Loreto Holuigue

The as-1 Promoter Element Is an Oxidative Stress-Responsive Element and Salicylic Acid Activates It via Oxidative Species

The activation sequence-1(as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35gene coding for a GST and (as-1)4 /β-glucuronidase(GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H2O2 levels by incubation with H2O2 or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)4 /GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)4 /GUSgene. These results suggest that oxidative species other than H2O2 mediate the activation of theas-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.

Early genomic responses to salicylic acid in Arabidopsis

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.

Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression

It is well established that salicylic acid (SA) plays a critical role in the transcriptional reprograming that occurs during the plant defense response against biotic and abiotic stress. In the course of the defense response, the transcription of different sets of defense genes is controlled in a spatio-temporal manner via SA-mediated mechanisms. Interestingly, different lines of evidence indicate that SA interplays with reactive oxygen species (ROS) and glutathione (GSH) in stressed plants. In this review we focus on the evidence that links SA, ROS, and GSH signals to the transcriptional control of defense genes. We discuss how redox modifications of regulators and co-regulators involved in SA-mediated transcriptional responses control the temporal patterns of gene expression in response to stress. Finally, we examine how these redox sensors are coordinated with the dynamics of cellular redox changes occurring in the defense response to biotic and abiotic stress.

NPR1-independent activation of immediate early salicylic acid-responsive genes in Arabidopsis

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.

Mitochondrial Complex II Is Essential for Gametophyte Development in Arabidopsis

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.

Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in Arabidopsis, have distinct cell-specific expression patterns and promoter activities.

Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex II (succinate dehydrogenase) in Arabidopsis (Arabidopsis thaliana), raising interesting questions about their origin and function. To find clues about their role, we have undertaken a detailed analysis of their expression. Two genes (SDH2-1 and SDH2-2) that likely arose via a relatively recent duplication event are expressed in all organs from adult plants, whereas transcripts from the third gene (SDH2-3) were not detected. The tissue- and cell-specific expression of SDH2-1 and SDH2-2 was investigated by in situ hybridization. In flowers, both genes are regulated in a similar way. Enhanced expression was observed in floral meristems and sex organ primordia at early stages of development. As flowers develop, SDH2-1 and SDH2-2 transcripts accumulate in anthers, particularly in the tapetum, pollen mother cells, and microspores, in agreement with an essential role of mitochondria during anther development. Interestingly, in contrast to the situation in flowers, only SDH2-2 appears to be expressed at a significant level in root tips. Strong labeling was observed in all cell layers of the root meristematic zone, and a cell-specific pattern of expression was found with increasing distance from the root tip, as cells attain their differentiated state. Analysis of transgenic Arabidopsis plants carrying SDH2-1 and SDH2-2 promoters fused to the β-glucuronidase reporter gene indicate that both promoters have similar activities in flowers, driving enhanced expression in anthers and/or pollen, and that only the SDH2-2 promoter is active in root tips. These β-glucuronidase staining patterns parallel those obtained by in situ hybridization, suggesting transcriptional regulation of these genes. Progressive deletions of the promoters identified regions important for SDH2-1 expression in anthers and/or pollen and for SDH2-2 expression in anthers and/or pollen and root tips. Interestingly, regions driving enhanced expression in anthers are differently located in the two promoters.

Glutaredoxin GRXS13 plays a key role in protection against photooxidative stress in Arabidopsis

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.

Enhanced resistance to bacterial infection by Erwinia Carotovora subsp. Atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes

Blackleg and soft rot diseases, caused by the bacteriumErwinia carotovora, are among the diseases that cause important losses in culture and storage of potato. In this paper, we introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, potato clones were transformed either with the gene encoding the acidic attacin protein fromHyalophora cecropia, or with the gene encoding the cecropin analog peptide SB37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strainErwinia carotovora subsp.atroseptica T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15–22%) showed increased resistance to bacterial infection, revealed by reduced severity of blackleg or soft rot symptoms. Expression of the transgenes was demonstrated in some of the clones by Northern blot analysis. This is the first report indicating that expression of the gene encoding for an attacin protein and for the cecropin SB-37 peptide in transgenic potato confers increased resistance to bacterial infection.

Splicing and editing of rps10 transcripts in potato mitochondria

The structure and expression of the potato mitochondrial gene rps10, encoding ribosomal protein S10, has been characterized. The RPS10 polypeptide of 129 amino acids is encoded by two exons of 307 bp and 80 bp respectively, which are separated by a 774-bp class-II intron. Editing of the complete rps10 coding region was studied by sequence analysis of spliced cDNAs. Four C residues are edited into U, resulting in the creation of a putative translational initiation codon, a new stop codon which eliminated ten carboxy-terminal residues, and two additional amino-acid alterations. All these changes increase the similarity between the potato and liverwort polypeptides. One additional C-to-U RNA editing event, observed in the intron sequence of unspliced cDNAs, improves the stability of the secondary structure in stem I (i) of domain I and may thus be required for the splicing reaction. All spliced cDNAs, and most unspliced cDNAs, were completely edited, suggesting that editing is an early step of rps10 mRNA processing and precedes splicing. Earlier work on potato rps10 (Zanlungo et al. 1994) is now known to comprise only a partial analysis of the gene, since the short downstream exon was not identified.

A Deficiency in the Flavoprotein of Arabidopsis Mitochondrial Complex II Results in Elevated Photosynthesis and Better Growth in Nitrogen-Limiting Conditions

Mitochondrial complex II (succinate dehydrogenase [SDH]) plays roles both in the tricarboxylic acid cycle and the respiratory electron transport chain. In Arabidopsis (Arabidopsis thaliana), its flavoprotein subunit is encoded by two nuclear genes, SDH1-1 and SDH1-2. Here, we characterize heterozygous SDH1-1/sdh1-1 mutant plants displaying a 30% reduction in SDH activity as well as partially silenced plants obtained by RNA interference. We found that these plants displayed significantly higher CO2 assimilation rates and enhanced growth than wild-type plants. There was a strong correlation between CO2 assimilation and stomatal conductance, and both mutant and silenced plants displayed increased stomatal aperture and density. By contrast, no significant differences were found for dark respiration, chloroplastic electron transport rate, CO2 uptake at saturating concentrations of CO2, or biochemical parameters such as the maximum rates of carboxylation by Rubisco and of photosynthetic electron transport. Thus, photosynthesis is enhanced in SDH-deficient plants by a mechanism involving a specific effect on stomatal function that results in improved CO2 uptake. Metabolic and transcript profiling revealed that mild deficiency in SDH results in limited effects on metabolism and gene expression, and data suggest that decreases observed in the levels of some amino acids were due to a higher flux to proteins and other nitrogen-containing compounds to support increased growth. Strikingly, SDH1-1/sdh1-1 seedlings grew considerably better in nitrogen-limiting conditions. Thus, a subtle metabolic alteration may lead to changes in important functions such as stomatal function and nitrogen assimilation.