ERF4 affects fruit ripening by acting as a JAZ interactor between ethylene and jasmonic acid hormone signaling pathways

The regulation of apple(Malus domestica)fruit texture during ripening is complex and a fundamental determinant of its commercial quality.In climacteric fruit,ripening-related processes are regulated by ethylene(ET),and jasmonate(JA)is also involved in the ethylene biosynthesis pathway,mainly through the transcription factor MYC2.However,the molecular genetic mechanism for fruit ripening processes between the JA and ET signaling pathways still needs to be elucidated.In order to explore how JA regulates apple fruit ripening through ERF4,we used’Gala’and’Ralls Janet’fruit at different developmental stages as experimental materials to determine the fruit firmness and related gene expression analysis.Meanwhile,we carried out different hormone treatments on’Gala’fruit at ripening stage.Here,we show that ERF4 is a core JA signaling hub protein JASMONATE ZIM-DOMAIN(JAZ)interactor that affects ethylene signaling pathways.During fruit development,ERF4 represses the expression of ACS1 and ACO1 by interacting with JAZ,as well as with the JA-activated transcription factor MYC2.Ripening is promoted in JAZ-suppressed apples.Thus,ERF4 acts as a molecular link between ethylene and JA hormone signals,and the natural variation of the ERF4Ethylene-responsive binding factor-associated amphiphilic repression(EAR)motif decreases repression of ethylene biosynthesis genes.

Transcriptional profiles underlying the effects of salicylic acid on fruit ripening and senescence in pear(Pyrus pyrifolia Nakai)

Salicylic acid(SA) plays a pivotal role in delaying fruit ripening and senescence. However, little is known about its underlying mechanism of action. In this study, RNA sequencing was conducted to analyze and compare the transcriptome profiles of SA-treated and control pear fruits. We found a total of 159 and 419 genes differentially expressed between the SA-treated and control pear fruits after 12 and 24 h of treatment, respectively. Among these differentially expressed genes(DEGs), 125 genes were continuously differentially expressed at both treatment times, and they were identified as candidate genes that might be associated with SA-regulated fruit ripening and senescence. Bioinformatics analysis results showed that 125 DEGs were mainly associated with plant hormone biosynthesis and metabolism, cell wall metabolism and modification, antioxidant systems, and senescence-associated transcription factors. Additionally, the expression of several candidate DEGs in ripening and senescent pear fruits after SA treatments were further validated by quantitative real-time PCR(qRT-PCR). This study provides valuable information and enhances the understanding of the comprehensive mechanisms of SA-meditated pear fruit ripening and senescence.

PaPYL9 is involved in the regulation of apricot fruit ripening through ABA signaling pathway

Abscisic acid(ABA)is a major regulator of non-climacteric fruit ripening;however,the role of ABA in the ripening of climacteric fruit is not clear.Here,as a typical climacteric fruit,apricots were used to investigate the role of ABA in fruit ripening.Based on weighted gene coexpression network analysis(WGCNA)of our previous transcriptome data,we treated‘Danxing’fruit with exogenous ABA and obtained ABA receptor genes,genes related to ABA biosynthesis and signal transduction,and analyzed the response of these candidate genes to exogenous ABA during fruit ripening.Subsequently,the full length of candidate PYLs genes were cloned,and their putative function were analyzed by phylogenetic analysis and protein structure domain analysis.And then the function of one candidate gene PaPYL9 was verified by using transgenic tomato.Furthermore,the response genes in transgenic tomato were screened by transcriptome sequencing,and ultimately the related regulatory network was proposed.The results showed that the injection of exogenous 1.89 mmol·L^(-1) ABA remarkably promoted fruit coloration,and increased the color index for red grapes(CIRG)and the total soluble solids(TSS)content,but significantly decreased the firmness and titratable acid(TA)content(p<0.01).Nordihydroguaiaretic acid(NDGA),the inhibitor of ABA,appeared to have the converse role in TA,TSS,CIRG and firmness,during the ripening process.One NCED(9-cis-epoxycarotenoiddioxygenase)and five ABA receptor genes related to signal transduction were mined from the transcriptome data of apricot fruit through WGCNA.Compared with the control,the expression levels of NCED1,PYL9(PYR/PYL/RCAR),SnRK2(SUCROSE NON-FERMENTING1(SNF1)-RELATED PROTEIN KINASE 2S),and ABF2(ABRE-binding bZIP transcription)were induced dramatically by ABA treatment(p<0.01),while NDGA treatment significantly inhibited their expression.Based on gene expression and protein domain analysis,we inferred that PaPYL9 is putatively involved in apricot fruit ripening.Overexpression of PaPYL9 in Micro-TOM tomatoes resulted in the promotion of early ripening.Simultaneously,the expression levels of genes related ethylene biosynthesis,chlorophyll degradation,fruit softening,flavor formation,pigment synthesis,and metabolism were all significantly induced in overexpression of PaPYL9 tomatoes.This indicates the central role of ABA in climacteric fruit ripening.A regulatory network was tentatively proposed,laying the foundation to unveil the molecular mechanism of the regulatory role of PaPYL9 in fruit ripening.

Characteristics of fruit ripening in tomato mutant epi

The characteristics of fruit ripening and expression of ripening-related genes were investigated in epi, an ethylene overproduction mutant of tomato (Lycopersicon esculentum Mill.). The epi produces apparently more ethylene than its wild type VFN8 at every stage of vegetative and fruit growth and ripening; compared to VFN8, the epi fruit showed higher CO2 evolution, faster descending of chlorophyll, slightly quicker increase of carotenoid and lycopene, and faster reduction in pericarp firmness during maturation and ripening; and the mRNAs of three ripening-related genes including E8, pTOM5 and pTOM6 were at higher levels in epi. The ripening-related characteristics changing of the fruit are consistent with the increase of ethylene production and ripening-related genes expression. These results suggest that epi mutation possibly did not affect the ethylene perception and signaling during fruit ripening, and that the modified characteristics of fruit ripening possibly resulted from the ethylene over- production and increased expression of ripening-related genes.

Over-expression of GhDWF4 gene improved tomato fruit quality and accelerated fruit ripening

Brassinosteroids（BRs）, a class of steroidal phytohormones are essential for many biological processes in plant. However, little is known about their roles in fruit development. Tomato is a highly valuable vegetable and has been adopted as the model species for studying fruit growth, development, and ripening. To understand the role of endogenous BRs in the development of tomato fruit, the expression patterns of three homologues of DWF4 gene were investigated and the transgenic tomato plants were generated in which the Gh DWF4 gene from upland cotton（Gossypium hirsutum L.） was ectopically expressed. The contents of main quality components were analyzed in fruits of transgenic tomato line and non-transgenic line（control plant, CP） when the fruit was mature. Sl CYP90B3 that possesses high homology with Gh DWF4 preferentially expressed in mature fruit. Significantly higher contents of soluble sugar, soluble proteins, and vitamin C were obtained in fruit of transgenic tomato lines compared with those in the CP. Furthermore, overexpressing Gh DWF4 promoted fruit growth and ripening. The weight per fruit was increased by about 23% in transgenic lines. In addition, overexpressing Gh DWF4 promoted the germination of transgenic tomato seeds and hypocotyl elongation of seedlings. These results indicated that overexpressing Gh DWF4 gene in tomato could increase the contents of many nutrients in fruit and accelerate fruit ripening. It is suggested that increased endogenous BRs in fruit affect the growth and development of tomato fruit and therefore improved the nutrient quality of tomato.

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid OxidaseⅠon the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase Ⅰ on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l-aminocyclopropane-1-carboxylic acid oxidase 1 gene （LeAC01）, 1- aminocyclopropane-l-carboxylic acid oxidase 3 gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5）, and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig （AC＋＋） and the LeAC01 co-suppression tomatoes （V1187 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carded out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.

Movement of ACC oxidase 3 mRNA from seeds to flesh promotes fruit ripening in apple

Xenia,the phenomenon in which the pollen genotype directly affects the phenotypic characteristics of maternal tissues(i.e.,fruit ripening),has applications in crop production and breeding.However,the underlying molecular mechanism has yet to be elucidated.Here,we investigated whether mobile mRNAs from the pollen affect the ripening and quality-related characteristics of the fruit using cross-pollination between distinct Malus domestica(apple)cultivars.We demonstrated that hundreds of mobile mRNAs originating fromthe seeds are deliveredto thefruit.Wefoundthat themovementof one of these mRNAs,AcC oxidase 3(MdAco3),is coordinated with fruit ripening.Salicylic acid treatment,which can cause plasmodesmal closure,blocks MdAcO3 movement,indicating that MdAcO3 transcripts may move through the plasmodesmata.To assess the role of mobile MdAcO3 transcripts in apple fruit,we created MdACO3-GFP-expressing apple seeds using MdACO3-GFP-overexpressing pollen for pollination and showed that MdACO3 transcripts in the transgenic seeds move to the flesh,where they promote fruit ripening.Furthermore,we demonstrated that MdAco3 can be transported from the seeds to fruit in the fleshy-fruited species tomato and strawberry.These results underscore the potential of mobile mRNAs from seeds to influence fruit characteristics,providing an explanation for the xenia phenomenon.Notably,our findings highlight the feasibility of leveraging diverse pollen genomic resources,without resorting to genome editing,to improve fruitquality.

Transcriptomics and metabolomics analyses provide insights into postharvest ripening and senescence of tomato fruit under low temperature

Tomato is one of the most important vegetable crops in the world and is a model plant used to study the ripening of climacteric fleshy fruit.During the ripening process of tomato fruit,flavor and aroma metabolites,color,texture and plant hormones undergo significant changes.However,low temperatures delayed the ripening process of tomato fruit,inhibiting flavor compounds and ethylene production.Metabolomics and transcriptomics analyses of tomato fruit stored under low temperature(LT,5°C)and room temperature(RT,25°C)were carried out to investigate the effects of storage temperature on the physiological changes in tomato fruit after harvest.The results of transcriptomics changes revealed that the differentially expressed genes(DEGs)involved in tomato fruit ripening,including several kinds of transcription factors(TFs)(TCP,WRKY,MYB and bZIP),enzymes involved in cell wall metabolism[beta-galactosidase(β-GAL),pectinesterase(PE)and pectate lyase(PL),cellulose and cellulose synthase(CESA)],enzymes associated with fruit flavor and aroma[acetyltransferase(AT),malic enzyme(ME),lipoxygenase(LOX),aldehyde dehydrogenase(ALDH),alcohol dehydrogenase(ADH)and hexokinase(HK)],genes associated with heat stress protein 70 and genes involved in the production of plant hormones such as Ethylene responsive factor 1(ERF1),Auxin/indoleacetic acids protein(AUX/IAA),gibberellin regulated protein.Based on the above results,we constructed a regulatory network model of the effects of different temperatures during the fruit ripening process.According to the analysis of the metabolomics results,it was found that the contents of many metabolites in tomato fruit were greatly affected by storage temperature,including,organic acids(L-tartaric acid,a-hydroxyisobutyric acid and 4-acetamidobutyric acid),sugars(melezitose,beta-Dlactose,D-sedoheptulose 7-phosphate,2-deoxyribose 1-phosphate and raffinose)and phenols(coniferin,curcumin and feruloylputrescine).This study revealed the effects of storage temperature on postharvest tomato fruit and provided a basis for further understanding of the molecular biology and biochemistry of fruit ripening.

The brassinosteroid signaling component SlBZR1 promotes tomato fruit ripening and carotenoid accumulation

The plant hormone ethylene is essential for climacteric fruit ripening, although it is unclear how other phytohormones and their interactions with ethylene might affect fruit ripening. Here, we explored how brassinosteroids(BRs) regulate fruit ripening in tomato(Solanum lycopersicum) and how they interact with ethylene. Exogenous BR treatment and increased endogenous BR contents in tomato plants overexpressing the BR biosynthetic gene SlCYP90B3promoted ethylene production and fruit ripening.Genetic analysis indicated that the BR signaling regulators Brassinazole-resistant1(SlBZR1) and BRI1-EMS-suppressor1(SlBES1) act redundantly in fruit softening. Knocking out Sl BZR1 inhibited ripening through transcriptome reprogramming at the onset of ripening. Combined transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing identified 73 Sl BZR1-repressed targets and 203 Sl BZR1-induced targets involving major ripening-related genes, suggesting that Sl BZR1 positively regulates tomato fruit ripening.Sl BZR1 directly targeted several ethylene and carotenoid biosynthetic genes to contribute to the ethylene burst and carotenoid accumulation to ensure normal ripening and quality formation. Furthermore,knock-out of Brassinosteroid-insensitive2(Sl BIN2),a negative regulator of BR signaling upstream of Sl BZR1, promoted fruit ripening and carotenoid accumulation. Taken together, our results highlight the role of Sl BZR1 as a master regulator of tomato fruit ripening with potential for tomato quality improvement and carotenoid biofortification.

MaBEL1 regulates banana fruit ripening by activating cell wall and starch degradation-related genes

Banana is a typical subtropical fruit, sensitive to chilling injuries and prone to softening disorder.However, the underlying regulatory mechanisms of the softening disorder caused by cold stress remain obscure. Herein, we found that BEL1-LIKE HOMEODOMAIN transcription factor 1(MaBEL1)and its associated proteins regulate the fruit softening and ripening process. The transcript and protein levels of MaBEL1 were up-regulated with fruit ripening but severely repressed by the chilling stress. Moreover, the MaBEL1 protein interacted directly with the promoters of the cell wall and starch degradation-related genes, such as MaAMY3, MaXYL32, and MaEXP-A8. The transient overexpression of MaBEL1 alleviated fruit chilling injury and ripening disorder caused by cold stress and promoted fruit softening and ripening of “Fenjiao” banana by inducing ethylene production and starch and cell wall degradation. The accelerated ripening was also validated by the ectopic overexpression in tomatoes.Conversely, MaBEL1-silencing aggravated the chilling injury and ripening disorder and repressed fruit softening and ripening by inhibiting ethylene production and starch and cell wall degradation. MaABI5-like and MaEBF1, the two positive regulators of the fruit softening process,interacted with MaBEL1 to enhance the promoter activity of the starch and cell wall degradationrelated genes. Moreover, the F-box protein MaEBF1 does not modulate the degradation of MaBEL1, which regulates the transcription of MaABI5-like protein. Overall, we report a novel MaBEL1-MaEBF1-MaABI5-like complex system that mediates the fruit softening and ripening disorder in “Fenjiao” bananas caused by cold stress.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation

Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence watermelon fruit ripening. However,the molecular mechanisms are unclear. Here,we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4. Furthermore,we determined that the pyrophosphate-dependent phosphofructokinase(ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase(Cl GA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4. Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein(ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9’-cis-epoxycarotenoid dioxygenase 3(Cl NCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4. Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

Ethylene control in fruit quality assurance:A material science perspective

The waste of resources associated with fruit decay is rapidly spreading globally,threatening the interests of relevant practitioners and the health of consumer groups,and demanding precise solutions.Controlling fruit ripening through ethylene reg-ulation is one of the most important strategies for providing high-quality fruits.However,current materials for ethylene regulation still have difficulty realizing their application potential due to high manufacturing costs and performance defi-ciencies.In this review,the ethylene-controlled release materials for ripening based on molecular encapsulation and the ethylene scavengers for preservation based on mechanisms such as oxidation,photodegradation,and adsorption are presented.We discuss and analyze a wide range of materials in terms of mechanism,perfor-mance,potential of applicability,and sustainability.The ethylene release behavior of encapsulating materials depends on the form in which the ethylene binds to the material as well as on environmental factors(humidity and temperature).For ethy-lene scavengers,there are a variety of scavenging mechanisms,but they generally require porous materials as adsorption carriers.We highlight the great opportunity of designing soft crystalline porous materials as efficient ethylene adsorbent due to their unique structural properties.We present this review,including a summary of practical characteristics and deficiencies of various materials,to establish a system-atic understanding of fruit quality assurance materials applied to ethylene regulation,anticipating a promising prospect for these new materials.

Fig fruit ripening is regulated by the interaction between ethylene and abscisic acid

Fleshy fruit ripening is typically regulated by ethylene in climacteric fruits and abscisic acid(ABA)in non-climacteric fruits.Common fig(Ficus carica)shows a dual-ripening mechanism,which is not fully understood.Here,we detected separate peaks of ethylene and ABA in fig fruits at the onset-and onripening stages,in conjunction with a sharp rise in glucose and fructose contents.In a newly-designed split-fruit system,exogenous ethylene failed to rescue fluridone-inhibited fruit ripening,whereas exogenous ABA rescued 2-amino-ethoxy-vinyl glycine(AVG)-inhibited fruit ripening.Transcriptome analysis revealed changes in the expression of genes key to both ABA and ethylene biosynthesis and perception during fig fruit ripening.At the degreening stage,downregulation of FcACO2 or FcPYL8 retarded ripening,but downregulation of FcETR1/2 did not;unexpectedly,downregulation of FcAAO3 promoted ripening,but it inhibited ripening only before the de-greening stage.Furthermore,we detected an increase in ethylene emissions in the FcAAO3-RNAi ripening fruit and a decrease in ABA levels in the FcACO2-RNAi unripening fruit.Importantly,FcPYL8 can bind to ABA,suggesting that it functions as an ABA receptor.Our findings support the hypothesis that ethylene regulates the fig fruit ripening in an ABA-dependent manner.We propose a model for the role of the ABA-ethylene interaction in climacteric/non-climacteric processes.

Natural variation in the NAC transcription factor NONRIPENING contributes to melon fruit ripening

ThThe NAC transcription factor NONRIPENING(NOR)is a master regulator of climacteric fruit ripening.Melon(Cucumis melo L.)has climacteric and nonclimacteric fruit ripening varieties and is an ideal model to study fruit ripening.Two natural CmNAC-NOR variants,the climacteric haplotype CmNAC-NOR^(S,N) and the non-climacteric haplotype CmNAC-NOR^(A,S),have effects on fruit ripening;however,their regulatory mechanisms have not been elucidated.Here,we report that a natural mutation in the transcriptional activation domain of CmNAC-NORS,Ncontributes to climacteric melon fruit ripening.CmNAC-NOR knockout in the climacteric-type melon cultivar“BYJH”completely inhibited fruit ripening,while ripening was delayed by 5-8 d in heterozygous cmnac-nor mutant fruits.CmN AC-NOR directly activated carotenoid,ethylene,and abscisic acid biosynthetic genes to promote fruit coloration and ripening.Furthermore,CmNAC-NOR mediated the transcription of the“CmNAC-NOR-CmNAC73-CmCWINV2”module to enhance flesh sweetness.The transcriptional activation activity of the climacteric haplotype CmNAC-NORS,Non these target genes was significantly higher than that of the nonclimacteric haplotype CmNAC-NOR^(A,S).Moreover,CmNAC-NORS,Ncomplementation fully rescued the non-ripening phenotype of the tomato(Solanum lycopersicum)cr-nor mutant,while CmNAC-NOR^(A,S) did not.Our results provide insight into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon.

Role of ethylene response factors(ERFs)in fruit ripening

The ethylene response factors(ERFs)belong to the APETALA2/ethylene response factor(AP2/ERF)superfamily and act downstream of the ethylene signalling pathway to regulate the expression of ethylene responsive genes.In different species,ERFs have been reported to be involved in plant development,flower abscission,fruit ripening,and defense responses.In this review,based on the new progress made by recent studies,we summarize the specific role and mode of action of ERFs in regulating different aspects of ripening in both climacteric and non-climacteric fruits,and provide new insights into the role of ethylene in non-climacteric fruit ripening.

RECENT ADVANCES IN THE REGULATION OF CLIMACTERIC FRUIT RIPENING:HORMONE,TRANSCRIPTION FACTOR AND EPIGENETIC MODIFICATIONS

Fruit ripening is a complex developmental process made up of genetically programmed physiological and biochemical activities.It culminates in desirable changes in the structural and textural properties and is governed by a complex regulatory network.Much is known about ethylene,one of the most important metabolites promoting the ripening of climacteric fruits.However,the dynamic interplay between phytohormones also plays an important part.Additional regulatory factors such as transcription factors(TFs)and epigenetic modifications also play vital role in the regulation of climacteric fruit ripening.Here,we review and evaluate the complex regulatory network comprising interactions between hormones and the action of TFs and epigenetic modifications during climacteric fruit ripening.

Elucidating the role of SlXTH5 in tomato fruit softening

Fruit softening in tomato(Solanum lycopersicum)is closely associated with cell wall disassembly,which is brought about through the action of a range of cell wall structure-related enzymes and other proteins such as expansins.Xyloglucan endotransglucosylase/hydrolase(XTH)(EC 2.4.1.207 and/or EC 3.2.1.151)has been proposed to be key player involved in xyloglucan metabolism.SlXTH5 showed the highest expression level among all SlXTHs during tomato ripening.In this study,the role of SlXTH5 involved in tomato softening was investigated in CRISPR-based knockout mutants of SlXTH5.Loss-of-function of SlXTH5 in transgenic tomato lines resulted in slightly firmer fruit pericarp,but significantly decreased their color index compared with azygous wild type(WT)control fruits.Increased paste viscosity was detected in CRISPR mutants,indicating that the activity of SlXTH5 is responsible for maintaining cell wall structural integrity.Immunocytochemistry studies were performed using the monoclonal antibody probe LM25 to examine the localization and distribution of xyloglucan in the pericarp cells of the CRISPR mutant fruits.The data indicated more xyloglucan was retained in the pericarp of CRISPR mutant fruit than in WT control fruit.This study revealed the link between SlXTH5 and xyloglucan metabolism and indicated the potential of manipulating SlXTH5 to regulate fruit softening.

Genome-wide binding analysis of the tomato transcription factor SlDof1 reveals its regulatory impacts on fruit ripening

The DNA binding with one finger(Dof)proteins are plant-specific transcription factors involved in a variety of biological processes.However,little is known about their functions in fruit ripening,a flowering-plant-specific process that is required for seed maturation and dispersal.Here,we found that the tomato Dof transcription factor SlDof1,is necessary for normal fruit ripening.Knockdown of SlDof1 expression by RNA interference delayed ripening-related processes,including lycopene synthesis and ethylene production.Transcriptome profiling indicated that SlDof1 influences the expression of hundreds of genes,and a chromatin immunoprecipitation sequencing revealed a large number of SlDof1 binding sites.A total of 312 genes were identified as direct targets of SlDof1,among which 162 were negatively regulated by SlDof1 and 150 were positively regulated.The SlDof1 target genes were involved in a variety of metabolic pathways,and follow-up analyses verified that SlDof1 directly regulates some well-known ripening-related genes including ACS2 and PG2A as well as transcriptional repressor genes such as SlIAA27.Our findings provide insights into the transcriptional regulatory networks underlying fruit ripening and highlight a gene potentially useful for genetic engineering to control ripening.

Comparative transcriptome analysis of the climacteric of apple fruit uncovers the involvement of transcription factors affecting ethylene biosynthesis

Apple(Malus domestica)fruit generally undergoes a climacteric.During its ripening process,there is a peak in ethylene release and its firmness simultaneously decreases.Although more in-depth research into the mechanism of climacteric-type fruit ripening is being carried out,some aspects remain unclear.In this study,we compared the transcriptomes of 0-Pre and 15-Post(pre-and post-climacteric fruit),and 15-Post and 15-MCP[fruit treated with 1-MCP(1-methylcyclopropene)].Various transcription factors,such as MADS-box,ERF,NAC,Dof and SHF were identified among the DEGs(differential gene expressions).Furthermore,these transcription factors were selected for further validation analysis by qRT-PCR.Moreover,yeast one hybrid(Y1H),β-glucuronidase(GUS)transactivation assay and dual-luciferase reporter assay showed that MdAGL30,MdAGL104,MdERF008,MdNAC71,MdDof1.2,MdHSFB2a and MdHSFB3 bound to MdACS1 promoter and directly regulated its transcription,thereby regulating ethylene biosynthesis in apple fruit.Our results provide useful information and new insights for research on apple fruit ripening.

Effects of calcium application on apple fruit softening during storage revealed by proteomics and phosphoproteomics

Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely unknown.In order to study the changes of proteins and enriched phosphopeptides,we conducted TMT labeling,bio-material-based PTM enrichment based on mass spectrometry in Ca-treated‘Golden Delicious’(GD)apple fruit(Malus×domestica).This dataset presents a comprehensive overview of the critical pathways involved in fruit ripening.A total of 47 proteins and 124 phosphoproteins significantly changed in Ca-treated fruit,which are crucial for regulating the cell wall and cytoskeleton,Ca-mediated signaling and transport,ethylene production,protein fate,especially ubiquitination-based protein degradation,and primary and secondary metabolisms.Our results indicated that Ca inhibited the abundance of polygalacturonase(PG)activity and increased the phosphorylation level of CSLD3.PG and phosphorylation were involved in cell wall degradation,thereby delaying fruit softening.As a secondary messenger,Ca-mediated signaling subsequently triggered downstream mitogen-activated protein kinases(MAPK)cascades and activated the membrane,transport,and ROS signaling.Moreover,MdEIN2,a key enzyme involved in the ubiquitin of protein modification,increased at Ser753 and Ser758 in Ca-treated fruit.Furthermore,diverse primary and secondary metabolisms including glycolysis,fatty acid metabolism,and oxidation respiratory chain were modulated to prevent fruit softening.These results provide basic information from protein and phosphorylation levels for apple fruit ripening during storage,which may be helpful for apple fruit storage control.