Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus  laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd2+) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd2+-sensitive phenotype. Further analyses showed that Cd2+ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd2+ tolerance.

SummaryPlant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKa. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2 DD plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H 2 O 2 in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.

;Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamatelike ligand through an anion channel and the binding of this ligand to a glutamate receptor.

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.

Electrical potentials in cell walls ( Wall ) and at plasma membrane surfaces ( PM ) are determinants of ion activities in these phases. The PM plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of Wall . Wall from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of Wall with effectiveness in the order AlThis order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of Wall , and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for Wall were in close agreement, usually, and we consider Wall to be at least proportional to the actual Donnan potentials. Wall and PM display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. Wall is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.The cell wall (CW) is composed of various crosslinked units (macrofibrils, microfibrils, micelles, cellulose units, and linked agents such as neutral sugars, pectin, proteins, and ions; Cosgrove, 1997; Buchanan et al., 2000). The CW behaves as an ion exchanger where the fixed CW charges interact with exchangeable ions in the surrounding solution (Briggs and Robertson, 1957; Gillet and Lefèbvre, 1981; Sentenac and Grignon, 1981; Irwin et al., 1985; Richter and Dainty, 1989a, 1989b, 1990a, 1990b Grignon and Sentenac, 1991). The net CW charge is negative and results from weakly dissociating acidic groups having pK a values similar to those of polyGalUA, the principal origin of the negative charges (Ritchie and Larkum, 1982; Saftner and Raschke, 1981; Richter and Dainty, 1989a; Buchanan et al., 2000). Some positive charges occur too, mainly associated with CW proteins (Cassab and Varner, 1988; Buchanan et al., 2000).The CW determines cell dimensions (Taiz, 1984) and intracellular volume. The volume of the CW is a consequence of the dimensions of its internal spaces, i.e. the distances between the intra-CW units (Shomer et al., 1984; Shomer and Levy, 1988), which are determined by the repulsive strengths of diffuse double layers (Shomer et al., 1991). The swelling of parenchyma CW is restrained by the increase of valence and concentration of the exchangeable ions and with the decrease of the dielectric constant of the bulk solution (Shomer et al., 1991; Shomer, 1995). Although cell expansion and CW extension have been studied comprehensively (Cosgrove, 1997), the properties governing the volume of the CW, from the point of view of the dimensions of its internal spaces...

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85-88). Activity of a 49-kD Mg2+-dependent and Caz+-independent kinase in tobacco leaves increased 50-fold 15 min after infiltration of harpin from Erwinia amylovora (harpin,,).Much less pronounced and more transient activation was detected in water-infiltrated leaves. Biochemical characteristics of the harpin,,-activated protein kinase (HAPK) activity are consistent with those of the mitogenactivated protein kinase family. HAPK is cytosolic and phosphorylates myelin basic protein on serine/threonine residues. Treatment with a protein tyrosine phosphatase completely eliminated HAPK activity, suggesting that tyrosine phosphorylation is required for posttranslational activation. Sustained HAPK activation after cycloheximide treatment implies that HAPK may be negatively regulated by a translation-dependent mechanism. The extracellular Ca2+ chelator ECTA or the protein kinase inhibitor K252a, infiltrated in planta together with harpinEa, partially blocked HAPK activation. The Ca2+-channel blocker La3+ had no effect on HAPK activation, suggesting that phosphorylation events precede and/or do not depend on the entry of extracellular CaZ+ into the cell. These results suggest that early signal transduction events during harpin,,-induced hypersensitive response elicitation depend in part on the activation of HAPK.