Molecular regulation of apple and grape ripening: exploring common and distinct transcriptional aspects of representative climacteric and non-climacteric fruits.

Fleshy fruits of angiosperms are organs specialized for promoting seed dispersal by attracting herbivores and enticing them to consume the organ and the seeds it contains. Ripening can be broadly defined as the processes serving as a plant strategy to make the fleshy fruit appealing to animals, consisting of a coordinated series of changes in color, texture, aroma, and flavor that result from an intricate interplay of genetically and epigenetically programmed events. The ripening of fruits can be categorized into two types: climacteric, which is characterized by a rapid increase in respiration rate typically accompanied by a burst of ethylene production, and non-climacteric, in which this pronounced peak in respiration is absent. Here we review current knowledge of transcriptomic changes taking place in apple (Malus × domestica, climacteric) and grapevine (Vitis vinifera, non-climacteric) fruit during ripening, with the aim of highlighting specific and common hormonal and molecular events governing the process in the two species. With this perspective, we found that specific NAC transcription factor members participate in ripening initiation in grape and are involved in restoring normal physiological ripening progression in impaired fruit ripening in apple. These elements suggest the existence of a common regulatory mechanism operated by NAC transcription factors and auxin in the two species.

Comparative transcriptome and metabolite survey reveal key pathways involved in the control of the chilling injury disorder superficial scald in two apple cultivars, 'Granny Smith' and 'Ladina'.

The low temperature normally applied to prevent fruit decay during the storage of apples, can also triggers the onset of a chilling injury disorder known as superficial scald. In this work, the etiology of this disorder and the mechanism of action of two preventing strategies, such as the application of 1-MCP (1-methylcyclopropene) and storage at low oxygen concentration in 'Granny Smith' and 'Ladina' apple cultivars were investigated. The metabolite assessment highlighted a reorganization of specific metabolites, in particular flavan-3-ols and unsaturated fatty acids, while the genome-wide transcriptomic analysis grouped the DEGs into four functional clusters. The KEGG pathway and GO enrichment analysis, together with the gene-metabolite interactome, showed that the treatment with 1-MCP prevented the development of superficial scald by actively promoting the production of unsaturated fatty acids, especially in 'Granny Smith'. 'Ladina', more susceptible to superficial scald and less responsive to the preventing strategies, was instead characterized by a higher accumulation of very long chain fatty acids. Storage at low oxygen concentration stimulated a higher accumulation of ethanol and acetaldehyde together with the expression of genes involved in anaerobic respiration, such as malate, alcohol dehydrogenase and pyruvate decarboxylase in both cultivars. Low oxygen concentration, likewise 1-MCP, through a direct control on ethylene prevented the onset of superficial scald repressing the expression of PPO, a gene encoding for the polyphenol oxidase enzyme responsible of the oxidation of chlorogenic acid. Moreover, in 'Granny Smith' apple, the expression of three members of the VII subgroups of ERF genes, encoding for elements coordinating the acclimation process to hypoxia in plants was observed. The global RNA-Seq pattern also elucidated a specific transcriptomic signature between the two cultivars, disclosing the effect of the different genetic background in the control of this disorder.

Transcriptomic Insights on the Preventive Action of Apple (cv Granny Smith) Skin Wounding on Superficial Scald Development.

Superficial scald is a post-harvest chilling storage injury leading to browning of the surface of the susceptible cv Granny Smith apples. Wounding of skins has been reported to play a preventive role on scald development however its underlying molecular factors are unknown. We have artificially wounded the epidermal and sub-epidermal layers of apple skins consistently obtaining the prevention of superficial scald in the surroundings of the wounds during two independent vintages. Time course RNA-Seq analyses of the transcriptional changes in wounded versus unwounded skins revealed that two transcriptional waves occurred. An early wave included genes up-regulated by wounding already after 6 h, highlighting a specific transcriptional rearrangement of genes connected to the biosynthesis and signalling of JA, ethylene and ABA. A later transcriptional wave, occurring after three months of cold storage, included genes up-regulated exclusively in unwounded skins and was prevented from its occurrence in wounded skins. A significant portion of these genes was related to decay of tissues and to the senescence hormones ABA, JA and ethylene. Such changes suggest a wound-inducible reversed hormonal balance during post-harvest storage which may explain the local inhibition of scald in wounded tissues, an aspect that will need further studies for its mechanistic explanation.

Dissection of the incidence and severity of purple spot physiological disorder in loquat fruit through a physiological and molecular approach.

Loquat (Eriobotrya japonica) fruit marketability is affected by the incidence and severity of purple spot (PS), a pre-harvest physiological disorder showing an evident skin discoloration with depressed surface. Despite its impact in limiting the cultivation and economic potential of loquat, the etiology of this disorder is still poorly understood. To this end, our study aimed to investigate and disclose possible mechanisms underlying PS development. The intensity and severity of PS in three loquat cultivars ('Morphitiki', 'Karantoki' and 'Obusa') was phenotypically monitored during successive on-tree fruit developmental stages. 'Obusa' fruits harvested at commercial maturity stage showed the highest incidence of purple spot (58.6%), while 'Morphitiki' fruits did not show any symptoms. 'Karantoki' fruits demonstrated an intermediate response, with 31.3% of the fruit being affected. Thereafter, fruits with 30-50% PS severity were selected and used for further analysis; peel tissue was removed from both symptomatic and asymptomatic tissue of the same fruit for all examined cultivars. 'Karantoki' fruit with PS were characterized by the highest accumulation of total soluble sugars, sucrose, glucose and fructose contents, while the concentration of these primary metabolites was the lowest in asymptomatic fruit of 'Obusa', exception made for the sucrose. The incidence of PS was also transcriptionally investigated by assessing the mRNA profile of important genes involved in polyphenolic (PAL1, PAL2 and PPO1) and carbohydrate (CWI2, CWI3, SPS1, SPS2, NI2, NI3, SuSy, HXK, FRK and VI) pathway. The enhanced expression levels of CWI3 and VI genes in symptomatic fruit of the highly susceptible cultivar 'Obusa' highlight a cultivar-specific type of response. Notably, SuSy registered significantly suppressed levels in symptomatic tissue of both 'Obusa' and 'Karantoki'. To what extent PPO is associated with PS incidence and whether the etiology of the disorder can be assigned to an oxidative process triggered and coordinated by its action need to be further elucidated. The aforementioned genes are suggested to be further examined as potential markers towards a more sophisticated and informed characterization of purple spot detection in loquat fruit.

Genomic prediction of fruit texture and training population optimization towards the application of genomic selection in apple.

Texture is a complex trait and a major component of fruit quality in apple. While the major effect of MdPG1, a gene controlling firmness, has already been exploited in elite cultivars, the genetic basis of crispness remains poorly understood. To further improve fruit texture, harnessing loci with minor effects via genomic selection is therefore necessary. In this study, we measured acoustic and mechanical features in 537 genotypes to dissect the firmness and crispness components of fruit texture. Predictions of across-year phenotypic values for these components were calculated using a model calibrated with 8,294 SNP markers. The best prediction accuracies following cross-validations within the training set of 259 genotypes were obtained for the acoustic linear distance (0.64). Predictions for biparental families using the entire training set varied from low to high accuracy, depending on the family considered. While adding siblings or half-siblings into the training set did not clearly improve predictions, we performed an optimization of the training set size and composition for each validation set. This allowed us to increase prediction accuracies by 0.17 on average, with a maximal accuracy of 0.81 when predicting firmness in the 'Gala' × 'Pink Lady' family. Our results therefore identified key genetic parameters to consider when deploying genomic selection for texture in apple. In particular, we advise to rely on a large training population, with high phenotypic variability from which a 'tailored training population' can be extracted using a priori information on genetic relatedness, in order to predict a specific target population.

Investigation of the transcriptomic and metabolic changes associated with superficial scald physiology impaired by lovastatin and 1-methylcyclopropene in pear fruit (cv. "Blanquilla").

To elucidate the physiology underlying the development of superficial scald in pears, susceptible "Blanquilla" fruit was treated with different compounds that either promoted (ethylene) or repressed (1-methylcyclopropene and lovastatin) the incidence of this disorder after 4 months of cold storage. Our data show that scald was negligible for the fruit treated with 1-methylcyclopropene or lovastatin, but highly manifested in untreated (78% incidence) or ethylene-treated fruit (97% incidence). The comparison between the fruit metabolomic profile and transcriptome evidenced a distinct reprogramming associated with each treatment. In all treated samples, cold storage led to an activation of a cold-acclimation-resistance mechanism, including the biosynthesis of very-long-chain fatty acids, which was especially evident in 1-methylcyclopropane-treated fruit. Among the treatments applied, only 1-methylcyclopropene inhibited ethylene production, hence supporting the involvement of this hormone in the development of scald. However, a common repression effect on the PPO gene combined with higher sorbitol content was found for both lovastatin and 1-methylcyclopropene-treated samples, suggesting also a non-ethylene-mediated process preventing the development of this disorder. The results presented in this work represent a step forward to better understand the physiological mechanisms governing the etiology of superficial scald in pears.

Heterogeneous networks integration for disease-gene prioritization with node kernels.

MOTIVATION: The identification of disease-gene associations is a task of fundamental importance in human health research. A typical approach consists in first encoding large gene/protein relational datasets as networks due to the natural and intuitive property of graphs for representing objects' relationships and then utilizing graph-based techniques to prioritize genes for successive low-throughput validation assays. Since different types of interactions between genes yield distinct gene networks, there is the need to integrate different heterogeneous sources to improve the reliability of prioritization systems. RESULTS: We propose an approach based on three phases: first, we merge all sources in a single network, then we partition the integrated network according to edge density introducing a notion of edge type to distinguish the parts and finally, we employ a novel node kernel suitable for graphs with typed edges. We show how the node kernel can generate a large number of discriminative features that can be efficiently processed by linear regularized machine learning classifiers. We report state-of-the-art results on 12 disease-gene associations and on a time-stamped benchmark containing 42 newly discovered associations. AVAILABILITY AND IMPLEMENTATION: Source code: https://github.com/dinhinfotech/DiGI.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GraphClust2: Annotation and discovery of structured RNAs with scalable and accessible integrative clustering.

BACKGROUND: RNA plays essential roles in all known forms of life. Clustering RNA sequences with common sequence and structure is an essential step towards studying RNA function. With the advent of high-throughput sequencing techniques, experimental and genomic data are expanding to complement the predictive methods. However, the existing methods do not effectively utilize and cope with the immense amount of data becoming available. RESULTS: Hundreds of thousands of non-coding RNAs have been detected; however, their annotation is lagging behind. Here we present GraphClust2, a comprehensive approach for scalable clustering of RNAs based on sequence and structural similarities. GraphClust2 bridges the gap between high-throughput sequencing and structural RNA analysis and provides an integrative solution by incorporating diverse experimental and genomic data in an accessible manner via the Galaxy framework. GraphClust2 can efficiently cluster and annotate large datasets of RNAs and supports structure-probing data. We demonstrate that the annotation performance of clustering functional RNAs can be considerably improved. Furthermore, an off-the-shelf procedure is introduced for identifying locally conserved structure candidates in long RNAs. We suggest the presence and the sparseness of phylogenetically conserved local structures for a collection of long non-coding RNAs. CONCLUSIONS: By clustering data from 2 cross-linking immunoprecipitation experiments, we demonstrate the benefits of GraphClust2 for motif discovery under the presence of biological and methodological biases. Finally, we uncover prominent targets of double-stranded RNA binding protein Roquin-1, such as BCOR's 3' untranslated region that contains multiple binding stem-loops that are evolutionary conserved.

ShaKer: RNA SHAPE prediction using graph kernel.

SUMMARY: SHAPE experiments are used to probe the structure of RNA molecules. We present ShaKer to predict SHAPE data for RNA using a graph-kernel-based machine learning approach that is trained on experimental SHAPE information. While other available methods require a manually curated reference structure, ShaKer predicts reactivity data based on sequence input only and by sampling the ensemble of possible structures. Thus, ShaKer is well placed to enable experiment-driven, transcriptome-wide SHAPE data prediction to enable the study of RNA structuredness and to improve RNA structure and RNA-RNA interaction prediction. For performance evaluation, we use accuracy and accessibility comparing to experimental SHAPE data and competing methods. We can show that Shaker outperforms its competitors and is able to predict high quality SHAPE annotations even when no reference structure is provided. AVAILABILITY AND IMPLEMENTATION: ShaKer is freely available at https://github.com/BackofenLab/ShaKer.

Multifaceted analyses disclose the role of fruit size and skin-russeting in the accumulation pattern of phenolic compounds in apple.

Fruits are nowadays considered important suppliers of anti-oxidant molecules. Apples are particularly rich in phenolic compounds, non-nutritional phytochemicals that play active roles in controlling severe chronic diseases. In this work, 19 phenolic compounds were investigated in both skin and pulp tissues of seven apple accessions across the Malus genus collected at two stages: during fruit development and at harvest. The primary difference in phenolic concentration between wild and domesticated accessions, especially in the pulp, could be explained by the larger growth rate of the domesticated varieties. The proposed dilution effect was also confirmed through the observation of the increased content of procyanidin B2+B4 and phloridzin in russet-skinned apples, known to have higher concentrations of these compounds. The metabolite screening was also accompanied by the expression analysis of 16 polyphenolic genes showing, for nine elements, a higher expression at harvest than during fruit development. Finally, a polyphenolic comparison with red-fleshed apples was also carried out, underlying a larger amount of procyanidins and quercetin-3rhamnoside in the white-fleshed accessions. The results presented and discussed in this work suggest that specific white-fleshed apples, especially with russeted-skin, may play an important role in ameliorating the nutraceutical potential of apple fruit.

Apple whole genome sequences: recent advances and new prospects.

In 2010, a major scientific milestone was achieved for tree fruit crops: publication of the first draft whole genome sequence (WGS) for apple (Malus domestica). This WGS, v1.0, was valuable as the initial reference for sequence information, fine mapping, gene discovery, variant discovery, and tool development. A new, high quality apple WGS, GDDH13 v1.1, was released in 2017 and now serves as the reference genome for apple. Over the past decade, these apple WGSs have had an enormous impact on our understanding of apple biological functioning, trait physiology and inheritance, leading to practical applications for improving this highly valued crop. Causal gene identities for phenotypes of fundamental and practical interest can today be discovered much more rapidly. Genome-wide polymorphisms at high genetic resolution are screened efficiently over hundreds to thousands of individuals with new insights into genetic relationships and pedigrees. High-density genetic maps are constructed efficiently and quantitative trait loci for valuable traits are readily associated with positional candidate genes and/or converted into diagnostic tests for breeders. We understand the species, geographical, and genomic origins of domesticated apple more precisely, as well as its relationship to wild relatives. The WGS has turbo-charged application of these classical research steps to crop improvement and drives innovative methods to achieve more durable, environmentally sound, productive, and consumer-desirable apple production. This review includes examples of basic and practical breakthroughs and challenges in using the apple WGSs. Recommendations for "what's next" focus on necessary upgrades to the genome sequence data pool, as well as for use of the data, to reach new frontiers in genomics-based scientific understanding of apple.

Wide transcriptional investigation unravel novel insights of the on-tree maturation and postharvest ripening of 'Abate Fetel' pear fruit.

To decipher the transcriptomic regulation of the on-tree fruit maturation in pear cv. 'Abate Fetel', a RNA-seq transcription analysis identified 8939 genes differentially expressed across four harvesting stages. These genes were grouped into 11 SOTA clusters based on their transcriptional pattern, of which three included genes upregulated while the other four were represented by downregulated genes. Fruit ripening was furthermore investigated after 1 month of postharvest cold storage. The most important variation in fruit firmness, production of ethylene and volatile organic compounds were observed after 5 days of shelf-life at room temperature following cold storage. The role of ethylene in controlling the ripening of 'Abate Fetel' pears was furthermore investigated through the application of 1-methylcyclopropene, which efficiently delayed the progression of ripening by reducing fruit softening and repressing both ethylene and volatile production. The physiological response of the interference at the ethylene receptor level was moreover unraveled investigating the expression pattern of 12 candidate genes, initially selected to validate the RNA-seq profile. This analysis confirmed the effective role of the ethylene competitor in downregulating the expression of cell wall (PG) and ethylene-related genes (ACS, ACO, ERS1, and ERS2), as well as inducing one element involved in the auxin signaling pathway (Aux/IAA), highlighting a possible cross-talk between these two hormones. The expression patterns of these six elements suggest their use as molecular toolkit to monitor at molecular level the progression of the fruit on-tree maturation and postharvest ripening.

Left atrial appendage closure guided by 3D computed tomography printing technology: A case control study.

BACKGROUND: We sought to evaluate the additional value of left atrial appendage (LAA) 3D printing derived from computed tomography (CCT) in determining the size for LAA occlusion (LAAO) devices as compared to standard measurement by using occurrence of LAA leak as endpoint. METHODS: We evaluated 6 patients with LAA leak (cases) and 14 matched patients without LAA leak (controls) after LAAO. For each group, a patient-specific 3D printed model of LAA was manufactured using CT pre-operative images. The size recommended by the 3D printed model was compared with the size of the implanted device. RESULTS: Compared to the 3D printed model, 55% of the devices were underestimated, the two sizing approaches agreed in 35% of the patients, while the 3D printed model overestimated the size in 10% of patients. The prevalence of LAA leak was significantly higher in the subset of patients with underestimation of prosthesis implanted with the standard approach as compared to the other patients (p = 0.019). CONCLUSION: 3D printing of the LAA may provide additional value to standard practice for LAAO device prosthesis sizing with the potential impact to reduce LAA leak.

GraphDDP: a graph-embedding approach to detect differentiation pathways in single-cell-data using prior class knowledge.

Cell types can be characterized by expression profiles derived from single-cell RNA-seq. Subpopulations are identified via clustering, yielding intuitive outcomes that can be validated by marker genes. Clustering, however, implies a discretization that cannot capture the continuous nature of differentiation processes. One could give up the detection of subpopulations and directly estimate the differentiation process from cell profiles. A combination of both types of information, however, is preferable. Crucially, clusters can serve as anchor points of differentiation trajectories. Here we present GraphDDP, which integrates both viewpoints in an intuitive visualization. GraphDDP starts from a user-defined cluster assignment and then uses a force-based graph layout approach on two types of carefully constructed edges: one emphasizing cluster membership, the other, based on density gradients, emphasizing differentiation trajectories. We show on intestinal epithelial cells and myeloid progenitor data that GraphDDP allows the identification of differentiation pathways that cannot be easily detected by other approaches.

Freiburg RNA tools: a central online resource for RNA-focused research and teaching.

The Freiburg RNA tools webserver is a well established online resource for RNA-focused research. It provides a unified user interface and comprehensive result visualization for efficient command line tools. The webserver includes RNA-RNA interaction prediction (IntaRNA, CopraRNA, metaMIR), sRNA homology search (GLASSgo), sequence-structure alignments (LocARNA, MARNA, CARNA, ExpaRNA), CRISPR repeat classification (CRISPRmap), sequence design (antaRNA, INFO-RNA, SECISDesign), structure aberration evaluation of point mutations (RaSE), and RNA/protein-family models visualization (CMV), and other methods. Open education resources offer interactive visualizations of RNA structure and RNA-RNA interaction prediction as well as basic and advanced sequence alignment algorithms. The services are freely available at http://rna.informatik.uni-freiburg.de.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

What makes for sound science?

The inclusive threshold policy for publication in BMC journals including BMC Plant Biology means that editorial decisions are largely based on the soundness of the research presented rather than the novelty or potential impact of the work. Here we discuss what is required to ensure that research meets the requirement of scientific soundness. BMC Plant Biology and the other BCM-series journals ( https://www.biomedcentral.com/p/the-bmc-series-journals ) differ in policy from many other journals as they aim to provide a home for all publishable research. The inclusive threshold policy for publication means that editorial decisions are largely based on the soundness of the research presented rather than the novelty or potential impact of the work. The emphasis on scientific soundness ( http://blogs.biomedcentral.com/bmcseriesblog/2016/12/05/vital-importance-inclusive/ ) rather than novelty or impact is important because it means that manuscripts that may be judged to be of low impact due to the nature of the study as well as those reporting negative results or that largely replicate earlier studies, all of which can be difficult to publish elsewhere, are available to the research community. Here we discuss the importance of the soundness of research and provide some basic guidelines to assist authors to determine whether their research is appropriate for submission to BMC Plant Biology.Prior to a research article being sent out for review, the handling editor will first determine whether the research presented is scientifically valid. To be valid the research must address a question of biological significance using suitable methods and analyses, and must follow community-agreed standards relevant to the research field.

The Peach RGF/GLV Signaling Peptide pCTG134 Is Involved in a Regulatory Circuit That Sustains Auxin and Ethylene Actions.

In vascular plants the cell-to-cell interactions coordinating morphogenetic and physiological processes are mediated, among others, by the action of hormones, among which also short mobile peptides were recognized to have roles as signals. Such peptide hormones (PHs) are involved in defense responses, shoot and root growth, meristem homeostasis, organ abscission, nutrient signaling, hormone crosstalk and other developmental processes and act as both short and long distant ligands. In this work, the function of CTG134, a peach gene encoding a ROOT GROWTH FACTOR/GOLVEN-like PH expressed in mesocarp at the onset of ripening, was investigated for its role in mediating an auxin-ethylene crosstalk. In peach fruit, where an auxin-ethylene crosstalk mechanism is necessary to support climacteric ethylene synthesis, CTG134 expression peaked before that of ACS1 and was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. In addition, the promoter of CTG134 fused with the GUS reporter highlighted activity in plant parts in which the auxin-ethylene interplay is known to occur. Arabidopsis and tobacco plants overexpressing CTG134 showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the sulfated peptide. Moreover, in tobacco, lateral root emergence and capsule size were also affected. In Arabidopsis overexpressing lines, molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin synthesis, transport and perception. These data support the role of pCTG134 as a mediator in an auxin-ethylene regulatory circuit and open the possibility to exploit this class of ligands for the rational design of new and environmental friendly agrochemicals able to cope with a rapidly changing environment.

An efficient graph kernel method for non-coding RNA functional prediction.

MOTIVATION: The importance of RNA protein-coding gene regulation is by now well appreciated. Non-coding RNAs (ncRNAs) are known to regulate gene expression at practically every stage, ranging from chromatin packaging to mRNA translation. However the functional characterization of specific instances remains a challenging task in genome scale settings. For this reason, automatic annotation approaches are of interest. Existing computational methods are either efficient but non-accurate or they offer increased precision, but present scalability problems. RESULTS: In this article, we present a predictive system based on kernel methods, a type of machine learning algorithm grounded in statistical learning theory. We employ a flexible graph encoding to preserve multiple structural hypotheses and exploit recent advances in representation and model induction to scale to large data volumes. Experimental results on tens of thousands of ncRNA sequences available from the Rfam database indicate that we can not only improve upon state-of-the-art predictors, but also achieve speedups of several orders of magnitude. AVAILABILITY AND IMPLEMENTATION: The code is available from http://www.bioinf.uni-freiburg.de/~costa/EDeN.tgz . CONTACT: f.costa@exeter.ac.uk.