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.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp.citri to achieve resistance to citrus canker disease

Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a serious bacterial disease that affects citrus production worldwide.Citron C-05(Citrus medica)is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc.However,it has not been determined when,where,and how Xcc is restricted in the tissues of Citron C-05 during the infection process.In the present study,we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc(eGFP-Xcc)strain in Citron C-05 along with five susceptible biotypes(i.e.,lemon,pummelo,sour orange,sweet orange,and ponkan mandarin)upon inoculation via the spraying or leaf infiltration of a bacterial suspension.The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes,Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection.Not surprisingly,resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death,as well as greater upregulation of several defense-related genes,including a pathogenesis-related gene(PR1)and a glutathione S-transferase gene(GST1),compared with sweet orange as a susceptible control.Taken together,our results not only provide further valuable details of the spatiotemporal dynamics of the host entry,propagation,and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants

Endogenous auxin is an important regulator of in vivo organ development,but its role in in vitro organogenesis is unclear.It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end.Here,we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis.Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene,we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport.Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis.An auxin transport inhibitor,N-1-naphthylphthalamic acid(NPA),can also enhance shoot organogenesis independent of its action on polar auxin transport.Finally,we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent.Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport,as well as auxin–cytokinin interactions,in in vitro shoot organogenesis.Meanwhile,our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

Green fl uorescent protein(GFP)has been widely used for monitoring gene expression and protein localization in diverse organisms.However,highly sensitive imaging equipment,like fl uorescence microscope,is usually required for the visualization of GFP,limitings its application to fi xed locations in samples.A reporter that can be visualized in realtime regardless the shape,size and location of the target samples will increase the fl exibility and ef fi ciency of research work.Here,we report the application of a GFP-like protein,called eYGFPuv,in both transient expression and stable transformation,in two herbaceous plant species(Arabidopsis and tobacco)and two woody plant species(poplar and citrus).We observed bright fl uorescence under UV light in all of the four plant species without any effects on plant growth or development.eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues.With a focus on in vitro transformation,we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identi fi ed visually during the callus stage and the shoot stage,enabling early and ef fi cient selection of transformants.Furthermore,whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants.In addition,our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics.This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

Molecular Detection and Characterization of Citrus Viroids

Total RNA was isolated from the phloem of young shoots and retro-transcripted to cDNA by reverse transcriptasepolymerase chain reaction （RT-PCR） with specific primers; the amplified cDNA fragments were sequenced for identification of subtypes. In this study, an effective RT-PCR technique was established for detecting citrus viroids, and three citrus viroids were recognized, namely, CEVd, CVd-II, and CVd-III. The latter two were identified for the first time in China. The sequences of CVd-Ⅱ were different from all the three subtypes reported in the GenBank and seemed to be a new subtype. CVd-Ⅲb detected in the present study could be further developed as a dwarfing agent.

Comparative analysis of Wanjincheng orange leaf and root responses to ‘Candidatus liberibacter asiaticus’ infection using leaf-disc grafting

Citrus Huanglongbing,associated with Candidatus Liberibacter asiaticus(Las),is the serious disease of citrus worldwide.Here,we compared differences between leaf and root responses in Wanjincheng Orange(Citrus sinensis Osbeck)to Las infection using leaf-disc grafting.Trees had no obvious symptoms in the first two months after grafting(MAG),but yellowing leaves,thickened midribs and decayed roots began to appear at 6 MAG.The Las growth rate in roots was greater than inmidribs from 2 to 6 MAG;however,by 10 MAG,it was significantly lower in roots than in midribs.Amicroscopic analysis revealed that starch accumulation,callose deposition,cellwall thickness and the number of cell layers increased in the phloem of infected trees.After Las infection,the starch content in the leaves was significantly higher than that in the roots,while the callose deposition in the midribs was 3–19 times that in the roots.A gene expression analysis showed that transcripts of callose-and starch metabolism-related genes were obviously affected by Las infection,and the pathogenesis-related genes PR1,PR2 and PR5 were significantly upregulated and downregulated in midrib and root,respectively.Our results indicated that the PR-mediated resistance response was repressed in root but activated in midrib,which may result in the more rapid Las growth in roots than in midribs during the early infection stage.

Genetic characterization of an almond germplasm collection and volatilome profiling of raw and roasted kernels

Almond is appreciated for its nutraceutical value and for the aromatic profile of the kernels.In this work,an almond collection composed of 96 Sicilian accessions complemented with 10 widely cultivated cultivars was phenotyped for the production of volatile organic compounds using a proton-transfer time-of-flight mass spectrometer and genotyped using the Illumina Infinium®18 K Peach SNP array.The profiling of the aroma was carried out on fresh and roasted kernels enabling the detection of 150 mass peaks.Sixty eight,for the most related with sulfur compounds,furan containing compounds,and aldehydes formed by Strecker degradation,significantly increased during roasting,while the concentration of fifty-four mass peaks,for the most belonging to alcohols and terpenes,significantly decreased.Four hundred and seventy-one robust SNPs were selected and employed for population genetic studies.Structure analysis detected three subpopulations with the Sicilian accessions characterized by a different genetic stratification compared to those collected in Apulia(South Italy)and the International cultivars.The linkage-disequilibrium(LD)decay across the genome was equal to r^(2)=0.083.Furthermore,a high level of collinearity(r^(2)=0.96)between almond and peach was registered confirming the high synteny between the two genomes.A preliminary application of a genome-wide association analysis allowed the detection of significant marker-trait associations for 31 fresh and 33 roasted almond mass peaks respectively.An accurate genetic and phenotypic characterization of novel germplasm can represent a valuable tool for the set-up of marker-assisted selection of novel cultivars with an enhanced aromatic profile.

Cloning and Expression Analysis of Citrus Genes Cs GH3.1 and Cs GH3.6Responding to Xanthomonas axonopodis pv. citri Infection

To study the functions of the early auxin-responsive genes Cs GH3.1 and Cs GH3.6 in citrus resistance against canker disease, we cloned Cs GH3.1and Cs GH3.6 in ‘Newhall' Navel Orange(Citrus sinensis Osbeck). They are 1 797 bp and 1 887 bp and encode 598 and 629 amino acids, respectively. In vitro mature leaves from susceptible ‘Newhall' and resistant Calamondin(C. madurensis) were inoculated by a Xanthomonas axonopodis pv. citri(Xac) bacterial suspension, and expression of Cs GH3.1 and Cs GH3.6 in the two varieties were analyzed using quantitative real-time PCR(q RT-PCR). ‘Newhall' leaves were treated with different hormones for 3 days, inoculated by Xac bacterial suspension, and then the symptoms in these leaves were investigated. We used q RT-PCR to analyze the effect of different hormones on Cs GH3.1 and Cs GH3.6 expression in ‘Newhall'leaves. The expression levels of both Cs GH3.1 and Cs GH3.6 were significantly induced by Xac in ‘Newhall' leaves, compared with levels in Calamondin leaves. 1-naphthy acetic acid(NAA) increased the hypertrophy of infection sites in ‘Newhall' leaves, while naphthyl-phthalamic acid(NPA)had no visible effect on lesion development. NAA hormone greatly improved expression of Cs GH3.1 in ‘Newhall', but not Cs GH3.6. These results indicate that the auxin primary-response gene Cs GH3.1 plays an important role in citrus susceptibility to Xac.