-sequencing results indicate a connection between UV radiation stress and plant-pathogen-like defense responses.
Project description:Genome-wide transcriptome analysis of Arabidopsis thaliana was performed to understand the role of auxin in the response of leaf growth to osmotic stress. We studied transcriptional changes in proliferating leaves of the seedlings grown in vitro on control medium, medium supplemented with 25mM mannitol, 0.1μM NAA and 0.1μM NAA + 25mM mannitol.
Project description:Chromatin, in addition to its purely structural functions, is considered a major regulatory system coordinating various genetic networks in eukaryotes. Constant changes of gene expression programs are especially important for plants, which have to respond to environment by modulating their growth and development during whole lifetime. External and developmental signals can be transmitted through signaling cascades to chromatin remodeling complexes like SWI/SNF, which alter chromatin structure by moving, ejecting or restructuring nucleosomes. Genetic studies in Arabidopsis thaliana revealed that SWI/SNF chromatin remodeling complexes are critical for proper plant development and growth. Especially, BRM, a catalytic subunit of the complex, was shown to directly regulate several genes with important functions in leaf development, flowering initiation, as well as gibberellin and abscisic acid signaling. In this study, we profiled BRM global binding regions in Arabidopsis genome by ChIP-chip analysis. We found that BRM can bind to thousands of genes, many of which have key functions in hormone and stress signaling.