Transcriptomic and biochemical investigations support the role of rootstock-scion interaction in grapevine berry quality.

BACKGROUND: In viticulture, rootstock genotype plays a critical role to improve scion physiology, berry quality and to adapt grapevine (Vitis vinifera L.) to different environmental conditions. This study aimed at investigating the effect of two different rootstocks (1103 Paulsen - P - and Mgt 101-14 - M) in comparison with not grafted plants - NGC - on transcriptome (RNA-seq and small RNA-seq) and chemical composition of berry skin in Pinot noir, and exploring the influence of rootstock-scion interaction on grape quality. Berry samples, collected at veraison and maturity, were investigated at transcriptional and biochemical levels to depict the impact of rootstock on berry maturation. RESULTS: RNA- and miRNA-seq analyses highlighted that, at veraison, the transcriptomes of the berry skin are extremely similar, while variations associated with the different rootstocks become evident at maturity, suggesting a greater diversification at transcriptional level towards the end of the ripening process. In the experimental design, resembling standard agronomic growth conditions, the vines grafted on the two different rootstocks do not show a high degree of diversity. In general, the few genes differentially expressed at veraison were linked to photosynthesis, putatively because of a ripening delay in not grafted vines, while at maturity the differentially expressed genes were mainly involved in the synthesis and transport of phenylpropanoids (e.g. flavonoids), cell wall loosening, and stress response. These results were supported by some differences in berry phenolic composition detected between grafted and not grafted plants, in particular in resveratrol derivatives accumulation. CONCLUSIONS: Transcriptomic and biochemical data demonstrate a stronger impact of 1103 Paulsen rootstock than Mgt 101-14 or not grafted plants on ripening processes related to the secondary metabolite accumulations in berry skin tissue. Interestingly, the MYB14 gene, involved in the feedback regulation of resveratrol biosynthesis was up-regulated in 1103 Paulsen thus supporting a putative greater accumulation of stilbenes in mature berries.

Molecular basis of autotrophic vs mixotrophic growth in Chlorella sorokiniana.

In this work, we investigated the molecular basis of autotrophic vs. mixotrophic growth of Chlorella sorokiniana, one of the most productive microalgae species with high potential to produce biofuels, food and high value compounds. To increase biomass accumulation, photosynthetic microalgae are commonly cultivated in mixotrophic conditions, adding reduced carbon sources to the growth media. In the case of C. sorokiniana, the presence of acetate enhanced biomass, proteins, lipids and starch productivity when compared to autotrophic conditions. Despite decreased chlorophyll content, photosynthetic properties were essentially unaffected while differential gene expression profile revealed transcriptional regulation of several genes mainly involved in control of carbon flux. Interestingly, acetate assimilation caused upregulation of phosphoenolpyruvate carboxylase enzyme, enabling potential recovery of carbon atoms lost by acetate oxidation. The obtained results allowed to associate the increased productivity observed in mixotrophy in C. sorokiniana with a different gene regulation leading to a fine regulation of cell metabolism.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis.

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

Investigation of orthologous pathogen recognition gene-rich regions in solanaceous species.

Pathogen receptor proteins such as receptor-like protein (RLP), receptor-like kinase (RLK), and nucleotide-binding leucine-rich repeat (NLR) play a leading role in plant immunity activation. The genome architecture of such genes has been extensively investigated in several plant species. However, we still know little about their elaborate reorganization that arose during the plant speciation process. Using recently released pepper and eggplant genome sequences, we were able to identify 1097 pathogen recognition genes (PRGs) in the cultivated pepper Zunla-1 and 775 in the eggplant line Nakate-Shinkuro. The retrieved genes were analysed for their tendency to cluster, using different methods to infer the means of grouping. Orthologous relationships among clustering loci were found, and interesting reshuffling within given loci was observed for each analysed species. The information obtained was integrated into a comparative map to highlight the evolutionary dynamics in which the PRG loci were involved. Diversification of 14 selected PRG-rich regions was also explored using a DNA target-enrichment approach. A large number of gene variants were found as well as rearrangements of sequences encoding single protein domain and changes in chromosome gene order among species. Gene duplication and transposition activity have clearly influenced plant genome R-gene architecture and diversification. Our findings contribute to addressing several biological questions concerning the parallel evolution that occurred between genomes of the family Solanaceae. Moreover, the integration of different methods proved a powerful approach to reconstruct the evolutionary history in plant families and to transfer important biology findings among plant genomes.

Application of the whole-transcriptome shotgun sequencing approach to the study of Philadelphia-positive acute lymphoblastic leukemia.

Although the pathogenesis of BCR-ABL1-positive acute lymphoblastic leukemia (ALL) is mainly related to the expression of the BCR-ABL1 fusion transcript, additional cooperating genetic lesions are supposed to be involved in its development and progression. Therefore, in an attempt to investigate the complex landscape of mutations, changes in expression profiles and alternative splicing (AS) events that can be observed in such disease, the leukemia transcriptome of a BCR-ABL1-positive ALL patient at diagnosis and at relapse was sequenced using a whole-transcriptome shotgun sequencing (RNA-Seq) approach. A total of 13.9 and 15.8 million sequence reads was generated from de novo and relapsed samples, respectively, and aligned to the human genome reference sequence. This led to the identification of five validated missense mutations in genes involved in metabolic processes (DPEP1, TMEM46), transport (MVP), cell cycle regulation (ABL1) and catalytic activity (CTSZ), two of which resulted in acquired relapse variants. In all, 6390 and 4671 putative AS events were also detected, as well as expression levels for 18 315 and 18 795 genes, 28% of which were differentially expressed in the two disease phases. These data demonstrate that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations potentially involved in ALL.

Methods for nitric oxide detection during plant-pathogen interactions.

Nitric oxide (NO) is involved in the transduction of numerous signals in living organisms, and its biological effects are often influenced by its concentration. Therefore, the ability to reliably detect and quantify NO is crucial to understanding its role in cellular processes. Many techniques are available to detect and quantify NO, but depending on the material and the aim of the analysis, specific adaptations are often required because its high chemical reactivity leads to the formation of numerous reactive nitrogen species that make the accurate determination of NO levels difficult. Moreover, the pathogen-induced hypersensitive response leads to high rates of reactive oxygen species production that react with NO and lead to the formation of its oxidized derivates. The aim of this chapter is to provide an overview of the methods that have so far been employed to detect and measure NO in plants during the hypersensitive disease resistance response.

Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response.

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusion-limited reaction with O(2)(-) to generate peroxynitrite (ONOO(-)), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O(2)- but with H(2)O(2) generated from O(2)(-) by superoxide dismutase. Increasing the level of O(2)(-) reduces NO-mediated toxicity, and ONOO(-) is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O(2)(-) dismutation to H(2)O(2) to minimize the loss of NO by reaction with O(2)(-) and to trigger hypersensitive cell death through NO/H(2)O(2) cooperation. However, O(2)(-) rather than H(2)O(2) is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O(2)(-) generated during the oxidative burst play a crucial role in the modulation and integration of NO/H(2)O(2) signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

Transformation of elite white poplar (Populus alba L.) cv. ' Villafranca' and evaluation of herbicide resistance.

Transgenic white poplar plants (Populus alba L.) expressing the nptII gene and the bar gene from Streptomyces hygroscopicus have been produced using Agrobacterium tumefaciens-mediated gene transfer. Eleven kanamycin-resistant plant lines were obtained with a transformation frequency of 7%. Successful genetic transformation was confirmed by Southern and northern analyses. The level of resistance to the commercial preparation of phosphinothricin (Basta; Roussel-Hoechst Agrovet) was evaluated by in vitro and in vivo assays. Using in vitro selective conditions for phosphinothricin, only plantlets from four kanamycin-resistant independent lines remained green and continued to grow and root. After transfer to the growth chamber, all selected transgenic lines were shown to be completely resistant to the herbicide Basta with doses equivalent to 6 l ha-1 (normal field dosage) and were tolerant at concentration of 12 l ha-1. This is the first report describing the genetic transformation of a P. alba clonal cultivar of commercial interest with a gene of agronomic value.

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants.

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.

Nitric oxide functions as a signal in plant disease resistance.

Recognition of an avirulent pathogen triggers the rapid production of the reactive oxygen intermediates superoxide (O2-) and hydrogen peroxide (H2O2). This oxidative burst drives crosslinking of the cell wall, induces several plant genes involved in cellular protection and defence, and is necessary for the initiation of host cell death in the hypersensitive disease-resistance response. However, this burst is not enough to support a strong disease-resistance response. Here we show that nitric oxide, which acts as a signal in the immune, nervous and vascular systems, potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen intermediates and functions independently of such intermediates to induce genes for the synthesis of protective natural products. Moreover, inhibitors of nitric oxide synthesis compromise the hypersensitive disease-resistance response of Arabidopsis leaves to Pseudomonas syringae, promoting disease and bacterial growth. We conclude that nitric oxide plays a key role in disease resistance in plants.

Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a beta-ketoacyl reductase required for the biosynthesis of cuticular waxes.

The gl8 locus of maize (Zea mays L.) was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles were isolated from stocks that carried the Mutator transposon system. A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned. DNA flanking the Mu8 transposon was shown via allelic cross-referencing experiments to represent the gl8 locus. The gl8 probe revealed a 1.4-kb transcript present in wild-type seedling leaves and, in lesser amounts, in other organs and at other developmental stages. The amino acid sequence deduced from an apparently full-length gl8 cDNA exhibits highly significant sequence similarity to a group of enzymes from plants, eubacteria, and mammals that catalyzes the reduction of ketones. This finding suggests that the GL8 protein probably functions as a reductase during fatty acid elongation in the cuticular wax biosynthetic pathway.