Identification of miRNA858 long-loop precursors in seed plants.

MicroRNAs (miRNAs) are a class of nonprotein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this subclass. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100 to 2,100 nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350 and 5,500 nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.

Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

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.

Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Molecular mechanisms underlying natural deficient and ultraviolet-induced accumulation of anthocyanin in the peel of 'Jinxiu' peach.

Peach varieties that differ in red coloration due to varied anthocyanin accumulation result from transcriptional regulation by PpMYB10s, a group of specific R2R3 MYBs. Here we investigated the mechanisms driving a lack of anthocyanin in yellow-skinned 'Jinxiu' peach peel, as well as accumulation induced by UV irradiance. It was found that PpMYB10.1, PpMYB10.2 and PpMYB10.3 were positive regulators of anthocyanin accumulation, but the stimulation by PpMYB10.2 was weak. Low expression of PpMYB10.1 causes natural anthocyanin deficiency in 'Jinxiu' peel. However, the promoter sequences of PpMYB10.1 were identical in 'Jinxiu' and a naturally red-coloured peach 'Hujingmilu'. Therefore, potential negative regulator(s) upstream of PpMYB10.1 were explored. A novel R2R3-MYB repressor termed PpMYB80 was identified through comparative transcriptomic analysis and then functionally confirmed via transiently overexpressing and silencing in peach fruit, as well as transformation in tobacco. PpMYB80 directly binds to the promoter of PpMYB10.1 and inhibits its expression, but does not affect PpMYB10.3. In UV-exposed 'Jinxiu' fruit, expression of PpMYB10.3 was upregulated, while PpMYB10.1 remained low and PpMYB80 enhanced, which results in accumulation of anthocyanin in peel. This study revealed a transcriptional cascade involving PpMYB activators and repressors in regulating basal and UV-induced anthocyanin accumulation in peach peel.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening.

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 SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-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, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

An EjbHLH14-EjHB1-EjPRX12 module is involved in methyl jasmonate alleviation of chilling-induced lignin deposition in loquat fruit.

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.

Three AP2/ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus.

Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)-naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference-induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration-responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB-RAV transcriptional complex regulating flavonoid production.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Transcription factors DkBZR1/2 regulate cell wall degradation genes and ethylene biosynthesis genes during persimmon fruit ripening.

BRASSINAZOLE RESISTANT (BZR) transcription factors are critical components of the brassinosteroid signalling pathway, but their possible roles in fruit ripening have rarely been reported. In this study, four BZR sequences were isolated from persimmon fruit. Among the four BZR genes, DkBZR1/2 were expressed in persimmon fruit; DkBZR1 protein amount decreased and dephosphorylated DkBZR2 gradually accumulated during the storage period. DkBZR1/2 proteins were localized in both the nucleus and cytoplasm and accumulated in the nucleus after 24-epibrassinolide treatment. DkBZR1 suppressed the transcription of Diospyros kaki endo-1,4-betaglucanase 1 (DkEGase1) and 1-aminocyclopropane-1-carboxylate synthase 1 (DkACS1) by binding to the BR response element (BRRE) in their promoters, and DkBZR2 activated the transcription of pectate lyase 1 (DkPL1) and 1-aminocyclopropane-1-carboxylate oxidase 2 (DkACO2) by binding to the E-box motif in their promoters. Transient overexpression of DkBZR2 promoted the conversion of acid-soluble pectin to water-soluble pectin and increased ethylene production in persimmon fruit. Our findings indicate that DkBZR1 and DkBZR2 serve as repressors and activators of persimmon fruit ripening, respectively.

Transcriptome co-expression network analysis identifies key genes and regulators of ripening kiwifruit ester biosynthesis.

BACKGROUND: Aroma is an important organoleptic quality for fruit and has a large influence on consumer preference. Kiwifruit esters undergo rapid and substantial changes contributing to the flavor during fruit ripening. Part of enzymes and their coding genes have been indicated potential candidates for flavor-related esters synthesis. However, there still exist obvious gaps in the biosynthetic pathways of esters and the mechanisms regulating ester biosynthesis in kiwifruit remain unknown. RESULTS: Using gas chromatography-mass spectrometry (GC-MS), volatile compounds of kiwifruit were quantified in response to ethylene (ETH, 100 µl/l, 24 h, 20 °C) and 1-methylcyclopropene (1-MCP, 1 µl/l, 24 h, 20 °C). The results indicated that esters showed the most substantial changes enhanced by ethylene and were inhibited by 1-MCP. Correlations between RNA-seq results and concentrations of esters, constructed using Weighted Gene Co-Expression Network Analysis (WGCNA) indicated that three structural genes (fatty acid desaturase, AdFAD1; aldehyde dehydrogenase, AdALDH2; alcohol acyltransferase, AdAT17) had similar expression patterns that paralled the changes in total ester content, and AdFAD1 transcripts exhibited the highest correlation. In order to search for potential regulators for ester biosynthesis, 14 previously reported ethylene-responsive transcription factors (TFs) were included in the correlation analysis with esters and their biosynthetic genes. Using dual-luciferase assay, the in vivo regulatory activities of TFs on ester biosynthetic gene promoters were investigated and the results indicated that AdNAC5 and AdDof4 (DNA binding with one finger) trans-activated and trans-suppressed the AdFAD1 promoter. CONCLUSIONS: The present study advanced the molecular basis of ripening-related ester biosynthesis in kiwifruit by identifying three biosynthetic related genes AdFAD1, AdALDH2 and AdAT17 by transcriptome analysis, and highlighted the function of two TFs by transactivation studies.

The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes.

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.

Genome-wide analysis of coding and non-coding RNA reveals a conserved miR164-NAC regulatory pathway for fruit ripening.

Kiwifruit (Actinidia spp.) is a climacteric fruit with high sensitivity to ethylene, influenced by multiple ethylene-responsive structural genes and transcription factors. However, the roles of other post-transcriptional regulators (e.g. miRNAs) necessary for ripening remain elusive. High-throughput sequencing sRNAome, degradome and transcriptome methods were used to identify further contributors to ripening control in the kiwifruit (A. deliciosa cv 'Hayward'). Two NAM/ATAF/CUC domain transcription factors (AdNAC6 and AdNAC7), both predicted targets for miR164, showed significant upregulation by exogenous ethylene. Gene expression analysis and luciferase reporter assays indicated that Ade-miR164 and one of its precursor miRNAs (Ade-MIR164b) were repressed by ethylene treatment and negatively correlated with AdNAC6/7 expression. Subsequent analysis indicated that both AdNAC6 and AdNAC7 proteins are transcriptional activators and physically bind the promoters of AdACS1 (1-aminocyclopropane-1-carboxylate synthase), AdACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), AdMAN1 (endo-ß-mannanase) and AaTPS1 (terpene synthase). Moreover, subcellular analysis indicated that the location of the AdNAC6/7 proteins was influenced by Ade-miR164. Multiple omics-based approaches revealed a novel regulatory link for fruit ripening that involved ethylene-miR164-NAC. The regulatory pathway for miR164-NAC is present in various fruit (e.g. Rosaceae fruit, citrus, grape), with implications for fruit ripening regulation.

High-CO2/Hypoxia-Responsive Transcription Factors DkERF24 and DkWRKY1 Interact and Activate DkPDC2 Promoter.

Identification and functional characterization of hypoxia-responsive transcription factors is important for understanding plant responses to natural anaerobic environments and during storage and transport of fresh horticultural products. In this study, yeast one-hybrid library screening using the persimmon (Diospyros kaki) pyruvate decarboxylase (DkPDC2) promoter identified three ethylene response factor (ERF) genes (DkERF23/DkERF24/DkERF25) and four WRKY transcription factor genes (DkWRKY/DdkWRKY5/DkWRKY6/DkWRKY7) that were differentially expressed in response to high CO2 (95%, with 4% N2 and 1% oxygen) and high N2 (99% N2 and 1% oxygen). Yeast one-hybrid assays and electrophoretic mobility shift assays indicated that DkERF23, DkERF24, DkERF25, DkWRKY6, and DkWRKY7 could directly bind to the DkPDC2 promoter. Dual-luciferase assays confirmed that these transcription factors were capable of transactivating the DkPDC2 promoter. DkERF24 and DkWRKY1 in combination synergistically transactivated the DkPDC2 promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays confirmed protein-protein interaction between DkERF24 and DkWRKY1. Transient overexpression of DkERF24 and DkWRKY1 separately and in combination in persimmon fruit discs was effective in maintaining insolubilization of tannins, concomitantly with the accumulation of DkPDC2 transcripts. Studies with Arabidopsis (Arabidopsis thaliana) homologs AtERF1 and AtWRKY53 indicated that similar protein-protein interactions and synergistic regulatory effects also occur with the DkPDC2 promoter. We propose that an ERF and WRKY transcription factor complex contributes to responses to hypoxia in both persimmon fruit and Arabidopsis, and the possibility that this is a general plant response requires further investigation.

High CO2/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes.

Most persimmon (Diospyros kaki) cultivars are astringent and require post-harvest deastringency treatments such as 95% CO2 (high-CO2 treatment) to make them acceptable to consumers. High-CO2 treatment can, however, also induce excessive softening, which can be reduced by adding 1-methylcyclopropene (1-MCP). Previous studies have shown that genes encoding the ETHYLENE RESPONSE FACTORS (ERFs) DkERF8/16/19 can trans-activate xyloglucan endotransglycosylase/hydrolase (DkXTH9), which encodes the cell wall-degrading enzyme associated with persimmon fruit softening. In this study, RNA-seq data between three treatments were compared, namely high-CO2, high-CO2+1-MCP, and controls. A total of 227 differentially expressed genes, including 17 transcription factors, were predicted to be related to persimmon post-deastringency softening. Dual-luciferase assays indicated that DkNAC9 activated the DkEGase1 promoter 2.64-fold. Synergistic effects on transcription of DkEGase1 that involved DkNAC9 and the previously reported DkERF8/16 were identified. Electrophoretic mobility shift assay indicated that DkNAC9 could physically bind to the DkEGase1 promoter. Bimolecular fluorescence complementation and firefly luciferase complementation imaging assays indicated protein-protein interactions between DkNAC9 and DkERF8/16. Based on these findings, we conclude that DkNAC9 is a direct transcriptional activator of DkEGase1 that can co-operate with DkERF8/16 to enhance fruit post-deastringency softening.

ETHYLENE RESPONSE FACTOR39-MYB8 complex regulates low-temperature-induced lignification of loquat fruit.

Flesh lignification is a specific chilling response that causes deterioration in the quality of stored red-fleshed loquat fruit (Eribotrya japonica) and is one aspect of wider chilling injury. APETALA2/ETHLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors are important regulators of plant low-temperature responses and lignin biosynthesis. In this study, the expression and action of 27 AP2/ERF genes from the red-fleshed loquat cultivar 'Luoyangqing' were investigated in order to identify transcription factors regulating low-temperature-induced lignification. EjERF27, EjERF30, EjERF36, and EjERF39 were significantly induced by storage at 0 °C but inhibited by a low-temperature conditioning treatment (pre-storage at 5 °C for 6 days before storage at 0 °C, which reduces low-temperature-induced lignification), and their transcript levels positively correlated with flesh lignification. A dual-luciferase assay indicated that EjERF39 could transactivate the promoter of the lignin biosynthetic gene Ej4CL1, and an electrophoretic mobility shift assay confirmed that EjERF39 recognizes the DRE element in the promoter region of Ej4CL1. Furthermore, the combination of EjERF39 and the previously characterized EjMYB8 synergistically transactivated the Ej4CL1 promoter, and both transcription factors showed expression patterns correlated with lignification in postharvest treatments and red-fleshed 'Luoyangqing' and white-fleshed 'Ninghaibai' cultivars with different lignification responses. Bimolecular fluorescence complementation and luciferase complementation imaging assays confirmed direct protein-protein interaction between EjERF39 and EjMYB8. These data indicate that EjERF39 is a novel cold-responsive transcriptional activator of Ej4CL1 that forms a synergistic activator complex with EjMYB8 and contributes to loquat fruit lignification at low temperatures.