NO signalling during the plant HR

Summary Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. Dur- ing the last few years NO has been detected in several plant species, and the increasing number of reports on its function in plants have implicated NO as a key molecular signal that participates in the regulation of several physiological processes; in particular, it has a significant role in plant resistance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defence genes. NO stimulates signal transduction pathways through protein kinases, cytosolic Ca 2

Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

<p>Abstract</p> <p>Background</p> <p>The next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.</p> <p>Results</p> <p>The method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through <it>de novo </it>reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in <it>Vitis vinifera </it>as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.</p> <p>Conclusion</p> <p>This approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.</p

Modulation of nitric oxide bioactivity by plant haemoglobins

Abstract Nitric oxide (NO) is a highly reactive signalling molecule that has numerous targets in plants. Both enzymatic and non-enzymatic synthesis of NO has been detected in several plant species, and NO functions have been characterized during diverse physiological processes such as plant growth, development, and resistance to biotic and abiotic stresses. This wide variety of effects reflects the basic signalling mechanisms that are utilized by virtually all mammalian and plant cells and suggests the necessity of detoxification mechanisms to control the level and functions of NO. During the last two years an increasing number of reports have implicated non-symbiotic haemoglobins as the key enzymatic system for NO scavenging in plants, indicating that the primordial function of haemoglobins may well be to protect against nitrosative stress and to modulate NO signalling functions. The biological relevance of plant haemoglobins during specific conditions of plant growth and stress, and the existence of further enzymatic and non-enzymatic NO scavenging systems, suggest the existence of precise NO modulation mechanisms in plants, as observed for different NO sources

The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation

<p>Abstract</p> <p>Background</p> <p>High levels of ascorbic acid (AsA) in tomato fruits provide health benefits for humans and also play an important role in several aspects of plant life. Although AsA metabolism has been characterized in detail, the genetic mechanisms controlling AsA accumulation in tomatoes are poorly understood. The transcriptional control of AsA levels in fruits can be investigated by combining the advanced genetic and genomic resources currently available for tomato. A comparative transcriptomic analysis of fruit tissues was carried out on an introgression line containing a QTL promoting AsA accumulation in the fruit, using a parental cultivar with lower AsA levels as a reference.</p> <p>Results</p> <p>Introgression line IL 12-4 (<it>S. pennellii </it>in a <it>S. lycopersicum </it>background) was selected for transcriptomic analysis because it maintained differences in AsA levels compared to the parental genotypes M82 and <it>S. pennellii </it>over three consecutive trials. Comparative microarray analysis of IL 12-4 and M82 fruits over a 2-year period allowed 253 differentially-expressed genes to be identified, suggesting that AsA accumulation in IL 12-4 may be caused by a combination of increased metabolic flux and reduced utilization of AsA. In particular, the upregulation of a pectinesterase and two polygalacturonases suggests that AsA accumulation in IL12-4 fruit is mainly achieved by increasing flux through the L-galactonic acid pathway, which is driven by pectin degradation and may be triggered by ethylene.</p> <p>Conclusions</p> <p>Based on functional annotation, gene ontology classification and hierarchical clustering, a subset of the 253 differentially-expressed transcripts was used to develop a model to explain the higher AsA content in IL 12-4 fruits in terms of metabolic flux, precursor availability, demand for antioxidants, abundance of reactive oxygen species and ethylene signaling.</p

Resistance to Plasmopara viticola in a grapevine segregating population is associated with stilbenoid accumulation and with specific host transcriptional responses

<p>Abstract</p> <p>Background</p> <p>Downy mildew, caused by the oomycete <it>Plasmopara viticola</it>, is a serious disease in <it>Vitis </it><it>vinifera</it>, the most commonly cultivated grapevine species. Several wild <it>Vitis </it>species have instead been found to be resistant to this pathogen and have been used as a source to introgress resistance into a <it>V. vinifera </it>background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity is closely related to the specific compound. The aim of this study was to assess the resistance response to <it>P. viticola </it>of the Merzling × Teroldego cross by profiling the stilbenoid content of the leaves of an entire population and the transcriptome of resistant and susceptible individuals following infection.</p> <p>Results</p> <p>A three-year analysis of the population's response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of <it>trans</it>-resveratrol, <it>trans</it>-piceid, <it>trans</it>-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine.</p> <p>Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response.</p> <p>A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis.</p> <p>A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-<it>P. viticola </it>incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport.</p> <p>Conclusions</p> <p>This study reports the results of a combined metabolic and transcriptional profiling of a grapevine population segregating for resistance to <it>P. viticola</it>. Some resistant individuals were identified and further characterized at the molecular level. These results will be valuable to future grapevine breeding programs.</p

A transcriptional analysis reveals an extensive range of genes responsible for increasing the tolerance of Carrizo citrange to oxygen deficiency

Little information is available on the Citrus genus and its relatives with regard to their ability to tolerate oxygen deficiency, establishing physiological and structural modifications. In order to gain insight into how citrus rootstocks respond to low-oxygen stress, a transcriptomic analysis (using a custom microarray) was performed on Carrizo citrange (CC) seedlings. These seedlings were transformed with OsMybleu transcription factor (TF), known for inducing tolerance to oxygen deficiency, and compared with CC wildtype. They were flushed for 24 h with N2 and microarray, carrying out expressed sequence tags of Citrus and relatives isolated from the roots, was hybridized with RNA of roots before and after hypoxia treatment. The genes involved in fermentation, Krebs cycle, sugar metabolism, cell wall metabolism, hormones, and TFs all resulted significantly altered in response to hypoxia in both samples. Quantitative expression analysis was performed on 42 selected genes to validate microarray results. The outcome was that most of them were confirmed. The main results lead to the conclusion that CC is naturally tolerant to oxygen limitation. Transformed CC responded to hypoxia by activating the main genes which are known in other plants to be responsible for this type of tolerance such as pyruvate decarboxylase and alcohol dehydrogenase. Among TFs, several were also induced, such as an HDZipIII homologous to AtHB15, target of mir166, itself overexpressed exclusively in transformed CC under hypoxia compared with all other samples. The present manuscript represents one of the very few investigative works focused on hypoxia-responsive transcriptional networks in citru

Functional transcriptome analysis in ARSACS KO cell model reveals a role of sacsin in autophagy

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS

General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species

<p>Abstract</p> <p>Background</p> <p>Downy mildew is a destructive grapevine disease caused by <it>Plasmopara viticola </it>(Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (<it>Vitis</it>) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood.</p> <p>Results</p> <p>Early transcriptional changes associated with <it>P. viticola </it>infection in susceptible <it>V. vinifera </it>and resistant <it>V. riparia </it>plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in <it>V. riparia</it>, as determined by microscopic analysis. Our data indicate that resistance in <it>V. riparia </it>is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in <it>V. vinifera</it>. More interestingly, resistance in <it>V. riparia </it>also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in <it>V. vinifera </it>represents a weak attempted defense response rather than the activation of compatibility-specific pathways.</p> <p>Conclusions</p> <p>Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in <it>V. riparia </it>resistance to <it>P. viticola</it>.</p