Current insights into posttranscriptional regulation of fleshy fruit ripening.

Fruit ripening is a complicated process that is accompanied by the formation of fruit quality. It is not only regulated at the transcriptional level via transcription factors or DNA methylation but also fine-tuned after transcription occurs. Here, we review recent advances in our understanding of key regulatory mechanisms of fleshy fruit ripening after transcription. We mainly highlight the typical mechanisms by which fruit ripening is controlled, namely, alternative splicing, mRNA N6-methyladenosine RNA modification methylation, and noncoding RNAs at the posttranscriptional level; regulation of translation efficiency and upstream open reading frame-mediated translational repression at the translational level; and histone modifications, protein phosphorylation, and protein ubiquitination at the posttranslational level. Taken together, these posttranscriptional regulatory mechanisms, along with transcriptional regulation, constitute the molecular framework of fruit ripening. We also critically discuss the potential usage of some mechanisms to improve fruit traits.

Transcriptional landscape of pathogen-responsive lncRNAs in tomato unveils the role of hydrolase encoding genes in response to Botrytis cinerea invasion.

LncRNAs have gained increasing attention owing to their important regulatory roles on growth and stress responses of plants. However, the mechanisms underlying the functions of lncRNAs in fruit-pathogen interaction are still largely unknown. In this study, a total of 273 lncRNAs responding to Botrytis cinerea infection were identified in tomato fruit, among which a higher percentage of antisense lncRNAs were targeted to the genes enriched in hydrolase activity. To ascertain the roles of these lncRNAs, seven hydrolase-related transcripts were transiently knocked-down by virus-induced gene silencing. Silencing of lncRNACXE20 reduced the expression level of a carboxylesterase gene, further enhancing the resistance of tomato to B. cinerea. In contrast, silencing of lncRNACHI, lncRNAMMP, lncRNASBT1.9 and lncRNAPME1.9 impaired the resistance to B. cinerea, respectively. Further RT-qPCR assay and enzymatic activity detection displayed that the attenuated resistance of lncRNAMMP and lncRNASBT1.9-silenced plants was associated with the inhibition on the expression of JA-related genes, while the decreased resistance of lncRNACHI-silenced plants resulted in reduced chitinase activity. Collectively, these results may provide references for deciphering the mechanisms underlying specific lncRNAs to interfere with B. cinerea infection by regulating the expression of defence-related genes or affecting hydrolase activity.

The pivotal ripening gene SlDML2 participates in regulating disease resistance in tomato.

Fruit ripening and disease resistance are two essential biological processes for quality formation and maintenance. DNA methylation, in the form of 5-methylcytosine (5mC), has been elucidated to modulate fruit ripening, but its role in regulating fruit disease resistance remains poorly understood. In this study, we show that mutation of SlDML2, the DNA demethylase gene essential for fruit ripening, affects multiple developmental processes of tomato besides fruit ripening, including seed germination, leaf length and width and flower branching. Intriguingly, loss of SlDML2 function decreased the resistance of tomato fruits against the necrotrophic fungal pathogen Botrytis cinerea. Comparative transcriptomic analysis revealed an obvious transcriptome reprogramming caused by SlDML2 mutation during B. cinerea invasion. Among the thousands of differentially expressed genes, SlßCA3 encoding a ß-carbonic anhydrase and SlFAD3 encoding a ω-3 fatty acid desaturase were demonstrated to be transcriptionally activated by SlDML2-mediated DNA demethylation and positively regulate tomato resistance to B. cinerea probably in the same genetic pathway with SlDML2. We further show that the pericarp tissue surrounding B. cinerea infection exhibited a delay in ripening with singnificant decrease in expression of ripening genes that are targeted by SlDML2 and increase in expression of SlßCA3 and SlFAD3. Taken together, our results uncover an essential layer of gene regulation mediated by DNA methylation upon B. cinerea infection and raise the possible that the DNA demethylase gene SlDML2, as a multifunctional gene, participates in modulating the trade-off between fruit ripening and disease resistance.

A receptor-like kinase SlFERL mediates immune responses of tomato to Botrytis cinerea by recognizing BcPG1 and fine-tuning MAPK signaling.

FERONIA (FER) is a receptor-like kinase showing versatile functions during plant growth, development, and responses to environmental stimuli. However, its functions during the interaction between fruit and necrotrophic fungal pathogens are still unclear. Combining reverse genetic approaches, physiological assays, co-immunoprecipitation, protein phosphorylation identification, and site-directed mutagenesis, we reported a tomato FER homolog SlFERL (Solanum lycopersicum FERONIA Like) involved in the immune responses to Botrytis cinerea invasion. The results indicated that SlFERL extracellular domain recognized and interacted with the secreted virulence protein BcPG1 from B. cinerea, further revealed that SlFERL triggered downstream signaling by phosphorylating SlMAP3K18 at Thr45, Ser49, Ser76, and Ser135. Moreover, we verified that SlMAP2K2 and SlMAP2K4 synergistically contributed to immune response of tomato to B. cinerea, in which SlFERL-SlMAP3K18 module substantially modulated protein level and/or kinase activity of SlMAP2K2/SlMAP2K4. These findings reveal a new pattern-triggered immune pathway, indicating that SlFERL participates in the immune responses to B. cinerea invasion via recognizing BcPG1 and fine-tuning MAPK signaling.

m6 A-mediated regulation of crop development and stress responses.

Dynamic chemical modifications in eukaryotic messenger RNAs (mRNAs) constitute an essential layer of gene regulation, among which N6 -methyladenosine (m6 A) was unveiled to be the most abundant. m6 A functionally modulates important biological processes in various mammals and plants through the regulation of mRNA metabolism, mainly mRNA degradation and translation efficiency. Physiological functions of m6 A methylation are diversified and affected by intricate sequence contexts and m6 A machineries. A number of studies have dissected the functional roles and the underlying mechanisms of m6 A modifications in regulating plant development and stress responses. Recently, it was demonstrated that the human FTO-mediated plant m6 A removal caused dramatic yield increases in rice and potato, indicating that modulation of m6 A methylation could be an efficient strategy for crop improvement. In this review, we summarize the current progress concerning the m6 A-mediated regulation of crop development and stress responses, and provide an outlook on the potential application of m6 A epitranscriptome in the future improvement of crops.

PeMetR-mediated sulfur assimilation is essential for virulence and patulin biosynthesis in Penicillium expansum.

Penicillium expansum, as the causal agent of blue mould and a main producer of mycotoxin patulin, is a global concern for economic and food safety. To date, the nutritional requirements of the pathogen during infection and patulin biosynthesis are poorly understood. Here, we genetically characterized the role of the bZIP transcription factor PeMetR in sulfur metabolism, virulence and patulin biosynthesis of P. expansum. The PeMetR regulator is crucial for normal germination and growth on inorganic S-sources but dispensable for utilization of organic S-sources. Accordingly, it is involved in regulating the expression of genes in sulfur assimilation pathway rather than methionine metabolic processes. Disruption of PeMetR resulted in a complete loss of virulence on various fruits. Additionally, the mutant showed a remarkably reduced ability to produce patulin. Exogenous methionine could partially or completely rescue the impaired phenotypes of the mutant. Inactivation of the sulfur assimilation pathway genes, PesA, PesB, PesC, PesF, generated growth, virulence and patulin production defects similar to those of ΔPeMetR. Overall, our study provides evidence that PeMetR-mediated sulfur assimilation is essential for growth and infection and shows for the first time that regulation of sulfur assimilation affects biosynthesis of an important mycotoxin patulin in P. expansum.

SlFERL Interacts with S-Adenosylmethionine Synthetase to Regulate Fruit Ripening.

Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato ( Solanum lycopersicum) FERONIA Like (SlFERL), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses. Here, we report that RIN and Tomato AGAMOUS-LIKE1 (TAGL1) directly bind to the promoter region of SlFERL and further activate its expression transcriptionally, suggesting a potential role of SlFERL in fruit ripening. Overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL resulted in delayed fruit ripening. Moreover, a surface plasmon resonance assay coupled with tandem mass spectrometry and a protein interaction assay revealed that SlFERL interacts with the key enzyme S-adenosyl-Met synthetase 1 (SlSAMS1) in the ethylene biosynthesis pathway, leading to increased S-adenosyl-Met accumulation and elevated ethylene production. Thus, SlFERL serves as a positive regulator of ethylene production and fruit ripening. This study provides clues to the molecular regulatory networks underlying fruit ripening.

SlREM1 Triggers Cell Death by Activating an Oxidative Burst and Other Regulators.

Programmed cell death (PCD), a highly regulated feature of the plant immune response, involves multiple molecular players. Remorins (REMs) are plant-specific proteins with varied biological functions, but their function in PCD and plant defense remains largely unknown. Here, we report a role for remorin in disease resistance, immune response, and PCD regulation. Overexpression of tomato (Solanum lycopersicum) REMORIN1 (SlREM1) increased susceptibility of tomato to the necrotrophic fungus Botrytis cinerea and heterologous expression of this gene triggered cell death in Nicotiana benthamiana leaves. Further investigation indicated that amino acids 173 to 187 and phosphorylation of SlREM1 played key roles in SlREM1-triggered cell death. Intriguingly, multiple tomato REMs induced cell death in N benthamiana leaves. Yeast two-hybrid, split luciferase complementation, and coimmunoprecipitation assays all demonstrated that remorin proteins could form homo- and heterocomplexes. Using isobaric tags for relative and absolute quantitative proteomics, we identified that some proteins related to cell death regulation, as well as N benthamiana RESPIRATORY BURST OXIDASE HOMOLOG B (which is essential for reactive oxygen species production), were notably upregulated in SlREM1-expressing leaves. Heterologous expression of SlREM1 increased reactive oxygen species accumulation and triggered other cell death regulators. Our findings indicate that SlREM1 is a positive regulator of plant cell death and provide clues for understanding the PCD molecular regulatory network in plants.

Regulatory network of fruit ripening: current understanding and future challenges.

Fruit ripening is a developmental process that is spatio-temporally tuned at multiple levels. Molecular dissections of the mechanisms underlying the ripening process have revealed a network encompassed by hormones, transcriptional regulators, epigenomic modifications and other regulatory elements that directly determine fruit quality and the postharvest commodity of fresh produce. Many studies have addressed the important roles of ethylene, abscisic acid (ABA) and other hormones in regulating fruit ripening. Recent studies have shown that some spontaneous mutants for tomato transcription factors (TFs) have resulted from loss-of-function or dominant-negative mutations. Unlike in DNA methylation variation, the histone mark H3K27me3 may be conserved and prevents the transcriptional feedback circuit from generating autocatalytic ethylene. These observations of a network of partially redundant component indicate the need to improve our current understanding. Here, we focussed on the recent advances and future challenges in investigations of the molecular mechanisms of fruit ripening. We also identified several issues that still need to be addressed in future studies.

Honokiol suppresses mycelial growth and reduces virulence of Botrytis cinerea by inducing autophagic activities and apoptosis.

Fungal pathogens lead to severe quality deterioration and yield loss, making it urgent to explore efficient measures to control fungal diseases at the preharvest and postharvest stages of plants. Therefore, studies on natural substances targeting alternative antimicrobial targets have become hot spots of research. Here, we show that honokiol, a polyphenolic compound obtained from Magnolia officinalis, significantly suppressed mycelial growth and reduced virulence of B. cinerea on harvested fruit by inducing autophagic activities and apoptosis. Moreover, honokiol was capable of abolishing the mitochondrial membrane potential and inducing the accumulation of reactive oxygen species. Some key genes involved in pathogenicity on fruit were also found significantly down-regulated. In summary, honokiol was effective as an alternative agent targeting autophagic and apoptotic machineries to control the incidence of gray mold, which may further enrich the toolkit of crop managers for fighting postharvest diseases caused by this and similar fungi.

Versatile Roles of the Receptor-Like Kinase Feronia in Plant Growth, Development and Host-Pathogen Interaction.

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

Molecular basis and regulation of pathogenicity and patulin biosynthesis in Penicillium expansum.

Penicillium expansum is a necrotrophic plant pathogen with a wide range of fruit hosts. It causes blue mold rot during fruit storage, transport, and sale, resulting in huge economic losses to the fruit industry. Moreover, this pathogen is also the main producer of patulin, a toxic secondary metabolite that contaminates fruit and fruit-derived products and impairs human health. Therefore, understanding molecular basis of the pathogenicity and patulin biosynthesis of the fungal pathogen has important scientific significance and also plays an important guiding role in the research and development of new control technologies. Here, we comprehensively summarize the recent research progress, particularly regarding the molecular aspects of pathogenicity, patulin biosynthesis, and the related regulatory mechanisms, as well as control technologies for blue mold rot in the fruit industry.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

Dissection of patulin biosynthesis, spatial control and regulation mechanism in Penicillium expansum.

The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.

Ectopic expression of ORANGE promotes carotenoid accumulation and fruit development in tomato.

Carotenoids are critically important to plants and humans. The ORANGE (OR) gene is a key regulator for carotenoid accumulation, but its physiological roles in crops remain elusive. In this study, we generated transgenic tomato ectopically overexpressing the Arabidopsis wild-type OR (AtORWT ) and a 'golden SNP'-containing OR (AtORHis ). We found that AtORHis initiated chromoplast formation in very young fruit and stimulated carotenoid accumulation at all fruit developmental stages, uncoupled from other ripening activities. The elevated levels of carotenoids in the AtOR lines were distributed in the same subplastidial fractions as in wild-type tomato, indicating an adaptive response of plastids to sequester the increased carotenoids. Microscopic analysis revealed that the plastid sizes were increased in both AtORWT and AtORHis lines at early fruit developmental stages. Moreover, AtOR overexpression promoted early flowering, fruit set and seed production. Ethylene production and the expression of ripening-associated genes were also significantly increased in the AtOR transgenic fruit at ripening stages. RNA-Seq transcriptomic profiling highlighted the primary effects of OR overexpression on the genes in the processes related to RNA, protein and signalling in tomato fruit. Taken together, these results expand our understanding of OR in mediating carotenoid accumulation in plants and suggest additional roles of OR in affecting plastid size as well as flower and fruit development, thus making OR a target gene not only for nutritional biofortification of agricultural products but also for alteration of horticultural traits.

Production, Signaling, and Scavenging Mechanisms of Reactive Oxygen Species in Fruit-Pathogen Interactions.

Reactive oxygen species (ROS) play a dual role in fruit-pathogen interaction, which largely depends on their different levels in cells. Fruit recognition of a pathogen immediately triggers an oxidative burst that is considered an integral part of the fruit defense response. ROS are also necessary for the virulence of pathogenic fungi. However, the accumulation of ROS in cells causes molecular damage and finally leads to cell death. In this review, on the basis of data regarding ROS production and the scavenging systems determining ROS homeostasis, we focus on the role of ROS in fruit defense reactions against pathogens and in fungi pathogenicity during fruit-pathogen interaction.

The pH-responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum.

The PacC (loss or reduction in phosphatase activity at acid but not at alkaline pH [Pac]) transcription factor regulates environmental adaptation, secondary metabolism and virulence in many fungal pathogens. Here, we report the functions of PacC in Penicillium expansum, a postharvest pathogenic fungus in horticultural crops, and ascertain that the gene expression and proteolytic processing of PePacC are strictly pH-dependent. Loss of PePacC resulted in an obvious decrease in growth and conidiation of P. expansum cultured in both acidic and alkaline conditions. The ΔPePacC mutant lost the ability of patulin production at pH values above 6.0 because expressions of all the genes in patulin cluster were significantly down-regulated. Additionally, virulence of the ΔPePacC mutant was obviously reduced in pear and apple fruits. Proteome analysis revealed that PePacC could function as an activator or repressor for different target proteins, including calreticulin (PeCRT) and sulfate adenylyltransferase (PeSAT), which were further proved to be involved in virulence of P. expansum. Our results demonstrate important roles for PePacC in patulin biosynthesis via limiting expressions of the genes in the cluster, and in pathogenesis via mediating a known virulence factor glucose oxidase (PeGOD) and new virulence factors, such as PeCRT and PeSAT.

Variable-angle epifluorescence microscopy characterizes protein dynamics in the vicinity of plasma membrane in plant cells.

BACKGROUND: The assembly of protein complexes and compositional lipid patterning act together to endow cells with the plasticity required to maintain compositional heterogeneity with respect to individual proteins. Hence, the applications for imaging protein localization and dynamics require high accuracy, particularly at high spatio-temporal level. RESULTS: We provided experimental data for the applications of Variable-Angle Epifluorescence Microscopy (VAEM) in dissecting protein dynamics in plant cells. The VAEM-based co-localization analysis took penetration depth and incident angle into consideration. Besides direct overlap of dual-color fluorescence signals, the co-localization analysis was carried out quantitatively in combination with the methodology for calculating puncta distance and protein proximity index. Besides, simultaneous VAEM tracking of cytoskeletal dynamics provided more insights into coordinated responses of actin filaments and microtubules. Moreover, lateral motility of membrane proteins was analyzed by calculating diffusion coefficients and kymograph analysis, which represented an alternative method for examining protein motility. CONCLUSION: The present study presented experimental evidence on illustrating the use of VAEM in tracking and dissecting protein dynamics, dissecting endosomal dynamics, cell structure assembly along with membrane microdomain and protein motility in intact plant cells.

The mode of action of remorin1 in regulating fruit ripening at transcriptional and post-transcriptional levels.

Remorins are plant-specific and plasma membrane-associated proteins that display a variety of functions in plant growth, development, biotic and abiotic stresses, and signal transduction. However, little information is available for understanding their role in fruit ripening. Here, remorin 1 (SlREM1) is cloned from tomato and its localization is examined by co-localization analysis and immunoblotting. Functions of SlREM1 in fruit ripening are characterized based on gene expression, co-immunoprecipitation coupled with mass spectroscopy and split luciferase complementation imaging assays in SlREM1 overexpression and RNA interference (RNAi) lines. The results indicate that SlREM1 is localized at the plasma membrane. Overexpression of SlREM1 in tomato stimulates fruit ripening with an increase in ethylene production and lycopene accumulation as compared to the wild-type. Consistently, these genes involved in ethylene and lycopene biosynthesis and ripening regulators also are upregulated in SlREM1 overexpression lines. SlREM1 can interact with ethylene biosynthesis proteins SAM1, ACO1 and ACS2 and is degraded by ubiquitin-mediated proteolysis. Our findings reveal that SlREM1 serves as a positive regulator of fruit ripening and provide novel cues for understanding of the molecular regulation network of fruit ripening.

A Tomato Vacuolar Invertase Inhibitor Mediates Sucrose Metabolism and Influences Fruit Ripening.

Fruit ripening is a complex process that involves a series of physiological and biochemical changes that ultimately influence fruit quality traits, such as color and flavor. Sugar metabolism is an important factor in ripening, and there is evidence that it influences various aspects of ripening, although the associated mechanism is not well understood. In this study, we identified and analyzed the expression of 36 genes involved in Suc metabolism in ripening tomato (Solanum lycopersicum) fruit. Chromatin immunoprecipitation and gel mobility shift assays indicated that SlVIF, which encodes a vacuolar invertase inhibitor, and SlVI, encoding a vacuolar invertase, are directly regulated by the global fruit ripening regulator RIPENING INHIBITOR (RIN). Moreover, we showed that SlVIF physically interacts with SlVI to control Suc metabolism. Repression of SlVIF by RNA interference delayed tomato fruit ripening, while overexpression of SlVIF accelerated ripening, with concomitant changes in lycopene production and ethylene biosynthesis. An isobaric tags for relative and absolute quantification-based quantitative proteomic analysis further indicated that the abundance of a set of proteins involved in fruit ripening was altered by suppressing SlVIF expression, including proteins associated with lycopene generation and ethylene synthesis. These findings provide evidence for the role of Suc in promoting fruit ripening and establish that SlVIF contributes to fruit quality and the RIN-mediated ripening regulatory mechanisms, which are of significant agricultural value.