Impact of citrus fruit and hesperidin intake on multiple health outcomes:An umbrella review

Citrus fruits are rich sources of several biologically active flavonoids such as hesperidin,naringin,and polymethoxylated flavones.We evaluated the evidence of associations between citrus fruit or hesperidin intake and multiple health outcomes.An umbrella review was conducted for studies performed in humans.Overall,246 articles were initially identified by searching in 4 databases.Twenty-two meta-analyses and systematic reviews with 28 health outcomes met the inclusion criteria.Citrus fruit intake had beneficial effects on all-cause mortality(relative risk[RR].0.90;95%confidence interval[95%CI],0.86 to 0.94),cardiovascular diseases(RR,0.78;95%CI,0.66 to 0.92),coronary heart disease(RR,0.91;95%CI,0.86 to 0.96),stroke(RR,0.74;95%CI,0.65 to 0.84),type 2 diabetes mellitus(RR,0.85;95%CI,0.78 to 0.92),and several cancers.Dose-response analyses indicated that each 100-g/d increase in citrus fruit intake could reduce the risks of all-cause mortality by 6%(RR,0.94;95%CI,0.88 to 1.00),stroke by 22%(RR,0.78;95%CI,0.69 to 0.90),and cardia gastric cancer by 40%(RR,0.60;95%CI,0.44 to 0.83).Citrus fruit intake also had beneficial effects on the lipid profile and body weight control(weighted mean difference,−1.28;95%CI,−1.82 to−0.74).Grapefruits could reduce the systolic blood pressure(weighted mean difference,−2.43,95%CI,−4.77 to−0.09).Hesperidin supplementation significantly improved inflammation.Citrus fruit intake was generally safe and beneficial for multiple health outcomes in humans.However,grapefruit and pomelo juice may affect the bioavailability of various medications,so care should be exercised before increasing the intake of these fruits or their juices.

Hesperidin ameliorates H_(2)O_(2)-induced bovine mammary epithelial cell oxidative stress via the Nrf2 signaling pathway

Background Hesperidin is a citrus flavonoid with anti-inflammatory and antioxidant potential. However, its protective effects on bovine mammary epithelial cells(b MECs) exposed to oxidative stress have not been elucidated.Results In this study, we investigated the effects of hesperidin on H_(2)O_(2)-induced oxidative stress in b MECs and the underlying molecular mechanism. We found that hesperidin attenuated H_(2)O_(2)-induced cell damage by reducing reactive oxygen species(ROS) and malondialdehyde(MDA) levels, increasing catalase(CAT) activity, and improving cell proliferation and mitochondrial membrane potential. Moreover, hesperidin activated the Keap1/Nrf2/ARE signaling pathway by inducing the nuclear translocation of Nrf2 and the expression of its downstream genes NQO1 and HO-1, which are antioxidant enzymes involved in ROS scavenging and cellular redox balance. The protective effects of hesperidin were blocked by the Nrf2 inhibitor ML385, indicating that they were Nrf2 dependent.Conclusions Our results suggest that hesperidin could protect b MECs from oxidative stress injury by activating the Nrf2 signaling pathway, suggesting that hesperidin as a natural antioxidant has positive potential as a feed additive or plant drug to promote the health benefits of bovine mammary.

CsABF3-activated CsSUT1 pathway is implicated in pre-harvest water deficit inducing sucrose accumulation in citrus fruit

Pre-harvest water deficit(PHWD)plays an important role in sugar accumulation of citrus fruit.However,the mechanism is not known well.Here,it was confirmed that PHWD promoted sucrose accumulation of citrus fruit,but had limited effect on fructose,glucose and total acid.A sucrose transporter,Cs SUT1,which localizes to the plasma membrane,was demonstrated to function in sucrose transport induced by PHWD.Compared to wild-type,Cs SUT1 overexpression in citrus calli stimulated sucrose,fructose and glucose accumulation,while its silencing in juice sacs reduced sucrose accumulation.Increased sugar accumulation in transgenic lines enhanced plant drought tolerance,and resulted in decreased electrolyte leakage,malondialdehyde and hydrogen peroxide contents,as well as increased superoxide dismutase activity and proline contents.An abscisic acid(ABA)-responsive transcription factor,Cs ABF3,was found with a same expression pattern with Cs SUT1 under PHWD.Yeast one-hybrid,electrophoretic mobility shift assay and dual-luciferase assays all revealed that Cs ABF3 directly bound with the Cs SUT1 promoter by ABA responsive elements.When Cs ABF3 was overexpressed in citrus calli,the sucrose,fructose and glucose concentration increased correspondingly.Further,transgenic studies demonstrated that Cs ABF3 could affect sucrose accumulation by regulating Cs SUT1.Overall,this study revealed a regulation of Cs ABF3 promoting Cs SUT1 expression and sucrose accumulation in response to PHWD.Our results provide a detail insight into the quality formation of citrus fruit.

Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

Improved observation of colonized roots reveals the regulation of arbuscule development and senescence by drought stress in the arbuscular mycorrhizae of citrus

Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.

High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Quantitative estimation of hesperidin by HPTLC in different varieties of citrus peels

Objective:To develop a simple,selective,sensitive and accurate high-performance thin layer chromatography(HPTLC)method to determine the quantity of hesperidin in different varieties of citrus fruits.Methods:The method was carried out in aluminum-backed silica gel 60 F_(254)plates with ethyl acetate-methanol-water 15:3:2(%,v/v)as mobile phase.Results:A compact band was obtained for hesperidin at R_f value of(0.40±0.04).The calibration plot was linear in the range of 100-800 ng/spot of hesperidin and the correlation coefficient of 0.9986was indicative of good linear dependence of peak area on concentration.Limit of detection(8.87ng/spot),limit of quantification(23.21 ng/spot),accuracy(less than 2%)and recovery(ranging from98.55-99.38)were found satisfactory.Conclusions:The method developed can be used for routine analysis of hesperidin in crude drug as well as in herbal and pharmaceutical dosage form containing citrus fruits as an ingredient.

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.

Targets and molecular mechanisms of a citrus flavonoid, hesperidin, against luminal breast cancer cells: an integrative bioinformatics analysis

Objective:To identify the potential target and mechanisms of hesperidin in MCF-7 estrogen receptor-positive breast cancer cells using bioinformatics approaches.Methods:Gene expression profiles were accessed from public database GSE85871.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was carried out with Database for Annotation,Visualization and Integrated Discovery.The protein-protein interaction network was analyzed by STRING-DB and visualized by Cytoscape.Transcription factor regulatory networks were constructed from TRED,TRRUST,Reg Network and visualized by Cytoscape.Drug association analysis was conducted by Web Gestalt.Results:GO and KEGG pathway enrichment analysis revealed biological processes,cellular components and molecular functions that were related to cancer and estrogen signaling pathways.KEGG pathway enrichment analysis of the genes in transcription factor-differential expression genes regulatory network showed regulation of cancer,estrogen signaling pathways,epidermal growth factor receptor tyrosine kinase inhibitor resistance,and endocrine resistance.Moreover,drug association analysis revealed that hesperidin affected the expression of the same gene as raloxifene.Conclusions:Hesperidin targets estrogen receptor signaling in estrogen receptor-positive breast cancer cells.Results of this study could trace the molecular mechanism of hesperidin in estrogen receptor-positive breast cancer cells and integrative bioinformatics analysis could accelerate drug discovery and development.

Transcriptomic analysis reveals the defense mechanisms of citrus infested with Diaphorina citri

Huanglongbing(HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid(ACP), Diaphorina citri, is the main pest at the new shoot stage and is the only natural vector of HLB pathogenic bacteria. Little is known about how plants perceive and defend themselves from this destructive pest. Here, we characterized changes in the expression of various genes in citrus plants that were continuously infested by D. citri for different durations(12, 24, and 48 h). A total of 5 219 differentially expressed genes(DEGs) and 643 common DEGs were identified across all time points. Several pathways related to defense were activated, such as peroxisome, alpha-linolenic acid metabolism, and phenylpropanoid and terpenoid biosynthesis, and some pathways related to growth and signal transduction were suppressed in response to D. citri infestation. The expression of genes including kinases(CML44, CIPK6, and XTH6), phytohormones(SAMT, LOX6, and NPR3), transcription factors(bHLH162, WRKY70, and WRKY40), and secondary metabolite synthesis-related genes(PAL, 4CL2, UGT74B1 and CYP82G1) was significantly altered in response to D. citri infestation. The findings of this study greatly enhance our understanding of the mechanisms underlying the defense response of citrus plants to D. citri infestation at the molecular level. Functional characterization of the candidate defense-related genes identified in this study will aid the molecular breeding of insect-resistant citrus varieties.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.)

Carotenoids directly influence citrus fruit color and nutritional value,which is critical to consumer acceptance.Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality.Despite the well-established carotenoid biosynthetic pathways,the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood.Our previous studies have reported that the Myc-type basic helix-loop-helix(bHLH)transcription factor(TF)regulates citrus proanthocyanidin biosynthesis.Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli,but its regulatory mechanism is still unclear.In the present study,we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR,CsHDS,CsHDR,CsPDS,CsLCYE,CsZEP,and CsNCED2,which was accompanied by changes in the contents of abscisic acid and gibberellin.The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR,CsHDS,and CsHDR,the keymetabolic enzymes of the methylerythritol 4-phosphate(MEP)pathway,thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes.In addition,CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes,CsPDS,CsLCYE,CsZEP,and CsNCED2,directly promoting carotenoid biosynthesis.This study reveals a novel network of carotenoid metabolism regulated by CsTT8.Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

Mapping of QTLs and candidate genes associated with multiple phenotypic traits for Huanglongbing tolerance in citrus

Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.

Hesperidin attenuates arsenic trioxide-induced cardiac toxicity in rats

Objective:To explore the cardioprotective effect of hesperidin against arsenic trioxide-induced cardiac toxicity in rats.Methods:Cardiac toxicity was induced by oral administration of 4 mg/kg arsenic trioxide for 30 days.Hematological,biochemical,electrocardiography,echocardiography,and histopathological examinations were performed.Results:Hesperidin decreased the neutrophil-to-lymphocyte ratio,calcium,creatine kinase-myoglobin binding,lactate dehydrogenase,IL-6,and lipid peroxidation,as well as increased sodium and potassium concentration and superoxide dismutase and catalase activity in arsenic trioxide-intoxicated rats.Moreover,it reduced peak systolic velocity and end-diastolic velocity while increasing heart rate.Arsenic trioxide-induced histopathological damage to cardiac tissue was prominently alleviated by hesperidin treatment.Conclusions:Hesperidin attenuates arsenic trioxide-induced cardiac toxicity in rats.Therefore,it can be further explored as a cardioprotective agent.

Omics analyses in citrus reveal a possible role of RNA translation pathways and Unfolded Protein Response regulators in the tolerance to combined drought, high irradiance, and heat stress

Environmental changes derived from global warming and human activities increase the intensity and frequency of stressful conditions for plants.Multiple abiotic factors acting simultaneously enhance stress pressure and drastically reduce plant growth,yield,and survival.Stress combination causes a specific stress situation that induces a particular plant response different to the sum of responses to the individual stresses.Here,by comparing transcriptomic and proteomic profiles to different abiotic stress combinations in two citrus genotypes,Carrizo citrange(Citrus sinensis×Poncirus trifoliata)and Cleopatra mandarin(Citrus reshni),with contrasting tolerance to different abiotic stresses,we revealed key responses to the triple combination of heat stress,high irradiance and drought.The specific transcriptomic response to this stress combination in Carrizo was directed to regulate RNA metabolic pathways and translation processes,potentially conferring an advantage with respect to Cleopatra.In addition,we found endoplasmic reticulum stress response as common to all individual and combined stress conditions in both genotypes and identified the accumulation of specific groups of heat shock proteins(HSPs),such as small HSPs and HSP70s,and regulators of the unfolded protein response,BiP2 and PDIL2-2,as possible factors involved in citrus tolerance to triple stress combination.Taken together,our findings provide new insights into the acclimation process of citrus plants to multiple stress combination,necessary for increasing crop tolerance to the changing climatic conditions.

Haplotype resolved chromosome level genome assembly of Citrus australis reveals disease resistance and other citrus specific genes

Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus.Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased,with the available genomes of varying accuracy and completeness.We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species;Citrus australis(round lime)using highly accurate PacBio HiFi long reads,complemented with Hi-C scaffolding.Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C.australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness(BUSCO).Repeat analysis showed that more than 50% of the genome contained interspersed repeats.Among them,LTR elements were the predominant type(21.0%),of which LTR Gypsy(9.8%)and LTR copia(7.7%)elements were the most abundant repeats.A total of 29464 genes and 32009 transcripts were identified in the genome.Of these,28222 CDS(25753 genes)had BLAST hits and 21401 CDS(75.8%)were annotated with at least one GO term.Citrus specific genes for antimicrobial peptides,defense,volatile compounds and acidity regulation were identified.The synteny analysis showed conserved regions between the two haplotypes with some structural variations in Chromosomes 2,4,7 and 8.This chromosome scale,and haplotype resolved C.australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.

Citrus β-carotene hydroxylase 2 (BCH2) participates in xanthophyll synthesis by catalyzing the hydroxylation of β-carotene and compensates for BCH1 in citrus carotenoid metabolism

Abstract As an essential horticultural crop,Citrus has carotenoid diversity,which affects its aesthetic and nutritional values.β,β-Xanthophylls are the primary carotenoids accumulated in citrus fruits,and non-heme di-iron carotene hydroxylase(BCH)enzymes are mainly responsible forβ,β-xanthophyll synthesis.Previous studies have focused on the hydroxylation of BCH1,but the role of its paralogous gene in citrus,BCH2,remains largely unknown.In this study,we revealed theβ-hydroxylation activity of citrus BCH2(CsBCH2)for the first time through the functional complementation assay using Escherichia coli,although CsBCH2 exhibited a lower activity in hydroxylatingβ-carotene intoβ-cryptoxanthin than citrus BCH1(CsBCH1).Our results showed that overexpression of CsBCH2 in citrus callus increased xanthophyll proportion and plastoglobule size with feedback regulation of carotenogenic gene expression.This study revealed the distinct expression patterns and functional characteristics of two paralogous genes,CsBCH1 and CsBCH2,and illustrated the backup compensatory role of CsBCH2 for CsBCH1 in citrus xanthophyll biosynthesis.The independent function of CsBCH2 and its cooperative function with CsBCH1 inβ-cryptoxanthin biosynthesis suggested the potential of CsBCH2 to be employed for expanding the synthetic biology toolkit in carotenoid engineering。

Promoter insertion leads to polyembryony in mango-a case of convergent evolution with citrus

Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture.In heterozygous plants,after the identification of a plant with desired traits,vegetative propagation(cloning)is the primary path to create genetically uniform plants.Another natural plant mechanism that creates genetically uniform plants(clones)is apomixis.In fruit crops like citrus and mango,sporophytic apomixis results in polyembryony,where seeds contain multiple embryos,one of which is sexually originated and the others are vegetative clones of the parent mother tree.Utilizing the mango genome and genetic analysis of a diverse germplasm collection,we identified MiRWP as the gene that causes polyembryony in mango.There is a strong correlation between a specific insertion in the gene’s promoter region and altered expression in flowers and developing fruitlets,inducing multiple embryos.The MiRWP gene is an ortholog of CitRWP that causes polyembryony in citrus.Based on the data,we speculate that promoter insertion events,which occurred independently in citrus and mango,induced nucellar embryogenesis.The results suggest convergent evolution of polyembryony in the two species.Further work is required to demonstrate the utility of these genes(mango and citrus)in other biological systems as a tool for the clonal production of other crops.

Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus

Cytoplasmic male sterility(CMS)has long been used to produce seedless fruits in perennial woody crops like citrus.A male-sterile somatic cybrid citrus(G1+HBP)was generated by protoplast fusion between a CMS callus parent‘Guoqing No.1’Satsuma mandarin(Citrus unshiu,G1)and a fertile mesophyll parent Hirado Buntan pummelo(Citrus grandis,HBP).To uncover the male-sterile mechanism of G1+HBP,we compared the transcriptome profiles of stamen organ and cell types at five stages between G1+HBP and HBP,including the initial stamen primordia,enlarged stamen primordia,pollen mother cells,tetrads,and microspores captured by laser microdissection.The stamen organ and cell types showed distinct gene expression profiles.A majority of genes involved in stamen development were differentially expressed,especially CgAP3.2,which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1+HBP compared with HBP.Jasmonic acid-and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia,and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1+HBP.In contrast,the content of auxin biosynthesis metabolites was lower in G1+HBP.The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia,meiocytes,and microspores,indicating the dysfunction of mitochondria in stamen organ and cell types of G1+HBP.Taken together,the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development,and thus lead to male sterility in the citrus cybrid.