Identification of NPR1-Dependent and Independent Genes Early Induced by Salicylic Acid Treatment in Arabidopsis

Abstract

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.

Early genomic responses to salicylic acid in Arabidopsis

“…Consistently, in Arabidopsis, SA was required to restrict the oxidative stress and foster plant acclimation after transient exposure to high light intensity (Mateo et al 2006). Interestingly, we and others found that SA uses a transient kinetics to activate genes coding for detoxifying or antioxidant enzymes such as glutathione S-transferases (GSTs) and glycosyltransferases (GTs) (Wagner et al 2002;Lieberherr et al 2003;Sappl et al 2004;Uquillas et al 2004;Blanco et al 2005;Langlois-Meurinne et al 2005). Besides activating stress defenses, SA is known to inhibit JA-and auxin-mediated responses, as part of the SA-mediated disease-resistance mechanism (Fujita et al 2006;Ndamukong et al 2007;Wang et al 2007).…”

“…For Northern blot analysis, 10 lg total RNA (isolated by using TrizolÒ) of the different samples were separated on formaldehyde-agarose gels, as previously described (Blanco et al 2005). Radioactive signals were either quantified by a Phosphorimager (Cyclone, Storage Phosphor screen, Packard Bioscience Company) or detected by autoradiography after 12 h exposure.…”

“…Sequences of the gene specific primers are described in Table 1. Probes were labeled by PCR as described (Blanco et al 2005). …”

“…Promoters analysis was performed essentially as described (Blanco et al 2005), except that the motif lengths (w) were set at 16 and 20 bp. For each search, the algorithm was iterated 2,000 times; independent outputs were merged and the 10 highest scored motifs were selected and ranked according to their log-likelihood scores.…”

“…Genes that showed reduced responsiveness to SA in the npr1-1 background compared with the wild type were included in the early NPR1-independent SAIGs. In a previous study, we identified 14 early SAIGs (Uquillas et al 2004;Blanco et al 2005). In the CATMAv2 chip, only 6 of these genes were represented by probes and, although 5 of them seemed to be induced by SA, the induction values were statistically significant for only 3 of these genes (At2g29420, At5g54610, At3g11280).…”