Prenylated Flavonoids of the Moraceae Family: A Comprehensive Review of Their Biological Activities.

Prenylated flavonoids (PFs) are natural flavonoids with a prenylated side chain attached to the flavonoid skeleton. They have great potential for biological activities such as anti-diabetic, anti-cancer, antimicrobial, antioxidant, anti-inflammatory, enzyme inhibition, and anti-Alzheimer's effects. Medicinal chemists have recently paid increasing attention to PFs, which have become vital for developing new therapeutic agents. PFs have quickly developed through isolation and semi- or full synthesis, proving their high value in medicinal chemistry research. This review comprehensively summarizes the research progress of PFs, including natural PFs from the Moraceae family and their pharmacological activities. This information provides a basis for the selective design and optimization of multifunctional PF derivatives to treat multifactorial diseases.

High variability of perezone content in rhizomes of Acourtia cordata wild plants, environmental factors related, and proteomic analysis.

With the aim of exploring the source of the high variability observed in the production of perezone, in Acourtia cordata wild plants, we analyze the influence of soil parameters and phenotypic characteristics on its perezone content. Perezone is a sesquiterpene quinone responsible for several pharmacological effects and the A. cordata plants are the natural source of this metabolite. The chemistry of perezone has been widely studied, however, no studies exist related to its production under natural conditions, nor to its biosynthesis and the environmental factors that affect the yield of this compound in wild plants. We also used a proteomic approach to detect differentially expressed proteins in wild plant rhizomes and compare the profiles of high vs. low perezone-producing plants. Our results show that in perezone-producing rhizomes, the presence of high concentrations of this compound could result from a positive response to the effects of some edaphic factors, such as total phosphorus (Pt), total nitrogen (Nt), ammonium (NH4), and organic matter (O. M.), but could also be due to a negative response to the soil pH value. Additionally, we identified 616 differentially expressed proteins between high and low perezone producers. According to the functional annotation of this comparison, the upregulated proteins were grouped in valine biosynthesis, breakdown of leucine and isoleucine, and secondary metabolism such as terpenoid biosynthesis. Downregulated proteins were grouped in basal metabolism processes, such as pyruvate and purine metabolism and glycolysis/gluconeogenesis. Our results suggest that soil parameters can impact the content of perezone in wild plants. Furthermore, we used proteomic resources to obtain data on the pathways expressed when A. cordata plants produce high and low concentrations of perezone. These data may be useful to further explore the possible relationship between perezone production and abiotic or biotic factors and the molecular mechanisms related to high and low perezone production.

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling.

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.

Factors Affecting the Bioproduction of Resveratrol by Grapevine Cell Cultures under Elicitation.

Here we present a study of the characterization and optimization of the production of trans-Resveratrol (t-R) in grape (Vitis vinifera cv. Gamay) cell cultures elicited with methyl jasmonate (MeJA) and dimethyl-ß-cyclodextrin (DIMEB). The aim of this study was to determine the influence of a number of factors of the grapevine cell culture on t-R production level in 250 mL shaken flasks that would enable the better control of this bioproduction system when it is upscaled to a 2 L stirred bioreactor. The factors included the optimal growth phase for elicitation, the concentration of elicitors and of biomass, the order of addition of elicitors, and the illumination regime and ageing of cells. We found out that the optimal biomass density for the production of t-R was 19% (w/v) with an optimal ratio of 0.5 g DIMEB/g biomass. The most productive concentrations of the elicitors tested were 50 mM DIMEB and 100 µM MeJA, reaching maximum values of 4.18 mg·mL-1 and 16.3 mg·g biomass-1 of t-R concentration and specific production, respectively. We found that the order of elicitor addition matters since, as compared with the simultaneous addition of both elicitors, the addition of MeJA 48 h before DIMEB results in ca. 40% less t-R production, whilst there is no significant difference when MeJA is added 48 h after DIMEB. Upon upscaling, the better conditions tested for t-R production were aeration at 1.7 vol/vol/min without agitation, 24 °C, and 30 g·L-1 sucrose, achieving production rates similar to those obtained in shaken flasks.

Down-Regulation of Phosphoenolpyruvate Carboxylase Kinase in Grapevine Cell Cultures and Leaves Is Linked to Enhanced Resveratrol Biosynthesis.

In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-ß-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs in both tissues. Moreover, STS transcripts were co-induced with MYB14 and WRKY24 in cell cultures and leaves. These genes have not previously been reported to respond to CD in grape leaves. Our findings thus support the hypothesis that PPCK is involved in diverting metabolism towards stilbene biosynthesis, both for in vitro cell culture and whole leaves. We thus provide new evidence for PEP being redirected between primary and secondary metabolism to support tR production and the stress response.

Proteome changes in pepper (Capsicum annuum L.) leaves induced by the green peach aphid (Myzus persicae Sulzer).

BACKGROUND: Aphid attack induces defense responses in plants activating several signaling cascades that led to the production of toxic, repellent or antinutritive compounds and the consequent reorganization of the plant primary metabolism. Pepper (Capsicum annuum L.) leaf proteomic response against Myzus persicae (Sulzer) has been investigated and analyzed by LC-MS/MS coupled with bioinformatics tools. RESULTS: Infestation with an initially low density (20 aphids/plant) of aphids restricted to a single leaf taking advantage of clip cages resulted in 6 differentially expressed proteins relative to control leaves (3 proteins at 2 days post-infestation and 3 proteins at 4 days post-infestation). Conversely, when plants were infested with a high density of infestation (200 aphids/plant) 140 proteins resulted differentially expressed relative to control leaves (97 proteins at 2 days post-infestation, 112 proteins at 4 days post-infestation and 105 proteins at 7 days post-infestation). The majority of proteins altered by aphid attack were involved in photosynthesis and photorespiration, oxidative stress, translation, protein folding and degradation and amino acid metabolism. Other proteins identified were involved in lipid, carbohydrate and hormone metabolism, transcription, transport, energy production and cell organization. However proteins directly involved in defense were scarce and were mostly downregulated in response to aphids. CONCLUSIONS: The unexpectedly very low number of regulated proteins found in the experiment with a low aphid density suggests an active mitigation of plant defensive response by aphids or alternatively an aphid strategy to remain undetected by the plant. Under a high density of aphids, pepper leaf proteome however changed significantly revealing nearly all routes of plant primary metabolism being altered. Photosynthesis was so far the process with the highest number of proteins being regulated by the presence of aphids. In general, at short times of infestation (2 days) most of the altered proteins were upregulated. However, at longer times of infestation (7 days) the protein downregulation prevailed. Proteins involved in plant defense and in hormone signaling were scarce and mostly downregulated.

Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue.

Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4 Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.

Changes in the free amino acid composition of Capsicum annuum (pepper) leaves in response to Myzus persicae (green peach aphid) infestation. A comparison with water stress.

Amino acids play a central role in aphid-plant interactions. They are essential components of plant primary metabolism, function as precursors for the synthesis of defense-related specialized metabolites, and are major growth-limiting nutrients for aphids. To quantify changes in the free amino acid content of pepper (Capsicum annuum L.) leaves in response to green peach aphid (Myzus persicae Sulzer) feeding, plants were infested with a low (20 aphids/plant) or a high (200 aphids/plant) aphid density in time-course experiments ranging from 3 hours to 7 days. A parallel experiment was conducted with pepper plants that had been subjected to water stress. Factor Analysis of Mixed Data revealed a significant interaction of time x density in the free amino acid response of aphid-infested leaves. At low aphid density, M. persicae did not trigger a strong response in pepper leaves. Conversely, at high density, a large increase in total free amino acid content was observed and specific amino acids peaked at different times post-infestation. Comparing aphid-infested with water-stressed plants, most of the observed differences were quantitative. In particular, proline and hydroxyproline accumulated dramatically in response to water stress, but not in response to aphid infestation. Some additional differences and commonalities between the two stress treatments are discussed.

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells.

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or ß-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.

A Focused Multiple Reaction Monitoring (MRM) Quantitative Method for Bioactive Grapevine Stilbenes by Ultra-High-Performance Liquid Chromatography Coupled to Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ).

Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol, trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-ε-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.

The role of proteomics in progressing insights into plant secondary metabolism.

The development of omics has enabled the genome-wide exploration of all kinds of biological processes at the molecular level. Almost every field of plant biology has been analyzed at the genomic, transcriptomic and proteomic level. Here we focus on the particular contribution that proteomic technologies have made in progressing knowledge and characterising plant secondary metabolism (SM) pathways since early expectations were created 15 years ago. We analyzed how three major issues in the proteomic analysis of plant SM have been implemented in various research studies. These issues are: (i) the selection of a suitable plant material rich in secondary metabolites of interest, such as specialized tissues and organs, and in vitro cell cultures; (ii) the proteomic strategy to access target proteins, either a comprehensive or a differential analysis; (iii) the proteomic approach, represented by the hypothesis-free discovery proteomics and the hypothesis-driven targeted proteomics. We also examine to what extent the most-advanced technologies have been incorporated into proteomic research in plant SM and highlight some cutting edge techniques that would strongly benefit the progress made in this field.

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.

Differential plant proteome analysis by isobaric tags for relative and absolute quantitation (iTRAQ).

Protein relative quantitation is one of the main targets in many proteomic experiments. Among the range of techniques available for both top-down and bottom-up approaches, isobaric tags for relative and absolute quantitation (iTRAQ) have gained positions within the top-rank techniques used for this purpose in the recent years. Briefly, each iTRAQ reagent consists of three different components: a reporter group (with a variable mass in the range of 114-117 amu), a balance group, and an amino-reactive group. The isobaric nature of iTRAQ-labeled peptides adds a signal to every peptide in the sample which is detectable in both MS and MS/MS spectra, thus enhancing the sensitivity of detection. During MS/MS, the reporter groups are released as singly charged ions with m/z ratios ranking from 114 to 117 amu, visible in the low mass region of MS/MS spectra. The iTRAQ technology can be used to analyze up to four different samples using the 4-plex kit (reporter groups 114-115 amu) or can be scaled up to eight different samples using the 8-plex kit (reporter groups 113-121 amu). In this chapter, we focus on the experimental procedures typically using 4-plex labeling, including tips leading to successful application of iTRAQ technology for the analysis of plant protein mixtures.

Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

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.

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.

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.

iTRAQ-based profiling of grape berry exocarp proteins during ripening using a parallel mass spectrometric method.

The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevine berry skin ripening, from pre-veraison to fully ripening. Mass spectrometric data were acquired from three replicated analyses using a parallel acquisition method in an Orbitrap instrument by combining collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) peptide ion fragmentations. As a result, the number of spectra suitable for peptide identification (either from CID or HCD) increased 5-fold in relation to those suitable for quantification (from HCD). Spectra were searched against an NCBInr protein database subset containing all the Vitis sequences, including those derived from whole genome sequencing. In general, 695 unique proteins were identified with more than one single peptide, and 513 of them were quantified. The sequence annotation and GO term enrichment analysis assisted by the automatic annotation tool Blast2GO permitted a pathway analysis which resulted in finding that biological processes and metabolic pathways de-regulated throughout ripening. A detailed analysis of the function-related proteins profiles helped discover a set of proteins of known Vitis gene origin as the potential candidates to play key roles in grapevine berry quality, growth regulation and disease resistance.

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.