Dihydrochalcone glycoside biosynthesis in Malus is regulated by two MYB-like transcription factors and is required for seed development.

Dihydrochalcones (DHCs) including phlorizin (phloretin 2'-O-glucoside) and its positional isomer trilobatin (phloretin 4'-O-glucoside) are the most abundant phenylpropanoids in apple (Malus spp.). Transcriptional regulation of DHC production is poorly understood despite their importance in insect- and pathogen-plant interactions in human physiology research and in pharmaceuticals. In this study, segregation in hybrid populations and bulked segregant analysis showed that the synthesis of phlorizin and trilobatin in Malus leaves are both single-gene-controlled traits. Promoter sequences of PGT1 and PGT2, two glycosyltransferase genes involved in DHC glycoside synthesis, were shown to discriminate Malus with different DHC glycoside patterns. Differential PGT1 and PGT2 promoter activities determined DHC glycoside accumulation patterns between genotypes. Two transcription factors containing MYB-like DNA-binding domains were then shown to control DHC glycoside patterns in different tissues, with PRR2L mainly expressed in leaf, fruit, flower, stem, and seed while MYB8L mainly expressed in stem and root. Further hybridizations between specific genotypes demonstrated an absolute requirement for DHC glycoside production in Malus during seed development which explains why no Malus spp. with a null DHC chemotype have been reported.

A deep learning model based on sparse auto-encoder for prioritizing cancer-related genes and drug target combinations.

Prioritization of cancer-related genes from gene expression profiles and proteomic data is vital to improve the targeted therapies research. Although computational approaches have been complementing high-throughput biological experiments on the understanding of human diseases, it still remains a big challenge to accurately discover cancer-related proteins/genes via automatic learning from large-scale protein/gene expression data and protein-protein interaction data. Most of the existing methods are based on network construction combined with gene expression profiles, which ignore the diversity between normal samples and disease cell lines. In this study, we introduced a deep learning model based on a sparse auto-encoder to learn the specific characteristics of protein interactions in cancer cell lines integrated with protein expression data. The model showed learning ability to identify cancer-related proteins/genes from the input of different protein expression profiles by extracting the characteristics of protein interaction information, which could also predict cancer-related protein combinations. Comparing with other reported methods including differential expression and network-based methods, our model got the highest area under the curve value (>0.8) in predicting cancer-related genes. Our study prioritized ~500 high-confidence cancer-related genes; among these genes, 211 already known cancer drug targets were found, which supported the accuracy of our method. The above results indicated that the proposed auto-encoder model could computationally prioritize candidate proteins/genes involved in cancer and improve the targeted therapies research.

Comparative analysis of miniature inverted-repeat transposable elements (MITEs) and long terminal repeat (LTR) retrotransposons in six Citrus species.

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) and long terminal repeat (LTR) retrotransposons are ubiquitous in plants genomes, and highly important in their evolution and diversity. However, their mechanisms of insertion/amplification and roles in Citrus genome's evolution/diversity are still poorly understood. RESULTS: To address this knowledge gap, we developed different computational pipelines to analyze, annotate and classify MITEs and LTR retrotransposons in six different sequenced Citrus species. We identified 62,010 full-length MITEs from 110 distinguished families. We observed MITEs tend to insert in gene related regions and enriched in promoters. We found that DTM63 is possibly an active Mutator-like MITE family in the traceable past and may still be active in Citrus. The insertion of MITEs resulted in massive polymorphisms and played an important role in Citrus genome diversity and gene structure variations. In addition, 6630 complete LTR retrotransposons and 13,371 solo-LTRs were identified. Among them, 12 LTR lineages separated before the differentiation of mono- and dicotyledonous plants. We observed insertion and deletion of LTR retrotransposons was accomplished with a dynamic balance, and their half-life in Citrus was ~ 1.8 million years. CONCLUSIONS: These findings provide insights into MITEs and LTR retrotransposons and their roles in genome diversity in different Citrus genomes.

Network analysis of postharvest senescence process in citrus fruits revealed by transcriptomic and metabolomic profiling.

Citrus (Citrus spp.), a nonclimacteric fruit, is one of the most important fruit crops in global fruit industry. However, the biological behavior of citrus fruit ripening and postharvest senescence remains unclear. To better understand the senescence process of citrus fruit, we analyzed data sets from commercial microarrays, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry and validated physiological quality detection of four main varieties in the genus Citrus. Network-based approaches of data mining and modeling were used to investigate complex molecular processes in citrus. The Citrus Metabolic Pathway Network and correlation networks were constructed to explore the modules and relationships of the functional genes/metabolites. We found that the different flesh-rind transport of nutrients and water due to the anatomic structural differences among citrus varieties might be an important factor that influences fruit senescence behavior. We then modeled and verified the citrus senescence process. As fruit rind is exposed directly to the environment, which results in energy expenditure in response to biotic and abiotic stresses, nutrients are exported from flesh to rind to maintain the activity of the whole fruit. The depletion of internal substances causes abiotic stresses, which further induces phytohormone reactions, transcription factor regulation, and a series of physiological and biochemical reactions.

Prediction of Protein-Protein Interactions by Evidence Combining Methods.

Most cellular functions involve proteins' features based on their physical interactions with other partner proteins. Sketching a map of protein-protein interactions (PPIs) is therefore an important inception step towards understanding the basics of cell functions. Several experimental techniques operating in vivo or in vitro have made significant contributions to screening a large number of protein interaction partners, especially high-throughput experimental methods. However, computational approaches for PPI predication supported by rapid accumulation of data generated from experimental techniques, 3D structure definitions, and genome sequencing have boosted the map sketching of PPIs. In this review, we shed light on in silico PPI prediction methods that integrate evidence from multiple sources, including evolutionary relationship, function annotation, sequence/structure features, network topology and text mining. These methods are developed for integration of multi-dimensional evidence, for designing the strategies to predict novel interactions, and for making the results consistent with the increase of prediction coverage and accuracy.

Carotenoid accumulation affects redox status, starch metabolism, and flavonoid/anthocyanin accumulation in citrus.

BACKGROUND: Carotenoids are indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we integrated genomic, biochemical, and cellular studies to gain deep insight into carotenoid-related biological processes in citrus calli overexpressing CrtB (phytoene synthase from Pantoea agglomerans). Fortunella hindsii Swingle (a citrus relative) and Malus hupehensis (a wild apple) calli were also utilized as supporting systems to investigate the effect of altered carotenoid accumulation on carotenoid-related biological processes. RESULTS: Transcriptomic analysis provided deep insight into the carotenoid-related biological processes of redox status, starch metabolism, and flavonoid/anthocyanin accumulation. By applying biochemical and cytological analyses, we determined that the altered redox status was associated with variations in O2 (-) and H2O2 levels. We also ascertained a decline in starch accumulation in carotenoid-rich calli. Furthermore, via an extensive cellular investigation of the newly constructed CrtB overexpressing Fortunella hindsii Swingle, we demonstrated that starch level reducation occurred in parallel with significant carotenoid accumulation. Moreover, studying anthocyanin-rich Malus hupehensis calli showed a negative effect of carotenoids on anthocyanin accumulation. CONCLUSIONS: In citrus, altered carotenoid accumulation resulted in dramatic effects on metabolic processes involved in redox modification, starch degradation, and flavonoid/anthocyanin biosynthesis. These findings provided new perspectives to understand the biological importance of carotenogenesis and of the developmental processes associated with the nutritional and sensory qualities of agricultural products that accumulate carotenoids.

Prediction and functional analysis of the sweet orange protein-protein interaction network.

BACKGROUND: Sweet orange (Citrus sinensis) is one of the most important fruits world-wide. Because it is a woody plant with a long growth cycle, genetic studies of sweet orange are lagging behind those of other species. RESULTS: In this analysis, we employed ortholog identification and domain combination methods to predict the protein-protein interaction (PPI) network for sweet orange. The K-nearest neighbors (KNN) classification method was used to verify and filter the network. The final predicted PPI network, CitrusNet, contained 8,195 proteins with 124,491 interactions. The quality of CitrusNet was evaluated using gene ontology (GO) and Mapman annotations, which confirmed the reliability of the network. In addition, we calculated the expression difference of interacting genes (EDI) in CitrusNet using RNA-seq data from four sweet orange tissues, and also analyzed the EDI distribution and variation in different sub-networks. CONCLUSIONS: Gene expression in CitrusNet has significant modular features. Target of rapamycin (TOR) protein served as the central node of the hormone-signaling sub-network. All evidence supported the idea that TOR can integrate various hormone signals and affect plant growth. CitrusNet provides valuable resources for the study of biological functions in sweet orange.

Comprehensive insights on how 2,4-dichlorophenoxyacetic acid retards senescence in post-harvest citrus fruits using transcriptomic and proteomic approaches.

Auxin-like 2,4-dichlorophenoxyacetic acid (2,4-D), a high-efficiency anti-stalling agent for the post-harvest fresh fruit industry, has had its use restricted due to environmental concerns. However, no other substitutes for 2,4-D are available to the post-harvest industry. Insights into the molecular mechanism underlying the effects of 2,4-D on fruit quality preservation will provide a theoretical basis for exploring new safe and effective anti-stalling agents. This study comprehensively analysed changes in the peel of Olinda Valencia orange [Citrus sinensis (L.) Osbeck] induced by 500 ppm 2,4-D using 'omic'-driven approaches. Transcriptional profiling revealed that transcriptional factor (mainly AP2/ERF, WRKY, and NAC family members), transport, and hormone metabolism genes were over-represented and up-regulated within 24h post-treatment (HPT). Stress defence genes were up-regulated, while cell wall metabolism genes were down-regulated after 48 HPT. However, secondary metabolism genes, especially phenylpropanoid and lignin biosynthesis-related genes, were over-represented at all the time points. Comparative proteomic analysis indicated that the expression of proteins implicated in stress responses (25%), hormone metabolism, and signal transduction (12%) significantly accumulated at the post-transcriptional level. Hormone levels detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) showed that abscisic acid, salicylic acid, and 2,4-D significantly increased, while ethylene production (detected by gas chromatography) decreased after 2,4-D treatment. In addition, lignin and water content in the fruit peel also increased and the epicuticle wax ultrastructure was modified. In conclusion, 2,4-D retarded fruit senescence by altering the levels of many endogenous hormones and by improving stress defence capabilities by up-regulating defence-related genes and proteins.

Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening.

Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post-translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome-wide mapping of in vivo phosphorylation sites in chromoplast-enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide-based affinity chromatography for phosphoprotein enrichment with LC-MS/MS. A total of 109 plastid-localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif-X analysis, two distinct types of phosphorylation sites, one as proline-directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P(3) DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high-level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.

DkWRKY transcription factors enhance persimmon resistance to Colletotrichum horii by promoting lignin accumulation through DkCAD1 promotor interaction.

Persimmon anthracnose, a severe disease caused by the hemibiotrophic fungus Colletotrichum horii, poses a substantial threat to China's persimmon industry. Previous research showed that 'Kangbing Jianshi' cultivar exhibits strong resistance to anthracnose. Notably, 'Kangbing Jianshi' branches exhibit greater lignification compared with the susceptible 'Fuping Jianshi' cultivar. In this study, higher lignin content was observed in 'Kangbing Jianshi' compared with 'Fuping Jianshi', and this difference was associated with disease resistance. Transcriptome and metabolome analyses revealed that the majority of differentially expressed genes and differentially accumulated metabolites were primarily enriched in the phenylpropanoid biosynthesis and lignin synthesis pathways. Furthermore, significant upregulation of DkCAD1, a pivotal gene involved in lignin metabolism, was observed in the resistant cultivar when inoculated with C. horii. Transient overexpression of DkCAD1 substantially increased lignin content and improved resistance to C. horii in a susceptible cultivar. Furthermore, through yeast one-hybrid (Y1H) assays, we identified two WRKY transcription factors, DkWRKY8 and DkWRKY10, which interacts with the DkCAD1 promoter and induces its activity. Overexpression of DkWRKY8 and DkWRKY10 not only increased leaf lignin content but also enhanced persimmon tolerance to C. horii. Moreover, the expression levels of DkCAD1, DkWRKY8, and DkWRKY10 were significantly increased in response to salicylic acid and jasmonic acid in the resistant cultivar. These findings enhance our understanding of the molecular functions of DkWRKY8, DkWRKY10, and DkCAD1 in persimmons, as well as their involvement in molecular breeding processes in persimmons.

Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis).

BACKGROUND: The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening. RESULTS: The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening. CONCLUSION: A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant.

Comparative transcriptomics and proteomics analysis of citrus fruit, to improve understanding of the effect of low temperature on maintaining fruit quality during lengthy post-harvest storage.

Fruit quality is a very complex trait that is affected by both genetic and non-genetic factors. Generally, low temperature (LT) is used to delay fruit senescence and maintain fruit quality during post-harvest storage but the molecular mechanisms involved are poorly understood. Hirado Buntan Pummelo (HBP; Citrus grandis × C. paradis) fruit were chosen to explore the mechanisms that maintain citrus fruit quality during lengthy LT storage using transcriptome and proteome studies based on digital gene expression (DGE) profiling and two-dimensional gel electrophoresis (2-DE), respectively. Results showed that LT up-regulated stress-responsive genes, arrested signal transduction, and inhibited primary metabolism, secondary metabolism and the transportation of metabolites. Calcineurin B-like protein (CBL)-CBL-interacting protein kinase complexes might be involved in the signal transduction of LT stress, and fruit quality is likely to be regulated by sugar-mediated auxin and abscisic acid (ABA) signalling. Furthermore, ABA was specific to the regulation of citrus fruit senescence and was not involved in the LT stress response. In addition, the accumulation of limonin, nomilin, methanol, and aldehyde, together with the up-regulated heat shock proteins, COR15, and cold response-related genes, provided a comprehensive proteomics and transcriptomics view on the coordination of fruit LT stress responses.

Identification and expression analysis of the lipid phosphate phosphatases gene family reveal their involvement in abiotic stress response in kiwifruit.

Lipid phosphate phosphatases (LPPs) are a key enzyme in the production and degradation of phosphatidic acid (PA), which plays an important role in plant growth, development, stress resistance and plant hormone response. Thus far, little is known about the LPP family genes in kiwifruit (Actinidia spp.). According to this study, 7 members in the AcLPP family were identified from the whole genome of kiwifruit, the subcellular localization predictions were mainly on the plasma membrane. Chromosomal localization analysis showed that the AcLPP genes were unevenly distributed on 5 chromosomes, it was determined to have undergone strong purifying selection pressure. There were 5 duplicate gene pairs and all underwent segmental duplication events. The LPP genes of kiwifruit were conserved when compared with other plants, especially in terms of evolutionary relationships, conserved motifs, protein sequences, and gene structures. Cis-regulatory elements mainly included hormone response elements and abiotic response elements. Functional annotation of GO revealed that AcLPP genes were closely related to phosphatase/hydrolase activity, phosphorus metabolism and dephosphorylation. AcLPP genes family were predicted to be targets of miRNA. Transcript level analysis revealed that the AcLPP family played diverse functions in different tissues and during growth, development, and postharvest storage stages. qPCR analysis showed that the members of AcLPP gene family might be regulated by ETH, ABA, GA3, and IAA hormone signals. The family members were regulated by the stress of salt stress, osmotic stress, cold stress, and heat stress. These results would provide a basis and reference for studying the agricultural characteristics of kiwifruit and improving its stress resistance.

Integration of morphological, physiological and multi-omics analysis reveals a comprehensive mechanism for cuticular wax during development of greasiness in postharvest apples.

Skin greasiness is a common postharvest disorder of apple (Malus × domestica). However, the molecular mechanism of skin greasiness is unclear. In this study, fruits of 'Golden Delicious' (GD), 'Granny Smith', and 'Fuji' with distinct characteristics of greasiness were used for greasiness scoring, wax morphology, wax metabolite, and RNA-seq analyses. Additionally, GD fruit were treated with 1-methylcyclopropene (1-MCP), which repressed greasiness. A partial least squares discriminant analysis (PLS-DA) revealed that wax esters were the critical wax fraction for skin greasiness. Among these wax esters, liquid linoleate esters of short-chain alcohols (C4-C6) and farnesol showed increased contents with increasing greasiness. Their concentrations were significantly correlated with greasiness score. To identify the genes encoding key enzymes for the synthesis of liquid linoleate esters, a weighted gene co-expression network analysis was conducted. MdDCR1, encoding an acyltransferase (defective in cuticular ridges, DCR), was selected as a candidate gene. MdDCR1 was significantly upregulated in greasy skin, and significantly suppressed by 1-MCP treatment. MdDCR1 silencing suppressed the accumulation of liquid linoleate esters of short-chain alcohols, including butyl linoleate, pentyl linoleate, and hexyl linoleate, in GD skin. These results provide insights into the molecular mechanisms of cuticular wax metabolism related to skin greasiness in apple. Our results show that transcriptional regulation of MdDCR1, encoding an acyltransferase that catalyzes the biosynthesis of liquid linoleate esters of short-chain alcohols (C4-C6) via an independent side branch of the C18:2 CoA pathway, regulates the formation of greasiness.

Target of rapamycin (TOR) regulates the response to low nitrogen stress via autophagy and hormone pathways in Malus hupehensis.

Target of rapamycin (TOR) is a highly conserved master regulator in eukaryotes; it regulates cell proliferation and growth by integrating different signals. However, little is known about the function of TOR in perennial woody plants. Different concentrations of AZD8055 (an inhibitor of TOR) were used in this study to investigate the role of TOR in the response to low nitrogen (N) stress in the wild apple species Malus hupehensis. Low N stress inhibited the growth of M. hupehensis plants, and 1 µM AZD alleviated this effect. Plants supplied with 1 µM AZD had higher photosynthetic capacity, which promoted the accumulation of biomass, as well as higher contents of N and anthocyanins and lower content of starch. Exogenous application of 1 µM AZD also promoted the development of the root system. Plants supplied with at least 5 µM AZD displayed early leaf senescence. RNA-seq analysis indicated that TOR altered the expression of genes related to the low N stress response, such as genes involved in photosystem, starch metabolism, autophagy, and hormone metabolism. Further analysis revealed altered autophagy in plants supplied with AZD under low N stress; the metabolism of plant hormones also changed following AZD supplementation. In sum, our findings revealed that appropriate inhibition of TOR activated autophagy and jasmonic acid signaling in M. hupehensis, which allowed plants to cope with low N stress. Severe TOR inhibition resulted in the excessive accumulation of salicylic acid, which probably led to programmed cell death in M. hupehensis.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck].

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

Microarray expression profiling of postharvest Ponkan mandarin (Citrus reticulata) fruit under cold storage reveals regulatory gene candidates and implications on soluble sugars metabolism.

Low temperature storage is widely applied to maintain citrus postharvest fruit quality. In this study, the transcriptional and metabolic changes in the pulp tissue of Citrus reticulata Blanco cv. "Ponkan" were studied for three successive months under cold storage by Affymetrix Citrus GeneChip and gas chromatography, respectively. As many as 2 161 differentially expressed transcripts were identified based on the bayesian hierarchical model. The statistical analysis of gene ontology revealed that defense/stress-related genes were induced quickly, while autophagy-related genes were overrepresented in the late sampling stages, suggesting that the functional shift may coincide with the subsequent steps of chilling development. We further classified the potential regulatory components and concluded that ethylene may play the crucial role in chilling development in this non-climacteric fruit. To cope with complex events, 53 upregulated transcription factors represented regulatory candidates. Within these, the AP2-EREBP, C2H2 and AS2 gene family were overrepresented. Cold storage also causes alterations in various metabolic pathways; a keen interest is paid in deciphering expression changes of soluble sugar genes as increased evidence that soluble sugars act as both osmolytes and metabolite signal molecules. Our results will likely facilitate further studies in this field with promising genetic candidates during chilling.

Regulation of Flowering Time by Improving Leaf Health Markers and Expansion by Salicylic Acid Treatment: A New Approach to Induce Flowering in Malus domestica.

In the external coincidence model, internal and external molecular signals, provided by the circadian clock and sunlight, respectively, are required to induce flowering. Salicylic acid (SA) applications during floral induction have multiple effects. In the current study, Malus × domestica plants were exposed to SA during the flower-induction stage to analyze the effect on various health markers and flowering. A total of 56 equal-sized Fuji/M9 trees that were about 7 years old were randomly divided into two groups. The first group (SA-treated) was sprayed with 4 mM SA solution, while the second group was sprayed with distilled water which served as control (CK). The SA applications increased various leaf pigments. Abiotic stress markers were increased in CK during the flower-induction stage. In the SA-treated group, non-enzymatic antioxidants increased, whereas in the control group, enzymatic antioxidants increased during the flower-induction stage. Histo-morphometric properties of leaves were significantly improved in the SA-treated group. The relative expression of the mRNA levels of MdMED80, -81, -3, and -41 were significantly increased in SA-treated leaves, leading to an early and increased flowering phenotype. Thus, SA increased leaf expansion and health-related marker levels, which lead to early induction of flowering in M. domestica. Overall, our work established a role for leaf health assessments in the regulation of flowering in M. domestica.

Comparative Proteomic Analysis Reveals Key Proteins Linked to the Accumulation of Soluble Sugars and Organic Acids in the Mature Fruits of the Wild Malus Species.

Soluble sugars and organic acids are the main determinants of fruit organoleptic quality. To investigate the genes responsible for the soluble sugar and organic acid contents of apple fruits, a label-free proteomic analysis involving liquid chromatography (LC)-mass spectrometry (MS)/MS was conducted with the fruits of two Malus species, M. sargentii and M. niedzwetzkyana, which exhibit significant differences in soluble sugar and organic acid contents. A total of 13,036 unique peptides and 1,079 differentially-expressed proteins were identified. To verify the LC-MS/MS results, five candidate proteins were further analyzed by parallel reaction monitoring. The results were consistent with the LC-MS/MS data, which confirmed the reliability of the LC-MS/MS analysis. The functional annotation of the differentially-expressed proteins, based on the gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, revealed that they were mainly related to biological processes and cellular components. Additionally, the main enriched KEGG pathways were related to metabolic processes. Moreover, 31 proteins involved in soluble sugar metabolism, organic acid metabolism, and H+-transport were identified. The results of this study may be useful for the comprehensive characterization of the complex mechanism regulating apple fruit-soluble sugar and organic acid contents.

Integrated transcriptomic and metabolomic analyses of a wax deficient citrus mutant exhibiting jasmonic acid-mediated defense against fungal pathogens.

Naturally, resistant crop germplasms are important resources for managing the issues of agricultural product safety and environment deterioration. We found a spontaneous mutant of 'Newhall' navel orange (Citrus sinensis Osbeck) (MT) with broad-spectrum protections against fungal pathogens in the orchard, postharvest-storage, and artificial inoculation conditions. To understand the defense mechanism of MT fruit, we constructed a genome-scale metabolic network that integrated metabolome and transcriptome datasets. The coordinated transcriptomic and metabolic data were enriched in two sub-networks, showing the decrease in very long chain fatty acid (by 41.53%) and cuticular wax synthesis (by 81.34%), and increase in the synthesis of jasmonic acid (JA) (by 95.23%) and JA-induced metabolites such as 5-dimethylnobietin (by 28.37%) in MT. Furthermore, cytological and biochemical analyses confirmed that the response to fungal infection in MT was independent of wax deficiency and was correlated with the levels of jasmonates, and the expression of plant defensin gene PDF1.2. Results of exogenous application of MeJA and JA inhibitors such as propyl gallate proved that JA-mediated defense contributes to the strong tolerance against pathogens in MT. Our results indicated that jasmonate biosynthesis and signaling are stimulated by the fatty acid redirection of MT, and participate in the tolerance of pathogenic fungi.