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Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO[sub 3, sup -] or NH[sub 4]NO[sub 3] as N source. [ABSTRACT FROM AUTHOR]

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]

Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown. Here, we report that combining mutations for CURLY LEAF (CLF) and LIKE HETEROCHROMATIN PROTEIN1 (LHP1), two Arabidopsis PcG proteins, results in deregulation of expression of active genes that are targeted by PcG proteins or enriched in associated chromatin marks. We show that this deregulation is associated with accumulation of small RNAs corresponding to massive degradation of active gene transcripts. We demonstrate that transcriptionally active genes and especially those targeted by PcG proteins are prone to RNA degradation, even though deregulation of RNA degradation following the loss of function of PcG proteins is not likely to be mediated by a PcG protein-mediated chromatin environment. Therefore, we conclude that PcG protein function is essential to maintain an accurate level of RNA degradation to ensure accurate gene expression. [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]

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]

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO-3 transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes. [ABSTRACT FROM AUTHOR]

Control of K+ and Na+ transport plays a central role in plant adaptation to salinity. In the halophyte Hordeum maritimum , we have characterized a transporter gene, named HmHKT2;1 , whose homolog HvHKT2;1 in cultivated barley, Hordeum vulgare , was known to give rise to increased salt tolerance when overexpressed. The encoded protein is strictly identical in two H. maritimum ecotypes, from two biotopes (Tunisian sebkhas) affected by different levels of salinity. These two ecotypes were found to display distinctive responses to salt stress in terms of biomass production, Na+ contents, K+ contents and K+ absorption efficiency. Electrophysiological analysis of HmHKT2;1 in Xenopus oocytes revealed distinctive properties when compared with HvHKT2;1 and other transporters from the same group, especially a much higher affinity for both Na+ and K+, and an Na+–K+ symporter behavior in a very broad range of Na+ and K+ concentrations, due to reduced K+ blockage of the transport pathway. Domain swapping experiments identified the region including the fifth transmembrane segment and the adjacent extracellular loop as playing a major role in the determination of the affinity for Na+ and the level of K+ blockage in these HKT2;1 transporters. The analysis (quantitative reverse transcription-PCR; qRT-PCR) of HmHKT2;1 expression in the two ecotypes submitted to saline conditions revealed that the levels of HmHKT2;1 transcripts were maintained constant in the most salt-tolerant ecotype whereas they decreased in the less tolerant one. Both the unique functional properties of HmHKT2;1 and the regulation of the expression of the encoding gene could contribute to H. maritimum adaptation to salinity. [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]