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In Arabidopsis thaliana , root high-affinity nitrate (NO3–) uptake depends mainly on NRT2.1, 2.4, and 2.5, which are repressed by high NO3– supply at the transcript level. For NRT2.1 , this regulation is due to the action of (i) feedback down-regulation by N metabolites and (ii) repression by NO3– itself mediated by the transceptor NRT1.1(NPF6.3). However, for NRT2.4 and NRT2.5 , the signalling pathway(s) remain unknown as do the molecular elements involved. Here we show that unlike NRT2.1 , NRT2.4 and NRT2.5 are not induced in an NO3– reductase mutant but are up-regulated following replacement of NO3– by ammonium (NH4+) as the N source. Moreover, increasing the NO3– concentration in a mixed nutrient solution with constant NH4+ concentration results in a gradual repression of NRT2.4 and NRT2.5 , which is suppressed in an nrt1.1 mutant. This indicates that NRT2.4 and NRT2.5 are subjected to repression by NRT1.1-mediated NO3– sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2 transporters, such as HHO1, HRS1, PP2C, LBD39, BT1, and BT2, are also regulated by NRT1.1-mediated NO3– sensing, and that several of them are involved in NO3– repression of NRT2.1 , NRT2.4 , and NRT2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3– regulation of all these genes. Altogether, these data led us to propose a regulatory model for high-affinity NO3– uptake in Arabidopsis, highlighting several NO3– transduction cascades downstream of the phosphorylated form of the NRT1.1 transceptor. [ABSTRACT FROM AUTHOR]

Methylation of biologically active molecules is achieved by methyltransferases (MTases). MTases can act on proteins through N- or O-carboxylmethylation reactions. Methylation of lysine and glutamic acid residues was recently described on the N-terminal tail of AtPIP2;1, a plasma membrane aquaporin of plants. In this study, we combine a bioinformatic and a biochemical screen and identify two MTases of Arabidopsis thaliana, SDG7 (At2g44150) and OMTF3 (At3g61990), as acting on the N-terminal tail of AtPIP2;1, at Lys3 and Glu6, respectively. Confocal microscopy imaging showed the two enzymes to be associated with the endoplasmic reticulum. An in vitro assay using various AtPIP2;1 N-terminal peptides as a bait allowed characterization of the enzymatic properties of recombinant SDG7 and OMTF3. The two enzymes showed minimal apparent Km values for their substrates, S-adenosylmethionine and peptide, in the range of 5–8 and 2–9 μM, respectively. SDG7 was shown to almost exclusively mono- or di-methylate Lys3. In contrast, OMTF3 specifically methylated Glu6, this methylation being dependent on the methylation profile of the neighboring Lys3 residue. In conclusion, this study allows the characterization of the first MTases able to methylate plant transmembrane proteins and provides the first identification of a glutamate-MTase in eukaryotes. [ABSTRACT FROM AUTHOR]

Plant growth under low K+ availability or salt stress requires tight control of K+ and Na+ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K+ Transporters), permeable either to K+ and Na+ or to Na+ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na+ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKTI;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na+ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na+-K+ symport, Na+ uniport, or inhibited states, depending on external Na+ and K+ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K+ and Na+ accumulation in monocots. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana mutants deficient for the NRTI.1 NO3- transporter display complex phenotypes, including lowered NO3- uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH4NO3 as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO3-transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO3- HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH4NO3-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO3-HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH4+ plus 0.1 mM NO3-, a situation where NRT1.1 is likely to mediate very low NO3- transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH4+. Two hypotheses are discussed to explain these results: (1) NRT2.1... [ABSTRACT FROM AUTHOR]