Metabolite profiling reveals the influence of grapevine genetic distance on the chemical signature of juices.

BACKGROUND: Yield, disease tolerance, and climate adaptation are important traits in grapevine genetic breeding programs. Selection for these characteristics causes unpredictable changes in primary and specialized metabolism, affecting the physicochemical properties and chemical composition of the berries and their processed products, juice, and wine. In this study, we investigated the influence of the genetic distance between grapevine genotypes on the chemical signatures of the juices, by integrating comprehensive metabolic profiling to genetic analyses. RESULTS: The studied grapevine cultivars exhibited low genetic diversity. Breeding for agronomic traits promoted higher contents of soluble sugars, total phenolics, and anthocyanins in the juices. Untargeted juice metabolomics identified a total of 147 metabolites, consisting of 30 volatiles, 21 phenolics, and 96 ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) features. Juices from grapes of the most recent cultivars exhibited increased levels of trans-resveratrol, catechin, and luteolin. The blend of volatiles from juices of later cultivars was also more complex, consisting of 29 distinct metabolites in 'BRS Magna'. Grapes from 'BRS Carmem', an intermediate cultivar, gave the most divergent UHPLC-MS juice profile. CONCLUSION: Contents of soluble solids, total phenolics, and anthocyanins in grape juices were increased by controlled crosses and hybrid selection. Integrative analyses demonstrated that the juices' metabolic profiles accurately represent the cultivars' genetic distances. Juices from 'BRS Violeta' and 'BRS Magna' show relevant positive association with health-related phenolics and a distinct set of odor volatiles, although these characteristics were specifically sought by breeding. © 2023 Society of Chemical Industry.

Physicochemical properties and transcriptional changes underlying the quality of 'Gala' apples (Malus × domestica Borkh.) under atmosphere manipulation in long-term storage.

BACKGROUND: The year-round availability of apples (Malus × domestica Borkh.) depends on post-harvest technologies, which are essential for the retention of fruit sensory and chemical properties by delaying senescence. The effectiveness of strategies for preserving the quality of apples depends on complex interactions between the storage environment and endogenous biological factors. In the current work, we integrated instrumental, sensory, and transcriptional data to determine the role of conservation technologies cold storage, controlled atmosphere, and 1-methylcyclopropene-mediated ethylene blockage on the long-term conservation of apples. RESULTS: The results demonstrated that inhibition of the consumer's perception of the apples' ethylene content is essential for long-term cold storage, and such quality conservation can be achieved by reducing oxygen pressure. Overall appreciation of apples after storage was determined mainly by their texture, with crispness and juiciness contributing favorably, and mealiness contributing negatively. Reduced oxygen pressure and inhibition of ethylene perception exerted distinct effects on the transcription of candidate genes associated with ripening in apple. Hexose and cell-wall carbohydrate metabolism genes exhibit distinct expression patterns under storage. CONCLUSION: Inhibition of ethylene perception and reduction of relative oxygen pressure under cold storage both promote similar conservation of apple sensory traits under long-term cold storage. Texture was the main contributor to global appreciation of apples subjected to long-term storage. The conditions that were investigated were able to delay, but not fully prevent, senescence, as evidenced by physicochemical and gene expression analyses. The expression of gene-encoding enzymes involved in hexose metabolism was mainly developmentally regulated, whereas storage conditions exerted a stronger effect on the expression of genes associated with cell-wall metabolism. © 2022 Society of Chemical Industry.

Comparative transcriptome analyses between cultivated and wild grapes reveal conservation of expressed genes but extensive rewiring of co-expression networks.

KEY MESSAGE: The transcriptomes of wild and cultivated grapes consists of similar expressed genes but distinct wiring of co-expressed modules associated with environmental conditions. Grapevine is an important fruit crop worldwide, with high economic value and widespread distribution. Commercial production is based on Vitis vinifera, and, to a lesser extent, on hybrids with American grapes, such as V. labrusca. Wild grape relatives are important sources of resistance against biotic and abiotic factors; however, their global gene expression patterns remain poorly characterized. We associated genome-wide transcript profiling to phenotypic analyses to investigate the responses of cultivated and wild vines to vineyard conditions. The expressed genes in the Vitis reference transcriptome are largely shared by wild grapes, V. labrusca hybrids and vinifera cultivars. In contrast, significant differential regulation between wild and vinifera genotypes represents 80% of gene expression variation, regardless of the environment. In wild grapes, genes associated to regulatory processes are downregulated, whereas those involved in metabolic pathways are upregulated, in comparison to vinifera. Photosynthesis-related ontologies are overrepresented in the induced genes, in agreement with higher contents of chlorophyll in wild grapes. Co-regulated gene network analyses provide evidence of more complex transcriptome organization in vinifera. In wild grapes, genes involved in signaling pathways of stress-related hormones are overrepresented in modules associated with the environment. Consensus network analyses revealed high preservation within co-regulated gene modules between cultivated and wild grapes, but divergent relationships among the expression clusters. In conclusion, the distinct phenotypes of wild and cultivated grapes are underlain by differences in gene expression, but also by distinct higher-order organization of the transcriptome and contrasting association of co-expressed gene clusters with the environment.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage.

BACKGROUND: Postharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as 'woolliness' that is caused by juice loss leading to a 'wooly' fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches. RESULTS: Approximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach. CONCLUSION: Overall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries.

A reverse genetics approach identifies novel mutants in light responses and anthocyanin metabolism in petunia.

Flower color and plant architecture are important commercially valuable features for ornamental petunias (Petunia x hybrida Vilm.). Photoperception and light signaling are the major environmental factors controlling anthocyanin and chlorophyll biosynthesis and shade-avoidance responses in higher plants. The genetic regulators of these processes were investigated in petunia by in silico analyses and the sequence information was used to devise a reverse genetics approach to probe mutant populations. Petunia orthologs of photoreceptor, light-signaling components and anthocyanin metabolism genes were identified and investigated for functional conservation by phylogenetic and protein motif analyses. The expression profiles of photoreceptor gene families and of transcription factors regulating anthocyanin biosynthesis were obtained by bioinformatic tools. Two mutant populations, generated by an alkalyting agent and by gamma irradiation, were screened using a phenotype-independent, sequence-based method by high-throughput PCR-based assay. The strategy allowed the identification of novel mutant alleles for anthocyanin biosynthesis (CHALCONE SYNTHASE) and regulation (PH4), and for light signaling (CONSTANS) genes.

Essential Oils of Aromatic Plant Species from the Atlantic Rainforest Exhibit Extensive Chemical Diversity and Antimicrobial Activity.

Microbial resistance, caused by the overuse or inadequate application of antibiotics, is a worldwide crisis, increasing the risk of treatment failure and healthcare costs. Plant essential oils (EOs) consist of hydrophobic metabolites with antimicrobial activity. The antimicrobial potential of the chemical diversity of plants from the Atlantic Rainforest remains scarcely characterized. In the current work, we determined the metabolite profile of the EOs from aromatic plants from nine locations and accessed their antimicrobial and biocidal activity by agar diffusion assays, minimum inhibitory concentration, time-kill and cell-component leakage assays. The pharmacokinetic properties of the EO compounds were investigated by in silico tools. More than a hundred metabolites were identified, mainly consisting of sesqui and monoterpenes. Individual plants and botanical families exhibited extensive chemical variations in their EO composition. Probabilistic models demonstrated that qualitative and quantitative differences contribute to chemical diversity, depending on the botanical family. The EOs exhibited antimicrobial biocidal activity against pathogenic bacteria, fungi and multiple predicted pharmacological targets. Our results demonstrate the antimicrobial potential of EOs from rainforest plants, indicate novel macromolecular targets, and contribute to highlighting the chemical diversity of native species.

Identification of a novel α-L-arabinofuranosidase gene associated with mealiness in apple.

In order to investigate the genetic bases of the physiological syndrome mealiness that causes abnormal fruit softening and juice loss in apples, an integrative approach was devised, consisting of sensory, instrumental, biochemical, genetic, and genomic methods. High levels of activity of α-L-arabinofuranosidase (α-AFase), a hydrolase acting on the pectic component of the cell walls, were found in individuals exhibiting the mealiness phenotype in a segregating population. The expression levels of the previously uncharacterized apple AF gene MdAF3 are higher in fruits from plants consistently showing mealiness symptons and high α-AFase activity. The transcription of MdAF3 is differentially regulated in distinct genomic contexts and appears to be independent of ethylene. Thus, it is likely to be controlled by endogenous developmental mechanisms associated with fruit ripening. The use of integrative approaches has allowed the identification of a novel contributor to the mealiness phenotype in apple and it has been possible to overcome the problems posed by the unavailability of near-isogenic lines to dissect the genetic bases of a complex physiological trait in woody perennial species.

Contrasting Transcriptional Programs Control Postharvest Development of Apples (Malus x domestica Borkh.) Submitted to Cold Storage and Ethylene Blockage.

Apple is commercially important worldwide. Favorable genomic contexts and postharvest technologies allow year-round availability. Although ripening is considered a unidirectional developmental process toward senescence, storage at low temperatures, alone or in combination with ethylene blockage, is effective in preserving apple properties. Quality traits and genome wide expression were integrated to investigate the mechanisms underlying postharvest changes. Development and conservation techniques were responsible for transcriptional reprogramming and distinct programs associated with quality traits. A large portion of the differentially regulated genes constitutes a program involved in ripening and senescence, whereas a smaller module consists of genes associated with reestablishment and maintenance of juvenile traits after harvest. Ethylene inhibition was associated with a reversal of ripening by transcriptional induction of anabolic pathways. Our results demonstrate that the blockage of ethylene perception and signaling leads to upregulation of genes in anabolic pathways. We also associated complex phenotypes to subsets of differentially regulated genes.

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Ethylene-dependent regulation of an α-L-arabinofuranosidase is associated to firmness loss in 'Gala' apples under long term cold storage.

Fruit texture changes impair the quality of apples submitted to long term storage, especially under cold. The changes are due to cell wall modifications during ripening and senescence and are associated to ethylene. We have investigated the activity of α-l-arabinofuranosidase, a glycosyl hydrolase acting on the side chains of pectin in the cell wall and middle lamella. The transcription of arabinofuranosidase coding sequences 1 and 3 was investigated in plant organs and in response to ethylene, employing hormone application and 1-methylcyclopropene. The transcription of arabinofuranosidase genes is not restricted to fruits, although upregulated by ripening and ethylene. Transcripts of the genes were detected under cold storage up to 180 days. Similarly, arabinofuranosidase activity increased with rising levels of ethylene and under cold storage. Levels of arabinofuranosidase3 transcripts were higher than those of arabinofuranosidase1, suggesting that the first is an important contributor to enzyme activity and texture changes during cold storage.

Transcriptional profile of genes involved in the biosynthesis of phytate and ferritin in Coffea.

The present work aimed to study the control of the biosynthesis of the antinutritional factor phytate and its associated Fe-rich protein family, ferritin, in coffee. Phytate has the ability to chelate Fe, making it unavailable to human absorption. The Coffea genome databases were queried for genes associated with phytate metabolism and ferritin genes. The genetic framework for phytate biosynthesis and its reverse pathway was identified in silico analyses and indicate that Coffea phosphatidyl inositol kinase and monophosphatase families play nonredundant roles in phytate metabolism. The transcriptional profiles of phytate biosynthesis key-genes MYO-INOSITOL(3)P1 SYNTHASE, two genes coding for PHOSPHATIDYL INOSITOL KINASE, and three FERRITIN genes were temporally evaluated by qPCR in coffee seeds from two crop locations, Adamantina-SP and Ouro-Fino-MG, the last one traditionally associated with high-quality coffee beverage grain. A targeted metabolome profile of phytic acid contents throughout three fruit maturation stages in association with the transcriptional analysis was also obtained. Taken together, our data indicate that the investigated local conditions did not cause significant alterations in phytate biosynthesis. Futhermore, the temporal transcriptional profiling revealed that candidate gene expression is regulated independently of phytate accumulation. In contrast, the expression profile of ferritin-unit genes is affected by environmental conditions and genetic background. The roles of the investigated genes are discussed concerning the quality of coffee beverage.

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system.

BACKGROUND: The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps. RESULTS: Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 µM 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 µM 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization. CONCLUSIONS: The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Identification of photoperception and light signal transduction pathways in citrus

Studies employing model species have elucidated several aspects of photoperception and light signal transduction that control plant development. However, the information available for economically important crops is scarce. Citrus genome databases of expressed sequence tags (EST) were investigated in order to identify genes coding for functionally characterized proteins responsible for light-regulated developmental control in model plants. Approximately 176,200 EST sequences from 53 libraries were queried and all bona fide and putative photoreceptor gene families were found in citrus species. We have identified 53 orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses although some important Arabidopsis phytochrome- and cryptochrome-signaling components are absent from citrus sequence databases. The main gene families responsible for phototropin-mediated signal transduction were present in citrus transcriptome, including general regulatory factors (14-3-3 proteins), scaffolding elements and auxin-responsive transcription factors and transporters. A working model of light perception, signal transduction and response-eliciting in citrus is proposed based on the identified key components. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

In silico analysis of the endogenous time-keeping mechanism in citrus

The endogenous time-keeping mechanism is responsible for organizing plant physiology and metabolism according to periodic environmental changes, such as diurnal cycles of light and dark and seasonal progression throughout the year. In plants, circadian rhythms control gene expression, stomatal opening, and the timing component of the photoperiodic responses, leading to enhanced fitness due to increased photosynthetic rates and biomass production. We have investigated the citrus genome databases of expressed sequence tags (EST) in order to identify genes coding for functionally characterized proteins involved in the endogenous time-keeping mechanism in Arabidopsis thaliana. Approximately 180,000 EST sequences from 53 libraries were investigated and 81 orthologs of clock components were identified. We found that the vast majority of Arabidopsis circadian clock genes are present in citrus species, although some important components are absent such as SRR1 and PRR5. Based on the identified transcripts, a model for the endogenous oscillatory mechanism of citrus is proposed. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

Identifying water stress-response mechanisms in citrus by in silico transcriptome analysis

Water deficit is one of the most critical environmental stresses to which plants are submitted during their life cycle. The evolutionary and economic performance of the plant is affected directly by reducing its survival in the natural environment and its productivity in agriculture. Plants respond to water stress with biochemical and physiological modifications that may be involved in tolerance or adaptation mechanisms. A great number of genes have been identified as transcriptionally regulated for water deficit. EST sequencing projects provide a significant contribution to the discovery of expressed genes. The identification and determination of gene expression patterns is important not only to understand the molecular bases of plant responses but also to improve water stress tolerance. In our citrus transcriptome survey we have attempted to identify homologs to genes known to be induced and regulated under water stress conditions. We have identified 89 transcripts whose deduced amino acid sequences share similarities with proteins involved in uptake and transport of water and ion, 34 similar to components of the osmolyte metabolism, 67 involved in processes of membranes and proteins protection and 115 homologs of reactive oxygen species scavenger. Many drought-inducible genes identified are known to be regulated by development, salt, osmotic and low temperature. Their possible roles in specific or general mechanisms of water stress citrus responses are discussed.

In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.