Function of the anion transporter AtCLC-d in the trans-Golgi network

Anion transporting proteins of the CLC type are involved in anion homeostasis in a variety of organisms. CLCs from Arabidopsis have been shown to participate in nitrate accumulation and storage. In this study, the physiological role of the functional chloride transporter AtCLC-d from Arabidopsis was investigated. AtCLC-d is weakly expressed in various tissues, including the root. When transiently expressed as a GFP fusion in protoplasts, it co-localized with the VHA-a1 subunit of the proton-transporting V-type ATPase in the trans -Golgi network (TGN). Stable expression in plants showed that it co-localized with the endocytic tracer dye FM4-64 in a brefeldin A-sensitive compartment. Immunogold electron microscopy confirmed the localization of AtCLC-d to the TGN. Disruption of the AtCLC-d gene by a T-DNA insertion did not affect the nitrate and chloride contents. The overall morphology of these clcd-1 plants was similar to that of the wild-type, but root growth on synthetic medium was impaired. Moreover, the sensitivity of hypocotyl elongation to treatment with concanamycin A, a blocker of the V-ATPase, was stronger in the clcd-1 mutant. These phenotypes could be complemented by overexpression of AtCLC-d in the mutant background. The results suggest that the luminal pH in the trans -Golgi network is adjusted by AtCLC-d-mediated transport of a counter anion such as Cl⁻ or NO₃⁻.     

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.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Molecular Cloning, Subcellular Localization and Functional Analysis of ThCLC-a from Thellungiella halophila

Often, nitrate is the major source of available nitrogen for plants. Nitrate can accumulate in central vacuoles via tonoplast transporters. In the present study, a gene termed ThCLC-a that encodes a chloride channel protein was isolated from Thellungiella halophila. Deduced amino acid sequence analysis revealed high identity with AtCLC-a. RT-PCR analysis showed that the ThCLC-a gene was expressed ubiquitously in all major organs and its expression was induced by nitrate treatment. Confocal microscopy using green fluorescent fusion proteins revealed that ThCLC-a was localized specifically to the tonoplast membrane. Furthermore, an RNAi construct expressing a ThCLC-a cDNA fragment was used to silence the endogenous ThCLC-a in T. halophila. HPLC analysis showed that the nitrate content in shoots or roots of silenced plants was 19–36 % lower than in wild-type plants. Transgenic Arabidopsis plants ectopically expressing the ThCLC-a gene could accumulate 15–21 % more nitrate content than wild type plants under limited nitrogen conditions. Finally, our results suggest ThCLC-a may play an important role in the transport of nitrate via the vacuolar membrane.     

Review. CLC-mediated anion transport in plant cells

Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO3-/1H+ exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure-function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.     

BcNRT1, a plasma membrane-localized nitrate transporter from non-heading Chinese cabbage

A nitrate transporter, BcNRT1, was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar 'Suzhouqing'. The full-length cDNA was obtained using the rapid amplification of cDNA ends technique and contains an open reading frame of 1,770?bp that predicts a protein of 589 acid residues that possesses 12 putative transmembrane domains. Using the GUS marker gene driven by the BcNRT1 promoter, we found BcNRT1 expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants. The YFP fused to BcNRT1 and transformed into cabbage protoplasts indicated that BcNRT1 was localized to the plasma membrane. The expression of BcNRT1 in roots was induced by exposure to 25?mM nitrate, and the BcNRT1 cRNA heterologously expressed in Xenopus laevis oocytes showed nitrate conductance when nitrate was included in the medium. Moreover, mutant chl1-5 plants harboring 35S::BcNRT1 showed sensitivity to chlorate treatment and exhibited restored nitrate uptake. In conclusion, the results indicate that BcNRT1 functions as a low affinity nitrate transporter in non-heading Chinese cabbage.     

T-DNA tagging and characterization of a cryptic root-specific promoter in Arabidopsis

From a T-DNA tagged Arabidopsis population, a line, M-57 showing GUS (beta-glucuronidase) expression in the vascular regions of young roots was identified. Southern analysis revealed presence of a single T-DNA insert. Using inverse PCR, the plant sequence flanking the T-DNA insertion was cloned. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l-Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. A 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies. This cryptic promoter element does not show the presence of any known root-specific promoter element.     

Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate Cl(-) approximately Br(-) > I(-) > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK(a) of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.     

An arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3' region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the de-regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.     

Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene

Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants. Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.     

Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.     

An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.     

The shoot-specific expression of gamma-glutamylcysteine synthetase directs the long-distance transport of thiol-peptides to roots conferring tolerance to mercury and arsenic

Thiol-peptides synthesized as intermediates in phytochelatin (PC) biosynthesis confer cellular tolerance to toxic elements like arsenic, mercury, and cadmium, but little is known about their long-distance transport between plant organs. A modified bacterial gamma-glutamylcysteine synthetase (ECS) gene, S1ptECS, was expressed in the shoots of the ECS-deficient, heavy-metal-sensitive cad2-1 mutant of Arabidopsis (Arabidopsis thaliana). S1ptECS directed strong ECS protein expression in the shoots, but no ECS was detected in the roots of transgenic plant lines. The S1ptECS gene restored full mercury tolerance and partial cadmium tolerance to the mutant and enhanced arsenate tolerance significantly beyond wild-type levels. After arsenic treatment, the root concentrations of gamma-glutamylcysteine (EC), PC2, and PC3 peptides in a S1ptECS-complemented cad2-1 line increased 6- to 100-fold over the mutant levels and were equivalent to wild-type concentrations. The shoot and root levels of glutathione were 2- to 5-fold above those in wild-type plants, with or without treatment with toxicants. Thus, EC and perhaps glutathione are efficiently transported from shoots to roots. The possibility that EC or other PC pathway intermediates may act as carriers for the long-distance phloem transport and subsequent redistribution of thiol-reactive toxins and nutrients in plants is discussed.