Competitive Immunoassay in a Microfluidic Biochip for In-Field Detection of Abscisic Acid in Grapes.

Viticulture and associated products are an important part of the economy in many countries. However, biotic and abiotic stresses impact negatively the production of grapes and wine. Climate change is in many aspects increasing both these stresses. Routine sample retrievals and analysis tend to be time-consuming and require expensive equipment and skilled personnel to operate. These challenges could be overcome through the development of a miniaturized analytic device for early detection of grapevine stresses in the field. Abscisic acid is involved in several plant processes, including the onset of fruit ripening and tolerance mechanisms against drought stress. This hormone can be detected through a competitive immunoassay and is found in plants in concentrations up to 10-1 mg/mL. A microfluidic platform is developed in this work which can detect a minimum of 10-11 mg/mL of abscisic acid in buffer. Grape samples were tested using the microfluidic system alongside benchmark techniques such as high-performance liquid chromatography. The microfluidic system could detect the increase to 10-5 mg/mL of abscisic acid present in real berry samples at the veraison stage of ripening.

Network of GRAS transcription factors in plant development, fruit ripening and stress responses.

The plant-specific family of GRAS transcription factors has been wide implicated in the regulation of transcriptional reprogramming associated with a diversity of biological functions ranging from plant development processes to stress responses. Functional analyses of GRAS transcription factors supported by in silico structural and comparative analyses are emerging and clarifying the regulatory networks associated with their biological roles. In this review, a detailed analysis of GRAS proteins' structure and biochemical features as revealed by recent discoveries indicated how these characteristics may impact subcellular location, molecular mechanisms, and function. Nomenclature issues associated with GRAS classification into different subfamilies in diverse plant species even in the presence of robust genomic resources are discussed, in particular how it affects assumptions of biological function. Insights into the mechanisms driving evolution of this gene family and how genetic and epigenetic regulation of GRAS contributes to subfunctionalization are provided. Finally, this review debates challenges and future perspectives on the application of this complex but promising gene family for crop improvement to cope with challenges of environmental transition.

Virulence-related metabolism is activated in Botrytis cinerea mostly in the interaction with tolerant green grapes that remain largely unaffected in contrast with susceptible green grapes.

Botrytis cinerea is responsible for the gray mold disease, severely affecting Vitis vinifera grapevine and hundreds of other economically important crops. However, many mechanisms of this fruit-pathogen interaction remain unknown. The combined analysis of the transcriptome and metabolome of green fruits infected with B. cinerea from susceptible and tolerant genotypes was never performed in any fleshy fruit, mostly because green fruits are widely accepted to be resistant to this fungus. In this work, peppercorn-sized fruits were infected in the field or mock-treated, and berries were collected at green (EL32) stage from a susceptible (Trincadeira) and a tolerant (Syrah) variety. RNAseq and GC-MS data suggested that Syrah exhibited a pre-activated/basal defense relying on specific signaling pathways, hormonal regulation, namely jasmonate and ethylene metabolisms, and linked to phenylpropanoid metabolism. In addition, putative defensive metabolites such as shikimic, ursolic/ oleanolic, and trans-4-hydroxy cinnamic acids, and epigallocatechin were more abundant in Syrah than Trincadeira before infection. On the other hand, Trincadeira underwent relevant metabolic reprogramming upon infection but was unable to contain disease progression. RNA-seq analysis of the fungus in planta revealed an opposite scenario with higher gene expression activity within B. cinerea during infection of the tolerant cultivar and less activity in infected Trincadeira berries. The results suggested an activated virulence state during interaction with the tolerant cultivar without visible disease symptoms. Together, this study brings novel insights related to early infection strategies of B. cinerea and the green berry defense against necrotrophic fungi.

A proteomic analysis shows the stimulation of light reactions and inhibition of the Calvin cycle in the skin chloroplasts of ripe red grape berries.

The role of photosynthesis in fruits still challenges scientists. This is especially true in the case of mature grape berries of red varieties lined by an anthocyanin-enriched exocarp (skin) almost impermeable to gases. Although chlorophylls are degraded and replaced by carotenoids in several fruits, available evidence suggests that they may persist in red grapes at maturity. In the present study, chloroplasts were isolated from the skin of red grape berries (cv. Vinhão) to measure chlorophyll levels and the organelle proteome. The results showed that chloroplasts (and chlorophylls) are maintained in ripe berries masked by anthocyanin accumulation and that the proteome of chloroplasts from green and mature berries is distinct. Several proteins of the light reactions significantly accumulated in chloroplasts at the mature stage including those of light-harvesting complexes of photosystems I (PSI) and II (PSII), redox chain, and ATP synthase, while chloroplasts at the green stage accumulated more proteins involved in the Calvin cycle and the biosynthesis of amino acids, including precursors of secondary metabolism. Taken together, results suggest that although chloroplasts are more involved in biosynthetic reactions in green berries, at the mature stage, they may provide ATP for cell maintenance and metabolism or even O2 to feed the respiratory demand of inner tissues.

The road to molecular identification and detection of fungal grapevine trunk diseases.

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regions.

Volatile Metabolism of Wine Grape Trincadeira: Impact of Infection with Botrytis cinerea.

The aroma of grapes is cultivar dependent and is influenced by terroir, vineyard practices, and abiotic and biotic stresses. Trincadeira is a non-aromatic variety associated with low phenolic content and high sugar and organic acid levels. This cultivar, widely used in Portuguese wines, presents high susceptibility to Botrytis cinerea. This work aimed to characterise the volatile profile of Trincadeira grapes and how it changes under infection with B. cinerea. Thirty-six volatile organic compounds were identified, from different functional groups, namely alcohols, ester acetates, fatty acid esters, fatty acids, aldehydes, and products of the lipoxygenase pathway. Both free and glycosidic volatile organic compounds were analysed by Gas Chromatography and Gas Chromatography coupled to Mass Spectrometry for component quantification and identification, respectively. A multivariance analysis showed a clear discrimination between healthy and infected grapes with 2-trans-hexenal and isoamyl-acetate among the compounds identified as negative and positive markers of infection, respectively. Ester acetates such as 2-phenylethyl acetate, isoamyl acetate, and 2-methylbutyl acetate were present in higher contents in infected samples, whereas the contents of several fatty acid esters, such as ethyl decanoate and ethyl dodecanoate, decreased. These data were integrated with quantitative PCR data regarding genes involved in volatile metabolism and showed up-regulation of a gene coding for Hydroperoxide Lyase 2 in infected grapes. Altogether, these changes in volatile metabolism indicate an impact on the grape quality and may be related to defence against B. cinerea. The presence/absence of specific compounds might be used as infection biomarkers in the assessment of Trincadeira grapes' quality.

Transcriptional, hormonal, and metabolic changes in susceptible grape berries under powdery mildew infection.

Grapevine (Vitis vinifera) berries are extremely sensitive to infection by the biotrophic pathogen Erysiphe necator, causing powdery mildew disease with deleterious effects on grape and wine quality. The combined analysis of the transcriptome and metabolome associated with this common fungal infection has not been previously carried out in any fruit. In order to identify the molecular, hormonal, and metabolic mechanisms associated with infection, healthy and naturally infected V. vinifera cv. Carignan berries were collected at two developmental stages: late green (EL33) and early véraison (EL35). RNA sequencing combined with GC-electron impact ionization time-of-flight MS, GC-electron impact ionization/quadrupole MS, and LC-tandem MS analyses revealed that powdery mildew-susceptible grape berries were able to activate defensive mechanisms with the involvement of salicylic acid and jasmonates and to accumulate defense-associated metabolites (e.g. phenylpropanoids, fatty acids). The defensive strategies also indicated organ-specific responses, namely the activation of fatty acid biosynthesis. However, defense responses were not enough to restrict fungal growth. The fungal metabolic program during infection involves secretion of effectors related to effector-triggered susceptibility, carbohydrate-active enzymes and activation of sugar, fatty acid, and nitrogen uptake, and could be under epigenetic regulation. This study also identified potential metabolic biomarkers such as gallic, eicosanoic, and docosanoic acids and resveratrol, which can be used to monitor early stages of infection.

Plant SWEETs: from sugar transport to plant-pathogen interaction and more unexpected physiological roles.

Sugars Will Eventually be Exported Transporters (SWEETs) have important roles in numerous physiological mechanisms where sugar efflux is critical, including phloem loading, nectar secretion, seed nutrient filling, among other less expected functions. They mediate low affinity and high capacity transport, and in angiosperms this family is composed by 20 paralogs on average. As SWEETs facilitate the efflux of sugars, they are highly susceptible to hijacking by pathogens, making them central players in plant-pathogen interaction. For instance, several species from the Xanthomonas genus are able to upregulate the transcription of SWEET transporters in rice (Oryza sativa), upon the secretion of transcription-activator-like effectors. Other pathogens, such as Botrytis cinerea or Erysiphe necator, are also capable of increasing SWEET expression. However, the opposite behavior has been observed in some cases, as overexpression of the tonoplast AtSWEET2 during Pythium irregulare infection restricted sugar availability to the pathogen, rendering plants more resistant. Therefore, a clear-cut role for SWEET transporters during plant-pathogen interactions has so far been difficult to define, as the metabolic signatures and their regulatory nodes, which decide the susceptibility or resistance responses, remain poorly understood. This fuels the still ongoing scientific question: what roles can SWEETs play during plant-pathogen interaction? Likewise, the roles of SWEET transporters in response to abiotic stresses are little understood. Here, in addition to their relevance in biotic stress, we also provide a small glimpse of SWEETs importance during plant abiotic stress, and briefly debate their importance in the particular case of grapevine (Vitis vinifera) due to its socioeconomic impact.

Microfluidic device for multiplexed detection of fungal infection biomarkers in grape cultivars.

Early diagnosis of fungal infections, which have seen an increase due to different environmental factors, is essential to an appropriate treatment of the plant by avoiding proliferation of the pathogen without excessive fungicide applications. In this work, we propose a microfluidic based approach to a multiplexed, point-of-need detection system capable of identifying infected grape cultivars. The system relies on the simultaneous detection of three plant hormones: salicylic, azelaic and jasmonic acids with a total assay time under 7 minutes, with LODs of 15 µM, 10 µM and 4.4 nM respectively. The three detection assays are based on optical transduction, with the detection of salicylic and azelaic acids using transmission measurements, while the detection of jasmonic acid is a fluorescence-based assay. The molecular recognition event for each metabolite is different: nanoparticle conjugation for salicylic acid, enzymatic reaction for azelaic acid and antibody-antigen recognition for jasmonic acid. In this work, two cultivars, Trincadeira and Carignan, presented infections with two fungal pathogens, Botrytis cinerea and Erysiphe necator. The grapes were tested using the microfluidic system alongside the benchmark techniques such as, high-performance liquid chromatography and enzyme-linked immunosorbent assay. The microfluidic system was not only capable of distinguishing infected from healthy samples, but also capable of distinguishing between different infection types.

VvERD6l13 is a grapevine sucrose transporter highly up-regulated in response to infection by Botrytis cinerea and Erysiphe necator.

The Early-Response to Dehydration six-like (ERD6l) is one of the largest families of sugar transporters in plants, however, is also one of the less studied with very few members characterized. In this work, we identified 18 members of the grapevine ERD6l family, analyzed their promoters and putative topology and additionally functionally characterized the member VvERD6l13. VvERD6l13 was strongly up-regulated in grape berries infected with Botrytis cinerea and Erysiphe necator in cv. Trincadeira and Carignan, respectively, suggesting an important role in grape berry-pathogen interaction, as we had hypothesized. In Cabernet Sauvignon Berry suspension cultured cells, VvERD6l13 was also up-regulated, by 4-fold, 48 h after elicitation with mycelium extract of B. cinerea. Besides being expressed in grape berries from various developmental stages, VvERD6l13 is also expressed in leaves, canes, flowers and, noticeably, in roots. Using tobacco and an hxt-null Saccharomyces cerevisiae strain as heterologous expression models, we showed that VvERD6l13 is localized at the plasma membrane and mediates the H+-dependent transport of sucrose (Km = 33 mM) thus confirming VvERD6l13 as a bona fide sugar transporter involved in sugar mobilization in grapevine and transcriptionally induced in response to biotic stress.

Evolutionary insights into FYVE and PHOX effector proteins from the moss Physcomitrella patens.

MAIN CONCLUSION: Genome-wide identification, together with gene expression patterns and promoter region analysis of FYVE and PHOX proteins in Physcomitrella patens, emphasized their importance in regulating mainly developmental processes in P. patens. Phosphatidylinositol 3-phosphate (PtdIns3P) is a signaling phospholipid, which regulates several aspects of plant growth and development, as well as responses to biotic and abiotic stresses. The mechanistic insights underlying PtdIns3P mode of action, specifically through effector proteins have been partially explored in plants, with main focus on Arabidopsis thaliana. In this study, we searched for genes coding for PtdIns3P-binding proteins such as FYVE and PHOX domain-containing sequences from different photosynthetic organisms to gather evolutionary insights on these phosphoinositide binding domains, followed by an in silico characterization of the FYVE and PHOX gene families in the moss Physcomitrella patens. Phylogenetic analysis showed that PpFYVE proteins can be grouped in 7 subclasses, with an additional subclass whose FYVE domain was lost during evolution to higher plants. On the other hand, PpPHOX proteins are classified into 5 subclasses. Expression analyses based on RNAseq data together with the analysis of cis-acting regulatory elements and transcription factor (TF) binding sites in promoter regions suggest the importance of these proteins in regulating stress responses but mainly developmental processes in P. patens. The results provide valuable information and robust candidate genes for future functional analysis aiming to further explore the role of this signaling pathway mainly during growth and development of tip growing cells and during the transition from 2 to 3D growth. These studies would identify ancestral regulatory players undertaken during plant evolution.

Microfluidic device for the point of need detection of a pathogen infection biomarker in grapes.

Bacterial, fungal and viral infections in plant systems are on the rise, most of which tend to spread quickly amongst crops. These pathogens are also gaining resistance to known treatments, which makes their early detection a priority to avoid extensive loss of crops and the spreading of disease to animal systems. In this work, we propose a microfluidic platform coupled with integrated thin-film silicon photosensors for the detection of pathogen infections in grapes. This detection was achieved by monitoring the concentration of Azelaic Acid (AzA). This small organic acid plays a significant role in the defense mechanism in plant systems. In this platform, the enzyme tyrosinase was immobilized on microbeads inside a microfluidic system. By colorimetric monitoring of the inhibitory effect of AzA on the enzyme tyrosinase in real time, it was possible, in under 10 minutes, to detect different concentrations of AzA in both buffer and spiked solutions of grape juice, in both cases with limits of detection in the 5-10 nM range. In addition, with this microfluidic device, it was possible to clearly distinguish infected from healthy grape samples at three different grape maturation points. Healthy grape samples showed AzA concentrations in the range of 10-20 nM (post-dilution) while infected samples have an estimated increase of AzA of 10-30×, results which were confirmed using HPLC. In both juice and grape samples an integrated sample preparation stage that decreases the phenol content of the solutions was required to achieve fit-for-purpose sensitivities to AzA.

Transcriptional Modulation of Polyamine Metabolism in Fruit Species Under Abiotic and Biotic Stress.

Polyamines are growth regulators that have been widely implicated in abiotic and biotic stresses. They are also associated with fruit set, ripening, and regulation of fruit quality-related traits. Modulation of their content confers fruit resilience, with polyamine application generally inhibiting postharvest decay. Changes in the content of free and conjugated polyamines in response to stress are highly dependent on the type of abiotic stress applied or the lifestyle of the pathogen. Recent studies suggest that exogenous application of polyamines or modulation of polyamine content by gene editing can confer tolerance to multiple abiotic and biotic stresses simultaneously. In this review, we explore data on polyamine synthesis and catabolism in fruit related to pre- and postharvest stresses. Studies of mutant plants, priming of stress responses, and treatments with polyamines and polyamine inhibitors indicate that these growth regulators can be manipulated to increase fruit productivity with reduced use of pesticides and therefore, under more sustainable conditions.

The study of hormonal metabolism of Trincadeira and Syrah cultivars indicates new roles of salicylic acid, jasmonates, ABA and IAA during grape ripening and upon infection with Botrytis cinerea.

Hormones play an important role in fruit ripening and in response to biotic stress. Nevertheless, analyses of hormonal profiling during plant development and defense are scarce. In this work, changes in hormonal metabolism in grapevine (Vitis vinifera) were compared between a susceptible (Trincadeira) and a tolerant (Syrah) variety during grape ripening and upon infection with Botrytis cinerea. Infection of grapes with the necrotrophic pathogen Botrytis cinerea leads to significant economic losses worldwide. Peppercorn-sized fruits were infected in the field and mock-treated and infected berries were collected at green, veraison and harvest stages for hormone analysis and targeted qPCR analysis of genes involved in hormonal metabolism and signaling. Results indicate a substantial reprogramming of hormonal metabolism during grape ripening and in response to fungal attack. Syrah and Trincadeira presented differences in the metabolism of abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonates during grape ripening that may be connected to fruit quality. On the other hand, high basal levels of salicylic acid (SA), jasmonates and IAA at an early stage of ripening, together with activated SA, jasmonates and IAA signaling, likely enable a fast defense response leading to grape resistance/ tolerance towards B. cinerea. The balance among the different phytohormones seems to depend on the ripening stage and on the intra-specific genetic background and may be fundamental in providing resistance or susceptibility. In addition, this study indicated the involvement of SA and IAA in defense against necrotrophic pathogens and gains insights into possible strategies for conventional breeding and/or gene editing aiming at improving grape quality and grape resistance against Botrytis cinerea.

VvSWEET7 Is a Mono- and Disaccharide Transporter Up-Regulated in Response to Botrytis cinerea Infection in Grape Berries.

The newly-identified SWEETs are high-capacity, low-affinity sugar transporters with important roles in numerous physiological mechanisms where sugar efflux is critical. SWEETs are desirable targets for manipulation by pathogens and their expression may be transcriptionally reprogrammed during infection. So far, few plant SWEET transporters have been functionally characterized, especially in grapevine. In this study, in the Botrytis-susceptible variety "Trincadeira," we thoroughly analyzed modifications in the gene expression profile of key SWEET genes in Botrytis cinerea-infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and Botrytis infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, B. cinerea infection down-regulated VvSWEET17a expression at the green stage, VvSWEET10 and VvSWEET17d expression at the veraison stage, and VvSWEET11 expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in Saccharomyces cerevisiae as a low-affinity, high-capacity glucose and sucrose transporter with a K m of 15.42 mM for glucose and a K m of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana epidermal cells and confocal microscopy allowed to observe that both proteins clearly localize to the plasma membrane. In sum, VvSWEETs transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to Botrytis infection.

Characterization of the cork formation and production transcriptome in Quercus cerris × suber hybrids.

Cork oak is the main cork-producing species worldwide, and plays a significant economic, ecological and social role in the Mediterranean countries, in particular in Portugal and Spain. The ability to produce cork is limited to a few species, hence it must involve specific regulation mechanisms that are unique to these species. However, to date, these mechanisms remain largely understudied, especially with approaches involving the use of high-throughput sequencing technology. In this study, the transcriptome of cork-producing and non-cork-producing Quercus cerris × suber hybrids was analyzed in order to elucidate the differences between the two groups of trees displaying contrasting phenotypes for cork production. The results revealed the presence of a significant number of genes exclusively associated with cork production, in the trees that developed cork. Moreover, several gene ontology subcategories, such as cell wall biogenesis, lipid metabolic processes, metal ion binding and apoplast/cell wall, were only detected in the trees with cork production. These results indicate the existence, at the transcriptome level, of mechanisms that seem to be unique and necessary for cork production, which is an advancement in our knowledge regarding the genetic regulation behind cork formation and production.

Transcriptional profiling of cork oak phellogenic cells isolated by laser microdissection.

MAIN CONCLUSION: The phenylpropanoid pathway impacts the cork quality development. In cork of bad quality, the flavonoid route is favored, whereas in good quality, cork lignin and suberin production prevails. Cork oaks develop a thick cork tissue as a protective shield that results of the continuous activity of a secondary meristem, the cork cambium, or phellogen. Most studies applied to developmental processes do not consider the cell types from which the samples were extracted. Here, laser microdissection (LM) coupled with transcript profiling using RNA sequencing (454 pyrosequencing) was applied to phellogen cells of trees producing low- and good quality cork. Functional annotation and functional enrichment analyses showed that stress-related genes are enriched in samples extracted from trees producing good quality cork (GQC). This process is under tight transcriptional (transcription factors, kinases) regulation and also hormonal control involving ABA, ethylene, and auxins. The phellogen cells collected from trees producing bad quality cork (BQC) show a consistent up-regulation of genes belonging to the flavonoid pathway as a response to stress. They also display a different modulation of cell wall genes resulting into a thinner cork layer, i.e., less meristematic activity. Based on the analysis of the phenylpropanoid pathway regulating genes, in GQC, the synthesis of lignin and suberin is promoted, whereas in BQC, the same pathway favors the biosynthesis of free phenolic compounds. This study provided new insights of how cell-specific gene expression can determine tissue and organ morphology and physiology and identified robust candidate genes that can be used in breeding programs aiming at improving cork quality.