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.

Combination of bone marrow mesenchymal stem cells and brain-derived neurotrophic factor for treating spinal cord injury

BACKGROUND： Because bone marrow mesenchymal stem cells （BMSCs） do not secrete sufficient brain-derived neurotrophic factor （BDNF）, the use of exogenous BDNF could improve microenvironments in injured regions for BMSCs differentiation. OBJECTIVE： To analyze recovery of the injured spinal cord following BMSCs venous transplantation in combination with consecutive injections of BDNF. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Central Laboratory of First Hospital and Anatomical Laboratory, Fujian Medical University from October 2004 to May 2006. MATERIALS： Human BDNF was purchased from Sigma, USA. METHODS： A total of 44 New Zealand rabbits were randomly assigned to model （n = 8）, BDNF （n = 12）, BMSC （n= 12）, and BMSC＋BDNF （n= 12） groups. Spinal cord （I-2）injury was established with the dropping method. The model group rabbits were injected with 1 mL normal saline via the ear margin vein; the BDNF group was subdurally injected with 100 μg/d human BDNF for 1 week; the BMSC group was injected with 1 mL BMSCs suspension （2 × 10^6/mL） via the ear margin vein; and the BMSC＋BDNF group rabbits were subdurally injected with 100 μg/d BDNF for 1 week, in addition to BMSCs suspension via the ear margin vein. MAIN OUTCOME MEASURES： BMSCs surface markers were detected by flow cytometry. BMSCs differentiation in the injured spinal cord was detected by immunofluorescence histochemistry. Functional and structural recovery, as well as morphological changes, in the injured spinal cord were respectively detected by Tarlov score, horseradish peroxidase retrograde tracing, and hematoxylin ＆ eosin staining methods at 1, 3, and 5 weeks following transplantation. RESULTS： Transplanted BMSCs differentiated into neuronal-like cells in the injured spinal cord at 3 and 5 weeks following transplantation. Neurological function and pathological damage improved following BMSC ＋ BDNF treatment compared with BDNF or BMSC alone （P 〈 0.01 or P 〈 0.05）. CONCLUSION： BMSCs venous transplantation in combination with BDNF subdural injection benefits neuronal-like cell differentiation and significantly improves structural and function of injured spinal cord compared with BMSCs or BDNF alone.

Non-cerebral vasospasm factors and cerebral vasospasm predict delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage

Aneurysmal subarachnoid hemorrhage(aSAH)is a typical neurosurgical emergency.The patient's prognosis is related to the severity of the initial illness and post-operative complications,especially delayed cerebral ischemia(DCI).DCI,which occurs in up to 30%of patients with subarachnoid hemorrhage(SAH),usually 2 weeks after hemorrhage,is one of the leading causes of death and disability in aSAH.

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.