SUMMARYNitrate acts as a potent signal to control global gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly after nitrate treatments in roots. Global gene expression analysis revealed 97% of the genes with altered expression in tga1 tga4 double mutant plants respond to nitrate treatments, indicating that these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. We found TGA1 and TGA4 regulate the expression of nitrate transporter genes NRT2.1 and NRT2.2. Specific binding of TGA1 to its cognate DNA sequence on NRT2.1 and NRT2.2 promoters was confirmed by chromatin immunoprecipitation assays. The tga1 tga4 double mutant plants exhibit nitrate-dependent lateral and primary root phenotypes. Lateral root initiation is affected in both tga1 tga4 and nrt1.2 nrt2.2 double mutants, suggesting TGA1 and TGA4 regulate lateral root development at least partly via NRT2.1 and NRT2.2. Additional root phenotypes of tga1 tga4 double mutants indicate that these transcription factors play an important role in root developmental responses to nitrate. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots.

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell-specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.

Salicylic acid (SA) is a stress-induced hormone involved in the activation of defense genes. Here we analyzed the early genetic responses to SA of wild type and npr1-1 mutant Arabidopsis seedlings, using Complete Arabidopsis Transcriptome MicroArray (CATMAv2) chip. We identified 217 genes rapidly induced by SA (early SAIGs); 193 by a NPR1-dependent and 24 by a NPR1-independent pathway. These two groups of genes also differed in their functional classification, expression profiles and over-representation of cis-elements, supporting differential pathways for their activation. Examination of the expression patterns for selected early SAIGs from both groups indicated that their activation by SA required TGA2/5/6 subclass of transcription factors. These genes were also activated by Pseudomonas syringae pv. tomato AvrRpm1, suggesting that they might play a role in defense against bacteria. This study gives a global idea of the early response to SA in Arabidopsis seedlings, expanding our knowledge about SA function in plant defense.

Casein kinase 2 (CK2) is a ubiquitous enzyme essential for the viability of eukaryotic cells. In the present work we analyzed the Arabidopsis thaliana genome in a search for the genes coding for all CK2 alpha and beta subunits. We found four alpha subunit and four beta subunit genes. Expression analysis showed that all CK2 subunit genes are expressed in inflorescences, stems, leaves and roots. The level of expression of these genes is very similar, except for the one that codes for an alpha subunit harboring a putative chloroplastic destination peptide (alphacp), which shows a slightly higher expression level in all tissues. Using transgenic plants and agroinfiltration, we have also characterized the subcellular localization of all proteins encoded by CK2 genes. Our results show that all alpha subunits are localized in the nucleus, with the exception of alphacp, which is only found in the chloroplasts. On the other hand, beta subunits have a more diverse distribution, with some of them localizing both to the nucleus and to the cytosol, while others are exclusively located in one of these compartments. Remarkably, no CK2beta subunit was found in the chloroplasts. Finally, by directly measuring its activity, we have demonstrated that purified Arabidopsis chloroplasts have active CK2 that can be regulated by external addition of CK2beta. This study represents a complete survey of the CK2 gene family in Arabidopsis and the first step for future studies on CK2 cellular function in this species.

Salicylic acid (SA) is a key signal for the activation of defense genes in response to stress. The activation of late defense genes by SA, such as PR-1, involves the participation of the NPR1 protein. This protein acts as coactivator of the TGA factors that recognize as-1-like elements in the PR-1 promoter. Considering that functional as-1-like elements are also found in the promoter of SA- and auxin-responsive immediate early genes, we tested the hypothesis that NPR1 is also required for activation of these genes. The expression of the immediate early genes glutathione S-transferase (GST6) and glucosyltransferase (EIGT) was studied in npr1 mutant and wild-type Arabidopsis plants. In the npr1 mutant background, SA and 2,4-dichlorophenoxyacetic acid were unable to promote transcription of PR-1 but effectively stimulated the expression of GST6 and EIGT. Furthermore, increased binding of proteins to the GST6 as-1-like promoter element was detected in nuclear extracts from npr1 and wild-type plants after treatment with SA. In summary, these results indicate that activation of immediate early genes by SA proceeds through an NPR1-independent pathway. Therefore, we propose that activation by SA of immediate early and late genes occur by different mechanisms.

Salicylic acid (SA) plays a crucial role in stress resistance in plants by modifying the expression of a battery of genes. In this paper, we report the identification of a group of early SA-regulated genes of Arabidopsis (activated between 0.5-2.5 h), using the cDNA-amplified fragment length polymorphism technique (cDNA-AFLP). Using 128 different primer combinations, we identified several genes based on their differential expression during SA treatment. Among these, we identified 12 genes up-regulated by SA whose patterns of induction were confirmed by Northern analysis. The identified genes can be grouped into two functional groups: Group 1: genes involved in cell protection (i.e. glycosyltransferases, glutathion S-transferases), and Group 2: genes involved in signal transduction (protein kinases and transcription factors). We also evaluated NPR1 requirement for the induction of the 12 up-regulated genes, and found that only those belonging to Group 2 require this co-activator for their expression. In silico analysis of the promoter sequences of the up-regulated genes, allowed us to identify putative cis-elements over-represented in these genes. Interestingly, as-1-like elements, previously characterized as SA-responsive elements, were specifically over-represented in Group 1 genes. The identification of early SA-regulated genes is an important step towards understanding the complex role of this hormone in plant stress resistance.

Mitochondrial complex II (succinate dehydrogenase [SDH]) is part of the tricarboxylic acid cycle and the respiratory electron transport chain. Its flavoprotein subunit is encoded by two nuclear genes, SDH1-1 and SDH1-2, in Arabidopsis (Arabidopsis thaliana). The SDH1-2 gene is significantly expressed only in roots, albeit at very low level, and its disruption has no effect on growth and development of homozygous mutant plants. In contrast, SDH1-1 transcripts are ubiquitously expressed, with highest expression in flowers. Disruption of the SDH1-1 gene results in alterations in gametophyte development. Indeed, heterozygous SDH1-1/sdh1-1 mutant plants showed normal vegetative growth, yet a reduced seed set. In the progeny of selfed SDH1-1/sdh1-1 plants, distorted segregation ratios were observed, and no homozygous mutant plants were obtained. Reciprocal test crosses with the wild type demonstrated that the mutated sdh1-1 allele is not transmitted through the male gametophyte and is only partially transmitted through the female gametophyte. Consistently, microscopic analysis showed that mutant microspores develop normally until the vacuolated microspore stage, but fail to undergo mitosis I, and then cell structures are degraded and cell content disappears. On the other hand, half the mutant embryo sacs showed arrested development, either at the two-nucleate stage or before polar nuclei fusion. Down-regulation of SDH1-1 by RNA interference results in pollen abortion and a reduced seed set, as in the insertional mutant. Altogether, our results show that SDH1-1, and therefore complex II, are essential for gametophyte development.

Glutaredoxins (GRXs) belong to the antioxidant and signalling network involved in the cellular response to oxidative stress in bacterial and eukaryotic cells. In spite of the high number of GRX genes in plant genomes, the biological functions and physiological roles of most of them remain unknown. Here the functional characterization of the Arabidopsis GRXS13 gene (At1g03850), that codes for two CC-type GRX isoforms, is reported. The transcript variant coding for the GRXS13.2 isoform is predominantly expressed under basal conditions and is the isoform that is induced by photooxidative stress. Transgenic lines where the GRXS13 gene has been knocked down show increased basal levels of superoxide radicals and reduced plant growth. These lines also display reduced tolerance to methyl viologen (MeV) and high light (HL) treatments, both conditions of photooxidative stress characterized by increased production of superoxide ions. Consistently, lines overexpressing the GRXS13.2 variant show reduced MeV- and HL-induced damage. Alterations in GRXS13 expression also affect superoxide levels and the ascorbate/dehydroascorbate ratio after HL-induced stress. These results indicate that GRXS13 gene expression is critical for limiting basal and photooxidative stress-induced reactive oxygen species (ROS) production. Together, these results place GRXS13.2 as a member of the ROS-scavenging/antioxidant network that shows a particularly low functional redundancy in the Arabidopsis GRX family.