The MADS-box protein SlTAGL1 regulates a ripening-associated SlDQD/SDH2 involved in flavonoid biosynthesis and resistance against Botrytis cinerea in post-harvest tomato fruit.

3-Dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites is still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers resistance against Botrytis cinerea attack in post-harvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomatoes.

Genome-wide characterization of the tomato GASA family identifies SlGASA1 as a repressor of fruit ripening.

Gibberellins (GAs) play crucial roles in a wide range of developmental processes and stress responses in plants. However, the roles of GA-responsive genes in tomato (Solanum lycopersicum) fruit development remain largely unknown. Here, we identify 17 GASA (Gibberellic Acid-Stimulated Arabidopsis) family genes in tomato. These genes encode proteins with a cleavable signal peptide at their N terminus and a conserved GASA domain at their C terminus. The expression levels of all tomato GASA family genes were responsive to exogenous GA treatment, but adding ethylene eliminated this effect. Comprehensive expression profiling of SlGASA family genes showed that SlGASA1 follows a ripening-associated expression pattern, with low expression levels during fruit ripening, suggesting it plays a negative role in regulating ripening. Overexpressing SlGASA1 using a ripening-specific promoter delayed the onset of fruit ripening, whereas SlGASA1-knockdown fruits displayed accelerated ripening. Consistent with their delayed ripening, SlGASA1-overexpressing fruits showed significantly reduced ethylene production and carotenoid contents compared to the wild type. Moreover, ripening-related genes were downregulated in SlGASA1-overexpressing fruits but upregulated in SlGASA1-knockdown fruits compared to the wild type. Yeast two-hybrid, co-immunoprecipitation, transactivation, and DNA pull-down assays indicated that SlGASA1 interacts with the key ripening regulator FRUITFULL1 and represses its activation of the ethylene biosynthesis genes ACS2 and ACO1. Our findings shed new light on the role and mode of action of a GA-responsive gene in tomato fruit ripening.

SlERF.F12 modulates the transition to ripening in tomato fruit by recruiting the co-repressor TOPLESS and histone deacetylases to repress key ripening genes.

Ethylene response factors (ERFs) are downstream components of ethylene-signaling pathways known to play critical roles in ethylene-controlled climacteric fruit ripening, yet little is known about the molecular mechanism underlying their mode of action. Here, we demonstrate that SlERF.F12, a member of the ERF.F subfamily containing Ethylene-responsive element-binding factor-associated Amphiphilic Repression (EAR) motifs, negatively regulates the onset of tomato (Solanum lycopersicum) fruit ripening by recruiting the co-repressor TOPLESS 2 (TPL2) and the histone deacetylases (HDAs) HDA1/HDA3 to repress the transcription of ripening-related genes. The SlERF.F12-mediated transcriptional repression of key ripening-related genes 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2 (ACS2), ACS4, POLYGALACTURONASE 2a, and PECTATE LYASE is dependent on the presence of its C-terminal EAR motif. We show that SlERF.F12 interacts with the co-repressor TPL2 via the C-terminal EAR motif and recruits HDAs SlHDA1 and SlHDA3 to form a tripartite complex in vivo that actively represses transcription of ripening genes by decreasing the level of the permissive histone acetylation marks H3K9Ac and H3K27Ac at their promoter regions. These findings provide new insights into the ripening regulatory network and uncover a direct link between repressor ERFs and histone modifiers in modulating the transition to ripening of climacteric fruit.

Integrative analyses of metabolome and genome-wide transcriptome reveal the regulatory network governing flavor formation in kiwifruit (Actinidia chinensis).

Soluble sugars, organic acids and volatiles are important components that determine unique fruit flavor and consumer preferences. However, the metabolic dynamics and underlying regulatory networks that modulate overall flavor formation during fruit development and ripening remain largely unknown for most fruit species. In this study, by integrating flavor-associated metabolism and transcriptome data from 12 fruit developmental and ripening stages of Actinidia chinensis cv Hongyang, we generated a global map of changes in the flavor-related metabolites throughout development and ripening of kiwifruit. Using this dataset, we constructed complex regulatory networks allowing to identify key structural genes and transcription factors that regulate the metabolism of soluble sugars, organic acids and important volatiles in kiwifruit. Moreover, our study revealed the regulatory mechanism involving key transcription factors regulating flavor metabolism. The modulation of flavor metabolism by the identified key transcription factors was confirmed in different kiwifruit species providing the proof of concept that our dataset provides a suitable tool for clarification of the regulatory factors controlling flavor biosynthetic pathways that have not been previously illuminated. Overall, in addition to providing new insight into the metabolic regulation of flavor during fruit development and ripening, the outcome of our study establishes a foundation for flavor improvement in kiwifruit.

Molecular basis of the formation and removal of fruit astringency.

Astringency is a dry puckering mouthfeel mainly generated by the binding of tannins with proteins in the mouth. Tannins confer benefits such as resistance to biotic stresses and have antioxidant activity, and moderate concentrations of tannins can improve the flavor of fruits or their products. However, fruits with high contents of tannins have excessive astringency, which is undesirable. Thus, the balance of astringency formation and removal is extremely important for human consumption of fruit and fruit-based products. In recent years, the understanding of fruit astringency has moved beyond the biochemical aspects to focus on the genetic characterization of key structural genes and their transcriptional regulators that cause astringency. This article provides an overview of astringency formation and evaluation. We summarize the methods of astringency regulation and strategies and mechanisms for astringency removal, and discuss perspectives for future exploration and modulation of astringency for fruit quality improvement.

Ethylene response factor AcERF91 affects ascorbate metabolism via regulation of GDP-galactose phosphorylase encoding gene (AcGGP3) in kiwifruit.

Kiwifruit is known as 'the king of vitamin C' because of the high content of ascorbic acid (AsA) in the fruit. Deciphering the regulatory network and identification of the key regulators mediating AsA biosynthesis is vital for fruit nutrition and quality improvement. To date, however, the key transcription factors regulating AsA metabolism during kiwifruit developmental and ripening processes remains largely unknown. Here, we generated a putative transcriptional regulatory network mediating ascorbate metabolism by transcriptome co-expression analysis. Further studies identified an ethylene response factor AcERF91 from this regulatory network, which is highly co-expressed with a GDP-galactose phosphorylase encoding gene (AcGGP3) during fruit developmental and ripening processes. Through dual-luciferase reporter and yeast one-hybrid assays, it was shown that AcERF91 is able to bind and directly activate the activity of the AcGGP3 promoter. Furthermore, transient expression of AcERF91 in kiwifruit fruits resulted in a significant increase in AsA content and AcGGP3 transcript level, indicating a positive role of AcERF91 in controlling AsA accumulation via regulation of the expression of AcGGP3. Overall, our results provide a new insight into the regulation of AsA metabolism in kiwifruit.

Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the subcellular sites of L-theanine biosynthesis in tea.

l-Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l-theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l-theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l-theanine synthetase and also to determine the subcellular localization of fluorescent-tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l-theanine biosynthesis, and cytosol-located CsTSI was the key l-theanine synthase. In tea shoot tissue, l-theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l-theanine synthases. In addition, l-theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.

Genome-Wide Identification of DNA Methylases and Demethylases in Kiwifruit (Actinidia chinensis).

DNA methylation plays an important role in a wide range of developmental and physiological processes in plants. It is primarily catalyzed and regulated by cytosine-5 DNA methyltransferases (C5-MTases) and a group of DNA glycosylases that act as demethylases. To date, no genome-scale analysis of the two kiwifruit (Actinidia chinensis) families has been undertaken. In our study, nine C5-MTases and seven DNA demethylase genes were identified in the kiwifruit genome. Through selective evolution analysis, we found that there were gene duplications in C5-MTases and demethylases, which may have arisen during three genome doubling events followed by selection during evolution of kiwifruit. Expression analysis of DNA methylases (C5-MTases) and demethylases identified changes in transcripts of DNA methylation and demethylation genes during both vegetative and reproductive development. Moreover, we found that some members of the two methylase/demethylase families may also be involved in fruit ripening and the regulation of softening. Our results help to better understand the complex roles of methylation/demethylation in plants and provide a foundation for analyzing the role of DNA methylation modification in kiwifruit growth, development and ripening.

Like Heterochromatin Protein 1b represses fruit ripening via regulating the H3K27me3 levels in ripening-related genes in tomato.

Polycomb group (PcG) proteins play vital roles in plant development via epigenetically repressing the transcription of target genes. However, to date, their function in fruit ripening is largely unknown. Combining reverse genetic approaches, physiological methods, yeast two-hybrid, co-immunoprecipitation, and chromatin immunoprecipitation assays, we show that Like Heterochromatin Protein 1b (SlLHP1b), a tomato Polycomb Repressive Complex 1 (PRC1)-like protein with a ripening-related expression pattern, represses fruit ripening via colocalization with epigenetic mark H3K27me3. RNA interference (RNAi)-mediated downregulation of SlLHP1b advanced ripening initiation, climacteric ethylene production, and fruit softening, whereas SlLHP1b overexpression delayed these events. Ripening-related genes were significantly upregulated in SlLHP1b RNAi fruits and downregulated in overexpressing fruits compared with wild-type. Furthermore, SlLHP1b protein interacts with ripening regulator MSI1, a subunit of the PRC2 complex. Moreover, SlLHP1b also binds the epigenetic histone mark H3K27me3 in vivo and chromatin immunoprecipitation-quantitative PCR results showed binding occurs preferentially to regions of ripening-associated chromatin marked by histone H3K27me3. Furthermore, the H3K27me3 levels in chromatin of ripening-related genes is negatively correlated with accumulation of their transcripts in SlLHP1b down or upregulated fruits during ripening. Our findings reveal a novel regulatory function of SlLHP1b in fruit and provide new insights into the PcG-mediated epigenetic regulation of climacteric fruit ripening.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution.

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for 'Youshi' (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembled D. oleifera genome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that the D. oleifera genome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO2 treatment to remove astringency. Using the D. oleifera genome as the reference, SLAF-seq indicated that 'Youshi' is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.

Effect of Ethylene on Cell Wall and Lipid Metabolism during Alleviation of Postharvest Chilling Injury in Peach.

Peach is prone to postharvest chilling injury (CI). Here it was found that exogenous ethylene alleviated CI, accompanied by an increased endogenous ethylene production. Ethylene treatment resulted in a moderately more rapid flesh softening as a result of stronger expression of genes encoding expansin and cell wall hydrolases, especially xylosidase and galactosidase. Ethylene treatment alleviated internal browning, accompanied by changes in expression of polyphenol oxidase, peroxidase and lipoxygenases. An enhanced content of phospholipids and glycerolipids and a reduced content of ceramide were observed in ethylene-treated fruit, and these were associated with up-regulation of lipid phosphate phosphatase, fatty acid alpha-hydroxylase, and golgi-localized nucleotide sugar transporter, as well as down-regulation of aminoalcoholphosphotransferases. Expression of two ethylene response factors (ERFs), ESE3 and ABR1, was highly correlated with that of genes involved in cell wall metabolism and lipid metabolism, respectively. Furthermore, the expression of these two ERFs was strongly regulated by ethylene treatment and the temperature changes during transfer of fruit into or out of cold storage. It is proposed that ERFs fulfill roles as crucial integrators between cell wall modifications and lipid metabolism involved in CI processes ameliorated by exogenous ethylene.

Genome-Wide Identification and Functional Analysis of Carboxylesterase and Methylesterase Gene Families in Peach (Prunus persica L. Batsch).

Carboxylesterases (CXE) and methylesterases (MES) are hydrolytic enzymes that act on carboxylic esters and are involved in plant metabolic processes and defense responses. A few functions of plant CXE and MES genes have been identified but very little information is available about the role of most members. We made a comprehensive study of this gene family in a commercially important species, peach (Prunus persica L. Batsch). A total of 33 peach CXE genes and 18 MES genes were identified and shown to be distributed unevenly between the chromosomes. Based on phylogenetic analysis, CXEs and MESs clustered into two different branches. Comparison of the positions of intron and differences in motifs revealed the evolutionary relationships between CXE and MES genes. RNA-seq revealed differential expression patterns of CXE/MESs in peach flower, leaf, and ripening fruit and in response to methyl jasmonate (MeJA) and ultraviolet B treatment. Transcript levels of candidate genes were verified by real-time quantitative PCR. Heterologous expression in Escherichia coli identified three CXEs that were involved in the hydrolysis of volatile esters in vitro. Furthermore, two recombinant MES proteins were identified that could hydrolyze MeJA and methyl salicylate. Our results provide an important resource for the identification of functional CXE and MES genes involved in the catabolism of volatile esters, responses to biotic and abiotic stresses and activation of signaling molecules such as MeJA and methyl salicylate.

Lycopene cyclases determine high α-/ß-carotene ratio and increased carotenoids in bananas ripening at high temperatures.

Bananas are a recommended food source to alleviate vitamin A deficiency because they contain a high ratio of provitamin A precursors. The objective of this study was to investigate carotenoid accumulation pattern in banana fruits during postharvest ripening and the mechanisms regulating this process. Ripe banana pulp had an unusually high α-/ß-carotene ratio (1.05), and the carotenoid contents increased (p ≤ 0.05) under light and high temperature. We analyzed the sequences, transcript levels, and functions of genes involved in carotenoid synthesis. The high ratio of α-/ß-carotene in ripe banana fruit was explained by the high flux to the α-carotene biosynthetic pathway, as reflected by high transcript levels of LCYE, and the weak flux to the ß-carotene branch of the biosynthetic pathway due to inactive MaLCYB1.2. High temperature during ripening up-regulated the transcript levels of genes involved in the α- and ß-carotene biosynthesis pathways and the activities of their encoded enzymes.

Microscopic Analyses of Fruit Cell Plastid Development in Loquat (Eriobotrya japonica) during Fruit Ripening.

Plastids are sites for carotenoid biosynthesis and accumulation, but detailed information on fruit plastid development and its relation to carotenoid accumulation remains largely unclear. Here, using Baisha (BS; white-fleshed) and Luoyangqing (LYQ; red-fleshed) loquat (Eriobotrya japonica), a detailed microscopic analysis of plastid development during fruit ripening was carried out. In peel cells, chloroplasts turned into smaller chromoplasts in both cultivars, and the quantity of plastids in LYQ increased by one-half during fruit ripening. The average number of chromoplasts per peel cell in fully ripe fruit was similar between the two cultivars, but LYQ peel cell plastids were 20% larger and had a higher colour density, associated with the presence of larger plastoglobules. In flesh cells, chromoplasts could be observed only in LYQ during the middle and late stages of ripening, and the quantity on a per-cell basis was higher than that in peel cells, but the size of chromoplasts was smaller. It was concluded that chromoplasts are derived from the direct conversion of chloroplasts to chromoplasts in the peel, and from de novo differentiation of proplastids into chromoplasts in flesh. The relationship between plastid development and carotenoid accumulation is discussed.

A critical evaluation of the role of ethylene and MADS transcription factors in the network controlling fleshy fruit ripening.

Contents Summary 1724 I. Introduction 1725 II. Ripening genes 1725 III. The importance of ethylene in controlling ripening 1727 IV. The importance of MADS-RIN in controlling ripening 1729 V. Interactions between components of the ripening regulatory network 1734 VI. Conclusions 1736 Acknowledgements 1738 Author contributions 1738 References 1738 SUMMARY: Understanding the regulation of fleshy fruit ripening is biologically important and provides insights and opportunities for controlling fruit quality, enhancing nutritional value for animals and humans, and improving storage and waste reduction. The ripening regulatory network involves master and downstream transcription factors (TFs) and hormones. Tomato is a model for ripening regulation, which requires ethylene and master TFs including NAC-NOR and the MADS-box protein MADS-RIN. Recent functional characterization showed that the classical RIN-MC gene fusion, previously believed to be a loss-of-function mutation, is an active TF with repressor activity. This, and other evidence, has highlighted the possibility that MADS-RIN itself is not important for ripening initiation but is required for full ripening. In this review, we discuss the diversity of components in the control network, their targets, and how they interact to control initiation and progression of ripening. Both hormones and individual TFs affect the status and activity of other network participants, which changes overall network signaling and ripening outcomes. MADS-RIN, NAC-NOR and ethylene play critical roles but there are still unanswered questions about these and other TFs. Further attention should be paid to relationships between ethylene, MADS-RIN and NACs in ripening control.

Transcriptomic changes triggered by carotenoid biosynthesis inhibitors and role of Citrus sinensis phosphate transporter 4;2 (CsPHT4;2) in enhancing carotenoid accumulation.

MAIN CONCLUSION: Carotenoid accumulation and chromoplast development in orange were perturbed by carotenoid inhibitors, and candidate genes were identified via transcriptomic analysis. The role of CsPHT4;2 in enhancing carotenoid accumulation was revealed. Carotenoids are important plant pigments and their accumulation can be affected by biosynthesis inhibitors, but the genes involved were largely unknown. Here, application of norflurazon (NFZ), 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) and clomazone for 30 days to in vitro cultured sweet orange juice vesicles caused over-accumulation of phytoene (over 1000-fold), lycopene (2.92 µg g-1 FW, none in control), and deficiency in total carotenoids (reduced to 22%), respectively. Increased carotenoids were associated with bigger chromoplasts with enlarged plastoglobules or a differently crystalline structure in NFZ, and CPTA-treated juice vesicles, respectively. Global transcriptomic changes following inhibitor treatments were profiled. Induced expression of 1-deoxy-D-xylulose 5-phosphate synthase 1 by CPTA, hydroxymethylbutenyl 4-diphosphate reductase by both NFZ and CPTA, and reduced expression of chromoplast-specific lycopene ß-cyclase by CPTA, as well as several downstream genes by at least one of the three inhibitors were observed. Expression of fibrillin 11 (CsFBN11) was induced following both NFZ and CPTA treatments. Using weighted correlation network analysis, a plastid-type phosphate transporter 4;2 (CsPHT4;2) was identified as closely correlated with high-lycopene accumulation induced by CPTA. Transient over-expression of CsPHT4;2 significantly enhanced carotenoid accumulation over tenfold in 'Cara Cara' sweet orange juice vesicle-derived callus. The study provides a valuable overview of the underlying mechanisms for altered carotenoid accumulation and chromoplast development following carotenoid inhibitor treatments and sheds light on the relationship between carotenoid accumulation and chromoplast development.

Cytological and molecular characterization of carotenoid accumulation in normal and high-lycopene mutant oranges.

Ripe Cara Cara sweet orange contains 25 times as much carotenoids in flesh as Newhall sweet orange, due to high accumulation of carotenes, mainly phytoene, lycopene and phytofluene. Only yellow globular chromoplasts were observed in Newhall flesh. Distinct yellow globular and red elongated crystalline chromoplasts were found in Cara Cara but only one type of chromoplast was present in each cell. The red crystalline chromoplasts contained lycopene as a dominant carotenoid and were associated with characteristic carotenoid sequestering structures. The increased accumulation of linear carotenes in Cara Cara is not explained by differences in expression of all 18 carotenogenic genes or gene family members examined, or sequence or abundance of mRNAs from phytoene synthase (PSY) and chromoplast-specific lycopene ß-cyclase (CYCB) alleles. 2-(4-Chlorophenylthio)-triethylamine hydrochloride (CPTA) enhanced lycopene accumulation and induced occurrence of red crystalline chromoplasts in cultured Newhall juice vesicles, indicating that carotenoid synthesis and accumulation can directly affect chromoplast differentiation and structure. Norflurazon (NFZ) treatment resulted in high accumulation of phytoene and phytofluene in both oranges, and the biosynthetic activity upstream of phytoene desaturase was similar in Newhall and Cara Cara. Possible mechanisms for high carotene accumulation and unique development of red crystalline chromoplasts in Cara Cara are discussed.

Transcriptomic and metabolic analyses provide new insights into chilling injury in peach fruit.

Low temperature conditioning (LTC) alleviates peach fruit chilling injury but the underlying molecular basis is poorly understood. Here, changes in transcriptome, ethylene production, flesh softening, internal browning and membrane lipids were compared in fruit maintained in constant 0 °C and LTC (pre-storage at 8 °C for 5 d before storage at 0 °C). Low temperature conditioning resulted in a higher rate of ethylene production and a more rapid flesh softening as a result of higher expression of ethylene biosynthetic genes and a series of cell wall hydrolases. Reduced internal browning of fruit was observed in LTC, with lower transcript levels of polyphenol oxidase and peroxidase, but higher lipoxygenase. Low temperature conditioning fruit also showed enhanced fatty acid content, increased desaturation, higher levels of phospholipids and a preferential biosynthesis of glucosylceramide. Genes encoding cell wall hydrolases and lipid metabolism enzymes were coexpressed with differentially expressed ethylene response factors (ERFs) and contained ERF binding elements in their promoters. In conclusion, LTC is a special case of cold acclimation which increases ethylene production and, operating through ERFs, promotes both softening and changes in lipid composition and desaturation, which may modulate membrane stability, reducing browning and contributing to alleviation of peach fruit chilling injury.