Genome-wide analysis of cytochrome P450 genes in Citrus clementina and characterization of a CYP gene encoding f lavonoid 3′-hydroxylase

Cytochrome P450s(CYPs)are the largest family of enzymes in plant and play multifarious roles in development and defense but the available information about the CYP superfamily in citrus is very limited.Here we provide a comprehensive genome-wide analysis of the CYP superfamily in Citrus clementina genome,identifying 301 CYP genes grouped into ten clans and 49 families.The characteristics of both gene structures and motif compositions strongly supported the reliability of the phylogenetic relationship.Duplication analysis indicated that tandem duplication was the major driving force of expansion for this superfamily.Promoter analysis revealed numerous cis-acting elements related to various responsiveness.RNA-seq data elucidated their expression patterns in citrus fruit peel both during development and in response to UV-B.Furthermore,we characterize a UV-B-induced CYP gene(Ciclev10019637m,designated CitF3′H)as a f lavonoid 3-hydroxylase for the first time.CitF3 H catalyzed numerous f lavonoids and favored naringenin in yeast assays.Virusinduced silencing of CitF3′ H in citrus seedlings significantly reduced the levels of 3′-hydroxylated f lavonoids and their derivatives.These results together with the endoplasmic reticulum-localization of CitF3 H in plant suggest that this enzyme is responsible for the biosynthesis of 3-hydroxylated f lavonoids in citrus.Taken together,our findings provide extensive information about the CYP superfamily in citrus and contribute to further functional verification.

Functional characterization of two flavone synthase Ⅱ members in citrus

Polymethoxylated flavones(PMFs),the main form of flavones in citrus,are derived from the flavone branch of the flavonoid biosynthesis pathway.Flavone synthases(FNSs)are enzymes that catalyze the synthesis of flavones from flavanones.However,the FNS in citrus has not been characterized yet.Here,we identified two type II FNSs,designated CitFNSII-1 and CitFNSII-2,based on phylogenetics and transcriptome analysis.Both recombinant CitFNSII-1 and CitFNSII-2 proteins directly converted naringenin,pinocembrin,and liquiritigenin to the corresponding flavones in yeast.In addition,transient overexpression of CitFNSII-1 and CitFNSII-2,respectively,in citrus peel significantly enhanced the accumulation of total PMFs,while virus-induced CitFNSII-1 and CitFNSII-2 genes silencing simultaneously significantly reduced the expression levels of both genes and total PMF content in citrus seedlings.CitFNSII-1 and CitFNSII-2 presented distinct expression patterns in different cultivars as well as different developmental stages.Methyl salicylate(MeSA)treatment reduced the CitFNSII-2 expression as well as the PMFs content in the peel of Citrus sinensis fruit but did not affect the CitFNSII-1 expression.These results indicated that both CitFNSII-1 and CitFNSII-2 participated in the flavone biosynthesis in citrus while the regulatory mechanism governing their expression might be specific.Our findings improved the understanding of the PMFs biosynthesis pathway in citrus and laid the foundation for further investigation on flavone synthesis regulation.

Biosynthetic labeling with 3-O-propargylcaffeyl alcohol reveals in vivo cell-specific patterned lignification in loquat fruits during development and postharvest storage

Lignification is a major cell wall modification that often results in the formation of sophisticated subcellular patterns during plant development or in response to environmental stresses.Precise localization of the spatiotemporal deposition of lignin is of great importance for revealing the lignification regulatory mechanism of individual cells.In loquat fruits,lignification typically increases the flesh lignin content and firmness,reducing their edibility and processing quality.However,the precise localization of the spatiotemporal active zones of lignification inside loquat fruit flesh remains poorly understood,and little is known about the contribution of patterned lignification to cell wall structure dynamics and the subsequent fruit-quality deterioration.Here,we performed an emerging bioorthogonal chemistry imaging technique to trace the in vivo patterned lignification dynamics in cells of loquat fruit flesh during development and storage.In developing fruits,lignified cells(LCs)and vascular bundles(VBs)were the zones of active lignification,and ring-like LCs deposited lignin at both the inner wall layer and the outer periphery sides.The domino effect of the generation of LCs was preliminarily visualized.In mature fruits,the newly formed lignin in the flesh of fruits during storage was specifically deposited in the corners and middle lamellae of parenchyma cells surrounding the VBs,resulting in the development of a reticular structure.Based on the findings,distinct spatiotemporal patterned lignification modes for different flesh cells in loquat fruits were proposed.These findings provide loquat lignification dynamics together with spatiotemporal data that can improve our understanding of the lignification process in planta.

The chemistry,distribution,and metabolic modifications of fruit flavonols

Fruits are considered as healthy foods because they provide a rich source of vitamins,antioxidants and other nutrients,including a range of essential bioactive flavonoid compounds.Flavonols,with diverse chemical properties and biological activities,are the most ubiquitous flavonoids that occur naturally in fruits and they are nutritionally important to animals and humans.Numerous investigations have emphasized that significant intake of dietary flavonols is associated with lower incidences of degenerative diseases.Here,we review current knowledge concerning the molecular structures,composition and distribution,regulation,and structural modification of fruit flavonols.In addition,we consider biotechnological approaches to enhance the levels of flavonols in plants or microorganism.An understanding of the factors determining production of flavonols in fruit crops will improve breeding programs and facilitate the production of fruits or bio-products with desirable contents of bioactive flavonols of benefit to humans.

Application of electronic nose and GC-MS for detection of strawberries with vibrational damage

Objectives: This study evaluated the potential of using electronic nose (e-nose) technology to non- destructively detect strawberry fruits with vibrational damage based on their volatile substances (VOCs). Materials and methods: Four groups of strawberries with different durations of vibrations (0, 0.5, 1, and 2 h) were prepared, and their e-nose signals were collected at 0, 1, 2, and 3 days after vibration treatment. Results: The results showed that when the samples from all four sampling days during storage were used for modelling, both the levels of vibrational damage and the day after the damage happened were accurately predicted. The best models had residual prediction deviation values of 2.984 and 5.478. The discrimination models for damaged strawberries also obtained good classification results, with an average correct answer rate of calibration and prediction of 99.24%. When the samples from each sampling day or vibration time were used for modelling, better results were obtained, but these models were not suitable for an actual situation. The gas chromatography-mass spectrophotometry results showed that the VOCs of the strawberries varied after experiencing vibrations, which was the basis for e-nose detection. Limitations: The changes in VOCs released by other forces should be studied in the future. Conclusions: The above results showed the potential use of e-nose technology to detect strawberries that have suffered vibrational damage.

Effects of fruit bagging on the physiochemical changes of grapefruit(Citrus paradisi)

Fruit bagging is a commonly used cultivation measure to protect citrus fruit from insects and adverse environments.The present study aimed to comprehensively investigate the effects of bagging on the physiochemical characteristics of grapefruit.The grapefruit were bagged at approximately 110–120 d after anthesis with a one-layer kraft paper bag with black coating inside(SL),a double-layer kraft paper bag with one black paper as the inner layer(DL),and a three-layer kraft paper bag with two black papers as inner layers(TL),respectively.Ultra performance liquid chromatography-high resolution mass spectrometry(UPLC-HRMS)technique was used to identify a total of 19 flavonoids,2 phenylpropanoids,9 coumarins,and 5 limonoids.By using UPLC,50 carotenoids were identified.Gas chromatography–mass spectrometry was used to identify 3 soluble sugars,3 organic acids,and 11 amino acids.In the quantitated components in the peel(albedo and flavedo),the chlorophylls and the carotenoids components(such as luteoxanthin,violaxanthin,9-cis-violaxanthin,xanthophyll,zeaxanthin andβ-carotene)were significantly downregulated by bagging,causing the surface color of bagged fruit to turn yellow earlier but paler than that of the unbagged control,particularly in the three-layer kraft bag treatment.Unlike the peel,the color and the carotenoid content of the juice sacs were less affected.The physiochem-ical compounds other than pigments,including soluble sugars,organic acids,amino acids,flavonoids,coumarins and limonoids,were minimally affected by bagging treatments.Our results indicated that bagging at approximately 110–120 d after anthesis exerted influence mainly on peel color,but less on sugars,acids,amino acids,flavonoids,limonoids and coumarins of grapefruit.