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Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold-induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT-induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis-element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co-activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 μg g -1 FW) than MrMYB5-MrbHLH2 (7.43 μg g -1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES ( LOB ) TF, SlLOB1 , up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 ( EXP1 ) are strongly suppressed in Sl LOB1 RNA interference lines, while EXP1 is induced in Sl LOB1 -overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, Sl LOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Flavor-associated volatile chemicals make major contributions to consumers' perception of fruits. Although great progress has been made in establishing the metabolic pathways associated with volatile synthesis, much less is known about the regulation of those pathways. Knowledge of how those pathways are regulated would greatly facilitate efforts to improve flavor. Volatile esters are major contributors to fruity flavor notes in many species, providing a good model to investigate the regulation of volatile synthesis pathways. Here we initiated a study of peach (Prunus persica L. Batsch) fruits, and identified that the alcohol acyltransferase PpAAT1 contributes to ester formation. We next identified the transcription factor (TF) PpNAC1 as an activator of PpAAT1 expression and ester production. These conclusions were based on in vivo and in vitro experiments and validated by correlation in a panel of 30 different peach cultivars. Based on homology between PpNAC1 and the tomato (Solanum lycopersicum) TF NONRIPENING (NOR), we identified a parallel regulatory pathway in tomato. Overexpression of PpNAC1 enhances ripening in a nor mutant and restores synthesis of volatile esters in tomato fruits. Furthermore, in the NOR-deficient mutant tomatoes generated by CRISPR/Cas9, lower transcript levels of SlAAT1 were detected. The apple (Malus domestica) homolog MdNAC5 also stimulates MdAAT1 expression via binding to this gene's promoter. In addition to transcriptional control, epigenetic analysis showed that increased expression of NACs and AATs is associated with removal of the repressive mark H3K27me3 during fruit ripening. Our results support a conserved molecular mechanism in which NAC TFs activate ripening-related AAT expression, which in turn catalyzes volatile ester formation in multiple fruit species., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

'Jimba', a well-known white flowered chrysanthemum cultivar, occasionally and spontaneously produces red colored petals under natural cultivation, but there is little information about the molecular regulatory mechanism underlying this process. We analysed the expression patterns of 91 MYB transcription factors in 'Jimba' and 'Turning red Jimba' and identified an R3 MYB, CmMYB#7, whose expression was significantly decreased in 'Turning red Jimba' compared with 'Jimba', and confirmed it is a passive repressor of anthocyanin biosynthesis. CmMYB#7 competed with CmMYB6, which together with CmbHLH2 is an essential component of the anthocyanin activation complex, for interaction with CmbHLH2 through the bHLH binding site in the R3 MYB domain. This reduced binding of the CmMYB6-CmbHLH2 complex and inhibited its ability to activate CmDFR and CmUFGT promoters. Moreover, using transient expression assays we demonstrated that changes in the expression of CmMYB#7 accounted for alterations in anthocyanin content. Taken together, our findings illustrate that CmMYB#7 is a negative regulator of anthocyanin biosynthesis in chrysanthemum., (© Society for Experimental Biology 2019.)

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., (© 2018 The Authors. New Phytologis © 2018 New Phytologist Trust.)

4-Hydroxy-2,5-dimethyl-3(2H)-furanone is a major contributor to the aroma of strawberry ( Fragaria × ananassa ) fruit, and the last step in its biosynthesis is catalyzed by strawberry quinone oxidoreductase (FaQR). Here, an ethylene response factor (FaERF#9) was characterized as a positive regulator of the FaQR promoter. Linear regression analysis indicated that FaERF#9 transcript levels were correlated significantly with both FaQR transcripts and furanone content in different strawberry cultivars. Transient overexpression of FaERF#9 in strawberry fruit significantly increased FaQR expression and furaneol production. Yeast one-hybrid assays, however, indicated that FaERF#9 by itself did not bind to the FaQR promoter. An MYB transcription factor (FaMYB98) identified in yeast one-hybrid screening of the strawberry cDNA library was capable of both binding to the promoter and activating the transcription of FaQR by ∼5.6-fold. Yeast two-hybrid assay and bimolecular fluorescence complementation confirmed a direct protein-protein interaction between FaERF#9 and FaMYB98, and in combination, they activated the FaQR promoter 14-fold in transactivation assays. These results indicate that an ERF-MYB complex containing FaERF#9 and FaMYB98 activates the FaQR promoter and up-regulates 4-hydroxy-2,5-dimethyl-3(2H)-furanone biosynthesis in strawberry., (© 2018 American Society of Plant Biologists. All rights reserved.)

The unique flavor of Citrus fruit depends on complex combinations of soluble sugars, organic acids, and volatile compounds. The monoterpene E-geraniol is an important volatile, contributing to flavor in sweet orange (Citrus sinensis Osbeck). Moreover, antifungal activity of E-geraniol has also been observed. However, the terpene synthase (TPS) responsible for its synthesis has not been identified in sweet orange. Terpene synthase 16 (CitTPS16) was shown to catalyze synthesis of E-geraniol in vitro, and transient overexpression of CitTPS16 in fruits and leaves of Newhall sweet orange resulted in E-geraniol accumulation in vivo. Having identified the responsible enzyme, we next examined transcriptional regulation of CitTPS16 in the fruit. Among cloned members of the AP2/ERF transcription factor gene family, CitERF71 showed a similar expression pattern to CitTPS16. Moreover, CitERF71 was able to activate the CitTPS16 promoter based on results from transient dual-luciferase assays and yeast one-hybrid assays. EMSAs showed that CitERF71 directly binds to ACCCGCC and GGCGGG motifs in the CitTPS16 promoter. These results indicate an important role for CitERF71 in transcriptional regulation of CitTP16 and, therefore, in controlling production of E-geraniol in Citrus fruit., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Heat shock transcription factors (Hsfs) play a role in plant responses to stress. Citrus is an economically important fruit whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for citrus. A genome-wide analysis was carried out in Citrus clementina to identify Hsf genes, named CcHsfs. Eighteen CcHsfs were identified and classified into three main clades (clades A, B and C) according to the structural characteristics and the phylogenetic comparison with Arabidopsis and tomato. MEME motif analysis highlighted the conserved DBD and HR-A/B domains, which were similar to Hsf protein structures in other species. Gene expression analysis in Ponkan (Citrus reticulata Blanco cv. Ponkan) fruit identified 14 Hsf genes, named CrHsf, as important candidates for a role in fruit development and ripening, and showed seven genes to be expressed in response to hot air stress. CrHsfB2a and CrHsfB5 were considered to be important regulators of citrate content and showed variation in both developmentally-related and hot air-triggered citrate degradation processes. In summary, the data obtained from this investigation provides the basis for further study to dissect Hsf function during fruit development as well as in response to heat stress and also emphasizes the potential importance of CrHsfs in regulation of citrate metabolism in citrus fruit., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Background: The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins., Results: A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones., Conclusions: The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

SUMMARY: Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in animal and human nutrition. Low temperature (LT) promotes anthocyanin biosynthesis in many species including blood orange. A retrotransposon in the promoter of Ruby1, which encodes an R2R3 MYB transcription factor, controls cold‐induced anthocyanin accumulation in blood orange flesh. However, the specific mechanism remains unclear. In this study, we characterized two LT‐induced ETHYLENE RESPONSE FACTORS (CsERF054 and CsERF061). Both CsERF054 and CsERF061 can activate the expression of CsRuby1 by directly binding to a DRE/CRT cis‐element within the retrotransposon in the promoter of CsRuby1, thereby positively regulating anthocyanin biosynthesis. Further investigation indicated that CsERF061 also forms a protein complex with CsRuby1 to co‐activate the expression of anthocyanin biosynthetic genes, providing a dual mechanism for the upregulation of the anthocyanin pathway. These results provide insights into how LT mediates anthocyanin biosynthesis and increase the understanding of the regulatory network of anthocyanin biosynthesis in blood orange. Significance Statement: Anthocyanins are natural pigments and dietary antioxidants that play multiple biological roles in plants and are important in human nutrition. Low temperature is a necessary condition for anthocyanin accumulation in blood orange and the retrotransposon promoter of Ruby1 controls this process. Our results provide upstream insights into its cold response and enrich the understanding of the regulatory network of anthocyanin biosynthesis in blood orange. These results are of fundamental importance and great practical significance. [ABSTRACT FROM AUTHOR]

Summary: Flavor‐imparting volatile chemicals accumulate as fruits ripen, making major contributions to taste. The NAC transcription factor nonripening (NAC‐NOR) and DNA demethylase 2 (SlDML2) are essential for tomato fruit ripening, but details of the potential roles and the relationship between these two regulators in the synthesis of volatiles are lacking.Here, we show substantial reductions in fatty acid and carotenoid‐derived volatiles in tomato slnor and sldml2 mutants. An unexpected finding is the redundancy and divergence in volatile profiles, biosynthetic gene expression, and DNA methylation in slnor and sldml2 mutants relative to wild‐type tomato fruit. Reduced transcript levels are accompanied by hypermethylation of promoters, including the NAC‐NOR target gene lipoxygenase (SlLOXC) that is involved in fatty acid‐derived volatile synthesis.Interestingly, NAC‐NOR activates SlDML2 expression by directly binding to its promoter both in vitro and in vivo. Meanwhile, reduced NAC‐NOR expression in the sldml2 mutant is accompanied by hypermethylation of its promoter. These results reveal a relationship between SlDML2‐mediated DNA demethylation and NAC‐NOR during tomato fruit ripening.In addition to providing new insights into the metabolic modulation of flavor volatiles, the outcome of our study contributes to understanding the genetics and control of fruit ripening and quality attributes in tomato. [ABSTRACT FROM AUTHOR]

High CO2 treatment is a widely used deastringency technology that causes the accumulation of acetaldehyde which precipitates the astringent soluble tannins from persimmon fruit, making them more attractive to consumers. The identification of DkADH1 and DkPDC2 (the key genes for acetaldehyde accumulation) and their regulators (e.g., ERFs), has significantly advanced our understanding of the fruit deastringency mechanism, but other TFs are also involved in the high CO2 response. Here, 32 DkDofs genes were identified from 'Gongcheng-shuishi' persimmon, with nine of them shown to differentially respond to high CO2 treatment. Dual luciferase assay indicated that DkDof3 and DkDof6 could repress the promoters of DkADH1 and DkPDC2, respectively. EMSA assay showed that DkDof3 and DkDof6 physically interacted with probes containing T/AAAAG elements from the DkADH1 promoter, whereas they failed to recognize similar elements from the DkPDC2 promoter. The expression of DkDof3 and DkDof6 was also found to be repressed in different persimmon cultivars in response to high CO2 treatment. It is proposed that DkDof3 and DkDof6 were involved in fruit deastringency by regulating the expression of DkADH1 and DkPDC2 in different persimmon cultivars. [ABSTRACT FROM AUTHOR]

Loquat fruit are susceptible to chilling injuries induced by postharvest storage at low temperature. The major symptoms are increased lignin content and flesh firmness, which cause a leathery texture. Pretreatment with methyl jasmonate (MeJA) can alleviate this low-temperature-induced lignification, but the mechanism is not understood. In this study, we characterized a novel class III peroxidase, EjPRX12, and studied its relationship to lignification. Transcript levels of EjPRX12 were attenuated following MeJA pretreatment, consistent with the reduced lignin content in fruit. In vitro enzyme activity assay indicated that EjPRX12 polymerized sinapyl alcohol, and overexpression of EjPRX12 in Arabidopsis promoted lignin accumulation, indicating that it plays a functional role in lignin polymerization. We also identified an HD-ZIP transcription factor, EjHB1, repressed by MeJA pretreatment, which directly bound to and significantly activated the EjPRX12 promoter. Overexpression of EjHB1 in Arabidopsis promoted lignin accumulation with induced expression of lignin-related genes, especially AtPRX64. Furthermore, a JAZ-interacting repressor, EjbHLH14, was characterized, and it is proposed that MeJA pretreatment caused EjbHLH14 to be released to repress the expression of EjHB1. These results identified a novel regulatory pathway involving EjbHLH14-EjHB1-EjPRX12 and revealed the molecular mechanism whereby MeJA alleviated lignification of loquat fruit at low temperature. [ABSTRACT FROM AUTHOR]

Sugars are the primary products of photosynthesis and play an important role in plant growth and development. They contribute to sweetness and flavor of fleshy fruits and are pivotal to fruit quality, and their translocation and allocation are mainly dependent on sugar transporters. Genome-wide characterization of Satsuma mandarin identified eighteen SWEET family members that encode transporters which facilitate diffusion of sugar across cell membranes. Analysis of the expression profiles in tissues of mandarin fruit at different developmental stages showed that CitSWEET11d transcripts were significantly correlated with sucrose accumulation. Further studies indicated that overexpression of CitSWEET11d in citrus callus and tomato fruit showed a higher sucrose level compared to wild-type, suggesting that CitSWEET11d could enhance sucrose accumulation. In addition, we identified an ERF transcription factor CitERF16 by yeast one-hybrid screening assay which could directly bind to the DRE cis -element on the promoter of CitSWEET11d. Overexpression of CitERF16 in citrus callus significantly induced CitSWEET11d expression and elevated sucrose content, suggesting that CitERF16 acts as a positive regulator to promote sucrose accumulation via trans-activation of CitSWEET11d expression. [ABSTRACT FROM AUTHOR]

Summary: The RAV (related to ABI3/viviparous 1) group of transcription factors (TFs) play multifaceted roles in plant development and stress responses. Here, we show that strawberry (Fragaria × ananassa) FaRAV1 positively regulates anthocyanin accumulation during fruit ripening via a hierarchy of activation processes. Dual‐luciferase assay screening of all fruit‐expressed AP2/ERFs showed FaRAV1 had the highest transcriptional activation of the promoter of FaMYB10, a key activator of anthocyanin biosynthesis. Yeast one‐hybrid and electrophoretic mobility shift assays indicated that FaRAV1 could directly bind to the promoter of FaMYB10. Transient overexpression of FaRAV1 in strawberry fruit increased FaMYB10 expression and anthocyanin production significantly. Correspondingly, transient RNA interference‐induced silencing of FaRAV1 led to decreases in FaMYB10 expression and anthocyanin content. Transcriptome analysis of FaRAV1‐overexpressing strawberry fruit revealed that transcripts of phenylpropanoid and flavonoid biosynthesis pathway genes were up‐regulated. Luciferase assays showed that FaRAV1 could also activate the promoters of strawberry anthocyanin biosynthetic genes directly, revealing a second level of FaRAV1 action in promoting anthocyanin accumulation. These results show that FaRAV1 stimulates anthocyanin accumulation in strawberry both by direct activation of anthocyanin pathway gene promoters and by up‐regulation of FaMYB10, which also positively regulates these genes. [ABSTRACT FROM AUTHOR]

Finger citron (Citrus medica var. sarcodactylis) is a popular ornamental tree and an important source of essential oils rich in terpenoids, but the mechanisms behind volatile formation are poorly understood. We investigated gene expression changes combined with volatile profiling of ten samples from three developing organs: flower, leaf, and fruit. A total of 62 volatiles were identified with limonene and γ-terpinene being the most abundant ones. Six volatiles were identified using partial least squares discriminant analysis (PLS-DA) that could be used as markers for distinguishing finger citron from other citrus species. RNA-Seq revealed 1,611,966,118 high quality clean reads that were assembled into 32,579 unigenes. From these a total of 58 terpene synthase (TPS) gene family members were identified and the spatial and temporal distribution of their transcripts was measured in developing organs. Transcript levels of transcription factor genes AP2/ERF (251), bHLH (169), bZIP (76), MYB (155), NAC (184), and WRKY (66) during finger citron development were also analyzed. From extracted subnetworks of three modules constructed by weighted gene co-expression network analysis (WGCNA), thirteen TPS genes and fifteen transcription factors were suggested to be related to volatile terpenoid formation. These results provide a framework for future investigations into the identification and regulatory network of terpenoids in finger citron. [ABSTRACT FROM AUTHOR]

• CitMYC3 activates CsTPS1 transcription to enhance valencene synthesis. • Ethylene modulates CitMYC3 and CsTPS1 transcript abundance in sweet orange. • CitMYC3 modulates CitAP2.10 and CitMYC3 transcription. CsTPS1 (Cs5g12900) is a gene encoding a sesquiterpene synthase involved in generating valencene, a key volatile compound in sweet orange fruit. Here, a citrus basic helix-loop-helix (bHLH) transcription factor (CitMYC3), is characterized as a positive regulator of CsTPS1. Dual-luciferase assays showed that CitMYC3 transcriptionally activates its own promoter, also the CsTPS1 promoter and, in addition, the promoter of a second transcription factor, CitAP2.10 , which in turn positively regulates the CsTPS1 expression. During the developmental period, CitMYC3 transcripts were detectable at 60 days after full bloom (DAFB) and increased gradually to 120 DAFB, while CsTPS1 mRNA rapidly increased from 90 to 150 DAFB. Exogenous ethylene accelerated the expression of CitMYC3 (peaking at 4 h) before CsTPS1 expression (at 24 h). Furthermore, CitMYC3 and CitAP2.10 were also shown to activate the promoter of Arabidopsis AtTPS11 , a homolog of CsTPS1 , which is in charge of the synthesis of over fifteen different sesquiterpenes, indicating that CitMYC3 and CitAP2.10 have a likely conserved function in the regulation of sesquiterpene genes across a variety of plant species. [Display omitted] [ABSTRACT FROM AUTHOR]

Lignin is an important component of secondary cell walls, and its content influences the utilization of plant products for food and fiber. Loquat is a typical fruit accumulating lignin during postharvest low temperature storage. In this research, two EjNAC genes were isolated and designated as EjNAC1 and EjNAC2 . Both EjNAC1 and EjNAC2 had transcriptional activity in yeast cells. The expression of EjNAC1 and EjNAC2 in lignin-rich stems was higher than in pulp. However, only EjNAC1 was induced in fruit by low temperature and inhibited by heat treatment (HT), which can alleviate lignification, while EjNAC2 expression was not correlated with fruit lignification. Further analysis indicated that EjNAC1 could trans-activate the gene promoters in loquat and Arabidopsis for genes in the lignin biosynthesis pathway. Transient over-expression of EjNAC1 in tobacco leaves resulted in accumulation of lignin and induction of expression of endogenous lignin biosynthesis genes. In conclusion, EjNAC1 was associated with fruit lignification by activating genes involved in lignin biosynthesis. [ABSTRACT FROM AUTHOR]

• PpERF61 binds to the promoters of PpTPS1 and PpTPS3 and activated their transcription. • Overexpressing PpERF61 in peach fruit significantly promoted linalool accumulation. • PpERF61 expression correlated with linalool contents during peach fruit ripening and across 30 cultivars. • Demethylation of PpERF61 accompanied with increased linalool contents during peach fruit ripening. The monoterpene linalool is a major contributor to flavor of multiple fruit species. Although great progress has been made in identifying genes related to linalool formation, transcriptional regulation for the pathway remains largely unknown. As a super transcription factor family, roles of AP2/ERF in regulating linalool production have not been elucidated. Peach linalool is catalyzed by terpene synthases PpTPS1 and PpTPS3. Here, we observed that expression of PpERF61 correlated with these two PpTPSs during fruit ripening by transcriptome co-expression analysis. Dual-luciferase assay and EMSA results indicated that PpERF61 activated the PpTPS1 and PpTPS3 transcription by binding to the DRE/CRT motif in their promoters. Transient overexpressing PpERF61 in peach fruit significantly increased PpTPS1 and PpTPS3 expression and linalool content. Further study revealed significant correlation between PpERF61 transcripts and linalool contents across 30 peach cultivars. Besides transcriptional regulation, accumulated linalool was associated with DNA demethylation of PpERF61 during peach fruit ripening. In addition, interactions between PpERF61 and PpbHLH1 were evaluated, indicating these two transcription factors were associated with linalool production during peach fruit ripening. Overall, our results revealed a new insight into the regulation of linalool synthesis in fruit. [ABSTRACT FROM AUTHOR]