Systematic analysis and functional verification of citrus glutathione S-transferases reveals that CsGSTF1 and CsGSTU18contribute negatively to citrus bacterial canker

Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.

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.

Crc1,6RhaT is involved in the synthesis of hesperidin of the main bioactive substance in the Citrus reticulata ‘Chachi' fruit

Hesperidin is a dihydroflavonoids, accounting for more than 50% of the total flavonoids in Citri Reticulatae Pericarpium(CRP) of traditional Chinese medicine. It is an effective antioxidant and free radical scavenger that has anti-inflammatory, antiviral and hypoglycemic properties.The latest studies reported that hesperidin has a potential for novel coronavirus resistance. However, little is known about the synthesis regulation and accumulation site of hesperidin in plants. In this study, hesperidin synthase gene Crc1,6RhaT was cloned, and the protein can be completely transformed flavanone-7-O-glucoside into hesperidin in vitro and in vivo. Studies on biological characteristics of ovary walls and exocarps showed that the relative expression levels of the Crc1,6RhaT gene and protein decreased gradually with the development of citrus fruits, and the relative content of hesperidin firstly increased, then sequentially decreased. In situ hybridization results further revealed that Crc1,6RhaT transcription was mainly concentrated in the secretory cavity cells, which are revealed to be the site of flavonoid synthesis.Immunocytochemistry localization results showed that the Crc1,6RhaT was mainly located in the endoplasmic reticulum, nucleus and vacuole of secretory cells. We inferred that the Crc1,6RhaT was synthesized in the endoplasmic reticulum, then was transported into the vacuoles through enlarged vesicles at the end of the endoplasmic reticulum. Our results not only revealed that Crc1,6RhaT may be involved in the synthesis of hesperidin of the main bioactive substance in the medicinal plant Citrus reticulata ‘Chachi' fruit, but also provided novel insights into the main subcellular sites of hesperidin biosynthesis in vacuoles.

Antiglycating effects of citrus flavonoids and associated mechanisms

Glycation of proteins and DNA forms advanced glycation end products(AGEs)causing cell and tissue dysfunction and subsequent various chronic diseases,in particular,metabolic and age-related diseases.Targeted AGE inhibition includes scavengers of reactive carbonyl species(RCS)such as methylglyoxal(MG),glyoxalase-1 enhancers,Nrf2/ARE pathway activators,AGE/RAGE formation inhibitors and other antiglycatng agents.Citrus flavonoids have demonstrated antioxidant and anti-inflammatory effects and are also found to be effective antiglycating agents.Herein,we reviewed the up to date progress of the antiglycation effects of citrus flavonoids and associated mechanisms.Major citrus flavonoids,hesperedin and its aglycone,hesperetin,inhibited glycation by scavenging MG forming mono-or di-flavonoid adducts with MG,enhanced the activity of glyoxase-1,activated Akt/Nrf2 signal pathway while inhibiting AGE/RAGE/NF-κB pathway,reduced the formation of Nε-(carboxylmethyl)lysine(CML)and pentosidine,inhibited aldol reductase activity and decreased the levels of fructosamine.The antiglycating activity and mechanisms of other flavonoids was also summarized in this review.In conclusion,citrus flavonoids possess effective antiglycating activity via different mechanisms,yet there are many challenging questions remaining to be studied in the near future such as in vivo testing and human study of citrus flavonoids for efficacy,effectiveness and adverse effects of citrus flavonoids as a functional food in managing high levels of AGEs and controlling AGE-induced chronic diseases,diabetic complications in particular.

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.

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.

Citrus aurantium L. extract alleviate depression by inhibiting gut microbiota-mediated inflammation in mice

Gut microbiota plays a crucial role in the pathophysiology of depression.This study aimed to explore the antidepressant effect of mature whole Citrus aurantium fruit extract(FEMC)in the chronic unpredictable mild stress(CUMS)model.The behavioral tests were applied to assess antidepressant effect and 16S rRNA sequencing was used to analyze the changes of gut microbiota.The results showed that the major components of FEMC were naringin and neohesperidin and significantly increased the sucrose preference index of the mice.FEMC also could reduce the feeding latency in an open field test and the rest time in a novelty suppressed feeding test.In addition,FEMC could increase CUMS-induced reduction in the levels of BDNF,PSD95,and SYN in the hippocampus.Moreover,FEMC intervention slightly decreased the ratio of Firmicutes to Bacteroidota.Meanwhile,FEMC reduced the abundance of the Prevotellaceae_Ga6A1_group,[Ruminococcus]_torques_group,which have been reported to be closely related to inflammation.Bioinformatics analysis revealed that mitogen-activated protein kinase(MAPK)signaling pathway and lipopolysaccharide biosynthesis were involved in the anti-inflammatory effect of FEMC in the CUMS animal model.Finally,the ELISA results showed that FEMC could significantly reduce the expression of pro-inflammatory cytokines IL-6 and TNF-αin the serum of depressive mice.Our results suggest FEMC can am eliorate depressive behavior by i nhibiting gut microbiota-mediated inflammation in mice.

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.

Effect of Various Citrus Rootstock-scion Combinations on Tree Growth,Fruit Quality,and Photosynthetic Characteristics under Red Loam Soil Conditions

[Objectives]To investigate the effects of 15 distinct citrus rootstock-scion combinations on tree growth,fruit quality,and photosynthetic characteristics under red loam soil conditions and provide a theoretical foundation for the selection of appropriate citrus rootstock-scion combinations in the Zhaoqing region.[Methods]A total of 15 citrus rootstock-scion combinations were utilized as test materials for a comprehensive analysis of their phenological periods(budding,flowering,and fruiting),tree growth indicators(tree height,crown diameter,and growth),and fruit quality(appearance quality and intrinsic quality).The photosynthetic characteristics of the test materials,including the net photosynthetic rate(Pn),transpiration rate(Tr),water use efficiency(WUE),apparent quantum yield(AQY),and carboxylation efficiency(CE),were analyzed to determine their significance.Additionally,the leaf photosynthetic physiological indicators,such as soluble protein,specific leaf weight,chlorophyll,and carotenoids,were evaluated.[Results]There were notable differences observed in the phenological period,growth potential of trees,fruit quality,and photosynthetic characteristics among various citrus rootstock-scion combinations.The phenological periods exhibited variation contingent on the grafting varieties.In terms of tree growth potential,the Citrus tangerina Tanaka‘Hongju’and C.haniana Hort.‘Suanju’rootstocks demonstrated greater tree height,crown growth,and overall tree strength;however,they were also prone to excessive growth.Conversely,the C.limonia Osbeck‘Hongningmeng’and C.sinensis×P.trifoliata‘Zhicheng’rootstocks displayed medium growth potential,while the Poncirus trifoliate(L)Raf.‘Zhike’rootstock resulted in shorter trees.In terms of fruit quality,the single fruit weight of C.flamea Hort.‘Shatangju’ranged from 33 to 50 g,exhibiting a flat and round shape.The total soluble solids and titratable acid content of‘Shatangju’grafted onto the‘Zhike’rootstock were notably high.In contrast,the single fruit weight of C.haniana Hort.‘Chuntianju’varied between 65 and 81 g,characterized by a high flat round shape.The‘Suanju’rootstock demonstrated a higher sugar and acid content compared to other rootstocks.Additionally,the single fruit weight of C.nobilis Lour.‘Gonggan’ranged from 62 to 145 g,with the fruit shape being either round or oval.The soluble sugar and total soluble solids content associated with the‘Zhike’rootstock was also elevated.In relation to photosynthetic characteristics,the photosynthetic performance of the‘Shatangju’variety was superior when grafted onto the‘Zhike’and‘Hongju’rootstocks.Similarly,the‘Chuntianju’variety exhibited enhanced photosynthetic performance on the‘Zhike’,‘Zhicheng’,and‘Hongju’rootstocks.Furthermore,the‘Gonggan’variety demonstrated improved photosynthetic performance when grafted onto the‘Zhike’and‘Suanju’rootstocks.[Conclusions]Based on the characteristics of the red loam soil in the Zhaoqing region,the rootstocks‘Zhike’and‘Hongju’are conducive to the cultivation of the‘Shatangju’variety.Additionally,the rootstocks‘Zhike’,‘Zhicheng’,and‘Hongju’are optimal for the growth of the‘Chuntianju’variety,while the rootstocks‘Zhike’and‘Suanju’are appropriate for the growth of the‘Gonggan’variety.

Survey and Detection of Disease and Pest Occurrence in Citrus Nurseries

[Objectives]The paper was to ascertain the prevalence of diseases and pests in a range of citrus nurseries situated in Guangdong Province and its neighboring provinces.[Methods]Citrus diseases and pests were systematically investigated,and citrus leaf samples were randomly collected from 15 citrus nurseries across 8 regions in Guangdong Province and its neighboring provinces.Quantitative polymerase chain reaction(qPCR)and reverse transcription polymerase chain reaction(RT-PCR)techniques were employed to detect diseases in the collected samples.Additionally,root and substrate samples were obtained,and root-knot nematodes were isolated using the Baermann funnel method.[Results]The positive detection rate of citrus huanglongbing(HLB)was recorded at 3%,indicating an increase in attention towards this disease compared to 2013.Additionally,the positive detection rate for citrus bacterial canker disease(CBCD)was found to be 16.5%.It was observed that the majority of nurseries with positive samples employed open field rearing practices without the use of mesh chambers,and the primary source of scions was self-propagation.The detection rate of citrus tristeza virus(CTV)was found to be the highest,with a positive detection rate of 63%,and the prevalence in disease-bearing nurseries reached as high as 90%.In comparison to 2013,there had been no improvement in the condition of seedlings affected by CTV.The positive detection rate of citrus yellow vein clearing virus(CYVCV)was found to be 38%,with 70%of the surveyed nurseries exhibiting the disease.The citrus varieties identified as carriers of the disease included‘Qicheng’,‘Shatangju’,‘Wogan’,and‘Gonggan’.Nematodes were isolated from the matrix and roots of seedlings grown in both container and open field environments.The susceptibility of container seedlings to nematodes was found to be 36.4%,while the susceptibility of open field seedlings was 38.6%.Statistical analysis indicated no significant difference in susceptibility between the two groups.[Conclusions]The disease detection rates associated with various seedling rearing methods and citrus varieties exhibited notable variability.Open field seedlings without the protection of mesh chambers demonstrated a higher susceptibility to disease.Additionally,the types of infectious diseases varied among the different citrus varieties.

Effects of Different Seasons on Cutting Propagation of Five Citrus Rootstocks

[Objectives]To explore the cutting propagation mode of citrus rootstock,improve the survival rooting rate of citrus rootstock,and provide theoretical guidance and technical reference for the rooting research of difficult-to-root plants such as citrus.[Methods]Five citrus rootstocks Citrus tangerina Tanaka‘Hongju’,Citrus haniana Hort‘Suanju’,Citrus limonia Osbeck‘Hongningmeng’,Citrus sinensis×Poncirus trifoliata‘Zhicheng’and Poncirus trifoliate(L)Raf.‘Zhike’were used as experimental materials to select the suitable cutting substrate for citrus rootstocks by measuring the physical properties of the substrate.Cutting was carried out in spring,summer,autumn and winter respectively.After cutting,the morphological changes of cuttings were observed regularly,and the callus rate,germination rate and rooting rate of cuttings were recorded.[Results]The best substrate for citrus rootstock cutting was peat soil,vermiculite and fine river sand(2:1:1).The callus of citrus rootstock in different cutting seasons began to appear in 10-22 d,and the callus rate reached 55%-100%.In terms of budding,the budding time was the earliest in summer and autumn,and slightly later in spring and winter;the germination rate of C.limonia Osbeck‘Hongningmeng’was the highest,and the germination rate of C.sinensis×P.trifoliata‘Zhicheng’and P.trifoliate(L)Raf.‘Zhike’was lower;in terms of rooting,C.limonia Osbeck‘Hongningmeng’had the earliest rooting time and the highest rooting rate and could reach 100%in all seasons;the rooting rate of C.tangerina Tanaka‘Hongju’was 50%-80%;the rooting rate of C.haniana Hort‘Suanju’was 60%-80%;C.sinensis×P.trifoliata‘Zhicheng’and P.trifoliate(L)Raf.‘Zhike’showed the earliest rooting time and the highest rooting rate in summer,and the latest rooting time and the lowest rooting rate in winter,which were only 14.5%.Therefore,different citrus rootstock varieties should choose the appropriate cutting time according to their own characteristics.[Conclusions]The results of this study can provide a scientific basis for a large number of cutting propagation of different citrus rootstocks,and have practical guiding significance for large-scale planting.

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.

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.

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.

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.

An endolysin gene from Candidatus Liberibacter asiaticus confers dual resistance to huanglongbing and citrus canker

The most damaging citrus diseases are Huanglongbing(HLB)and citrus canker,which are caused by Candidatus Liberibacter asiaticus(CaLas)and Xanthomonas citri pv.citri(Xcc),respectively.Endolysins from bacteriophages are a possible option for disease resistance in plant breeding.Here,we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas.LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc,according to inhibition zone analyses.The two genes,driven by a strong promoter from Cauliflower mosaic virus,35S,were integrated into Carrizo citrange via Agrobacterium-mediated transformation.More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB,allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term.LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc,indicating that LasLYS2 had dual resistance to HLB and citrus canker.A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations,which might boost the citrus defense response,according to transcriptome analysis.We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2.Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2.Overall,the work shed light on the mechanisms of resistance of CaLas-derived endolysins,providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.

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。

Deep Transfer Learning Based Detection and Classification of Citrus Plant Diseases

Citrus fruit crops are among the world’s most important agricultural products,but pests and diseases impact their cultivation,resulting in yield and quality losses.Computer vision and machine learning have been widely used to detect and classify plant diseases over the last decade,allowing for early disease detection and improving agricultural production.This paper presented an automatic system for the early detection and classification of citrus plant diseases based on a deep learning(DL)model,which improved accuracy while decreasing computational complexity.The most recent transfer learning-based models were applied to the Citrus Plant Dataset to improve classification accuracy.Using transfer learning,this study successfully proposed a Convolutional Neural Network(CNN)-based pre-trained model(EfficientNetB3,ResNet50,MobiNetV2,and InceptionV3)for the identification and categorization of citrus plant diseases.To evaluate the architecture’s performance,this study discovered that transferring an EfficientNetb3 model resulted in the highest training,validating,and testing accuracies,which were 99.43%,99.48%,and 99.58%,respectively.In identifying and categorizing citrus plant diseases,the proposed CNN model outperforms other cuttingedge CNN model architectures developed previously in the literature.

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.