Biochemical Systematic and Ecology 38 (2010) 971-980 Contents lists available at ScienceDirect Biochemical Systematics and Ecology ~ ELSEVIER journal homepage: www.elsevier.com/locate/biochemsyseco cDNA-AFLP analysis on transcripts associated with hydroxysafflor yellow A(HSYA) biosynthetic pathway in Carthamus tinctorius Na Feng, Yakui Li, Jie Tang, Yan Wang, Meili Guo* Department of Pharmacognosy, Collegeof Pharmacy, Second Miltary Medical University, Shanghai 200433, China ARTICLE INFO ABSTRACT ‘ce stone Hydroxysaffor yellow A(HSYA), an important active compound, is uniquely presentin florets Received 10 May 2010, of Carthamus tinctorius. In current study, we applied cDNA amplified fragment length ‘Accepted 1 September 2010 polymorphism (cDNA-AFLP) to screen genes expressed differently between plants with and ‘Available online 29 September 2010 without HSVA. One hundred and thirty-two primer combinations produced 6751 fragments, SM _ seven transcript-derived fragments (TDs) showed consistent cifference between two gene “elon pools. An independent RT-PCR expression analysis validated the expression pattern for 3 Bulked Segregant analysis (A) Curthanses tts [| ‘TDEs (TDF-8, TDF-9 and TDF-27). The 3 TDFs were only ranscripted in plants that contained CONCAHE HSYA, implying that they were associated with the formation of HSYA. In addition, a full Evolution analysis length of TDF-8 was achieved, which termed as WV-prl. Bioinformatics analysis showed that Fiydroxyaftor yellow A (HSYA) itcartied a sequence highly homologous to the Broad bean wilt virus. Our study revealed that Rapid amplification of cDNA ends (RACE) the three genes are involved in the pracess of response to some virus infection and func- tioning in HSYA production, © 2010 Elsevier Ltd, All rights reserved. 1. Introduction Flavonoids are important secondary metabolites in the plant kingdom. Plants produce a wide variety of flavonoid compounds, which play important roles in the survival of plants in their ecosystem. Plant flavonoids are therefore involved in resistance against pests, pathogens and diseases, attraction of pollinators, formation of anthocyanidins, and interaction with symbiotic microorganisms (Dixon and Paiva, 1995; Dixon, 2001). As flavonoid compounds provide many pharmacologically active agents with antioxidant, anticarcinogenic, anti-inflammatory and cardiovascular protective activities with low toxicity (Dixon and Steel, 1999), they have attracted much attention over the past decade (Barnes et al, 1994; Krakauer et al, 2001; ‘Takahashi et al, 2001). Many investigations have disclosed the chemical structures of flavonoids in Carthamus tinctorius L (2n = 2x = 24) (Meselhy et al, 1993; Hang and Tang, 1995; Li and He, 2002), commonly known as safflower. Hydroxysafflor yellow A (HSYA), an important active member of flavonoids uniquely existing in the organ rather in the leaf, stem or root of safflower, is found to have a variety of biological actions (Zang et al, 2002; in etal, 2004; Sato et al, 2005). For example, HSYA is able to raise hypoxia tolerance, dilate the coronary artery, increase coronary blood flow, and inhibit ADP-induced platelet aggregation in rabbits (Zhu et al, 2003). But as safflower has a long history of plantation and good adaptation to the envi- ronment, intraspecific variation accurs and the content of HSYA in safflower varies greatly among different varieties (Guo et al, 2006a,b). In most cases, the natural yields of HSA are very low, or even absent in partition of varieties. ‘Abbreviations: Bp, base pair(s ORF, open reading frame; HSYA, Hydroxsafor yellow A; HPLC, high performance liquil chromatography: cDNA-ARLP, cDNA amplified fragment length polymorphism; BSA, Bulked segregant analysis; TDFs,anscrip-derived fragments; RACE, rapid amplification of eDNA ends, * Corresponding author. Telyfax: +86 21 25074576, Esmail address: miguo@smmuedu.cn (M. Guo) (0305-1978)5 - see front matter © 2010 Elsevier Lt. All rights reserved, ois 101016)} bse 2010.09.001om 1. Feng eal. (Biochemical Systematics and Ecology 38 (2010) 971-980 Recently, various approaches such as introducing genes encoding the key biosynthetic enzymes or antisense genes to block competitive pathways, or genes encoding regulatory proteins to overcome the specific rate-limiting steps have been used to manipulate biosynthesis of flavonoids (Tanaka et al., 1998; Yu et al, 2000; Vom Endt et al, 2002). However, in comparison to other medical plants, very little research has been done on safflower with respect to transcriptome wide information of specific gene expression patterns based on various molecular tools, much less genes in relation to the formation of HSYA. Analysis of the molecular mechanism controlling the expression and differentiation of HSVA by more efficient investigations is immensely important for exploitation and development of potential new medicines. cDNA amplified fragment length polymorphism (cDNA-AFLP) is a reliable, stable and highly reproducible AFLP-based ‘mRNA fingerprinting technique (Bachem et al., 1996) and has been widely used in displaying gene differential expression and isolating genes of plants (Breyne et al,, 2003; Albertini et al, 2004; Sarosh and Meijer, 2007). Recently, successful combination of the cDNA-AFLP technique with bulked segregant analysis (BSA) (Michelmore et al,, 1991) was used to detect expressed tags (ESTs) and clone candidate genes (Barcaccia et al, 2001; Murata et al, 2006), demonstrating that the CDNA-AFLP based BSA approach could reveal the naturally existing genetic polymorphisms between two parental varieties contrasting in a given trait, which can be used to get candidate genes. Since 1997, we have undertaken studies on a molecular marker assisted breeding program targeting on HSYA analysis of safflower. We found that there was great genetic diversity in safflower populations by AFLP (Zhang et al, 2006) and RAPD (Guo et al, 2003). Also, pharmacologic studies with respect to chemical components of safflower showed that HSYA in safflower varies with different varieties, suggesting that the difference in HSYA content is mainly decided by heredity (Guo et al, 2006a). Zhang et al. used AFLP technology to screen the HSya-related genes. Four HSya-related genes from genomic DNA of Safflower have been identified and converted into SCAR markers (Zhang et al., 2009). To understand the mechanism of HSYA formation, we utilized the cDNA-AFLP based BSA approach to reveal the genetic polymorphisms in expressed cDNA sequences between an HSYA present (H) pool and an HSYA absent (HO) pool and obtained a stisp gene which suppressed the expression of HSya (Tang et al, 2009). Furthermore, we achieved three other genes (TDF-8, TDF-9 and TDF-27) by the same ‘method as Tang etal. (2009), Analysis of TDFs has revealed that TDFs as well as WV-pri gene share high homology with Broad bean wilt virus. Further investigation shows that these three genes may involve in the metabolic pathway of flavonoids and induce the expression of HSya. The following is our first open report about this study. 2. Materials and methods 21, Plant materials ‘Two parental strains (No,0016 and No.0025) were selected from Chinese populations by our laboratory. The former (P1) was in the presence of HSVA with a content of 2.11% + 0.09% (n = 83), and the latter (P2) was in the absence of HSVA with 2 content of 0.00% + 0.00% (n = 89). The reciprocal crosses (Pj P2, P) x P}) were made and Fy seeds (87, 93) were hand- harvested in the summer of 2003 from the medicinal plant garden of the Second Military Medical University (Shanghai, China), The F) seeds of the crosses were produced in the field of Sanya, Hainan Province by bagging F; plants in paper bags prior to the flowering period during 2003 and 2004. A segregating F population was obtained by shifting a single Fy (P1 xP) plant the next year in the medicinal plant garden of the said university. Two hundred and sixty-six segregating F2 individuals ‘were obtained. F2 populations possessed the same genetic background except for HSYA difference. 22, Determination of HSYA content HSYA standard sample (Co7H270)6) was extracted from Flos Carthami, the purity of which was 99.5% by HPLC analysis and the structure of which is illustrated in Fig. 1 (Guo et al, 2006b). Chromatography was performed on Agilent 1100 (USA) model 510 binary gradient equipment, and an Agilent 1100 chromatography workstation equipped with an injection valve with 20 iL sample loop. HSYA was separated on a 250 mm x 46 mm id., 5 jim particle, ZORBAX SB-Cjg column (Agilent Company). Optimum HPLC separation was achieved by use of 10% aqueous acetonitrile at a 1.0 mL min ' low rate. The detection wavelength was 403 nm and the temperature was 22 °C. Dry safflower florets (approx. 0.5 g) were weighed accurately into 2250 mL tube, extracted with 100 ml water by soaking overnight, ultrasonicated for 20 min in a sealed container, and filtered through a 0.45 Im Nylon syringe filter (Millex-HN, Ireland) before injection for HPLC analysis. HO. Ge Ho. OHFeng etl iochemical Systematics and Ecology 38 (2010) 971-980, on Tablet ‘80%) with known genes. Nucleotide sequence analysis showed that TDF-8 shared high homology with BBWV-2 RNA1 putative RNA polymerase, and that TDF-9 was a homologue of BBWV-2 mRNA. TDF-27 showed great homology with Patchouli Mild Mosaic Virus RNAI for polyprotein. ‘The full length cDNA of WV-prl was presumed to contain a 1257 bp open reading frame (ORF), starting with an GTG codon at position 366-368 and terminating with an TAA codon at position 1620-1622, encoding a protein of 419 amino acids with a calculated molecular mass of 48011.34D (pI 7.07). Homologous analysis at the protein level showed that WV-prl carried a highly homology (97%) to Broad bean wilt virus. These results indicated that WV-prl was a member of the genus Fabavirus. Broad bean wilt virus is a member of the genus Fabavirus of the family Comoviridae (Fauquet et al, 2005), and contains 2 cofactor required for proteinase (Co-pro), putative helicase (Hel), genome-linked protein (VPg), proteinase (Pro), and RNA-dependent RNA polymerase (RdRp) conserved domains. Alignments of WV-pri and Broad bean wilt virus2 indicated that WV-pil is of high identity to RdRp, ranging from 1349 bp to 1638 bp (Fig. 7). 4. Discussion ur previous studies showed that sHSP only expressed in HSYA-absent lines and it might be directly or indirectly disturb the HSVA biosynthetic pathway (Tang et al, 2009). The present study further investigated differentially expressed TDFs of o com tet hy he RE 1220 * 240 * 1260 + 1380 >BOW2 + EWILDYPCOKLPSVLTRGDPRLAGTVIADYDPFASGNSKYAKEAGPFDAASLKQVCSGIVEIME : 1280 OWeprl: ————VCSGIVEINE : 10 VCSGIVEIVE = 1300 * 1320 = 1340 BEW2 + DASADFPMDEVDLDTAIN--GLENVEFFDALVLGTSEGFPYRLDRGPGOKGKSRYVSGESGNLK : 1342 We>prL:. DASADFPMDEVDLDTAINGYGLENVEFRDALVLGTSEGPPYRLDRGPGDKGKSRYVSGESGNLK : 74 DDASADFPMDEVDLDTAIN GLENVEPFDALVLGTSEGPPYRLDRGPGDKGKSRY VSGESGNLK. «1360 = 1380 * 1400 >BBWV2 : ITDEGYLSDIAWFEEVSKTOVPDLYCIECYKDERLPIRKVLHEPKSRLFTVLPASYNIVIRKKF. 1406 OWY-prl:_ITDEGYLSDIAWFEEVSKTOVPDLYCIECYKDERLPIRKVLHEPKSRLFTVLPMSYNIVIRKKF 138 | TDEGLSDLAWFEEVSKTQVPDLYCIECYKDERLPIRKVLHEPKSRLFTVLPMSYNIVIRKKF * 1420 «10 «1460 * >BBW2 + LAFVRFFIKRRDVLPAQGINPYSREWIRIANKLISKGANILCCDYSRFOGFLPKCIINETGNM 1470, OWprl:_ LNFVRFFINKRRDVLPAQVGINPYSREWTRIANKLISKGANILOCDYSRFOGFLPKCINETGNM : 202 LLNFVREFIRRROVL AQVGINPYSREWTRIAAKLLSKGNNTLCCDYSRFDGFLPKCTINE. Xt 1480 = 1500 * 1520 * BOWE + TARLMVDEVSRAQIKNLMLACTSRYAMCARVLYRVENGIPSGEPLTVIVRSILNEILVKYANW : 1534 Wp: TARLMKADEVSKTQIKNLALACTSRYANCARVLYRVENGIPSGEPLTVIVASILNEILVKYAYW ; 266, TARLAK. DEVS. QIKNLMLACTSRYANCNRVLYRVENGIPSGFPLTVIVNSILNETLVRYAYW 1540 = 1560 1580 * 1600 BBV: HCFEDNPSVGSNFDAHYSHVVYGDDNLISVSDATSSKFDGSFLVSFNEGLGIKVTDGIOKTAVG : 1598 DW-prl:HCREDNPSVQSNPDAHVSHVVYGDONLISVSDAISSRFDGNFLVSFHEGLGIKVTOGIDKTKIG : 330, HCREONPSVQSNFDANVSMVVYGODNL ISVSDAISSKFDG FLVSPMEGLGIKVTDGIOKTRGG «1620 «1640 «1660 BOW : TEFRRLENCDFLKRSPRNSPOGTHRSPUSKESLRPOLHFVKARKLEMAEAYINNCRVILRELWL 1662 Wp: TEFRRLENCDFLKRSFRNSPDGTHRSPUSKESLBPOLHPVKARKLEMAEAYINNCRNILRELWL : 294 [TEFRRLENCDFLRRSFKOSPOGTWRSPUSKESLWPQLHFVKAKKLEMAEAYINYCNTLRELWL * 1680 = 1700 * 1720 BBV: HOVKEAKEFRNKVLRNLRWVGHEGLLNIQQL AVERSEGHNGYSDFLSTCVIVOSIPLIDPLYPG : 1726 SW-prl:_HOVKEAREFRNKVLRNLRWIG— HDVKEAKEFRNKVLRNLRWGG Fig. 7. A Lines and lage boxes represent non-coding Sequence and long open reading frames, respectively. Vertical lines through the boxes indicate putative cleavage sits. Calelated relative molecular mas values for each protein and postions of consensus sequences for cofactor required fr proteinase (Co), haicase(He), genome linked protein(VP. proteinase (Pra, and RNA-dependent RNA polymerase( Rp) are indicated. B Alignments of Fpl and Broad bean vrsFeng etl iochemical Systematics and Ecology 38 (2010) 971-980, 79 interest on the basis of BSA-AFLP analysis, using RACE to get the full-length gene of TDF-8. We compared the differential gene expression patterns between HSYA present and absent segregating bulks. 266 F individual identification and RT-PCR vali- dation were also processed. The results showed that the three TDFs were present only in the female parent and in HSYA- present lines, implying that proteins encoded by these fragments may be involved in regulation of HSYA. Homologous analysis of the TDFs sequences generated significant matches to sequence databases. Interestingly, the TDFs were highly homologous with the gene coding protein of Broad bean wilt virus and Patchouli mild mosaic virus. Broad bean wilt virus 1 (BBWV-1), BBWV-2, Patchouli mild mosaic virus (PatMMV) and Lamium mild mosaic virus (LMMV) are four recognized species that compose the genus Fabavirus of the family Comoviridae (Wellink et al., 2000). Fabaviruses have icosahedral virions composed of two coat proteins and bipartite, infecting a wide range of host plants worldwide, including economically important horticultural and ornamental species, and aphid-transmitted in a nonpersistent mode (Vittoria and Guido, 1996). BBWV, in particular, infested a lot of beans, complicated with ring spot, stunting, wilting or withering. Besides, secondary metabolites such as flavonoids could be mobilized to protect the plant from pathogen attacks. Flavonoids are a diverse group of compounds with a wide range of biological effects, especially anti-viral activity against a range of plant viruses, for example, high concentrations of flavonoids in fruits often go parallel with a low incidence by pathogens (Lattanzio et al, 1994; Lattanzio, 2003). Tobacco mosaic virus infectivity was reduced by a range of flavonoids. Quercetin and morin were also reported to prevent the formation of lesion in quinoa effected by potato virus X in a low concentration (1g-mL') (French and ‘Towers, 1992). Beckman reviewed the possible role of preformed phenolics in periderm formation in wilt disease resistance in a time-space model of host-parasite interactions (Beckman, 2000). The connection between the amount of flavonoids and the degree of wilt-resistance of the plants such as cotton, kenaf was also observed (Navrezova et al, 1986). Flavonoids could inactivate recognition sites on the viral coat protein which interact with host recognition sequences required for the initiation of infection (French and Towers, 1992). However, the replication of viruses was not inhibited by flavonoids treated such as in Tobacco mosaic virus (TMV) situations (Chen et al, 2003). In China, after Chinese soybeans were infested by soybean mosaic virus, the content of flavonoids was significant higher in resistant species than susceptible species (Zhu et al, 2001). All the opinions were supported by Ryder et al. (1987) with the similar conclusion that the CHS gene could be induced by fungus infested and mechanical wounding. It also happened in CHS gene of Phaseolus vulgaris (Mehdy and Lamb, 1987), All the evidence implies that safflower might be infested by BBWV, mosaic virus or other virus or fungus long time ago, which induced the production of HSYA with antivirus activity to inhibit the virus at the moment, but cannot prevent the replication of virus. Finally, safflower allowed the microbe to enter the symbiotic modus and caused systemic acquired resistance (SAR). More interestingly, we also obtained a full length of TDF-8 (WV-pri gene) by RACE that carried a highly homologous sequence (97% identity) to the Broad bean wilt virus from safflower. In conclusion, the study clearly shows various genes involved in HSYA production. Although further clues are needed to understand physiological roles of these genes, the findings obtained suggest that these three genes are absolutely involved in a process of response to some virus infection and functioning in HSYA production. 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