Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

Proteomic study of 'Moncada' mandarin buds from on- versus off-crop trees.

A proteomic analysis of buds from mandarin trees with contrasting fruit load (on- and off-crop trees) was carried out during the onset of low-temperature induction. The aim of the study was to find out more about the molecular mechanism relating to alternate bearing in Citrus and its relationship with flowering. The 'Moncada' variety (Clementine 'Oroval'x'Kara' mandarin), displaying remarkable behaviour in alternate production, was used in this study. From 2D DIGE gel, 192 spots were isolated: 97 showed increased expression in the off-crop buds as compared to the on-crop buds, while 95 exhibited enhanced expression in the on-crop buds versus the off-crop buds. These spots were identified by MALDI-MS or LC-MS-MS. The largest groups of proteins up-expressed in the off-crop buds were the proteins involved in carbohydrate and amino acid metabolism, and the proteins expressed in response to stimuli such as reactive oxygen species. The largest groups of proteins up-expressed in the on-crop buds were related to primary metabolism, oxidative stress and defence responses. Depending on their function, some of these proteins can stimulate the flowering, such as fructose-bisphosphate aldolase or leucine-rich repeat transmembrane protein kinase, while others can inhibit it, such as cytochrome c oxidase subunit II. Twenty-two other proteins with unknown functions were up-expressed in the on- or off-crop buds.

A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato.

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a ß-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens.

Proteomic analysis of "Moncada" mandarin leaves with contrasting fruit load.

A proteomic approach was used to know more about the molecular mechanism related to Citrus alternate bearing. To this end, we researched protein expression differences between on-crop and off-crop "Moncada" [Clementine 'Oroval' (Citrus clementina Hort ex Tanaka) x 'Kara' mandarin (Citrus unshiu Marc. x Citrus nobilis Lou.)] mandarin leaves. This variety usually shows a remarkable behaviour in alternate production. Samples were collected in the period during which the fruit affect flowering induction. From 2D DIGE gel, 110 spots were isolated: 43 showed increased expression in the off-crop samples compared to on-crop samples, while 67 showed increased expression in the on-crop samples against off-crop samples. These spots were identified by MALDI-MS or LC-MS-MS. According to the up-expressed proteins in off-crop leaves such as proteins related to nutrient reservoir activity or to the pentose phosphate pathway, the primary metabolism was more active in off-crop trees than in on-crop trees. In contrast, the proteins up-expressed in on-crop samples such as catalase were related to the oxidoreductase activity and, therefore, the redox state seemed different for off-crop and for on-crop leaves. Other proteins with unknown functions were isolated, which could be also related to the alternate bearing and to the flowering induction.

Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells.

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.

Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid.

The importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many incompatible plant-pathogen interactions, but less is known about signalling in compatible interactions. In this type of interaction, tomato plants have been found to accumulate high levels of 2,5-dihydroxybenzoic acid (gentisic acid, GA), a metabolic derivative of SA. Exogenous GA treatments induce in tomato plants a set of PR proteins that differ from those induced by salicylic acid. While SA accumulates in tomato plants mainly as 2-O-ß-D-glucoside, GA has only been found as 5-O-ß-D-xyloside. To characterize this step of the GA signalling pathway further, the present work focuses on the study of the GA-conjugating activity in tomato plants. A gentisate glycosyltransferase (GAGT) cDNA has been isolated and overexpressed in Pichia pastoris, and GA-conjugating activity was confirmed by detecting the xylosylated GA. The purified plant protein is highly specific for GA, showing no activity toward many other phenolic compounds, including SA. In addition, it shows an outstanding selectivity for UDP-xylose as the sugar donor, which differentiates this enzyme from most glycosyltransferases. Both the GA-conjugating activity and the corresponding mRNA show a strong, rapid, and transient induction upon treatment of tomato plants with GA or SA. Furthermore, its expression is rapidly induced by compatible infections. However, neither the gene nor the activity seems to respond to incompatible infections or wounding. The unique properties of this new glycosyltransferase suggest a specific role in regulating the free GA levels in compatible plant-pathogen interactions.