ABSTRACT
Stress response and adaptation are important physiological mechanisms in plants. As plants are not able to avoid stressful environments by moving away, as animals, they have developed diverse mechanisms to respond to stressful situations. One of the genes involved in these mechanisms is NRP (Asparagine-rich protein or N-rich protein). In this study, NRP expression, protein localization and nrp knockout plants were investigated for further understanding of NRP function. NaCl-induced salt stress, oxidative stress (ozone exposure) and mechanical perturbation (touch treatment) were used to induce abiotic stress. NRP expression was up-regulated in the early phase of stress response to all three elicitors. Stressed nrp knockout seedlings revealed a more pronounced growth inhibition compared to wildtype (salt and osmotic stress). Seedlings showed NRP-GFP expression in the apical meristem, leaf veins, central cylinder, root hair zone and root tip. Analyses of NRP-GFP localization in root cells and protoplasts revealed cytosolic distribution under non-stress conditions and translocation of NRP-GFP to mitochondria due to stress response. Summarizing, our findings point to a contribution of NRP in signal transduction of the initial phase of general stress response in Arabidopsis thaliana.
Subject(s)
Adaptation, Physiological/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Intracellular Signaling Peptides and Proteins/metabolism , Stress, Physiological/genetics , Animals , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Genes, Plant/physiology , Intracellular Signaling Peptides and Proteins/genetics , Mutation , Oxidative Stress/genetics , Ozone , Plant Structures/metabolism , Salt Tolerance/genetics , Stress, Mechanical , Up-RegulationABSTRACT
Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.
Subject(s)
Arabidopsis/enzymology , Inositol Oxygenase/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Cloning, Molecular , Inositol Oxygenase/chemistry , Kinetics , Lilium/enzymology , Models, Biological , Molecular Sequence Data , Nucleotides/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phylogeny , Polymers/chemistry , Recombinant Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min(-1). Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.
