Quantitative trait loci analysis of nitrate storage in Arabidopsis leading to an investigation of the contribution of the anion channel gene, AtCLC-c, to variation in nitrate levels

Storage of excess nitrate in the vacuole and its subsequent remobilization is an important aspect of a plant's nitrogen economy, but the genes controlling the underlying processes have not all been identified and characterized. Cape Verdi Island (Cvi)/Landsberg erecta (Ler) and Columbia (Col)/Landsberg erecta recombinant inbred line (RIL) populations of Arabidopsis thaliana were used to identify quantitative trait loci (QTL) controlling natural variation in nitrate concentrations. One major and two minor QTLs were found for the Cvi/Ler population and one minor QTL for the Col/Ler RIL. These were designated NA1 to NA4. The major Cvi/Ler QTL (NA3) was located at the bottom of chromosome 5. No interaction among the QTLs was found by two-way ANOVA. By comparing in silico the locations of the QTLs with a physical map of the Arabidopsis genome, candidate genes for each QTL were identified. Several of these were anion channels of the AtCLC family. One of these, AtCLC-c, coincided with NA3 and its role was investigated using a mutant with a transposon insertion in AtCLC-c. Mutant plants homozygous for the insertion (designated clcc-1) had less than 5% of AtCLC-c mRNA compared with wild-type (WT) shoots. They also had significantly lower nitrate concentrations when grown at a range of external nitrate concentrations. The concentrations of chloride, malate, and citrate were also affected in the mutant. In wild-type plants, expression of AtCLC-c was down-regulated in the presence of nitrate, but ammonium had a much smaller effect while chloride and sulphate did not affect expression. These and published results suggest that multiple genes affect nitrate concentrations in plants and that AtCLC-c and other members of the AtCLC gene family play some role in this.     

Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis

The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1-VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC-green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.     

Salt-dependent regulation of a CNG channel subfamily in Arabidopsis

Background  

In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies.     

The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds

In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. beta-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.     

A mechanosensitive anion channel in Arabidopsis thaliana mesophyll cells

Mechanosensitive ion channels are expected to play important roles in transducing mechanical stimuli into intracellular signals during the development and morphogenesis of higher plants. We have identified a novel mechanosensitive anion channel in the protoplast of Arabidopsis thaliana mesophyll cells by using the patch-clamp technique. The channel in the outside-out patches could be activated by positive pressure in the pipette while negative pressure had no effect. The amphipathic membrane crenator trinitrophenol, which is supposed to preferentially insert in the outer leaflet of the lipid bilayer of the plasma membrane, synergized with mechanical membrane stretch to activate the channel. These results suggest that the channel activation is mediated by a convex curvature of the plasma membrane. Therefore, activation of this channel may play an important role when cell volume is increasing during cell growth or hypo-osmotic challenge, which is accompanied by membrane stretch with increasingly convex curvature.     

Disruption of a DNA topoisomerase I gene affects morphogenesis in Arabidopsis

The genesis of phyllotaxis, which often is associated with the Fibonacci series of numbers, is an old unsolved puzzle in plant morphogenesis. Here, we show that disruption of an Arabidopsis topoisomerase (topo) I gene named TOP1alpha affects phyllotaxis and plant architecture. The divergence angles and internode lengths between two successive flowers were more random in the top1alpha mutant than in the wild type. The top1alpha plants sporadically produced multiple flowers from one node, and the number of floral organ primordia often was different. The mutation also caused the twisting of inflorescences and individual flowers and the serration of leaf margins. These morphological abnormalities indicate that TOP1alpha may play a critical role in the maintenance of a regular pattern of organ initiation. The top1alpha mutant transformed with the RNA interference construct for TOP1beta, another topo I gene arrayed tandemly with TOP1alpha, was found to be lethal at young seedling stages, suggesting that topo I activity is essential in plants.     

A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.

Light acts both directly as a signal and indirectly through photosynthesis to regulate the expression of genes encoding nitrate reductase (NR). Here, we report the isolation and characterization of a novel chlorate-resistant mutant that is defective in the regulation of NR gene expression. The response of NR2, but not NR1 or the gene encoding nitrate reductase (NiR), to light signals was impaired in this Arabidopsis mutant, designated cr88. In addition to NR2, the light regulation of the genes encoding the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) was also impaired in this mutant. These results suggest that the pathway through which light regulates the expression of NR2, CAB, and RBCS genes is different from those that regulate the expression of NR1 and NiR. An examination of the deetiolation process under different light spectrum showed that cr88 is defective in red light-mediated deetiolation. Complementation tests with various long hypocotyl (hy) mutants indicated that CR88 identifies a new HY locus. The possible functions of CR88 are discussed.     

A role for SPINDLY gene in the regulation of oxidative stress response in Arabidopsis

SPINDLY (SPY) gene encodes a putative O-linked N-acetyl-glucosamine transferase, and yeast two-hybrid assay identified GIGANTEA (GI) as a SPY-interacting partner in Arabidopsis. GIGANTEA gene was previously shown to be involved in the regulation of oxidative stress response; however, it is unclear whether SPY gene is also involved in oxidative stress response. Here we showed that SPY plays a role in the regulation of the oxidative stress response. The spy-1 mutant was more tolerant to paraquat (PQ)-or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of the spy-1 mutant to PQ-stress was not due to defects in the PQ uptake or the PQ sequestration from its site of action but rather the spy-1 mutation alleviated oxidative damage of plant cells upon PQ stress. Higher constitutive activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in spy-1 are more likely to be due to activation of both CSD2 gene encoding chloroplast Cu/Zn SOD and APX1 gene. Taken together, these results suggest that enhanced tolerance of the spy-1 mutant to oxidative stress is associated, at least in part, with constitutive activation of CSD2 and APX1. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 604–611. The text was submitted by the authors in English.     

Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth

Acyl-acyl carrier protein thioesterases determine the amount and type of fatty acids that are exported from the plastids. To better understand the role of the FATB class of acyl-acyl carrier protein thioesterases, we identified an Arabidopsis mutant with a T-DNA insertion in the FATB gene. Palmitate (16:0) content of glycerolipids of the mutant was reduced by 42% in leaves, by 56% in flowers, by 48% in roots, and by 56% in seeds. In addition, stearate (18:0) was reduced by 50% in leaves and by 30% in seeds. The growth rate was reduced in the mutant, resulting in 50% less fresh weight at 4 weeks compared with wild-type plants. Furthermore, mutant plants produced seeds with low viability and altered morphology. Analysis of individual glycerolipids revealed that the fatty acid composition of prokaryotic plastid lipids was largely unaltered, whereas the impact on eukaryotic lipids varied but was particularly severe for phosphatidylcholine, with a >4-fold reduction of 16:0 and a 10-fold reduction of 18:0 levels. The total wax load of fatb-ko plants was reduced by 20% in leaves and by 50% in stems, implicating FATB in the supply of saturated fatty acids for wax biosynthesis. Analysis of C(18) sphingoid bases derived from 16:0 indicated that, despite a 50% reduction in exported 16:0, the mutant cells maintained wild-type levels of sphingoid bases, presumably at the expense of other cell components. The growth retardation caused by the fatb mutation was enhanced in a fatb-ko act1 double mutant in which saturated fatty acid content was reduced further. Together, these results demonstrate the in vivo role of FATB as a major determinant of saturated fatty acid synthesis and the essential role of saturates for the biosynthesis and/or regulation of cellular components critical for plant growth and seed development.     

Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development

Phytohormones play crucial roles in regulating many aspects of plant development. Although much has been learned about the effects of individual hormones, cross-talk between and integration of different hormonal signals are still not well understood. We present a study of MINI ZINC FINGER 1 (MIF1), a putative zinc finger protein from Arabidopsis, and suggest that it may be involved in integrating signals from multiple hormones. MIF1 homologs are highly conserved among seed plants, each characterized by a very short sequence containing a central putative zinc finger domain. Constitutive overexpression of MIF1 caused dramatic developmental defects, including dwarfism, reduced apical dominance, extreme longevity, dark-green leaves, altered flower morphology, poor fertility, reduced hypocotyl length, spoon-like cotyledons, reduced root growth, and ectopic root hairs on hypocotyls and cotyledons. In addition, 35S::MIF1 seedlings underwent constitutive photomorphogenesis in the dark, with root growth similar to that in the light. Furthermore, 35S::MIF1 seedlings were demonstrated to be non-responsive to gibberellin (GA) for cell elongation, hypersensitive to the GA synthesis inhibitor paclobutrazol (PAC) and abscisic acid (ABA), and hyposensitive to auxin, brassinosteroid and cytokinin, but normally responsive to ethylene. The de-etiolation defect could not be rescued by the hormones tested. Consistent with these observations, genome-scale expression profiling revealed that 35S::MIF1 seedlings exhibited decreased expression of genes involved in GA, auxin and brassinosteroid signaling as well as cell elongation/expansion, and increased expression of ABA-responsive genes. We propose that MIF1, or the protein(s) with which MIF1 interacts, is involved in mediating the control of plant development by multiple hormones.     

Functional characterization of a putative nitrate transporter gene promoter from rice

Drought is one of the most significant abiotic stresses that influence plant growth anddevelopment.Expression analysis revealed that OsNRT1.3,a putative nitrate transporter gene in rice,wasinduced by drought.To confirm if the OsNRT1.3 promoter can respond to drought stress,a 2019 bpupstream sequence of OsNRT1.3 was cloned.Three OsNRT1.3 promoter fragments were generated by5′-deletion,and fused to the β-glucuronidase (GUS) gene.The chimeric genes were introduced into riceplants.NRT2019::GUS,NRT1196::GUS and NRT719::GUS showed similar expression patterns in seeds,roots,leaves and flowers in all transgenic rice,and GUS activity conferred by different OsNRT1.3 promoterfragments was significantly upregulated by drought stress,indicating that OsNRT1.3 promoter responds todrought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.     

Disruption of a gene encoding a putative gamma-butyrolactone-binding protein in Streptomyces tendae affects nikkomycin production

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several Gamma-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.     

The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.     

A Cl channel in Ascaris suum selectivity conducts dicarboxylic anion products of glucose fermentation and suggests a role in removal of waste organic anions

The permeability of organic anions (produced from anaerobic fermentation of glucose) through a nonselective membrane Cl channel was examined. Single channel recording techniques were used to study the permeabilities of the anions: oxalate, succinate, oxaloacetate, malate, lactate and pyruvate in Ascaris muscle cell membranes. All of the anions, except malate, were found to be conducted through the channel. The relative permeability of most anions could be predicted from the component structure of the anions. The failure of the channel to conduct malate prevents an energy drain on the cell. These studies further the hypothesis that a Cl channel functions to transport waste organic anions across the cell membrane. This mechanism does not require specific exchange carriers for the anions. Channels with properties like the nonselective anion channels of Ascaris, are suitable for transport of carboxylic anions through cell membranes, down a concentration or potential gradient.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.).     

Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C. At the restrictive temperature, ypt1ts mutants accumulated ER membranes, small vesicles, and unprocessed invertase, and they exhibited cytoskeletal defects and an enhanced 45Ca2+ uptake. Similar alterations were seen in YPT1-depleted cells. The ypt1ts mutant cells could be rescued from growth arrest by increasing extracellular Ca2+, and, even at the permissive temperature, they displayed increased trifluoperazine sensitivity.