Roque Bru-Martínez

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.

Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors.

In plant cells, elicitors induce defense responses that resemble those triggered by pathogen attack, such as the synthesis of phytoalexins and pathogen-related proteins which accumulate in the extracellular space. In the search for the particular proteins involved in defense responses, we investigated the changes in the extracellular proteome of a grapevine (Vitis vinifera cv. Gamay) cell suspension in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA). Twenty-five of the 39 spots differentially expressed in 2-D gels were identified and found to be encoded by 10 different genes: three secretory peroxidases, chitinase-III, beta-1,3-glucanase, thaumatin-like, SGNH plant lipase-like, NtPR27-like, xyloglucan endotransglycosylase and subtilisin-like protease. Most of them belong to the pathogenesis-related type proteins. A new class III secretory basic peroxidase and chitinase III were strongly induced in cultures treated with MBCD alone or combined with MeJA, while cultures treated with MeJA alone displayed a general repression of most of the extracellular proteins. Some of the proteins induced in grapevine cell cultures by MBCD are induced in other species by activators of systemic acquired resistance (SAR), a form of plant immunity. Collectively, the results suggest that treatment with MBCD resembles the effect of SAR induction agents in cell cultures.

iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

BackgroundGrapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components such as sugars, acids, flavors, anthocyanins, tannins, etc., accumulate in the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance in our understanding of berry development and ripening processes.ResultsWe report the developmental analysis of Vitis vinifera cv. Muscat Hamburg berries at the protein level from fruit set to full ripening. An iTRAQ-based bottom-up proteomic approach followed by tandem mass spectrometry led to the identification and quantitation of 411 and 630 proteins in the green and ripening phases, respectively. Two key points in development relating to changes in protein level were detected: end of the first growth period (7 mm-to-15 mm) and onset of ripening (15 mm-to-V100, V100-to-110). A functional analysis was performed using the Blast2GO software based on the enrichment of GO terms during berry growth.ConclusionsThe study of the proteome contributes to decipher the biological processes and metabolic pathways involved in the development and quality traits of fruit and its derived products. These findings lie mainly in metabolism and storage of sugars and malate, energy-related pathways such as respiration, photosynthesis and fermentation, and the synthesis of polyphenolics as major secondary metabolites in grape berry. In addition, some key steps in carbohydrate and malate metabolism have been identified in this study, i.e., PFP-PFK or SuSy-INV switches among others, which may influence the final sugar and acid balance in ripe fruit. In conclusion, some proteins not reported to date have been detected to be deregulated in specific tissues and developmental stages, leading to formulate new hypotheses on the metabolic processes underlying grape berry development. These results open up new lines to decipher the processes controlling grape berry development and ripening.

DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-β-cyclodextrin and methyl jasmonate elicitors.

We had previously shown that Vitis vinifera cv. Gamay cell suspension accumulates extracellularly large amounts of the phytoalexin trans-resveratrol (tR) in response to elicitation with methylated cyclodextrins (MBCD), which can be triplicated when the elicitor is combined with methyl jasmonate (MeJA). In parallel, new pathogenesis-related proteins accumulated in the apoplast-like extracellular space. The aim of this study was to investigate changes in the grapevine cell proteome potentially related to tR accumulation in response to the above elicitors. The DIGE technique was used to detect statistically significant changes in the cells proteome. A total number of 1031 unique spots were detected, 67 of which were de-regulated upon elicitation. Sixty-four spots were successfully identified by nLC-MS/MS database search analysis. The tR biosynthetic pathway enzymes were up-regulated by MBCD alone or combined with MeJA, but not by treatment with MeJA alone, in agreement with tR accumulation pattern. Seven spots contained stilbene synthase encoded by four different isogenes. Likewise, four glutathione-S-transferases, potentially involved in tR trafficking within the cell and across membranes, were up-regulated in the same fashion as stilbene synthases. The relation of other de-regulated proteins with other effects caused by elicitors on grapevine cells, namely defense response and cell growth inhibition, is discussed.

Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post-translational modifications

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre‐mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms “protein species” and “protein isoform.” We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel‐based, gel‐free, bottom‐up and top‐down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α‐1 and α‐2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene.

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 μg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Summary Grapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (Vv ROMT) or human cytochrome P450 hydroxylase 1B1 (Hs CYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and Vv ROMT or Hs CYP1B1. Grapevine cell cultures transformed with Vv ROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with Hs CYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.

Alterations in Energy Metabolism, Neuroprotection and Visual Signal Transduction in the Retina of Parkinsonian, MPTP-Treated Monkeys

Parkinson disease is mainly characterized by the degeneration of dopaminergic neurons in the central nervous system, including the retina. Different interrelated molecular mechanisms underlying Parkinson disease-associated neuronal death have been put forward in the brain, including oxidative stress and mitochondrial dysfunction. Systemic injection of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to monkeys elicits the appearance of a parkinsonian syndrome, including morphological and functional impairments in the retina. However, the intracellular events leading to derangement of dopaminergic and other retinal neurons in MPTP-treated animal models have not been so far investigated. Here we have used a comparative proteomics approach to identify proteins differentially expressed in the retina of MPTP-treated monkeys. Proteins were solubilized from the neural retinas of control and MPTP-treated animals, labelled separately with two different cyanine fluorophores and run pairwise on 2D DIGE gels. Out of >700 protein spots resolved and quantified, 36 were found to exhibit statistically significant differences in their expression levels, of at least ±1.4-fold, in the parkinsonian monkey retina compared with controls. Most of these spots were excised from preparative 2D gels, trypsinized and subjected to MALDI-TOF MS and LC-MS/MS analyses. Data obtained were used for protein sequence database interrogation, and 15 different proteins were successfully identified, of which 13 were underexpressed and 2 overexpressed. These proteins were involved in key cellular functional pathways such as glycolysis and mitochondrial electron transport, neuronal protection against stress and survival, and phototransduction processes. These functional categories underscore that alterations in energy metabolism, neuroprotective mechanisms and signal transduction are involved in MPTP-induced neuronal degeneration in the retina, in similarity to mechanisms thought to underlie neuronal death in the Parkinson’s diseased brain and neurodegenerative diseases of the retina proper.

Development and Validation of MRM Methods to Quantify Protein Isoforms of Polyphenol Oxidase in Loquat Fruits

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using external calibrants.

Proteomics of Multigenic Families from Species Underrepresented in Databases: The Case of Loquat (Eriobotrya japonica Lindl.) Polyphenol Oxidases

Here, we approach the problem of obtaining accurate and reliable information about the gene origin of a protein belonging to a multigenic family, polyphenol oxidase, from an underrepresented species, Eriobotrya japonica. De novo sequencing was a key approach to obtain broad sequence coverage. Alignment of peptides on their most similar homologous protein revealed divergent amino acid positions that lead to hypothesize the minimal number of genes encoding for the proteins analyzed.