Identification of key sensory and chemical factors determining flavor quality of Xinyu mandarin during ripening and storage.

Xinyu mandarin is popular for its good flavor, but its flavor deteriorates during postharvest storage. To better understand the underlying basis of this change, the dynamics of the sensory profiles were investigated throughout fruit ripening and storage. Sweetness and sourness, determined especially by sucrose and citric acid content, were identified as the key sensory factors in flavor establishment during ripening, but not in flavor deterioration during storage. Postharvest flavor deterioration is mainly attributed to the reduction of retronasal aroma and the development of off-flavor. Furthermore, sugars, acids and volatile compounds were analyzed. Among the 101 detected volatile compounds, 10 changed significantly during the ripening process. The concentrations of 15 volatile components decreased during late postharvest storage, among which α-pinene and d-limonene were likely to play key roles in the reduction of aroma. Three volatile compounds were found to increase during storage, associated with off-flavor development.

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.

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.

The effect of NH4+ on phosphoenolpyruvate carboxykinase gene expression, metabolic flux and citrate content of citrus juice sacs.

Citrate is one of the most important metabolites determining the flavour of citrus fruit. It has been reported that nitrogen supply may have an impact on acid level of fruit. Here, the relationship between nitrogen metabolism and citrate catabolism was studied in pumelo juice sacs. Differences in metabolites, gene expression and flux distributions were analyzed in juice sacs incubated in medium with and without NH4+. Compared with those incubated with NH4+, juice sacs under nitrogen deficiency exhibited enhanced flux through phosphoenolpyruvate carboxykinase (PEPCK) and accelerated consumption of citrate, while the other two TCA cycle efflux points, through malic enzyme (ME) and glutamate dehydrogenase (GDH), were both repressed. Consistent with the estimated fluxes, the expression of PEPCK1 was upregulated under nitrogen deficiency, while that of GDH1, GDH2, NAD-ME1 and NADP-ME2 were all repressed. Thus, we propose that PEPCK1 contributes to citrate degradation under nitrogen limitation.

Citrus CitERF6 Contributes to Citric Acid Degradation via Upregulation of CitAclα1, Encoding ATP-Citrate Lyase Subunit α.

Citric acid is the most abundant organic acid in citrus fruit, and the acetyl-CoA pathway potentially plays an important role in citric acid degradation, which occurs during fruit ripening. Analysis of transcripts during fruit development of key genes in the acetyl-CoA pathway and transient overexpression assay in citrus leaves indicated that CitAclα1 could be a potential target gene involved in citrate degradation. In order to understand more about CitAclα1, 23 transcription factors coexpressed with CitAclα1 in citrus fruit were identified by RNA-seq. Using dual-luciferase assays, CitERF6 was shown to trans-activate the promoter of CitAclα1 and electrophoretic mobility shift assays (EMSAs) showed that CitERF6 directly bound to a 5'-CAACA-3' motif in the CitAclα1 promoter. Furthermore, citric acid content was significantly reduced when CitERF6 was overexpressed in transgenic tobacco leaves. Taken together, these results indicate an important role for CitERF6 in transcriptional regulation of CitAclα1 and control of citrate degradation.

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.

Auto- and mutual-regulation between two CitERFs contribute to ethylene-induced citrus fruit degreening.

Citrus fruit postharvest degreening is a critical stage in marketing, carried out by exposure to ethylene or ethephon. Genome-wide screening of the AP2/ERF superfamily indicated that a novel ERF-II (CitERF6) was shown to trans-activate the CitPPH promoter. Expression of CitERF6 is associated with both developmental and postharvest degreening in citrus fruit. Transient and stable over-expression of CitERF6 in Nicotiana tabacum leaves and 'Ponkan' fruit also results in rapid chlorophyll degradation. Auto- and mutual-regulation was also found between CitERF6 and the previously characterized CitERF13 using the dual-luciferase and yeast one-hybrid assays. Moreover, substitution of the 35S promoter for endogenous promoters showed that both pCitERF6::CitERF6 and pCitERF13::CitERF13 were effective in trans-activating their promoters or triggering chlorophyll degradation. It is proposed that ethylene is one of the triggers activating promoters of CitERF6 and CitERF13, and subsequent auto- and mutual-regulation between CitERF6 and CitERF13 might facilitate the effect of ethylene, leading to fruit degreening.

Ternary complex EjbHLH1-EjMYB2-EjAP2-1 retards low temperature-induced flesh lignification in loquat fruit.

Many transcription factors (TFs), including NACs and MYBs, are involved in regulation of lignin biosynthesis during plant development and in responses to biotic and abiotic stresses. The lignin biosynthesis gene Ej4CL1 has been identified as a target for cold-induced TFs. We isolated a bHLH gene from loquat, EjbHLH1, the expression of which was negatively correlated with cold-induced fruit lignification. During low temperature storage (0 °C), EjbHLH1 transcripts were stable but accumulated during low-temperature conditioning (LTC) treatment, an acclimation process that reduces lignification during subsequent storage at 0 °C. Dual luciferase assays showed EjbHLH1 could repress Ej4CL1 promoter, but yeast one hybrid assay indicated EjbHLH1 is not able to bind to the Ej4CL1 promoter. Bimolecular fluorescence complementation (BIFC) indicated that EjbHLH1 could interact with EjAP2-1 and EjMYB2, two previously characterized fruit lignification related transcription factors and firefly luciferase complementation imaging assay indicated EjbHLH1, EjMYB2 and EjAP2-1 could form a ternary complex which enhanced repression of transcription from the Ej4CL1 promoter, reducing lignification at 0 °C.

Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3.

Citric acid is the predominant organic acid of citrus fruit. Degradation of citric acid occurs during fruit development, influencing fruit acidity. Associations of CitAco3 transcripts and citric acid degradation have been reported for citrus fruit. Here, transient overexpression of CitAco3 significantly reduced the citric acid content of citrus leaves and fruits. Using dual luciferase assays, it was shown that CitNAC62 and CitWRKY1 could transactivate the promoter of CitAco3. Subcellular localization results showed that CitWRKY1 was located in the nucleus and CitNAC62 was not. Yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays indicated that the two differently located transcription factors could interact with each other. Furthermore, BiFC showed that the protein-protein interaction occurred only in the nucleus, indicating the potential mobility of CitNAC62 in plant cells. A synergistic effect on citrate content was observed between CitNAC62 and CitWRKY1. Transient overexpression of CitNAC62 or CitWRKY1 led to significantly lower citrate content in citrus fruit. The combined expression of CitNAC62 and CitWRKY1 resulted in lower citrate content compared with the expression of CitNAC62 or CitWRKY1 alone. The transcript abundance of CitAco3 was consistent with the citrate content. Thus, we propose that a complex of CitWRKY1 and CitNAC62 contributes to citric acid degradation in citrus fruit, potentially via modulation of CitAco3.

EjMYB8 Transcriptionally Regulates Flesh Lignification in Loquat Fruit.

Transcriptional regulatory mechanisms underlying lignin metabolism have been widely studied in model plants and woody trees, but seldom in fruits such as loquat, which undergo lignification. Here, twelve EjMYB genes, designed as EjMYB3-14, were isolated based on RNA-seq. Gene expression indicated that EjMYB8 and EjMYB9 were significantly induced in fruit with higher lignin content resulting from storage at low temperature (0°C), while two treatments (low temperature conditioning, LTC; heat treatment, HT) both alleviated fruit lignification and inhibited EjMYB8 and EjMYB9 expression. Dual-luciferase assays indicated that EjMYB8, but not EjMYB9, could trans-activate promoters of lignin-related genes EjPAL1, Ej4CL1 and Ej4CL5. Yeast one-hybrid assay indicated that EjMYB8 physically bind to Ej4CL1 promoter. Furthermore, the putative functions of EjMYB8 were verified using transient over-expression in both N. tabacum and loquat leaves, which increased lignin content. Moreover, combination of EjMYB8 and previously isolated EjMYB1 generated strong trans-activation effects on the Ej4CL1 promoter, indicating that EjMYB8 is a novel regulator of loquat fruit lignification.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.