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The Primary in Vivo Steroidal Alkaloid Glucosyltransferase from Potato

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The Primary in Vivo Steroidal Alkaloid Glucosyltransferase from Potato The Primary In Vivo Steroidal Alkaloid Glucosyltransferase From Potato Kent F. McCuea,*, Paul V. Allena, Louise V.T. Shepherdb, Alison Blakeb, Jonathan Whitworthc, M. Malendia Maccreea, David R. Rockholda, Derek Stewartb, Howard V. Daviesb, & William R. Belknapa aUSDA, Agricultural Research Service, Crop Improvement and Utilization Research Unit, 800 Buchanan St., Albany, CA 94710 USA bScottish Crop Research Institute, Quality, Health and Nutrition Programme, Dundee DD2 5DA UK. cUSDA, Agricultural Research Service, Small Grains & Potato Germplasm Research Unit, 1693 S 2700 W, Aberdeen, ID 83210 USA Wounded Tubers Wounded Leaves A. 100 A. Abstract α-solanine α-chaconine WT 1603 1605 1608 1614 1615 1616 1619 1621 1622 To assist control of the steroidal glycoalkaloid (SGA) Time (h) Time (h) 0 2 6 16 24 48 0 2 5 16 75 pathway in potato we have investigated the steroidal alkaloid 3 kb- glycosyltransferase (Sgt) gene family. The committed step in the Sgt1 50 SGA pathway is the glycosylation of solanidine by either UDP- 2 kb- glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. In this study we identify the Sgt2 gene, and the 25 Sgt2 function of the gene product (SGT2), that serves as the primary SGAs (mg/100g dry wt) UDP-glucose:solanidine glucosyltransferase in vivo. The Sgt2 0 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. Sgt1 has glucosyltransferase RNA 1619 1608 1615 1605 1622 1614 1621 1616 1603 L WT activity in vitro, but in vivo serves as the UDP- galactose:solanidine galactosyltransferase. Accumulation of α- Lenape Line B. solanine was enhanced and α-chaconine was inhibited in the Fig. 4. Expression of Sgt1 and Sgt2 RNA in control and wounded potato B. 40 tubers of transgenic potatoes (Solanum tuberosum) cvs. Lenape tubers and leaves. Potato cv. Lenape tubers and leaves were wounded for α-solanine α-chaconine and Desirée expressing an antisense Sgt2 gene construct. Studies the times indicated and total RNA (20µg/lane tubers, 10µg/lane leaves) 30 with recombinant SGT2 protein expressed in yeast and purified was probed with Sgt1 and Sgt2. The ethidium bromide stained gel is by metal ion affinity chromatography show that SGT2 shown to reference RNA loading and integrity. 2 kb- glycosylation activity is highly specific for UDP-glucose as a 20 sugar donor. 1 kb- Coomassie Stained Anti-V5 Western 10 - 80 kDa SGAs (mg/100g dry wt) solanidine 0 SGT1 SGT2 66 kDa - UDP-gal UDP-glu SGT2 C. 2-18 2-11 2-17 2-03 2-13 2-08 2-21 2-20 2-10 D WT γ-solanine γ-chaconine 45 kDa - Desirée Line β- β- - 39 kDa solanine chaconine 36 kDa - Fig. 6. SGA content of tubers from transgenic potatoes expressing the Sgt2 antisense transgene. Levels of α-solanine and α-chaconine in 3 kb- α-solanine α-chaconine selected transgenic and wild type (WT) control lines of A) Lenape and B) Desirée. The plant lines are arranged in the graphs sorted by 2 kb- Fig. 1. Glycosylation steps of the SGA biosynthetic pathway. decreasing α-solanine levels. Values are the average of 3 slices from 3 Solanidine is the branch point for synthesis of the two field-grown tubers (Lenape) or 2 glasshouse-grown minitubers predominant potato glycoalkaloids catalyzed by SGT1, the UDP- 1 kb- (Desirée). Error bars show s.d. galactose:solanidine galactosyltransferase and SGT2 the UDP- glucose:solanidine glucosyltransferase. L F W E L F W E Fig. 8. Steady state RNA expression of Sgt2 transcripts in Lenape lines. Analysis of steady state levels of total RNA Fig. 5. Recombinant SGT2 protein isolation. Coomassie stained gel and WT 1603 1605 1608 1614 1615 1616 1619 1621 1622 A. (30µg/lane) in control and transgenic lines of Lenape: A) Sgt2 Western blot with anti-V5 antibody The steps in isolation of protein: L- messenger levels, B) NptII messenger levels, and C) ethidium load, F-flow through, W-wash, and E-eluted protein. SGT1 1 MVATCNSGEILHVLFLPFLSAGHFIPLVNAARLFASR-GVKATILTTPHNALLFRSTIDD bromide stained gel. SGT2 1 ---MDNGSKQLHVLFLPYFATGHIIPLVNAARLFASRDGVKVTILTTHHNASLFRSSIDN SGT1 60 DVRISGFPISIVTIKFPSAEVGLPEGIESFNSATSPEMPHKIFYALSLLQKPMEDKIREL SGT2 58 SL------ISIVTLKFPSTEVGLPEGIENFSSASSTEIAGKVFGGTYLLQKPMEDKIREI SGT1 120 RPDCIFSDMYFPWTVDIADELHIPRILYNLSAYMCYSIMHNLKVYRPHK----QPNLDES Table I. SGT2 UDP-sugar and aglycone preference. 12 kbp - SGT2 112 HPDCIFSDMYFPWTVDIALELKIPRLLFNQSSYMYNSILYNLRLYKPHEKLINQMEYSKS SGT1 176 QSFVVPGLPDEIKFKLSQLTDDLRKSDDQKTVFDELLEQVEDSEERSYGIVHDTFYELEP Substrate UDP-[3H]Glucose UDP-[3H]Galactose Conclusions SGT2 172 TNFSVPDLPDKIEFKLSQLTDDLVRPADERNAFDELLDRTRESEDLSYGIVHDTFYELEP (nkat mg-1) (nkat mg-1) SGT1 236 AYVDYYQKLKKPKCWHFGPLSHFASKIRS-KELIS---EHNNNEIVIDWLNAQKPKSVLY Two Sgt2 sequences (Sgt2.1 and Sgt2.2) were isolated from the S. SGT2 232 AYADYYQKMKKTKCWQIGPISYFSSKLSPRKELINSSDESNSSAVVVEWLNKHKHKSVLY 6 kbp – tuberosum cDNA library and likely represent two major alleles expressed in SGT1 292 VSFGSMARFPESQLNEIAQALDASNVPFIFVLRPNEETAS-WLPVGNLEDKTKKGLYIKG SGT2 292 VSFGSTIRFPEEQLAEIAKALEASTVPFIWVVNKDQLAKTTWLPES-LFDE-KKCLIIKG Solanidine 1,060 ± 25 NA the heterozygous tetraploid genome. Extensive homology between the two SGT1 351 WVPQLTIMEHSATGGFMTHCGTNSVLEAITFGVPMITWPLYADQFYNEKVVEVRGLGIKI alleles suggests that both alleles would be simultaneously down-regulated in SGT2 350 WAPQLSILDHSAVGGFMTHCGWNSVLEAIIAGVPLVTWPVFAEQFYNEKLVEVMGLGVKV plants expressing effective antisense genotypes. The genomic DNA blot SGT1 411 GIDVWN--EGIEITGPVIESAKIREAIERLMISNGSEEIINIRDRVMAMSKMAQNATNEG 4 kbp - SGT2 410 GAEVYNTNGGAEISTPVLRSEKIKEAIERLMESQ------KIREKAVSMSKMAKNAVEEG Solasodine 880 ± 29 NA analysis allows an estimation of gene copy number. The presence of no more SGT1 469 GSSWNNLTALIQHIKNYNLN------ SGT2 464 GSSSNNLTALIDDIKNFTSSSLKIMD than two major bands indicates that Sgt2 is a low copy gene in S. tuberosum. Tomatidine 930 ± 152 NA This in combination with 2 distinct coding sequences from PCR and two Fig. 2. Alignment of SGT2 and SGT1 deduced amino acid representative tentative consensus sequences in the TIGR database suggests sequences showing regions of identity (black) and similarity (gray). that there are only two major active alleles. Glycosyltransferase activity of the recombinant SGT2:his fusion protein purified from yeast. Values represent the average of duplicate assays ± s.d. 2 kbp – Potato SGAs are knows to accumulate in tubers upon wounding. As (nkat mg-1 = moles of product per second per mg protein x 10-9). expected the expression of Sgt1 and Sgt2 is coordinately regulated in response to wounding in the tubers with nearly identical patterns of mRNA NA = No activity, for reactions with dpm counts less than 2-fold the assay blank. B. BamHI EcoRI HinDIII XbaI accumulation. Although SGAs are also found in leaves, the pattern of expression is quite different. Sgt1 is not wound-induced while Sgt2 shows induction. 12 kbp - Table II. Effect of UDP-galactose or triose end products, α-solanine or α- The two solanidine glycosyltransferases represent the committed steps for -9 kbp chaconine, on recombinant SGT2 glucosyltransferase activity. carbon flow into the SGA pathway. Down-regulation of either Sgt1 or Sgt2 tends to cause an increase in the accumulation of the end product of the other -6 kbp Substrate UDP-galactose α-solanine α-chaconine 6 kbp – branch, α-chaconine οr α-solanine, respectively. SGT2 is specific for UDP- Concentration (nkat mg-1) (nkat mg-1) (nkat mg-1) glucose as the sugar donor. UDP-galactose at concentrations from 33 to 1,000 µM did not affect glucosyltransferase activity. The presence of 1,000 µM α- -3 kbp 4 kbp - solanine had a small affect and 1,000 µM α-chaconine completely inhibited 33µM 380 ± 7.4 410 ± 102 490 ± 4.8 the reaction. Based on these data we assign the function of the gene product SGT2 as E.C. 2.4.1.173, a UDP-glucose 3-β sterol glucosyltransferase. 100µM 500 ± 84 430 ± 33 520 ± 10.6 The effective Lenape antisense lines show a correlation with multiple T- DNA inserts and reduction in steady state levels of Sgt2 RNA (1605, 1608 and 1,000µM 390 ± 23 250 ± 31 NA - 1 kbp 1619, and either reductions in α-chaconine (1605, 2-11 and 2-17) and or an 2 kbp – increase in α-solanine (1608, 1619 and 2-18) accumulation. All of this is Fig. 7. Integration patterns of antisense Sgt2 transgene in Lenape consistent with the conclusion that SGT2 is the primary in vivo solanidine Fig. 3. Occurrence of Sgt2 in the potato genome. Genomic DNA Glucosyltransferase activity of the recombinant SGT2:his fusion protein purified lines. Genomic DNA blot analysis of T-DNA insertion in control glucosyltransferase in a dedicated branch of SGA biosynthesis specific for the analysis using 10µg of DNA from S. tuberosum cv. Lenape cut from yeast in the presence of added UDP-galactose or steroidal alkaloid triose and transgenic lines of Lenape. Genomic DNA (20 µg/lane) was formation of α-chaconine. Future research will focus on the combined down- with the restriction endonucleases as indicated and probed with end products. Values represent the average of duplicate assays ± s.d. digested with HinDIII and probed with the A) the complete NptII regulation of Sgt1 and Sgt2 in concert to reduce the accumulation of both of NA = No activity, for reactions with dpm counts less than 2-fold the assay blank. the 1,066 bp amino terminal fragment of Sgt2. coding sequence, and B) the Sgt2 amino terminal CDS. these toxic compounds in the tubers of commercial potato cultivars..
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