Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts

Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Tonoplast-localized transporters catalyze the import and release of these molecules. The mechanisms determining the targeting of these transporters to the tonoplast are largely unknown. Using the paralogous Arabidopsis thaliana inositol transporters INT1 (tonoplast) and INT4 (plasma membrane), we performed domain swapping and mutational analyses and identified a C-terminal di-leucine motif responsible for the sorting of higher plant INT1-type transporters to the tonoplast in Arabidopsis mesophyll protoplasts. We demonstrate that this motif can reroute other proteins, such as INT4, SUCROSE TRANSPORTER2 (SUC2), or SWEET1, to the tonoplast and that the position of the motif relative to the transmembrane helix is critical. Rerouted INT4 is functionally active in the tonoplast and complements the growth phenotype of an int1 mutant. In Arabidopsis plants defective in the β-subunit of the AP-3 adaptor complex, INT1 is correctly localized to the tonoplast, while sorting of the vacuolar sucrose transporter SUC4 is blocked in cis-Golgi stacks. Moreover, we demonstrate that both INT1 and SUC4 trafficking to the tonoplast is sensitive to brefeldin A. Our data show that plants possess at least two different Golgi-dependent targeting mechanisms for newly synthesized transporters to the tonoplast.     

Arabidopsis INOSITOL TRANSPORTER4 mediates high-affinity H+ symport of myoinositol across the plasma membrane

Four genes of the Arabidopsis (Arabidopsis thaliana) monosaccharide transporter-like superfamily share significant homology with transporter genes previously identified in the common ice plant (Mesembryanthemum crystallinum), a model system for studies on salt tolerance of higher plants. These ice plant transporters had been discussed as tonoplast proteins catalyzing the inositol-dependent efflux of Na(+) ions from vacuoles. The subcellular localization and the physiological role of the homologous proteins in the glycophyte Arabidopsis were unclear. Here we describe Arabidopsis INOSITOL TRANSPORTER4 (AtINT4), the first member of this subgroup of Arabidopsis monosaccharide transporter-like transporters. Functional analyses of the protein in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes characterize this protein as a highly specific H(+) symporter for myoinositol. These activities and analyses of the subcellular localization of an AtINT4 fusion protein in Arabidopsis and tobacco (Nicotiana tabacum) reveal that AtINT4 is located in the plasma membrane. AtINT4 promoter-reporter gene plants demonstrate that AtINT4 is strongly expressed in Arabidopsis pollen and phloem companion cells. The potential physiological role of AtINT4 is discussed.     

Arabidopsis INOSITOL TRANSPORTER2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane

Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices. In contrast, AtINT2 codes for a functional transporter. AtINT2 localization in the plasma membrane was demonstrated by transient expression of an AtINT2-GREEN FLUORESCENT PROTEIN fusion in Arabidopsis and tobacco (Nicotiana tabacum) epidermis cells and in Arabidopsis protoplasts. Its functional and kinetic properties were determined by expression in yeast (Saccharomyces cerevisiae) cells and Xenopus laevis oocytes. Expression of AtINT2 in a Deltaitr1 (inositol uptake)/Deltaino1 (inositol biosynthesis) double mutant of bakers' yeast complemented the deficiency of this mutant to grow on low concentrations of myoinositol. In oocytes, AtINT2 mediated the symport of H(+) and several inositol epimers, such as myoinositol, scylloinositol, d-chiroinositol, and mucoinositol. The preference for individual epimers differed from that found for AtINT4. Moreover, AtINT2 has a lower affinity for myoinositol (K(m) = 0.7-1.0 mm) than AtINT4 (K(m) = 0.24 mm), and the K(m) is slightly voltage dependent, which was not observed for AtINT4. Organ and tissue specificity of AtINT2 expression was analyzed in AtINT2 promoter/reporter gene plants and showed weak expression in the anther tapetum, the vasculature, and the leaf mesophyll. A T-DNA insertion line (Atint2.1) and an Atint2.1/Atint4.2 double mutant were analyzed under different growth conditions. The physiological roles of AtINT2 are discussed.     

Vacuoles release sucrose via tonoplast-localised SUC4-type transporters

Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.     

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine‐based motif

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP‐fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild‐type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N‐terminal dileucine‐based motif. The NRAMP3 or NRAMP4 N‐terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4‐dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N‐terminal dileucine‐based motif as well as AP4.      

Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II

Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. An Arabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. Diploid Arabidopsis plants containing two psbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component in npq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll a fluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein.     

Arabidopsis actin gene ACT7 plays an essential role in germination and root growth

Arabidopsis contains eight actin genes. Of these ACT7 is the most strongly expressed in young plant tissues and shows the greatest response to physiological cues. Adult plants homozygous for the act7 mutant alleles show no obvious above-ground phenotypes, which suggests a high degree of functional redundancy among plant actins. However, act7-1 mutant plants are at a strong selective disadvantage when grown in competition with wild-type plants and therefore must have undetected physical defects. The act7-1 and act7-4 alleles contain T-DNA insertions just after the stop codon and within the first intron, respectively. Homozygous mutant seedlings of both alleles showed less than 7% of normal ACT7 protein levels. Mutants displayed delayed and less efficient germination, increased root twisting and waving, and retarded root growth. The act7-4 mutant showed the most dramatic reduction in root growth. The act7-4 root apical cells were not in straight files and contained oblique junctions between cells suggesting a possible role for ACT7 in determining cell polarity. Wild-type root growth was fully restored to the act7-1 mutant by the addition of an exogenous copy of the ACT7 gene. T-DNA insertions just downstream of the major polyadenylation sites (act7-2, act7-3) appeared fully wild type. The act7 mutant phenotypes demonstrate a significant requirement for functional ACT7 protein during root development and explain the strong negative selection component seen for the act7-1 mutant.     

On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum

Class B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2-like and DAL12-like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild-type plants and in different class B mutants. In wild-type plants and in a conditional mutant grown at a permissive temperature, gain-of-function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss-of-function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non-permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain-of-function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.     

An Arabidopsis mutant with deregulated ABA gene expression: implications for negative regulator function

The physiological acclimation of plants to osmotic stresses involves a complex programme of gene regulation. In one signalling pathway, elevated levels of abscisic acid (ABA) activate a subset of stress genes. Because ABA responses lack a definable morphological phenotype, we have screened for mutants that exhibit deregulated ABA-responsive gene expression. To monitor this ABA response, a line of Arabidopsis thaliana carrying a transgene composed of the ABA-responsive Arabidopsis kin2 promoter fused to the coding sequence for the firefly luciferase gene, kin2::luc, was generated. Patterns of ABA-responsive luciferase activity were monitored by photon counting. In contrast to wild-type plants which display a transient activation of kin2::luc, an ABA deregulated gene expression mutant (ade1) exhibits both sustained and enhanced levels of transgene activity. Levels of kin2, cor47 and rab18 expression in ade1 plants are also enhanced and prolonged indicating that the molecular mechanism(s) altered in ade1 plants affects the regulation of other ABA-responsive genes. The mutant phenotype is specific for the ABA response as cold-inducible kin2 expression is unaltered in ade1 plants. Genetic analyses indicate that the ade1 mutant is a monogenic recessive trait. A role for negative regulator function in ABA signalling is discussed.     

Nitric oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis

Nitric oxide (NO) has emerged as a key molecule involved in many physiological processes in plants. To characterize roles of NO in tolerance of Arabidopsis (Arabidopsis thaliana) to salt stress, effect of NaCl on Arabidopsis wild-type and mutant (Atnoa1) plants with an impaired in vivo NO synthase (NOS) activity and a reduced endogenous NO level was investigated. Atnoa1 mutant plants displayed a greater Na+ to K+ ratio in shoots than wild-type plants due to enhanced accumulation of Na+ and reduced accumulation of K+ when exposed to NaCl. Germination of Atnoa1 seeds was more sensitive to NaCl than that of wild-type seeds, and wild-type plants exhibited higher survival rates than Atnoa1 plants when grown under salt stress. Atnoa1 plants had higher levels of hydrogen peroxide than wild-type plants under both control and salt stress, suggesting that Atnoa1 is more vulnerable to salt and oxidative stress than wild-type plants. Treatments of wild-type plants with NOS inhibitor and NO scavenger reduced endogenous NO levels and enhanced NaCl-induced increase in Na+ to K+ ratio. Exposure of wild-type plants to NaCl inhibited NOS activity and reduced quantity of NOA1 protein, leading to a decrease in endogenous NO levels measured by NO-specific fluorescent probe. Treatment of Atnoa1 plants with NO donor sodium nitroprusside attenuated the NaCl-induced increase in Na+ to K+ ratio. Therefore, these findings provide direct evidence to support that disruption of NOS-dependent NO production is associated with salt tolerance in Arabidopsis.     

LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana

To gain insight into the processes involved in photosystem II (PSII) biogenesis and maintenance, we characterized the low psii accumulation1 (lpa1) mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant, while other plastid-encoded proteins were translated at rates similar to the wild type. In addition, turnover rates of the PSII core proteins CP47, CP43, D1, and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant. LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1 but not with D2, cytochrome b6, or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

Degradation of sphingoid long-chain base 1-phosphates (LCB-1Ps): functional characterization and expression of AtDPL1 encoding LCB-1P lyase involved in the dehydration stress response in Arabidopsis

Sphingoid long-chain base (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C(16) fatty aldehydes and phosphoethanolamine. Here, we confirmed that the At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase. Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants more than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress.     

Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.     

ECA3, a Golgi-localized P2A-type ATPase, plays a crucial role in manganese nutrition in Arabidopsis

Calcium (Ca) and manganese (Mn) are essential nutrients required for normal plant growth and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not eca2 mutants, displayed a striking difference from wild-type plants. After approximately 8 to 9 d on this medium, eca3 mutants became chlorotic, and root and shoot growth were strongly inhibited compared to wild-type plants. These severe deficiency symptoms were suppressed by low levels of Mn, indicating a crucial role for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than wild-type plants and eca2 mutants on medium lacking Ca; however, the differences were not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the growth defect of K616 on low Ca. Promoter:beta-glucuronidase studies show that ECA3 is expressed in a range of tissues and cells, including primary root tips, root vascular tissue, hydathodes, and guard cells. When transiently expressed in Nicotiana tabacum, an ECA3-yellow fluorescent protein fusion protein showed overlapping expression with the Golgi protein GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly functioning in the transport of these ions into the Golgi. ECA3 is the first P-type ATPase to be identified in plants that is required under Mn-deficient conditions.