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.

Global analysis of basic leucine zipper transcription factors in trifoliate orange and the function identification of PtbZIP49 in salt tolerance

As one of the most widely distributed and highly conserved transcription factors in eukaryotes,basic leucine zipper proteins(bZIPs)are involved in a variety of biological processes in plants,but they are largely unknown in citrus.In this study,56 bZIP family members were identified genome-wide from an important citrus rootstock,namely trifoliate orange(Poncirus trifoliata L.Raf.),and these putative bZIPs were named PtbZIP1—PtbZIP56.All PtbZIPs were classified into 13 subgroups by phylogenetic comparison with Arabidopsis thaliana bZIPs(AtbZIPs),and they were randomly distributed on nine known(50 genes)chromosomes and one unknown(6 genes)chromosome.Sequence analysis revealed the detailed characteristics of PtPZIPs,including their amino acid length,isoelectric point(pI),molecular weight(MW),predicted subcellular localization,gene structure,and conserved motifs.Prediction of promoter elements suggested the presence of drought,low-temperature,wound,and defense and stress responsive elements,as well as multiple hormone-responsive cis-acting elements.Spatiotemporal expression analysis showed the transcriptional patterns of PtbZIPs in different tissues and under dehydration,high salt,ABA,and IAA treatments.In addition,21 PtbZIPs were predicted to have direct or indirect protein—protein interactions.Among these,PtbZIP49 was experimentally proven to interact with PtbZIP1 or PtbZIP11 by using a yeast two-hybrid assay and bimolecular fluorescence complementation(BiFC).Subcellular localization analysis further revealed that PtbZIP1,PtbZIP11,and PtbZIP49 were localized in the nucleus.Moreover,PtbZIP49 was functionally identified as having an important role in salt stress via ectopic expression in A.thaliana and silenced in trifoliate orange using virus-induced gene silencing(VIGS).This study provided comprehensive information on PtbZIP transcription factors in citrus and highlights their potential functions in abiotic stress.

High-Performance Organic Field-Effect Transistors Based on Two-Dimensional Vat Orange 3 Crystals

The exploration and research of low-cost,environmentally friendly,and sustainable organic semiconductor materials are of immense significance in various fields,including electronics,optoelectronics,and energy conversion.Unfortunately,these semiconductors have almost poor charge transport properties,which range from∼10^(−4) cm^(2)·V^(−1)·s^(−1) to∼10^(−2) cm^(2)·V^(−1)·s^(−1).Vat orange 3,as one of these organic semiconductors,has great potential due to its highly conjugated structure.We obtain high-quality multilayered Vat orange 3 crystals with two-dimensional(2D)growth on h-BN surfaces with thickness of 10–100 nm using physical vapor transport.Raman’s results confirm the stability of the chemical structure of Vat orange 3 during growth.Furthermore,by leveraging the structural advantages of 2D materials,an organic field-effect transistor with a 2D vdW vertical heterostructure is further realized with h-BN encapsulation and multilayered graphene contact electrodes,resulting in an excellent transistor performance with On/Off ratio of 104 and high field-effect mobility of 0.14 cm^(2)·V^(−1)·s^(−1).Our results show the great potential of Vat orange 3 with 2D structures in future nano-electronic applications.Furthermore,we showcase an approach that integrates organic semiconductors with 2D materials,aiming to offer new insights into the study of organic semiconductors.

Green Synthesis of Silver Nanoparticles Using Aqueous Orange and Lemon Peel Extract and Evaluation of Their Antimicrobial Properties

Green synthesis of silver nanoparticles (AgNPs) using aqueous extracts of orange and lemon peels, as a reducing agent, and silver nitrate salts as a source of silver ions is a promising field of research due to the versatility of biomedical applications of metal nanoparticles. In this paper, AgNPs were synthetized at different reaction parameters such as the type and concentration of the extracts, metal salt concentration, temperature, speed stirring, and pH. The antibacterial properties of the obtained silver nanoparticles against E. coli, as well as the physical and chemical characteristics of the synthesized silver nanoparticles, were investigated. UV-Vis spectroscopy was used to confirm the formation of AgNPs. In addition to green biogenic synthesis, chemical synthesis of silver nanoparticles was also carried out. The optimal temperature for extraction was 65˚C, while for the synthesis of AgNPs was 35˚C. The synthesis is carried out in an acidic environment (pH = 4.7 orange and pH = 3.8 lemon), neutral (pH = 7) and alkaline (pH = 10), then for different concentrations of silver nitrate solution (0.5 mM - 1 mM), optimal time duration of the reaction was 60 min and optimal stirring speed rotation was 250 rpm on the magnetic stirrer. The physical properties of the synthesized silver nanoparticles (conductivity, density and refractive index) were also studied, and the passage of laser light through the obtained solution and distilled water was compared. Positive inhibitory effect on the growth of new Escherichia coli colonies have shown AgNPs synthesized at a basic pH value and at a 0.1 mM AgNO3 using orange or lemon peel extract, while for a 0.5 mM AgNO3 using lemon peel extract.

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.

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.

Design of an Interconnection Architecture and Sizing of Two (2) EPC Core Networks: The Case of Orange-Guinea

With the arrival of the 4G and 5G,the telecommunications networks have experienced a large expansion of these networks.That enabled the integration of many services and adequate flow,thus enabling the operators to respond to the growing demand of users.This rapid evolution has given the operators to adapt,their methods to the new technologies that increase.This complexity becomes more important,when these networks include several technologies to access different from the heterogeneous network like in the 4G network.The dimensional new challenges tell the application and the considerable increase in demand for services and the compatibility with existing networks,the management of mobility intercellular of users and it offers a better quality of services.Thus,the proposed solution to meet these new requirements is the sizing of the EPC(Evolved Packet Core)core network to support the 5G access network.For the case of Orange Guinea,this involves setting up an architecture for interconnecting the core networks of Sonfonia and Camayenne.The objectives of our work are of two orders:(1)to propose these solutions and recommendations for the heart network EPC sizing and the deployment to be adopted;(2)supply and architectural interconnection in the heart network EPC and an existing heart network.In our work,the model of traffic in communication that we use to calculate the traffic generated with each technology has link in the network of the heart.

Dynamic Changes of Sugar Concentrations in Pulp of ‘Cara cara' Navel Orange(Citrus. sinensis L. Osbeck) after Application by Exogenous ABA and GA_3

The dynamic concentrations of glucose, fructose, sucrose and total sugar were determined through exogenous ABA and GA3 treatments during young period of fruit and before fruit coloring in pulp of ‘Cara cara’ navel orange. The results are as fol ows： 10 mg/L ABA increased glucose, fructose and total sugar concen-tration significantly or very significantly, ABA treatment of 50 mg/L increased sucrose concentration very significantly, but ABA treatment of 100 mg/L decreased glucose concentration very significantly. GA3 treatment of lower and middle concentrations（10, 50 and 250 mg/L） increased sucrose concentration very significantly, GA3 treatment of 10 mg/L had no remarkable effect on glucose and fructose concentration but in-creased total sugar concentration very significantly, GA3 treatment of 50, 250 and 500 mg/L decreased glucose, fructose and total sugar concentration very significant-ly. Therefore, ABA treatment of lower concentration could increase one or several kinds of sugar concentration, but GA3 treatment of higher concentration （250 and 500 mg/L） restrained sugar concentration in pulp of ‘Cara cara’ navel orange seri-ously.

Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.

Production of Transgenic Anliucheng Sweet Orange(Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.

CsWAKL08,a pathogen-induced wall-associated receptor-like kinase in sweet orange,confers resistance to citrus bacterial canker via ROS control and JA signaling

Citrus bacterial canker(CBC)is a disease resulting from Xanthomonas citri subsp.citri(Xcc)infection and poses a grave threat to citrus production worldwide.Wall-associated receptor-like kinases(WAKLs)are proteins with a central role in resisting a range of fungal and bacterial diseases.The roles of WAKLs in the context of CBC resistance,however,remain unclear.Here,we explored the role of CsWAKL08,which confers resistance to CBC,and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance.Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis,CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety.CsWAKL08 can also be induced by the phytohormones salicylic acid(SA)and methyl jasmonic acid(MeJA)and spans the plasma membrane.Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges,whereas silencing of CsWAKL08 resulted in susceptibility to CBC.The peroxidase(POD)and superoxide dismutase(SOD)activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants,thereby mediating reactive oxygen species(ROS)homeostasis in the transgenic plants.Moreover,the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection.Based on these findings,we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling.These results further highlight the importance of this kinase family in plant pathogen resistance.

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Transcription factor WRKY22 regulates canker susceptibility in sweet orange (Citrus sinensis Osbeck) by enhancing cell enlargement and CsLOB1 expression

Pathological hypertrophy(cell enlargement)plays an important role in the development of citrus canker,but its regulators are largely unknown.Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis,rice and pepper,the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp.citri(Xcc)in Wanjincheng orange(Citrus sinensis Osbeck).Here,we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator.CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc,whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc.Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll.Transcriptome analysis showed that cell growth-related pathways,such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways,were significantly upregulated upon CsWRKY22 overexpression.Interestingly,CsWRKY22 activated the expression of CsLOB1,which is a key gene regulating susceptibility to citrus canker.We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro.We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression.Thus,our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.

Ca(NO3)2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange(Citrus sinensis Osbeck)

This study aimed to provide a theoretical basis for adopting suitable cultivation measures to tackle calcium （Ca） deficiency in citrus leaves. The Newhall navel orange （Citrus sinensis Osbeck） canopy was sprayed with 20.0 mmol L-1 of Ca（NO3）2 during physiological fruit drop period, fruit expanding period, and fruit maturing period on 30, 90, and 210 days after full bloom （DAFB）, respectively, and its effects on leaf gas exchange parameters and leaf mineral nutrition and fruit quality were analyzed. The results showed that： （1） The photosynthetic rate （ACO2） at 9：00 a.m. and 16：00 p.m. of fruit expanding period with 30 and 90 DAFB Ca（NO3）2treatments slightly or significantly improved mainly by decreasing stomatal limitation and nonstomatal limitation, respectively. （2） Compared with control （CK）, the Ca concentration in leaves with 30, 90, and 240 DAFB Ca（NO3）2 treatments increased by 127.16; 97.53, and 33.33%, respectively, and the leaf magnesium concentra- tion also increased by more than 32.26%. However, Ca（NO3）2 canopy spraying on 30 DAFB significantly reduced the leaf potassium concentration, by 22.14% compared with CK. （3） Ca（NO3）2 canopy spraying on 30 DAFB decreased the second fruit drop rate by 30.55% and increased the weight per fruit by 25.04%, thus resulting in a significant increase in citrus yield. （4） Spraying Ca（NO3） on 30 DAFB mainly affected the metabolism of titratable acid （TA） to improve the maturity of citrus fruits. Whilst it improved the external quality and the coloring of citrus fruit significantly. Therefore, Ca（NO3）2 canopy spraying during physiological fruit drop period has a better influence on the tree character and fruit quality of Newhall navel orange （Citrus sinensis Osbeck）.

Effect of harvest time on the chemical composition and antioxidant capacity of Gannan navel orange(Citrus sinensis L. Osbeck 'Newhall')juice

The present study investigates the chemical composition and antioxidant capacity of juice from the Gannan navel orange, which is harvested at one-to two-week intervals during the ripening period. The total soluble solid(TSS), total polyphenol content(TPC), total flavonoid content(TFC), sucrose and hesperidin contents gradually increase with the ripening of the fruit, followed by slight declines at the late maturity stage. Contrary to these observations, the contents of titratable acid(TA), vitamin C(Vc), and limonin trend downward throughout the ripening period. However, the contents of fructose, glucose, and narirutin fluctuate throughout the harvest time. Three in vitro antioxidant assays consistently indicate that the harvest time exerts no significant influence(P>0.01) on the antioxidant capacity. Furthermore, principal component analysis(PCA) and Pearson’s correlation test are performed to provide an overview of the complete dataset.This study provides valuable information for evaluating the fruit quality and determining when to harvest the fruit in order to meet the preferences of consumers. Meanwhile, our observations suggest that the fruits subjected to juice processing should be harvested at the late maturity stage to alleviate the "delayed bitterness" problem without compromising the antioxidant capacity and the flavonoid content in the juice.

Enhancement and Preservation of Fresh Orange Juice Using Citrus Essential Oils

Nowadays, consumers are demanding for minimally processed, additive-free, high quality, fresh-like andmicrobiologically safe foods. However, as food deterioration is the constant threat along the entire food chain, food preservationremains as necessary today as in the past. In this paper, several citrus essential oils, including sweet orange, lemon and lime essentialoil applied directly in fresh orange juice as a natural preservative, were analyzed for their antimicrobial activity. The antimicrobialactivity of each citrus essential oil was assessed against Saccharomyces cerevisiae, Lactobacillus plantarum and Aspergillus nigerusing broth macrodilution. Among all citrus essential oils tested, sweet orange essential oil with 0.10% concentration showedsignificant antimicrobial activity against microorganisms tested. Hence, it was chosen to be applied in the fresh orange juice. Then,shelf life analysis was conducted during 7 d of storage at 5 ℃ and during 2 d of storage at 25 ℃ using selected concentration of theselected citrus essential oil in the fresh orange juice. Based on the results obtained, the addition of sweet orange essential oil with0.10% concentration was not able to prolong the shelf life of the fresh orange juice according to the Indonesian National Standard（SNI）. However, the formulation of fresh orange juice itself was able to improve its shelf life when compared to the shelf life of thecommercial orange juice.

Two Lycopene β-Cyclases Genes from Sweet Orange (Citrus sinensis L. Osbeck) Encode Enzymes With Different Functional Efficiency During the Conversion of Lycopene-to-Provitamin A

Citrus fruits are rich in carotenoids.In the carotenoid biosynthetic pathway,lycopene β-cyclase（LCYb,EC：1.14.-.-） is a key regulatory enzyme in the catalysis of lycopene to β-carotene,an important dietary precursor of vitamin A for human nutrition.Two closely related lycopene β-cyclase cDNAs,designated CsLCYb1 and CsLCYb2,were isolated from the pulp of orange fruits（Citrus sinensis）.The expression level of CsLCYb genes is lower in the flavedo and juice sacs of a lycopeneaccumulating genotype Cara Cara than that in common genotype Washington,and this might be correlated with lycopene accumulation in Cara Cara fruit.The CsLCYb1 efficiently converted lycopene into the bicyclic β-carotene in an Escherichia coli expression system,but the CsLCYb2 exhibited a lower enzyme activity and converted lycopene into the β-carotene and the monocyclic γ-carotene.In tomato transformation studies,expression of CsLCYb1 under the control of the cauliflower mosaic virus（CaMV） 35S constitutive promoter resulted in a virtually complete conversion of lycopene into β-carotene,and the ripe fruits displayed a bright orange colour.However,the CsLCYb2 transgenic tomato plants did not show an altered fruit colour during development and maturation.In fruits of the CsLCYb1 transgenic plants,most of the lycopene was converted into β-carotene with provitamin A levels reaching about 700 μg g-1DW.Unexpectedly,most transgenic tomatoes showed a reduction in total carotenoid accumulation,and this is consistent with the decrease in expression of endogenous carotenogenic genes in transgenic fruits.Collectively,these results suggested that the cloned CsLCYb1 and CsLCYb2 genes encoded two functional lycopene β-cyclases with different catalytic efficiency,and they may have potential for metabolite engineering toward altering pigmentation and enhancing nutritional value of food crops.

Physiological changes and expression characteristics of ZIP family genes under zinc def iciency in navel orange(Citrus sinensis)

Zinc （Zn） deficiency is widespread among citrus plants, but information about the mechanisms for Zn deficiency response in these plants is scarce. In the present study, different navel orange （Citrus sinensis （L.） Osbeck） leaves with various yellowing levels were sampled in our experimental orchard, and upon estimation of nutrient contents, Zn deficiencies were diagnosed as mild, moderate, and severe. Further analysis of chlorophyll content, photosynthetic characteristics, antioxidant enzyme activities, and expression levels of Zn/Iron-regulated transporter-like protein （ZIP） family genes were conducted in the sampled Zn-deficient leaves. The results showed that chlorophyll contents and net photosynthetic rate （Pn） seemed to decrease with reduced Zn contents. In addition, comparison of severe Zn-deficient and normal leaves revealed that activities of peroxidase （POD） and catalase （CAT） increased significantly, whereas that of Zn-containing enzymes such as Cu/Zn superoxide dismutase （Cu/Zn-SOD） significantly reduced with decreasing Zn contents. As expected, expression of the ZIP family genes, ZIP1, ZIP3, and ZIP4, was induced by Zn deficiencies. These results deepen our understanding of Zn deficiency in citrus plants as well as provide useful preliminary information for further research.