Mol Breeding (2014) 34:2139–2149 DOI 10.1007/s11032-014-0169-1
Isolation and characterization of soybean chalcone reductase cDNA, which encodes the key enzyme for the biosynthesis of 4,20 ,40 -trihydroxychalcone in legumes Zhuo Zhang • Yongping Fu • Jian Ma Chao Zhang • Piwu Wang
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Received: 31 March 2014 / Accepted: 31 July 2014 / Published online: 9 September 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract In plants, phytoalexins induced by pathogen attack play an important role in disease resistance. In soybean [Glycine max L. (Merr.)], attack by pathogenic bacteria induces the synthesis of isoflavonoids, especially daidzein. Chalcone reductase (CHR) is the key enzyme in the biosynthesis of daidzein. Along with chalcone synthase, it catalyzes the formation of isoliquiritigenin, which is a necessary substrate for daidzein biosynthesis. In this study, a CHR gene, Gmchr2 (GenBank code: KF758395), was isolated from the soybean cultivar Jinong 17. The cDNA consisted of a 1,417-bp fragment that included an open reading frame of 969 bp. The gene is located on chromosome 9 of the soybean genome. Phytophthora sojae was inoculated onto soybean roots, and changes in the transcript levels of the chr genes and the catalytic activity of CHR were investigated in different soybean tissues by real-time fluorescence quantitative PCR and high-performance liquid chromatography (HPLC), respectively. The results showed that the isoliquiritigenin content in roots significantly increased after pathogen inoculation. The
Electronic supplementary material The online version of this article (doi:10.1007/s11032-014-0169-1) contains supplementary material, which is available to authorized users. Z. Zhang Y. Fu J. Ma C. Zhang P. Wang (&) Center for Plant Biotechnology, College of Agronomy, Jilin Agricultural University, No. 2888 Xincheng Street, Changchun 130118, People’s Republic of China e-mail:
[email protected];
[email protected]
Gmchr2 gene was transformed into tobacco, and the presence of isoliquiritigenin in the transformants was confirmed by HPLC. Expression of the Gmchr2 gene under the control of the 35S CaMV promoter was also confirmed. This characterization of a chr gene encoding a soybean CHR helps to shed light on the biological synthesis and regulation of soybean isoflavones and will be useful for manipulating the phenylpropanoid pathway leading to isoflavonoid phytoalexins. Keywords Soybean Chalcone reductase Isoliquiritigenin Daidzein Gene cloning and characterization
Introduction Daidzein, genistein, and glycitein make up the most common group of isoflavonoids in soybean [Glycine max L. (Merr.)]. These secondary metabolites contribute to protecting soybeans against attack by pathogenic microorganisms (Dixon 2001; Marienhagen and Bott 2013). Studies have shown that daidzein is not only involved in protecting plants from diseases and herbivores, but also provides resistance to strong ultraviolet radiation and promotes the accumulation of anthocyanidins and pollen germination (Winkel-Shirley 2001; Schijlen et al. 2004). Other isoflavonoids such as genistein play roles in regulating the
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transportation and distribution of phytohormones in plants and induce the expression of the rhizobium nod gene to promote the formation of nodules on the roots (Kampko¨tter et al. 2008; Forkmann and Martens 2001; Subramanian et al. 2006). In soybean, there is a wide diversity in the structure and functions of isoflavonoids and complicated regulation of various genes encoding components of the isoflavonoid biosynthetic pathway. Thus, soybean is an ideal research model to study the interactions between chalcone reductase (CHR) and chalcone synthase (CHS) in this secondary metabolic pathway. In soybean, the biosynthetic pathway for isoflavonoids is relatively complicated, but a great deal has been learned (Fig. S1). The precursors required for the biosynthesis of isoflavonoids are 4,20 ,40 ,60 -tetrahydroxychalcone (chalcone) and 4,20 ,40 -trihydroxychalcone (deoxychalcone). The synthesis of chalcone requires CHS (Welle et al. 1991; Bomati et al. 2005), while the synthesis of deoxychalcone relies on the joint catalytic actions of CHR and CHS. Under the individual catalytic action of CHS, three molecules of propanediyl coenzyme A combine with one molecule of coumaroyl coenzyme A to form one chalcone molecule. After CHR binds NADPH, it can act in concert with CHS to form a 4,20 ,40 -trihydroxychalcone compound, namely isoliquiritigenin (Weisshaar and Jenkins 1998; Shimada et al. 2000; Martens et al. 2003; Lozovaya et al. 2004). CHR is the key enzyme in the biosynthesis of the phytoalexin deoxychalcone. In addition, a number of reductases that generate other secondary metabolic products are closely related to CHR, such as codeinone reductase, an enzyme involved in the biosynthesis of the alkaloid morphine; dihydroflavonol reductase, which is involved in anthocyanin biosynthesis; and methylecgonone reductase, an enzyme involved in tropine alkaloid biosynthesis. These proteins show high sequence similarity to CHRs (Viljoen et al. 2013; Jirschitzka et al. 2012). Ayabe et al. (1998) first obtained CHR after using an elicitor from yeast to stimulate Glycyrrhiza echinata tissue and experimentally verified its catalytic ability. Later, CHRs and their corresponding nucleotide sequences were isolated from other leguminous species, including soybean, Medicago sativa, Pueraria montana, Astragalus membranaceus, Cicer arietinum, and Medicago truncatula (Welle et al. 1991; Ayabe et al. 1998; Ballance and Dixon 1995; Joung et al. 2003; Xu et al. 2012; Thill et al. 2012). Sequence
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analysis of these CHRs revealed that they belong to the aldo–ketoreductase (AKR) superfamily. The common characteristics of members of this superfamily are that each monomer contains an (a/b)8-barrel structure; each monomer contains an average of 320 amino acid residues; and NADPH is required to constitute the zymoprotein with complete physiological function (Steele et al. 1999). By screening the genetic library of M. sativa, Sallaud et al. (1995) isolated seven genes that could encode CHR and also demonstrated that the chr gene existed in polygenic form in leguminous plants. This was also demonstrated in the genomes of G. echinata, A. membranaceus, and M. truncatula. In soybean, only one chr gene has been confirmed so far; this has restricted research on the function of the soybean CHR, its joint mechanism of action with CHS, and the mechanism regulating its expression. In this study, following on from the studies of Graham (1990), Graham et al. (2007), we scanned the soybean expression sequence tag (EST) library to obtain the chr gene Gmchr2 (GenBank code: KF758395). We used rapid amplification of cDNA ends (RACE) technology to amplify the complete 1,417-bp cDNA sequence, containing a 969-bp open reading frame. We analyzed the conserved region in the gene sequence and the copy number of the chr gene in the soybean genome. Using real-time fluorescent quantitative PCR (Q-PCR), we found that the transcript levels of chr in soybean root showed the largest changes after stimulation by the Phytophthora root rot pathogen. Using high-performance liquid chromatography (HPLC), we analyzed the catalytic product of CHR, 4,20 ,40 -trihydroxychalcone, also known as isoliquiritigenin. The average isoliquiritigenin concentration in root tissues was 3.972 lmol/g DW. We also compared the newly isolated gene with the known soybean chr gene, Gmchr1. The cloned gene Gmchr2 was transformed into tobacco, and the catalytic function of the gene expression product was verified by Q-PCR and HPLC.
Materials and methods Plant materials We used the soybean cultivar ‘‘Jinong 17,’’ developed by Center for Plant Biotechnology at Jilin Agricultural University. This cultivar is highly resistant to
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Phytophthora root rot and has a high isoflavone content, suggesting that it might carry the gene controlling the daidzein pathway. We also used the tobacco cultivar ‘‘NC 89,’’ which was developed by Chinese Academy of Agricultural Sciences. Seeds were planted in a greenhouse at room temperature. At 15 days after planting, the soybean plants were inoculated with Phytophthora sojae (produced by Institute of Plant Protection of Chinese Academy of Agricultural Sciences) using the hypocotyledonaryaxis infection method. We inoculated 22 plants and used un-inoculated seedlings as the controls. After inoculation, a moisturizing treatment was performed at room temperature (20–25 °C) and relative humidity higher than 90 %. The fresh soybean leaves, stems, and root tissues were collected for RNA extraction every 12 h after inoculation, a total of five times. RNA extraction and preparation for cDNA generation The RNA was extracted using an RNA iso kit (Takara, Otsu, Japan) according to the manufacturer’s instructions. The transcript levels of relevant genes were determined by real-time PCR. RNase–free DNase I was used to digest and treat the RNA to prevent DNA contamination. The corresponding cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Fermentas Company, Hanover, MD, USA), according to the manufacturer’s instructions. The cDNAs were stored at -20 °C until use. Full-length sequence clone of the target gene After RNA extraction, the corresponding cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit. Based on the results reported by Graham (1990), Graham et al. (2007), the corresponding DNA sequence (GenBank No. TC203399) was used to search the soybean EST library on the NCBI website. BLAST analysis identified candidate genes with incomplete gene sequences as compared to the known sequence. The nested primers GSP1/GSP2 and GSP3/GSP4 were designed (Table S1). The corresponding segment was amplified by RT-PCR. The target sequence was then cloned and sequenced to verify that the correct target segment had been amplified. The 30 -end of the cloned fragment was amplified with a RACE kit (Takara). RNA from fresh soybean
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leaves was used as the template, and the 30 RACE adaptor primer from the kit was used for the reverse transcription reaction to synthesize first-strand cDNA. The GSP1 primer and 30 RACE outer primer were used for the outer PCR, and the GSP2 primer and the 30 RACE inner primer were used for the inner PCR. Amplification was performed to obtain the 30 -end fragment of the gene. The 50 -end of the cloned fragment was amplified with a RACE kit (Takara). Soybean leaf RNA was used as the template; the 50 RACE adaptor in the kit was used to evaluate the mRNA, and the multistep enzyme was used to speed up the reaction. Random 9-mers were used for reverse transcription to synthesize cDNA. The GSP1 primer and 50 RACE outer primer were used for the outer PCR. The reaction product was used as the template for the inner PCR with primer GSP2 and the 50 RACE inner primer. Amplification was performed to obtain the 50 -end fragment of the gene. The 30 RACE and 50 RACE products were aligned, and specific primers were designed to amplify the fulllength gene segment (Table S1). The cDNA from the total RNA from soybean root was used as the template, and the segment corresponding to the chr gene was amplified by RT-PCR. Gene copy number in the genome and phylogenetic tree analysis Southern imprinting technology was used to analyze the copy number of the newly isolated gene in the soybean genome. The gene sequence obtained from the RACE experiment was used as a probe, and the CTAB method was used to extract genomic soybean DNA using the DIG DNA Labelling and Detection Kit (Roche, Indianapolis, IN, USA). The restriction enzymes NheI, PstI, BamHI, and XbaI were used to digest the soybean genomic DNA. The sequence was uploaded into SoyBase and the Soybean Breeder’s Toolbox website (http://www. soybase.org/). The tools at this website were used to locate the position of the target gene in the soybean genome. The sequences of all of the known CHRs were downloaded from the NCBI website, and the online analytical procedure ORF FINDER (http://www.ncbi. nlm.nih.gov/gorf.html) was then used to perform readable frame analysis for the target gene sequence. DNAMAN software (Version 5.0, Lynnon Biosoft Company, Quebec, Canada) was used to translate the
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ORF of the target gene into the amino acid sequence and to perform multiple sequence alignment. MEGA (Ver 4.0; Tamura et al. 2007) software was used to analyze protein homology and create a phylogenetic tree. Real-time quantification of soybean chr gene transcripts at different sampling times The cDNA sequence was used to design the quantitative PCR primers QCHR2 and QACHR2 (Table S1). The sequence of the known soybean chr gene Gmchr1 (GenBank accession number: X55730) was used to design the quantitative PCR primers QCHR1 and QACHR1 (Table S1). The soybean b-actin gene (GenBank accession number: NM001252731) (Hu et al. 2009; Jian et al. 2008) was selected as the reference gene to design the quantitative PCR primers QFACT and QRACT (Table S1). We used the Stratagene Mx3005P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) for real-time PCR analysis of the Gmchr2 mRNA in soybean roots at different sampling times. Based on the protocol for the SYBR Premix Ex TaqTM kit (Takara), the 25-ll reaction mixtures included 12.5 ll 29 SYBR Premix Ex Taq (TLI RNase plus), 1 ll QGMCHR2 primer (10 lM), 1 ll QAGMCHR2 primer (10 lM), and 2 ll template. A two-step PCR was used, starting with predenaturation at 94 °C for 30 s followed by 40 cycles of denaturation at 94 °C for 5 s, and then extension at 60 °C for 30 s. The relative transcript level of the target gene was analyzed using the 2DDCt method. To make a stock solution of isoliquiritigenin, 3.58 g isoliquiritigenin (Sigma-Aldrich, St Louis, MO, USA) was dissolved in 100 % methanol to a volume of 5 ml as measured by volumetric flask. The isoliquiritigenin/ methanol solution had a concentration of 0.716 mg/ml. The stock solution was used to make standard solutions of 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 lmol/ml in methanol. The standard solutions filtered through 0.22 lm organic filters to obtain reference isoliquiritigenin solutions. A Shimadzu LC-20AT High Performance Liquid analysis system (Shimadzu Company, Japan) was used to analyze the samples. The HPLC was equipped with a GL Sciences Corporation (Japan) C18 chromatographic column (5 lm, 4.6 9 150 mm), which was used to separate compounds in the sample. The mobile phase had an 80:20 ratio of methanol– water with a flow rate 0.8 ml/min and 290/355 nm double wavelength detection. The retention time of
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isoliquiritigenin was 5.4 (±0.1) min. A regression equation for the retention time of isoliquiritigenin was generated from analyses of the standards. Five days after inoculation with the pathogen, soybean plants were treated with high-temperature drying methods at 80 °C. Soybean plants under the same treatment without inoculation served as controls. For each sample, 0.5 g tissue was ground into a powder in liquid nitrogen and dissolved in 90 % methanol solution (9:1, volume ratio). The leaf histamine was destroyed with ultrasonication, and the mixture was soaked in 90 % methanol solution overnight. The insoluble impurities were filtered, and the extracted solution was evaporated at room temperature in a fume cupboard. Then, acetate buffer (2 mol/l ammonium acetate, pH 5.0) was added to fully dissolve the extract and to ensure that the pH of the solution was 5. Ethyl acetate was used to extract isoliquiritigenin, and the ethyl acetate was removed by drying under N2. The sample was dissolved in methanol, and 10 ll of each sample was filtered through a 0.22 lm organic filter before HPLC analysis to identify and quantify the phytoalexins. Transformation and expression analysis of the Gmchr2 gene in tobacco The target gene was inserted into XbaI and BamHI sites in the expression vector PBI121, based on the nucleotide sequence of the Gmchr2 gene. The successfully constructed expression vector was transformed into Agrobacterium 105A, and then the target gene was transformed into cells for external implantation of tobacco with an Agrobacterium-mediated method (Mathis and Hinchee 1994). The transformed tissues were grown into tobacco plants. The presence of the transgene in transformed plants was confirmed by PCR. The transcript levels of the transgene and the function of the gene product in the transgenic tobacco plants were analyzed as described above.
Results Cloning of full-length Gmchr2 cDNA The 30 RACE and 50 RACE procedures yielded nucleotide sequences of 1,187 and 299 bp,
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respectively (Figures S2, S3). The sequencing results were aligned, and the duplicated sequences were removed. A specific primer to amplify the full-length gene product was designed, and the full-length cDNA, 1,417 bp in length, was obtained via reverse transcription PCR (RT-PCR) (Figure S4). To differentiate this gene from the known soybean chr gene, the target gene was named Gmchr2. Based on online analyses, the ORF of the target gene was determined to be 969 bp, encoding 322 amino acid residues in total. The analysis showed that the molecular weight of the expressed protein was 35,889.42 Da. The theoretical isoelectric point was 5.98. The protein included an aldo–ketoreductase family signature, an EGF-like domain signature, 2Fe–2S ferredoxin-type iron–sulfur-binding region signature, and a von Willebrand factor type C (VWFC) domain. Validation Gmchr2 copy number in the soybean genome and phylogenetic tree Four restriction endonucleases with different recognition sequences were used to digest the soybean genomic DNA to ensure that a single copy of the target gene was present in the randomly excised segments of the soybean genome. To avoid targeting the known gene, primers were designed as a 30 -probe based on the target gene sequence. When different restriction endonucleases were used to digest the genomic DNA, the hybridization results of the Southern blot showed only a single band (Figure S5), indicating that the target gene was present as a single copy in the soybean genome. The sequence of the gene Gmchr2 was used to scan the whole soybean genome on SoyBase (http://soybase.org). This analysis confirmed that the target gene is located on chromosome 9 in soybean. After analysis with DNAMAN software, the target gene Gmchr2 and the known soybean gene Gmchr1 were found to have 69.13 % homology at the nucleotide sequence level and 46 % at the amino acid sequence level (Figure S6). The Gmchr2 gene consists of four typical EGF-like domains, two VWFC domains, and one 2Fe–2S ferredoxin-type iron–sulfur-binding region. An analysis of the amino acid sequence revealed two aldo–ketoreductase family signature sites. The amino acid sequences of all of the known CHRs in leguminous plants were downloaded from the NCBI website, and CHR protein
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phylogenetic trees were constructed using MEGA4. The neighbor-joining method was used to divide the evolutionary analysis results into three groups (Groups I–III) based on the sequence homology of the CHR proteins (Figure S7). The soybean CHR2 protein showed high homology to Group I proteins and a shorter genetic distance to both the M. sativa CHR protein and the M. truncatula CHR protein than to the soybean CHR1, indicating that the evolutionary history of CHR1 differs from that of CHR. Real-time quantification of soybean chr gene transcripts at different sampling times According to the Q-PCR results (Figs. 1, 2, 3), the transcript levels of Gmchr2 differed among the various sampling times after inoculation and showed large differences after inoculation compared with its transcript level in the control. The experimental results were analyzed with the 2DDCt method, and the expression of Gmchr1 in the soybean root immediately before inoculation (sampling time, 0) was set to 1. As shown in Fig. 1, during the 60-h sampling period, there were no obvious changes in the mRNA levels of Gmchr1 and Gmchr2 in the control. Because the pathogens attack the roots directly, the mRNA levels of Gmchr1 and Gmchr2 increased a little in the roots of the inoculated plants from 0 to 24 h after inoculation. After 24 h, the transcript levels increased dramatically, and the transcript level of Gmchr1 was higher than that of Gmchr2. The transcript levels of Gmchr1 and Gmchr2 continued to increase in root tissues over the 60 h after inoculation. In stem tissue (Fig. 2), the transcript levels of Gmchr1 and Gmchr2 increased over the experimental period. The level of target mRNA in the control was almost same as that at the first sampling time. Except for sampling at 48 h, there may be some error occurred so that decreased level of mRNA for Gmchr1. Relative to the transcript level of Gmchr1, the increase in the transcript level of Gmchr2 in stem tissue was greater than that in the root, while the actual transcript levels in stem tissue were far lower than those in the root. In stem and leaf tissues, the level of target mRNAs tended to decrease by 60 h after inoculation, probably because metabolic processes turned to failure in the inoculated plants were affected by pathogen attack. In leaf tissue, both mRNAs of Gmchr1 and Gmchr2 reached peak levels at 48 h, the level of Gmchr1 mRNA had increased by 3
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Fig. 1 Expression profiles of the Gmchr1/Gmchr2 genes in soybean root tissue after inoculation. Sixty plants of ‘‘Jinong 17’’ were infected with Phytophthora sojae. The pathogen of Phytophthora root rot. Another 60 plants were detected as the control in the experiment. After the inoculation, ten roots were sampled every 12 h and performed Q-PCR assay. X-axis represents relative quantification of transcript levels of Gmchr1 and Gmchr2, and Y-axis represents the various sampling times after inoculation. Error bars represent confidence interval for the 10 replicates
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Fig. 2 Expression profiles of the Gmchr1/Gmchr2 genes in soybean stem tissue after inoculation. Sixty plants of ‘‘Jinong 17’’ were infected with Phytophthora sojae. The pathogen of Phytophthora root rot. Another 60 plants were detected as the control in the experiment. After the inoculation, ten stems were sampled every 12 h and performed Q-PCR assay. X-axis represents relative quantification of transcript levels of Gmchr1 and Gmchr2, and Y-axis represents the various sampling times after inoculation. Error bars represent confidence interval for the 10 replicates
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Fig. 3 Expression profiles of the Gmchr1/Gmchr2 genes in soybean leaf tissue after inoculation. Sixty plants of ‘‘Jinong 17’’ were infected with Phytophthora sojae. The pathogen of Phytophthora root rot. Another 60 plants were detected as the control in the experiment. After the inoculation, ten leaves were sampled every 12 h and performed Q-PCR assay. X-axis represents relative quantification of transcript levels of Gmchr1 and Gmchr2, and Y-axis represents the various sampling times after inoculation. Error bars represent confidence interval for the 10 replicates
times, and that of Gmchr2 had increased by 5.5 times (Fig. 3). In the leaf of control plants, the levels of the two target mRNAs remained stable throughout the experiment at close to the initial value. These results indicated that infection by P. sojae resulted in increased expression of the soybean chr gene through signal transduction, and the level of Gmchr2 transcripts was correlated with the response to the pathogen. Furthermore, the amount of Gmchr2 transcripts and the trends in changes in the Gmchr2 transcript level showed large differences in different soybean tissues, indicating the complexity of regulation of chr gene expression in soybean.
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Isoliquiritigenin content in soybean after inoculation A total of 10 ll of the standard was used for HPLC analysis according to the methods described in the Materials and methods section. The regression equation Y = 2.528 9 106X ? 0.223 (r = 0.9999) was obtained by plotting the amount of isoliquiritigenin (lmol) as the X-axis and the peak area (lV s) in the HPLC chromatogram as the Y-axis. The average content of isoliquiritigenin (Fig. 4) in soybean after
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Fig. 4 HPLC chromatogram of isoliquiritigenin content in inoculated soybean plants. HPLC analysis of liquiritigenin biosynthesis in soybean plants which were infected with Phytophthora sojae. X-axis represents the retention time of individual separation (min), and Y-axis UV-detector responses of individual separation (lV). The peak indicated by the red
arrow represents isoliquiritigenin in the sample. HPLC assay of extracted sample from inoculated t plants showed absorbance at 290/355 nm. The peak with retention time and spectrum corresponding to isoliquiritigenin is labeled. The retention time of the peak is 5.325 min, the area is 1,482,595 lV s, and the height is 118,671 lV s. (Color figure online)
inoculation was calculated using the regression equation and was compared with the amount of isoliquiritigenin in the controls (Figure S8). The amount was normalized to the dry weight of the soybean plants. The average isoliquiritigenin content in inoculated soybean plants was 3.972 lmol/g DW, significantly higher (p \ 0.01) than the average content in the control plants (1.597 lmol/g DW). Clearly, the isoliquiritigenin content significantly increased in inoculated soybean plants, proving that the increase in the expression of the chr gene in soybeans increased the amount of CHR protein, leading to a large increase in the isoliquiritigenin content. This finding also indicated that increased expression of the chr gene in soybean can increase the concentrations of products of the daidzein biosynthesis pathway, such as isoliquiritigenin.
the root, leaf, flower, and stem tissues were sampled for RNA extraction. The b-actin gene in tobacco was used as the reference gene, and the experimental results were analyzed using the 2DDCt method. In untransformed tobacco, the transcript level of Gmchr2 gene was almost undetectable. In transformed tobacco, the transcript level of Gmchr2 gene differed among the different tissue types (Figure S9). The highest transcript level of Gmchr2 gene was in the leaf; the transcript level was 4.1 times higher than that of b-actin. The lowest transcript level of Gmchr2 was in the stem; the Gmchr2 gene transcript level was 1.3 times that of b-actin. The transcript level in the flower was similar to that in the leaf. Because we used an Agrobacterium-mediated transformation method, not all the cells in the transformed tobacco contained the Gmchr2 gene. This could explain the variations in the Q-PCR results. The isoliquiritigenin content in the flower tissue of tobacco was calculated using the regression equation defined previously. According to Jung et al. (2000), the precursor for the reaction catalyzed by CHS can be synthesized in tobacco inflorescences. However, in the tobacco genome, there are no genes encoding CHS or related isoenzymes, so isoliquiritigenin cannot be produced naturally in tobacco plants. HPLC analysis showed that tobacco flower tissues transformed with Gmchr2 gene contained isoliquiritigenin (average
Expression analysis of Gmchr2 and isoliquiritigenin content in transformed tobacco Gmchr2 gene was inserted into the plant expression vector pBI121 and then transformed into the tobacco genome using an Agrobacterium-mediated method. The 35S CaMV promoter was used in the pBI121 expression vector. The transformed tobacco plants were grown until inflorescences formed. At that stage,
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Fig. 5 HPLC chromatogram of isoliquiritigenin content in transgenic tobacco plants. HPLC chromatograms showing suppression of daidzein metabolites in Gmchr2 gene transformed tobacco plants. Note the obvious increase in isoliquiritigenin among in transgenic tobacco plants. UV spectral scans confirmed that the peak indicated by the red arrow represents
isoliquiritigenin in the sample. HPLC assay of extracts from transformed plants showed absorbance at 290/355 nm. The peak with retention time and spectrum corresponding to isoliquiritigenin is labeled. The retention time of the peak is 5.496 min, the area is 15,148,941 lV s, and the height is 602,236 lV s. (Color figure online)
content, 38.524 lmol/g; Fig. 5). No isoliquiritigenin was detected in untransformed tobacco. Therefore, the product of the Gmchr2 gene was able to catalyze the production of isoliquiritigenin. Both the Q-PCR and HPLC assay results confirmed that Gmchr2 gene encoded CHR.
foreign substrates (Jez and Penning 2001; Hyndman et al. 2003). All of the members of this family have an (a/b)8-barrel motif and contain an average of 320 amino acids per monomer. These enzymes use NADPH as a cofactor and form a zymoprotein with complete physiological function. The sequence of the GmCHR2 protein contained the AKR motifs mentioned above. There are at least 21 AKR proteins in the model plant Arabidopsis thaliana (Simpson et al. 2009). There may be other members of the AKR superfamily in the soybean genome. The phylogenetic tree also showed that the evolutionary distance between Gmchr1 and Gmchr2 in soybean is greater than that between the Gmchr2 gene in soybean and the chr genes in alfalfa and M. truncatula. This finding highlights the polymorphic nature of the gene and illustrates the relative conservation of the chr genes in soybean and those in the genomes of other leguminous plants. Based on the amino acid sequence and structure of the AKR protein superfamily, Hyndman et al. (2003) divided its members into 16 families and even more subfamilies. Both Gmchr2 and Gmchr1 belong to the AKR4 family, but belong to different subfamilies, which correspond with the information in the phylogenetic tree. The product of the chr gene is a key enzyme in the biosynthesis of soybean phytoalexins, and soybean phytoalexins can increase plant resistance to various
Discussion Ballance and Dixon (1995) and Sallaud et al. (1995) independently found seven different CHRs in M. sativa. In addition, Young et al. (2011) isolated two chr genes from M. truncatula, and Wu et al. (2011) cloned two chr genes from the A. membranaceus genome. However, only one chr gene had been found and confirmed in soybean. Given that other leguminous plants have multiple chr genes, there was the possibility that at least one unknown chr gene was present in the soybean genome. Based on the sequence analysis of the gene identified in this study, the new Gmchr2 gene has the same type of structural domain and the same or similar properties as those of Gmchr1 in soybean. Sequence analysis of the expression product of Gmchr2 gene showed that the GmCHR2 protein is a typical AKR. The AKR family is one of three enzyme superfamilies whose members catalyze the oxido-reduction of a wide variety of natural and
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pathogenic bacteria. Graham et al. (2007) reported that there were marked changes in the transcript levels of the chr gene within 72 h of root tissue infection with P. sojae. In this present study, the transcript levels of both Gmchr2 and Gmchr1 gene increased significantly after infection with P. sojae. In addition, in the root, the changes in the transcript levels of Gmchr1 were greater than those of Gmchr2. Also, the transcript level of Gmchr1 was higher than that of Gmchr2, consistent with the results of Subramanian et al. (2005, 2007). However, the expression level of chr genes in other soybean tissues also increased over time after pathogen infection. This finding indicates that after infection by pathogenic bacteria, signal molecules are transported to other tissues. Interestingly, in the stem and leaf tissues, the accumulated transcript level of Gmchr2 exceeded that of Gmchr1. This may illustrate that the system regulating the expression of Gmchr2 is less efficient, but more sensitive, than that regulating the expression of Gmchr1. The different evolutionary branches of Gmchr2 and Gmchr1 suggest that there could be significant differences in the regulation of the expression of these genes, perhaps through a cis-acting element or transcription factor. As the transcript levels of chr genes increased, so did the isoliquiritigenin content in soybean tissues. This confirmed that there was more CHR protein catalyzing the production of isoliquiritigenin after pathogen infection. However, the increasing isoliquiritigenin level did not show a direct linear relationship with the transcript levels of chr genes, indicating that the metabolism of isoflavone compounds may be a more complex process than is currently thought. Isoliquiritigenin and daidzein were shown to be metabolized into phytoalexins, as described by Shimada et al. (2003), although the details of this process have not been completely determined. Jung et al. (2000) cloned the gene encoding isoflavone synthetase (IFS) and transformed it into tobacco. Genistein was detected in the flowers of the transformed tobacco plants by HPLC. These results indicated that in tobacco flowers, there are probably other enzymes and substrates required for the biosynthesis of isoflavones. Wild-type tobacco lacks the key enzymes involved in isoflavone biosynthesis, like CHR and IFS, and so it cannot naturally produce daidzein and genistein. However, when the chr gene was transformed into tobacco, isoliquiritigenin was detected, as shown in this study. This result confirmed
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that the cloned gene encodes a functional CHR protein. Because of the high expression level of the transgene and the lack of metabolic pathways utilizing isoliquiritigenin in tobacco, isoliquiritigenin accumulated to high levels. Also, isoliquiritigenin absorbs at two ultraviolet absorption peaks (at 290 and 355 nm). Therefore, these wavelengths can be used to detect the purity and concentration of isoliquiritigenin in samples simultaneously. In soybean, the catalytic process involving CHS plays a key role in the synthesis of flavonoid compounds. Together with CHR, it synthesizes isoliquiritigenin, the necessary precursor for daidzein, glyceollin, and other phytoalexins. The complete catalytic process involving CHS includes three steps: decarboxylation condensation, thioester cyclization, and aromatization (Havsteen 2002; Katsuyama et al. 2007; Zernova et al. 2009; Yi et al. 2010). First, the decarboxylic reaction generates a thioester-based compound. The coumaroyl-CoA thioester is regarded as the basic molecule. Decarboxylic malonyl-CoA undergoes Claisen condensation, a polyketone is synthesized, and coenzyme A is released simultaneously. After three condensations, the cyclization reaction yields a tetraketo compound, and then the aromatization reaction generates the chalcone B ring in the benzene ring structure, yielding chalcone. In the presence of CHR and NADPH, intermediate products in the CHS catalytic process, such as di-, tri- ,and tetraketo compounds, and finally 4,20 ,40 -trihydroxychalcone, also known as isoliquiritigenin, are generated (Yu et al. 2000, 2003; Liu et al. 2007). Therefore, catalysis by CHR is the key process in the biosynthesis of deoxychalcone phytoalexins. However, the joint actions of CHR and CHS have not been demonstrated experimentally. It is therefore unclear which intermediate is the CHR substrate. In this study, we describe an approach to manipulate the expression of Gmchr2 in legumes. This could be used to improve pathogen and stress responses and produce valuable plant products or nutritionally enhanced foods. Our results showed that transgenic plants expressing the soybean Gmchr2 gene produced a functional enzyme in a species that does not naturally produce 4,20 ,40 -trihydroxychalcone. The transformants were also able to produce isoliquiritigenin and other chalcone-derived compounds. These findings demonstrate the potential for producing isoflavones in nonisoflavonoid-producing crop species.
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2148 Acknowledgments This research was funded by the grants from China National Natural Science Foundation (Project: 31171568) and the ‘948’ program from the Chinese Ministry of Agriculture (Project: 2013-Z47).
References Ayabe SI, Udagawa A, Furuya T (1998) NAD (P) H-dependent 60 -deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract. Arch Biochem Biophys 26:458–462 Ballance GM, Dixon RA (1995) Medicago sativa cDNAs encoding chalcone reductase. Plant Physiol 107:1027–1028 Bomati EK, Austin MB, Bowman ME, Dixon RA, Noel JP (2005) Structural elucidation of chalcone reductase and implications for deoxychalcone biosynthesis. J Biol Chem 280:30496–30503 Dixon RA (2001) Natural products and plant disease resistance. Nature 411:843–847 Forkmann G, Martens S (2001) Metabolic engineering and applications of flavonoids. Curr Opin Biotechnol 12: 155–160 Graham TL (1990) Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant Physiol 95:594–603 Graham TL, Graham MY, Subramanian S, Yu O (2007) RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and hypersensitive cell death in Phytophthora sojae infected tissues. Plant Physiol 144:728–740 Havsteen BH (2002) The biochemistry and medical significance of the flavonoids. Pharmacol Thera 96:67–202 Hu RB, Fan CM, Li HY, Zhang QZ, Fu YF (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10:1–12 Hyndman D, Bauman DR, Heredia VV, Penning TM (2003) The aldo–keto reductase superfamily homepage. Chem Biol Interact 143–144:621–631 Jez JM, Penning TM (2001) The aldo–keto reductase (AKR) superfamily: an update. Chem Biol Interact 130–132:499–525 Jian B, Liu B, Bi YR, Hou WS, Wu CX, Han TF (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59–62 Jiang HX, Wood KV, Morgan JA (2005) Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 71:2962–2969 Jirschitzka J, Schmidt GW, Reichelt M, Schneider B, Gershenzon J, D’Auria JC (2012) Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae. PNAS 109:10304–10309 Joung JY, Kasthuri GM, Park JY, Kang WJ, Kim HS, Yoon BS, Joung H, Jeon JH (2003) An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 50 -deoxyflavonoids in transgenic tobacco. Biochem Biophys Res Commun 303:326–331 Jung W, Yu O, Lau SC, O’Keefe DP, Odell J, Fader G, McGonigle B (2000) Identification and expression of isoflavone
123
Mol Breeding (2014) 34:2139–2149 synthase, the key enzyme for biosynthesis of isoflavones in legumes. Nature 18:208–212 Kampko¨tter A, Chovolou Y, Kulawik A, Bovy AG (2008) Isoflavone daidzein possesses no antioxidant activities in cell-free assays but induces the antioxidant enzyme catalase. Nutr Res 28:620–628 Katsuyama Y, Funa N, Miyahisa I, Horinouchi S (2007) Synthesis of unnatural flavonoids and stilbenes by exploiting the plant biosynthetic pathway in Escherichia coli. Chem Biol 14:613–620 Liu R, Hu Y, Li J, Lin Z (2007) Production of soybean isoflavone genistein in non-legume plants via genetically modified secondary metabolism pathway. Metab Eng 9:1–7 Lozovaya VV, Lygin AV, Zernova OV, Li S, Hartman GL, Widholm JM (2004) Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani. Plant Phys Biochem 42:671–679 Marienhagen J, Bott M (2013) Metabolic engineering of microorganisms for the synthesis of plant natural products. J Biotechnol 163:166–178 Martens S, Knott J, Seitz CA, Janvari L, Yu SN, Forkmann G (2003) Impact of biochemical pre-studies on specific metabolic engineering strategies of flavonoid biosynthesis in plant tissues. Biochem Eng J 14:227–235 Mathis NL, Hinchee MAW (1994) Agrobacterium inoculation techniques for plant tissues. Plant Mol Biol Manual B6:1–9 Sallaud C, El-Turk J, Breda C, Buffard D, Kozak I, Esnault R, Kondorosi A (1995) Differential expression of cDNA coding for chalcone reductase, a key enzyme of the 5-deoxyflavonoid pathway, under various stress conditions in Medicago sativa. Plant Sci 109:179–190 Schijlen EGWM, Vos CHR, Tunen AJ, Bovy AG (2004) Modification of flavonoid biosynthesis in crop plants. Phytochemistry 65:2631–2648 Shimada N, Akashi T, Aoki T, Ayabe S (2000) Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in phytoalexin biosynthesis. Plant Sci 160:37–47 Shimada N, Aoki T, Shusei S, Nakamura Y, Tabata S, Ayabe SI (2003) A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicas. Plant Physiol 131:941–951 Simpson P, Tantitadapitak C, Reed AM, Mather OC, Bunce CM, White SA, Ride JP (2009) Characterization of two novel aldo–keto reductases from Arabidopsis: expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress. J Mol Biol 392:465–480 Steele CL, Gijzen M, Qutob D, Dixon RA (1999) Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. Arch Biochem Biophys 367:146–150 Subramanian S, Graham MY, Yu O, Graham TL (2005) RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthora sojae. Plant Physiol 137:1345–1353 Subramanian S, Stacey G, Yu O (2006) Endogenous isoflavones are essential for the establishment of symbiosis between
Mol Breeding (2014) 34:2139–2149 soybean and Bradyrhizobium japonicum. Plant J 48: 261–273 Subramanian S, Stacey G, Yu O (2007) Distinct, crucial roles of flavonoids during legume nodulation. Trends Plant Sci 12:282–285 Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 Thill J, Regos I, Farag MA, Ahmad AF, Kuesek J, Castro A, Schlangen K, Carbonero CH, Gadjev IZ, Smith LM, Halbwirth H, Treutter D, Stich K (2012) Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin). Phytochemistry 82:67–80 Viljoen CD, Snyman MC, Spies JJ (2013) Identification and expression analysis of chalcone synthase and dihydroflavonol 4-reductase in Clivia miniata. S Afr J Bot 87:18–21 Weisshaar B, Jenkins GI (1998) Phenylpropanoid biosynthesis and its regulation. Curr Opin Plant Biol 1:251–257 ¨ der G, Schiltz E, Grisebach H, SchrO ¨ der J Welle R, SchrO (1991) Induced plant responses to pathogen attack Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy63). Eur J Biochem 196:423–430 Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493
2149 Xu RY, Nan P, Pan H, Zhou T, Chen J (2012) Molecular cloning, characterization and expression of a chalcone reductase gene from Astragalus membranaceus Bge. var. mongholicus. Mol Biol Rep 39:2275–2283 Yi J, Derynck MR, Chen L, Dhaubhadel S (2010) Differential expression of CHS7 and CHS8 genes in soybean. Planta 231:741–753 Young ND, Fre´de´ric D, Oldroyd GE et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524 Yu O, Jung W, Shi J, Croes RA, Fader GM, McGonigle B, Odell JT (2000) Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues. Plant Physiol 124:781–793 Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63:753–763 Zernova OV, Lygin AV, Widholm JM, Lozovaya VV (2009) Modification of isoflavones in soybean seeds via expression of multiple phenolic biosynthetic genes. Plant Physiol Biochem 47:769–777
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