Cloning and characterization of a soluble acid invertase-encoding gene from muskmelon

Cloning and characterization of a soluble acid invertase-encoding gene from muskmelonHongmei Tian ÆQingguo Kong ÆYanqing Feng ÆXiyan YuReceived:12December 2007/Accepted:21February 2008/Published online:4March 2008ÓSpringer Science+Business Media B.V.2008Abstract Soluble acid invertase (S-AIV;EC 3.2.1.26)is thought to play a critical role in sucrose hydrolysis in muskmelon (Cucumis melo L.)fruit.A full-length cDNA clone encoding S-AIV was isolated from muskmelon by RT-PCR and RACE.The clone,designated as CmS-AIV1,contains 2178nucleotides with an open reading frame of 1908nucleotides.The deduced 636amino acid sequence showed high identities with other plant soluble acid in-vertases.Northern blot analysis indicated that CmS-AIV1was expressed in flowers and fruit,but was not detected in roots,stems or leaves.Moreover,the mRNA accumulation of CmS-AIV1showed its maximum level at 10days after pollination (DAP)and decreased gradually during fruit development until its minimum level at mature fruit.Interestingly,the sucrose content was very low in fruit before 20DAP but increased dramatically between 20and 30DAP during fruit development.In contrast to sucrose content,the activities of S-AIV was very high in fruit before 20DAP and decreased apparently between 20and 30DAP,suggesting that sucrose metabolism may be linked to the CmS-AIV1transcript level in muskmelon fruit.Keywords Cucumis melo ÁCmS-AIV1ÁGene expression ÁSucrose metabolismIntroductionSugars are the most important biochemical components for fruit quality.The kind and amount of sugars directly influence fruit flavor components,such as sweetness.As the first step toward the genetic improvement of the quality of muskmelon fruit,it is necessary to determine the sugar components accumulated in fruit,elucidate enzymes involved in sugar metabolism and clarify the relationship between the content of accumulated sugar and the activity of some related enzymes [1–3].At the middle stage of fruit development,muskmelon fruit undergoes a metabolic transition marked by both physical and compositional changes such as netting of the exocarp,mesocarp softening,and the onset of sucrose accumulation [4].Attempts to elucidate the changes in metabolism that lead to accumulation of sucrose have focused on sucrose metabolizing enzymes during fruit growth and development [5–8].It was reported that both acid invertase (EC 3.2.1.26)and sucrose phosphate syn-thase (EC 2.4.1.14)are determinants of sucrose accumulation in muskmelon fruit.Moreover,because sucrose accumulates in vacuoles,a reduction in acid invertase activity would be a prerequisite for muskmelon sucrose accumulation [9].We have cloned a sucrose phosphate synthase-encoding gene from muskmelon and it was shown to be expressed predominantly in mature fruit [10].Recently,acid invertase genes have been isolated from a number of higher plant species [11–16].In this report,we present the molecular cloning of CmS-AIV1from muskmelon.In addition,the differential expression of CmS-AIV1was determined in various tissues and fruit development stages of Cucumis melo .H.Tian ÁQ.Kong ÁY.Feng ÁX.Yu (&)State Key Laboratory of Crop Biology,College of Horticulture Science and Engineering,Shandong Agricultural University,Taian 271018,Chinae-mail:yuxiyan@Mol Biol Rep (2009)36:611–617DOI 10.1007/s11033-008-9219-2Materials and methodsPlant material and tissue samplingMuskmelon inbred line M01-3was grown in a greenhouse on the experimental farm of Shandong Agricultural Uni-versity in Tai’an,China March through June2007.Plant spacing was50cm between plants with120cm between rows.Average day/night temperatures were about30°C/ 20°C.Average daylight was about12h.Fertilizer was applied at two stages,a preplant broadcast application of 900kg ha-1of14N-14P-36K,followed by a sidedress application of150kg ha-1N atflowering stage.Irrigation by furrows was applied as needed.To identify fruit of known age freshly opened femaleflowers were tagged on the day of hand-pollination and one fruit per plant was allowed to develop.Different developing stage(5,10,15, 20and25DAP)and mature fruit were harvested;30DAP is considered commercial maturity.Five fruit from each stage were pooled to isolate total RNA.A portion of each fruit was diced into small pieces and used for S-AIV activity assay and sucrose determination.The S-AIV activity assays and sucrose determination were carried out on duplicate samples.The experiments were repeated three times.For each replicate,five fruit were used.Vegetative tissues,including leaves,stems,and roots were sampled along withflowers at bloom and harvested simultaneously fromfive7-to8-week-old plants.All tissues were quickly frozen in liquid nitrogen and stored at-80°C until late use. Extraction and purification of RNATotal RNA was isolated using the Guanidine Isothiocyanate-phenol-chloroform method as described by Sambrook et al.[17]with modification.Frozen tissue samples(5–10g)were ground to a powder in liquid N2using a mortar and pestle. Samples of the homogenized,powdered tissue were trans-ferred to50-ml screw-cap centrifuge tubes prechilled on the ice,followed by immediate addition of15ml solution D (4M Guanidine Isothiocyanate,0.025M trisodium citrate dehydrate,0.5%lauryl sarcosine)and of1.5ml of2M sodium acetate,pH4.0,15ml of phenol equilibrated with distilled water and3ml of49chloroform:1isopropyl alcohol(V/V).After vortexing and incubation for15min on the ice,the samples were processed according to Sam-brook’s method.The crude RNA preparations were then treated with DNase(TaKaRa Bio,Kyoto,Japan)to degrade genomic DNA,extracted with1phenol:1chloroform(V/V), and total RNA was precipitated by addition of1volume isopropyl alcohol plus0.1volume of3M sodium acetate, pH5.5.The precipitated RNA was pelleted by centrifuga-tion,washed with cold70%ethanol,and resuspended in diethyl pyrocarbonate-treated water.Quality of the extracted RNA was checked by agarose gel electrophoresis and RNA was quantified spectrophotomerically(UV2450,Shimadzu Corp.,Kyoto,Japan).Cloning of CmS-AIV1complete cDNATotal RNA isolated from5DAP fruit of C.melo M01-3 plants was used for the initial cloning of soluble acid invertase cDNA fragments,including products of30and50 RACE(rapid amplification of cDNA ends).As the initial step in cloning a C.melo soluble acid invertase gene, degenerate primers(P1:50-GACCCGACYACTGCTTGG-30,P2:50-CGATGTTATSACTGTTCT-30,Y=T+C, S=C+G)were designed based on the conserved domain of soluble acid invertase genes from other plants in Gen-Bank.RT-PCR was performed according to the protocol of RNA PCR Kit(AMV)Ver.3.0(TaKaRa Bio).Thefirst cDNA was synthesized by reverse transcription using reverse transcriptase with1l g RNA as template and with Oligo(dT)-Adaptor primer asfirst strand primer.The RT reaction program is42°C for30min,99°C for5min and 5°C for5min,1cycle.PCR was performed by running the following program:94°C pre-denature for5min;94°C for 1min,50°C for1min and72°C for1.5min,30cycles;at last,72°C for10min.The PCR product was separated by electrophoresis in1.0%agarose gel.The isolated fragment was cloned by using PMD18-T vector(TaKaRa Bio)for sequencing.A total of1l g RNA from muskmelon fruit was taken to convert mRNAs into cDNAs using a30RACE kit(TaKa-Ra)provided with AMV reverse transcriptase and a universal oligo(dT)containing adapter primer(50-CTGATCTAGAGGTACCGGATCCT17-30).The partial CmS-AIV1gene from the30end was then amplified by a pair of PCR primers:the gene specific forward primer1 (50-TACTCCGTGTTGACTCAGCTGCAG-30)and reverse primer(50-CTGATCTAGAGGTACCGGATCC-30)pro-vided with the kit.According to the manufacturer’s instructions,PCR was performed under the following conditions:cDNA was denatured at94°C for2min,fol-lowed by35cycles of amplification(94°C for30s,55°C for30s and72°C for1min)and7min at72°C.The PCR product was purified and cloned into pMD18-T vector for sequencing.Based on the sequence of the initial C.melo soluble acid invertase gene,the specific primers2,3,4(primer250-CGGGTTATCGGGTGTCCAT-30,primer350-CCCGGC CCGTTATATGATG-30,primer450-GCATGCAGCACT CCATCCA-30)were designed to amplify the50end of CmS-AIV1gene using50RACE system for rapid amplifi-cation of cDNA ends(version 2.0;Invitrogen Corp., Carlsbad,CA).The50RACE was performed according to the manufacturer’s instructions.The PCR product waspurified and cloned into pMD18-T vector for sequencing. DNAman 4.0(Lynnon Biosoft,Quebec,Canada),AN-THEPROT[version5.0;18],and NCBI web were used to analysis the DNA and protein sequences.Northern Blot analysisFor RNA gel-blot analysis,20l g of total RNA were sep-arated on 1.2%agarose gels containing formaldehyde. RNA gel blot analysis was performed according to the DIG Northern Starter Kit Manual(Roche,Penzberg,Germany). 30UTR fragment of CmS-AIV1cDNA(about300bp)was labeled with digoxygenin and used as the probe.RNA transfer andfixation were performed by capillary transfer with209SSC overnight and baking membrane at80°C for2h.Prehybridization was done at68°C in DIG Easy Hyb Buffer for30min with gentle agitation.Hybridization was carried out in the same buffer overnight at68°C.The blot was washed twice with29SSC and0.1%SDS at15–25°C under constant agitation for5min.Then the blot was washed twice in0.19SSC and0.1%SDS at68°C under constant agitation for15min.The hybridized probes are immunodetected with anti-digoxigenin-AP,Fab fragments and then visualized with the chemiluminescence substrate CDP-Star.Enzymatic dephosphorylation of CDP-Star by alkaline phosphatase leads to a light emission at a maxi-mum wavelength of465nm which is recorded on X-ray films.Exposure time was30min.Sucrose content measurementSucrose was extracted by grindingflesh tissues(10g fresh weight)in80%ethanol,adjusted to pH7.0with0.1N NaOH,and heated for5min at80°C.The extraction was repeated three times andfive fruit were used for each replicate.The extract corresponding to0.5g fresh weight was dried in vacuo and redissolved in water.An aliquot (10l l)of the solubilized material was analyzed by high performance liquid chromatography(Shimadzu Corp.)on a Sio2–NH2Lichrosorb column equipped with a RID–10A detector and a SCL-10AVP data acquisition system.Soluble acid invertase extraction and assayS-AIV extraction and assay were performed in a manner similar to that previously described by Lowell et al.[19] and Hubbard et al.[9]with some minor modifications. S-AIV was extracted from about1g of minced frozen muskmelon tissue by homogenizing for1min on ice in 200mM Hepes/NaOH(pH7.5),5mM MgCl2,1mM EDTA,0.5mg ml-1of BSA,0.05%Triton X-100 (Sigma-Aldrich Corp.,St.Louis,MO),1M NaCl,1% insoluble PVP,and5mM DTT.Homogenates were quickly desalted at4°C by centrifuging through a5-ml Sephadex G-50spin column[20].Desalted buffer con-tained20mM Hepes/NaOH(pH7.5),0.25mM MgCl2, 0.01%2-Mercaptoethanol,1mM EDTA,0.05%BSA, 0.2%glycerine.Desalted extract was assayed immediately.S-AIV was assayed in50mM Na-citrate buffer(pH5.0), using50mM sucrose as a substrate.Control was boiled for 10min immediately after the addition of the desalted enzyme extract.The reactions were incubated for30min at37°C and was terminated by addition of1ml3,5-dinitrosalicylic reagent containing0.01%(W/V)3,5-dinitrosalicylic acid, 1.6%(W/V)NaOH and30%(W/V)sodium potassium tart-arate[21].The reaction mixture was boiled for10min and the amounts of reducing sugars were measured with a microplate reader(3550-UV Bio-Rad Lab Inc.,Hercules,CA)at570nm and compared to glucose standards.ResultsCloning and characterization of CmS-AIV1To isolate S-AIV cDNA from muskmelon,wefirst designed a pair of degenerated primers according to the conserved domain of S-AIV amino acid sequences from other plant species.A1038bp cDNA fragment was amplified by RT-PCR.A900bp and a800bp cDNA fragments were pro-duced by50RACE and30RACE,respectively.After sequencing confirmation,a2178bp cDNA clone including a full-length coding region was isolated from muskmelon and named CmS-AIV1(GenBank accession EU260044).The deduced amino acid sequence of CmS-AIV1contains636 amino acid residues with a predicted signal sequence cleavage site between residue47and residue48.The protein domain includes the amino acid residues of b-fructosidase motif(NDPNG)and the catalytic domain conserved in vacuolar invertase(MWECVD).Therefore,based on the features of the deduced polypeptide,we conclude that CmS-AIV1encodes a vacuolar acid invertase.The alignment analysis showed that CmS-AIV1shared overall amino acid identities of99.53%,95.15%,85.71%,68.95%and61.50% to tomato(Lycoperscicon esculentum L.),potato(Solanum tuberosum L.)pepper(Capsicum annuum L.),sweet potato (Ipomoea batatas L.)and carrot(Daucus carota L.)soluble acid invertase,respectively(Fig.1).CmS-AIV1differentially expressed in different muskmelon tissuesTo determine the pattern of CmS-AIV1expression in dif-ferent muskmelon organs,a northern blot hybridization analysis was performed.The result showed that CmS-AIV1 transcripts were easily detected in theflowers and fruit,butcouldn’t be detected at all in roots,stems and leaves (Fig.2),suggesting that CmS-AIV1might play a role in flower and fruit development in muskmelon.Regulation of CmS-AIV1mRNA abundance during fruit development and ripeningTo further investigate the developmental regulation of CmS-AIV1in fruit mesocarp tissues,an RNA blot analysiswas carried out using different mesocarp tissues from 5DAP to ripening.The result showed that CmS-AIV1mRNA showed its maximum level at 10DAP,then decreased gradually during fruit development until its minimum level at mature fruit,indicating that CmS-AIV1may be involved in sucrose metabolism in muskmelon fruit (Fig.3).Sucrose content and S-AIV activity during fruit development and ripeningTo demonstrate the functions of CmS-AIV1in regulating fruit quality,sucrose content and S-AIV activitywereFig.1Alignment of predicted amino acid sequences of S-AIV genes from different plants.The GenBank accession numbers for the plant S-AIV sequences are:Cucumis melo ,(had been submitted);Lycoperscicon esculentum ,CAA78060;Solanum tuberosum ,ABF18956;Capsicum annuum ,AAB48484;Ipomoea batatas ,AAK71504;Daucus carota ,CAA53098.This alignment was produced using DNAman 4.0program.Conserved residues are shaded in black.Dark grey shading indicates similarresidues in five out of six of the sequences and clear grey shading indicates similarresidues in three out of six of the sequences.Amino acid residues of b -fructosidase motif (NDPNG)and the catalytic domain conserved in vacuolar invertase (MWECVD)are boxedMatureFruitRoot Stem Leaf FlowerCmS-AIV1rRNAFig.2RNA blot analysis of CmS-AIV1spatial expression in muskmelon plants.About 20l g of total RNA from root,stem,leaf,flower and mature fruit of muskmelon inbred line M01-3respectively,were separated on 1.2%agarose gels containing formaldehyde.Root,stem,leaf and flower tissues were harvested simultaneously from five 7-to 8-week-old plants and mature fruit were harvested as they mature from five 11-to 12-week-old plants.The blot was hybridized with a CmS-AIV1-specific probe.The ethidium bromide-stained rRNA band in the agarose gel is shown as a loading controlCmS-AIV1rRNA5DAP 10DAP 15DAP 20DAP 25DAP Mature fruitFig.3RNA blot analysis of CmS-AIV1expression in developmental muskmelon fruit.About 20l g of total RNA from muskmelon inbred line M01-3fruit of 5,10,15,20,25DAP and mature fruit respectively,were separated on 1.2%agarose gels containing formaldehyde.Five fruit of each stage were pooled to isolate RNA.The blot was hybridized with a CmS-AIV1-specific probe.The ethidium bromide-stained rRNA band in the agarose gel is shown as a loading controlanalyzed.The results showed that very low concentration of sucrose was detected in young and unripe muskmelons. However,a rapid accumulation of sucrose in fruit was observed between20and30DAP(Fig.4a).In contrast to sucrose content,S-AIV activity showed very high levels in fruit before20DAP,but a rapid decrease between20and 30DAP during fruit development(Fig.4b),indicating an essential role of S-AIV in sucrose metabolism in musk-melon fruit.DiscussionSoluble acid invertase(S-AIV)has important biological functions relating to sucrose import[22–24],and sugar signals[25–27]particularly during the expansion growth of diverse sink structures[22–24,26,28–30].These roles for S-AIV are additional to previously recognized functions including turgor regulation and the control of sugar balance in fruit tissues and mature tubers[23,26,31].The sucrose import and potential signaling influences of S-AIV arise from their role in the primary path of cleavage for sucrose entering growing/expanding sink tissues.The transfer of sucrose to vacuoles for initial metabolism has a dual advantage in,first,giving a two-for-one return on the cost of transporting osmotically active solutes into the vacuole for expansion and,second,adjusting organ expansion relative to carbohydrate supply.Such contributions by S-AIV depend,however,on continued capacity for cell wall expansion[32].Sturm et al.[33]reported that S-AIV had two isoforms in carrot,sI and sII.The expression of the genes for sI and sII seemed to be under spatial and temporal control.The sI gene is expressed in primary root and leaves at the early growth stage,and sII is expressed prominently up to the middle stage of tap root enlargement,indicating their involvement in the partitioning,utilization and storage of sucrose.In this study,on the one hand,we demonstrated that CmS-AIV1was expressed inflower and fruit but not in root,stem and leaf tissues,implying that CmS-AIV1may be also involved in the partitioning,utilization and storage of sucrose in muskmelon.On the other hand,we showed that CmS-AIV1transcript level was high in the young fruit,then decrease gradually during fruit development,suggesting that CmS-AIV1may be linked to the cell division and dif-ferentiation during early fruit development.The availability of the complete plant genomes of rice and Arabidopsis allowed the identification of two genes encoding soluble acid invertase in rice and Arabidopsis[16].Two cDNAs of PsS-AIV1and PsS-AIV2isoforms were cloned from Japanese pear fruit[34].In this study,we cloned and characterized one S-AIV gene from muskmelon, CmS-AIV1.However,it is not clear how many S-AIV genes exist in muskmelon.We are constructing cDNA libraries from muskmelon fruit and trying to isolate other S-AIV gene family members involved in sucrose metabolism in muskmelon by screening the cDNA libraries.The inverse relationship between S-AIV activity and sucrose concentration has been reported in muskmelon and other fruit[9,28,35–37]as well as in other sink tissues such as carrot root[23]and tuber of potato[38].Recently, S-AIV-encoding genes have been isolated and character-ized from some higher plant species[12,35,39–41].To date,however,there have been only a few studies of S-AIV gene expression in relation to fruit tissues.Davies and Robinson[11]reported that transcripts of GIN1and GIN2, which are isogenes of S-AIV in grape berry,are expressed actively before the veraison stage and then decrease duringfruit maturation.In Japanese pear fruit(Cyrus pyrifolia Nakai cv.‘Hosui’),the levels of PsS-AIV1transcript had a maximum level at34days after full bloom and decreased rapidly during fruit development[34].Transgenic tomato containing the antisense gene of vacuolar invertase accu-mulates more sucrose than the control tomato during fruit maturation,implying that vacuolar invertase has an important role in determining sucrose content in fruit[28]. Our present results showed that the CmS-AIV1transcripts showed its maximum level at10DAP fruit,then decreased gradually during fruit development until its minimum level at mature fruit(Fig.3).This pattern of CmS-AIV1 expression in mesocarp tissues is in accordance with the alteration of S-AIV activity,but in contrast to the sucrose accumulation during fruit development of muskmelon (Fig.4).Taken together,these data led us to conclude that the sucrose content in plant sink organs may be regulated by the level of S-AIV expression.Therefore,decreasing S-AIV expression might be an important regulatory event of sweetening during melon fruit ripening.Currently we are suppressing CmS-AIV1expression by the antisense approach,which will help reveal the potential application of CmS-AIV1in controlling the quality of muskmelon fruit. 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