Proteomic and Low-Polar Metabolite Profiling Reveal Unique Dynamics in Fatty Acid Metabolism during Flower and Berry Development of Table Grapes.

Grapevine development and ripening are complex processes that involve several biochemical pathways, including fatty acid and lipid metabolism. Fatty acids are essential components of lipids, which play crucial roles in fruit maturation and flavor development. However, the dynamics of fatty acid metabolism in grape flowers and berries are poorly understood. In this study, we present those dynamics and investigate the mechanisms of fatty acid homeostasis on 'Thompson Seedless' berries using metabolomic and proteomic analyses. Low-polar metabolite profiling indicated a higher abundance of fatty acids at the pre-flowering and pre-veraison stages. Proteomic analyses revealed that grape flowers and berries display unique profiles of proteins involved in fatty acid biosynthesis, triacylglycerol assembly, fatty acid ß-oxidation, and lipid signaling. These findings show, for the first time, that fatty acid metabolism also plays an important role in the development of non-oil-rich tissues, opening new perspectives about lipid function and its relation to berry quality.

Metabolite Profiling Reveals the Effect of Cold Storage on Primary Metabolism in Nectarine Varieties with Contrasting Mealiness.

Chilling injury is a physiological disorder caused by cold storage in peaches and nectarines. The main symptom of chilling injury is mealiness/wooliness, described as a lack of juice in fruit flesh. In this work, we studied two nectarine varieties (Andes Nec-2 and Andes Nec-3) with contrasting susceptibility to mealiness after cold storage. A non-targeted metabolomic analysis was conducted by GC-MS to understand if changes in metabolite abundance are associated with nectarine mealiness induced by cold storage. Multivariate analyses indicated that in unripe nectarines, cold storage promoted a higher accumulation of amino acids in both varieties. Interestingly, for ripe nectarines, cold storage induced an accumulation of fewer amino acids in both varieties and showed an increased abundance of sugars and organic acids. A pathway reconstruction of primary metabolism revealed that in ripe nectarines, cold storage disrupted metabolite abundance in sugar metabolism and the TCA cycle, leading to a differential accumulation of amino acids, organic acids, and sugars in mealy and juicy nectarines.

Proteomic and metabolomic integration reveals the effects of pre-flowering cytokinin applications on central carbon metabolism in table grape berries.

Consumers around the world prefer high quality table grapes. To achieve higher quality traits at ripening, grapevine producers apply different plant growth regulators. The synthetic cytokinin forchlorfenuron N-(2-chloro-4-pyridinyl)-N'-phenylurea (CPPU) is widely used, its effect on grape quality is poorly understood. We hypothesized that the use of CPPU in pre-flowering can lead to changes in the metabolism that affects grape quality at harvest. Therefore, we investigated the role of CPPU applications on the quality of grapes by integrating proteomics and metabolomics. CPPU-treated grapevines showed a significant increase in berry size and firmness. Proteomic analyses indicated that CPPU-treated berries accumulated enzymes associated with carbohydrate metabolism, glycolysis, and tricarboxylic acid (TCA) cycle at harvest. Metabolomic analyses showed shifts in the abundance of compounds associated with carbohydrate metabolism and TCA cycle in CPPU-treated grapes. These findings suggest that CPPU applications modulate central carbon metabolism, improving grape berry quality.

A multiomics integrative analysis of color de-synchronization with softening of 'Hass' avocado fruit: A first insight into a complex physiological disorder.

Exocarp color de-synchronization with softening of 'Hass' avocado is a relevant recurrent problem for the avocado supply chain. This study aimed to unravel the mechanisms driving this de-synchronization integrating omics datasets from avocado exocarp of different storage conditions and color phenotypes. In addition, we propose potential biomarkers to predict color synchronized/de-synchronized fruit. Integration of transcriptomics, proteomics and metabolomics and network analysis revealed eight transcription factors associated with differentially regulated genes between regular air (RA) and controlled atmosphere (CA) and twelve transcription factors related to avocado fruit color de-synchronization control in ready-to-eat stage. CA was positively correlated to auxins, ethylene, cytokinins and brassinosteroids-related genes, while RA was characterized by enrichment of cell wall remodeling and abscisic acid content associated genes. At ready-to-eat higher contents of flavonoids, abscisic acid and brassinosteroids were associated with color-softening synchronized avocados. In contrast, de-synchronized fruit revealed increases of jasmonic acid, salicylic acid and auxin levels.

A sustainable application for the extraction of lichen metabolites from Usnea cornuta: nontargeted metabolomics and antioxidant activity.

In this study, isolation and purification of lichen substances from Usnea cornuta were performed using conventional solvents, green solvents and green technologies. In addition, several lichen compounds were tentatively identified by UHPLC/ESI/MS/MS and usnic acid, diffractaic and galbinic acids were quantified as well. Limonene, ethyl lactate and methanol, were compared regarding their extraction properties and antioxidant capacities, determined by DPPH, ORAC, and FRAP assays. In the ethyl lactate, methanol and limonene extracts, 28 compounds in all, were detected for the first time by high resolution UHPLC-MS/MS fingerprinting. Untargeted metabolomics tentatively identified 14 compounds from the methanolic extract, 4 from limonene extract, and 20 metabolites from ethyl lactate extract. The green extract of ethyl lactate showed a similar antioxidant capacity to toxic methanol extract, except at ORAC assay where it was higher. Therefore, ethyl lactate can replace methanol, to provide more sustainable green chemistry methods.

Transcriptome and Gene Regulatory Network Analyses Reveal New Transcription Factors in Mature Fruit Associated with Harvest Date in Prunus persica.

Harvest date is a critical parameter for producers and consumers regarding agro-industrial performance. It involves a pleiotropic effect controlling the development of other fruit quality traits through finely controlling regulatory mechanisms. Fruit ripening is a process in which various signals and biological events co-occur and are regulated by hormone signaling that produces the accumulation/degradation of multiple compounds. However, the regulatory mechanisms that control the hormone signaling involved in fruit development and ripening are still unclear. To investigate the issue, we used individuals with early, middle and late harvest dates from a peach segregating population to identify regulatory candidate genes controlling fruit quality traits at the harvest stage and validate them in contrasting peach varieties for this trait. We identified 467 and 654 differentially expressed genes for early and late harvest through a transcriptomic approach. In addition, using the Arabidopsis DAP-seq database and network analysis, six transcription factors were selected. Our results suggest significant hormonal balance and cell wall composition/structure differences between early and late harvest samples. Thus, we propose that higher expression levels of the transcription factors HB7, ERF017 and WRKY70 in early harvest individuals would induce the expression of genes associated with the jasmonic acid pathway, photosynthesis and gibberellins inhibition. While on the other hand, the high expression levels of LHY, CDF3 and NAC083 in late harvest individuals would promote the induction of genes associated with abscisic acid biosynthesis, auxins and cell wall remodeling.

Response Mechanisms of "Hass" Avocado to Sequential 1-methylcyclopropene Applications at Different Maturity Stages during Cold Storage.

1-Methylcyclopropene (1-MCP) is used for extending the postharvest life of the avocado during storage. Evaluated the effect of 1-MCP application at different times after harvest, i.e., 0, 7, 14, and 21 d at 5 °C, to identify the threshold of the ethylene inhibition response in "Hass" avocado. Our results showed that fruits from two maturity stages at harvest: low dry matter (20-23%) and high dry matter (27%). Changes in ethylene production rates and transcript accumulation of genes involved in ethylene metabolism were measured at harvest and during storage. 1-MCP treated fruit up to 14 d of storage showed similar values of firmness and skin color as fruit treated at harvest time. In contrast, when the application was performed after 21 d, the fruit showed ripening attributes similar to those of the untreated ones. To further understand the molecular mechanisms responsible for the lack of response to 1-MCP at 21 d of storage, transcriptomic analysis was performed. Gene ontology analyses based on the DEG analysis showed enrichment of transcripts involved in the 'response to ethylene' for both maturity stages. All genes evaluated showed similar expression profiles induced by cold storage time, with a peak at 21 d of storage and an increased softening of the fruit and peel color. This was a two-year field study, and results were consistent across the two experimental years. Our results should help growers and markets in selecting the optimal timing of 1-MCP application in "Hass" avocados and should contribute to a deeper understanding of the molecular mechanisms of the avocado ripening process.

Metabolomic and biochemical analysis of mesocarp tissues from table grape berries with contrasting firmness reveals cell wall modifications associated to harvest and cold storage.

Tissue texture influences the grape berry consumers acceptance. We studied the biological differences between the inner and outer mesocarp tissues in hard and soft berries of table grapes cv NN107. Texture analysis revealed lower levels of firmness in the inner mesocarp as compared with the outer tissue. HPAEC-PAD analysis showed an increased abundance of cell wall monosaccharides in the inner mesocarp of harder berries at harvest. Immunohistochemical analysis displayed differences in homogalacturonan methylesterification and cell wall calcium between soft and hard berries. This last finding correlated with a differential abundance of calcium measured in the alcohol-insoluble residues (AIR) of the inner tissue of the hard berries. Analysis of abundance of polar metabolites suggested changes in cell wall carbon supply precursors, providing new clues in the identification of the biochemical factors that define the texture of the mesocarp of grape berries.

Pre-Anthesis Cytokinin Applications Increase Table Grape Berry Firmness by Modulating Cell Wall Polysaccharides.

The use of plant growth regulators (PGRs) is widespread in commercial table grape vineyards. The synthetic cytokinin CPPU is a PGR that is extensively used to obtain higher quality grapes. However, the effect of CPPU on berry firmness is not clear. The current study investigated the effects of pre-anthesis applications (BBCH15 and BBCH55 stages) of CPPU on 'Thompson Seedless' berry firmness at harvest through a combination of cytological, morphological, and biochemical analyses. Ovaries in CPPU-treated plants presented morphological changes related to cell division and cell wall modification at the anthesis stage (BBCH65). Moreover, immunofluorescence analysis with monoclonal antibodies 2F4 and LM15 against pectin and xyloglucan demonstrated that CPPU treatment resulted in cell wall modifications at anthesis. These early changes have major repercussions regarding the hemicellulose and pectin cell wall composition of mature fruits, and are associated with increased calcium content and a higher berry firmness at harvest.

Unravelling the Molecular Regulation Mechanisms of Slow Ripening Trait in Prunus persica.

Fruit development is a complex process that involves the interplay of cell division, expansion, and differentiation. As a model to study fruit development, nectarines incapable of ripening were described as slow ripening. Slow ripening fruits remained firm and exhibited no rise in CO2 or ethylene production rates for one month or more at 20 °C. Different studies suggest that this trait is controlled by a single gene (NAC072). Transcriptome analysis between normal and slow ripening fruits showed a total of 157, 269, 976, and 5.224 differentially expressed genes in each fruit developmental stage analyzed (T1, T2, T3, and T7, respectively), and no expression of NAC072 was found in the slow ripening individuals. Using this transcriptomic information, we identified a correlation of NAC072 with auxin-related genes and two genes associated with terpene biosynthesis. On the other hand, significant differences were observed in hormonal biosynthetic pathways during fruit development between the normal and slow ripening individuals (gibberellin, ethylene, jasmonic acid and abscisic acid). These results suggest that the absence of NAC072 by the direct or indirect expression control of auxins or terpene-related genes prevents normal peach fruit development.

Identification of DNA Methylation and Transcriptomic Profiles Associated With Fruit Mealiness in Prunus persica (L.) Batsch.

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.

Cell Wall Calcium and Hemicellulose Have a Role in the Fruit Firmness during Storage of Blueberry (Vaccinium spp.).

The firmness of blueberry is one of its most significant quality attributes. Modifications in the composition of the cell wall have been associated with changes in the fruit firmness. In this work, cell wall components and calcium concentration in two blueberry cultivars with contrasting firmness phenotypes were evaluated at harvest and 30 days cold storage (0 °C). High performance anion-exchange chromatography with pulse amperometric detector (HPAEC-PAD) analysis was performed using the "Emerald" (firmer) and "Jewel" (softer) blueberry cultivars, showing increased glucose in the firmer cultivar after cold storage. Moreover, the LM15 antibody, which recognizes xyloglucan domains, displayed an increased signal in the Emerald cultivar after 30 d cold storage. Additionally, the antibody 2F4, recognizing a homogalacturonan calcium-binding domain, showed a greater signal in the firmer Emerald blueberries, which correlates with a higher calcium concentration in the cell wall. These findings suggest that xyloglucan metabolism and a higher concentration of cell wall calcium influenced the firmness of the blueberry fruit. These results open new perspectives regarding the role of cell wall components as xyloglucans and calcium in blueberry firmness.

Cell wall and metabolite composition of sweet cherry fruits from two cultivars with contrasting susceptibility to surface pitting during storage.

Surface pitting is a serious postharvest physiological disorder in sweet cherries that is observed as skin depressions developed days after bruising. This work aims to compare two cultivars displaying different pitting susceptibilities ('Kordia': relatively resistant; 'Sweetheart': relatively susceptible) using metabolomics profiling and cell wall sugar characterization at different developmental stages and during postharvest storage. Kordia was significantly firmer than Sweetheart, with 1.4-fold more alcohol-insoluble residues (AIRs). A significant correlation was observed between AIRs and deformation, indicating that the highest yields of cell wall material are positively correlated with the resistance to rupture. Additionally, free d-galacturonic acid was higher in pitted Sweetheart samples, likely indicating greater pectin degradation in this susceptible cultivar. Higher contents of the p-coumaric acid derivatives L-5-oxoproline and d-galactose in Sweetheart cherries were found. The metabolic changes during storage and cell wall composition could influence the susceptibility to surface pitting.

Identification of Metabolite and Lipid Profiles in a Segregating Peach Population Associated with Mealiness in Prunus persica (L.) Batsch.

The peach is the third most important temperate fruit crop considering fruit production and harvested area in the world. Exporting peaches represents a challenge due to the long-distance nature of export markets. This requires fruit to be placed in cold storage for a long time, which can induce a physiological disorder known as chilling injury (CI). The main symptom of CI is mealiness, which is perceived as non-juicy fruit by consumers. The purpose of this work was to identify and compare the metabolite and lipid profiles between two siblings from contrasting populations for juice content, at harvest and after 30 days at 0 °C. A total of 119 metabolites and 189 lipids were identified, which showed significant differences in abundance, mainly in amino acids, sugars and lipids. Metabolites displaying significant changes from the E1 to E3 stages corresponded to lipids such as phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG) and lysophosphatidylcholines (LPC), and sugars such as fructose 1 and 1-fructose-6 phosphate. These metabolites might be used as early stage biomarkers associated with mealiness at harvest and after cold storage.

Continental and Antarctic Lichens: isolation, identification and molecular modeling of the depside tenuiorin from the Antarctic lichen Umbilicaria antarctica as tau protein inhibitor.

Alzheimer´s disease (AD) is the most common form of dementia involving Aß and tau protein. So far, AD cure remains elusive, but considering that AD progresses throughout tau pathology, which turns tau protein an appropriate target, besides tau is also included in other neurodegenerative disorders named as tauopathies. Here, we have isolated seventeen compounds belonging to six lichens species. Due to scarce of spectroscopic data of the compound 5,7-dihydroxy-6-methylphthalide, we explained their structural elucidation based on NMR data. In this study, we show that only tenuiorin from Umbilicaria antarctica inhibited 50% of tau 4R at 100 µM. Then, we shown that molecular interactions of tenuiorin with the steric zipper model of the hexapeptide 306VQIVYK311 were studied by docking calculations and the results suggested that tenuiorin forms both hydrogen bonds with lysine and glutamine side chains and forms several hydrophobic interactions with valine and lysine from 306VQIVYK311 motif.

Expression QTL (eQTLs) Analyses Reveal Candidate Genes Associated With Fruit Flesh Softening Rate in Peach [Prunus persica (L.) Batsch].

Significant differences in softening rate have been reported between melting flesh in peach and nectarine varieties. This trait seems to be controlled by several genes. We aimed to identify candidate genes involved in fruit softening rate by integrating quantitative trait loci (QTL) and expression QTL (eQTL) analyses, comparing siblings with contrasting softening rates. We used a segregating population derived from nectarine cv. 'Venus' selfing, which was phenotyped for softening rate during three seasons. Six siblings with high (HSR) and six with low softening rate (LSR) were sequenced using RNA-Seq. A group of 5,041 differentially expressed genes was identified. Also, we found a QTL with a LOD (logarithm of odds) score of 9.7 on LG4 in all analyzed seasons. Furthermore, we detected 1,062 eQTLs, of which 133 were found co-localizing with the identified QTL. Gene Ontology (GO) analysis showed 'Response to auxin' as one the main over-represented categories. Our findings suggest over-expression of auxin biosynthetic related genes in the HSR group, which implies a higher expression and/or accumulation of auxin, thereby triggering fast softening. Conversely, the LSR phenotype might be explained by an altered auxin-homeostasis associated with low auxin levels. This work will contribute to unraveling the genetic mechanisms responsible for the softening rate in peaches and nectarines and lead to the development of molecular markers.

Primary Metabolism in Avocado Fruit.

Avocado (Persea americana Mill) is rich in a variety of essential nutrients and phytochemicals; thus, consumption has drastically increased in the last 10 years. Avocado unlike other fruit is characterized by oil accumulation during growth and development and presents a unique carbohydrate pattern. There are few previous and current studies related to primary metabolism. The fruit is also quite unique since it contains large amounts of C7 sugars (mannoheptulose and perseitol) acting as transportable and storage sugars and as potential regulators of fruit ripening. These C7 sugars play a central role during fruit growth and development, but still confirmation is needed regarding the biosynthetic routes and the physiological function during growth and development of avocado fruit. Relatively recent transcriptome studies on avocado mesocarp during development and ripening have revealed that most of the oil is synthesized during early stages of development and that oil synthesis is halted when the fruit is harvested (pre-climacteric stage). Most of the oil is accumulated in the form of triacylglycerol (TAG) representing 60-70% in dry basis of the mesocarp tissue. During early stages of fruit development, high expression of transcripts related to fatty acid and TAG biosynthesis has been reported and downregulation of same genes in more advanced stages but without cessation of the process until harvest. The increased expression of fatty acid key genes and regulators such as PaWRI1, PaACP4-2, and PapPK-ß-1 has also been reported to be consistent with the total fatty acid increase and fatty acid composition during avocado fruit development. During postharvest, there is minimal change in the fatty acid composition of the fruit. Almost inexistent information regarding the role of organic acid and amino acid metabolism during growth, development, and ripening of avocado is available. Cell wall metabolism understanding in avocado, even though crucial in terms of fruit quality, still presents severe gaps regarding the interactions between cell wall remodeling, fruit development, and postharvest modifications.

Integration of proteomics and metabolomics data of early and middle season Hass avocados under heat treatment.

Ripening heterogeneity of Hass avocados results in inconsistent quality fruit delivered to the triggered and ready to eat markets. This research aimed to understand the effect of a heat shock (HS) prior to controlled atmosphere (CA) storage on the reduction of ripening heterogeneity. HS prior to CA storage reduces more drastically the ripening heterogeneity in middle season fruit. Via correlation network analysis we show the different metabolomics networks between HS and CA. High throughput proteomics revealed 135 differentially expressed proteins unique to middle season fruit triggered by HS. Further integration of metabolomics and proteomics data revealed that HS reduced the glycolytic throughput and induced protein degradation to deliver energy for the alternative ripening pathways. l-isoleucine, l-valine, l-aspartic and ubiquitin carboxyl-terminal hydrolase involved in protein degradation were positively correlated to HS samples. Our study provides new insights into the effectiveness of HS in synchronizing ripening of Hass avocados.

De novo assembly of Persea americana cv. 'Hass' transcriptome during fruit development.

BACKGROUND: Avocado (Persea americana Mill.) is a basal angiosperm from the Lauraceae family. This species has a diploid genome with an approximated size of ~ 920 Mbp and produces a climacteric, fleshy and oily fruit. The flowering and fruit set are particularly prolonged processes, lasting between one to three months, generating important differences in physiological ages of the fruit within the same tree. So far there is no detailed genomic information regarding this species, being the cultivar 'Hass' especially important for avocado growers worldwide. With the aim to explore the fruit avocado transcriptome and to identify candidate biomarkers to monitore fruit development, we carried out an RNA-Seq approach during 4 stages of 'Hass' fruit development: 150 days after fruit set (DAFS), 240 DAFS, 300 DAFS (harvest) and 390 DAFS (late-harvest). RESULTS: The 'Hass' de novo transcriptome contains 62,203 contigs (x̅=988 bp, N50 = 1050 bp). We found approximately an 85 and 99% of complete ultra-conserved genes in eukaryote and plantae database using BUSCO (Benchmarking Universal Single-Copy Orthologs) and CEGMA (Core Eukaryotic Gene Mapping Approach), respectively. Annotation was performed with BLASTx, resulting in a 58% of annotated contigs (90% of differentially expressed genes were annotated). Differentially expressed genes analysis (DEG; with False Discovery Rate ≤ 0.01) found 8672 genes considering all developmental stages. From this analysis, genes were clustered according to their expression pattern and 1209 genes show correlation with the four developmental stages. CONCLUSIONS: Candidate genes are proposed as possible biomarkers for monitoring the development of the 'Hass' avocado fruit associated with lipid metabolism, ethylene signaling pathway, auxin signaling pathway, and components of the cell wall.

A Prunus persica genome-wide RNA-seq approach uncovers major differences in the transcriptome among chilling injury sensitive and non-sensitive varieties.

Chilling injury represents a major constrain for crops productivity. Prunus persica, one of the most relevant rosacea crops, have early season varieties that are resistant to chilling injury, in contrast to late season varieties, which display chilling symptoms such as mealiness (dry, sandy fruit mesocarp) after prolonged storage at chilling temperatures. To uncover the molecular processes related to the ability of early varieties to withstand mealiness, postharvest and genome-wide RNA-seq assessments were performed in two early and two late varieties. Differences in juice content and ethylene biosynthesis were detected among early and late season fruits that became mealy after exposed to prolonged chilling. Principal component and data distribution analysis revealed that cold-stored late variety fruit displayed an exacerbated and unique transcriptome profile when compared to any other postharvest condition. A differential expression analysis performed using an empirical Bayes mixture modeling approach followed by co-expression and functional enrichment analysis uncover processes related to ethylene, lipids, cell wall, carotenoids and DNA metabolism, light response, and plastid homeostasis associated to the susceptibility or resistance of P. persica varieties to chilling stress. Several of the genes related to these processes are in quantitative trait loci (QTL) associated to mealiness in P. persica. Together, these analyses exemplify how P. persica can be used as a model for studying chilling stress in plants.