Deficiency in the amino aldehyde dehydro

Plant Biotechnology Journal (2011) 9, pp 75–87 doi: 10.1111/j.1467-7652.2010.00533.x Deficiency in the amino aldehyde dehydrogenase encoded by GmAMADH2, the homologue of rice Os2AP, enhances 2-acetyl-1-pyrroline biosynthesis in soybeans (Glycine max L.) Siwaret Arikit1,2,†, Tadashi Yoshihashi3,†, Samart Wanchana4, Tran T Uyen3, Nguyen T T Huong3, Sugunya Wongpornchai5 and Apichart Vanavichit1,6,* Rice Science Center and Rice Gene Discovery, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand Interdisciplinary Graduate Program in Genetic Engineering, Kasetsart University, Bangkok, Thailand Postharvest Science and Technology Division, Japan International Research Center for Agricultural Sciences, Tsukuba Ibaraki, Japan International Rice Research Institute, Los Ban˜os, Laguna, Philippines Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand Department of Agronomy, Kasetsart Univerisity Kamphaeng Saen, Nakhon Pathom, Thailand Received 30 September 2009; accepted April 2010 *Correspondence (fax +66 34 355197; e-mail vanavichit@gmail.com) † These authors contributed equally to this work Summary 2-Acetyl-1-pyrroline (2AP), the volatile compound that provides the ‘popcorn-like’ aroma in a large variety of cereal and food products, is widely found in nature Deficiency in amino aldehyde dehydrogenase (AMADH) was previously shown to be the likely cause of 2AP biosynthesis in rice (Oryza sativa L.) In this study, the validity of this mechanism was investigated in soybeans (Glycine max L.) An assay of AMADH activity in soybeans revealed that the aromatic soybean, which contains 2AP, also lacked AMADH enzyme activity Two genes, GmAMADH1 and GmAMADH2, which are homologous to the rice Os2AP gene that encodes AMADH, were characterized The transcription level of GmAMADH2 was lower in aromatic varieties than in nonaromatic varieties, whereas the expression of GmAMADH1 did not differ A double nucleotide (TT) deletion was found in exon 10 of GmAMADH2 in all aromatic varieties This variation caused a frame-shift mutation and a premature stop codon Suppression of GmAMADH2 by introduction of a GmAMADH2-RNAi construct into the calli of the two nonaromatic wild-type varieties inhibited the synthesis of AMADH and induced the biosynthesis of 2AP These results suggest that deficiency in the Keywords: AMADH, 2-acetyl-1-pyrr- GmAMADH2 product, AMADH, plays a similar role in soybean as in rice, which is to oline, vegetable soybean, GABA, promote 2AP biosynthesis This phenomenon might be a conserved mechanism polyamine metabolism, Os2AP among plant species Introduction 2-Acetyl-1-pyrroline (2AP) is a volatile compound that preference in positive terms (Fitzgerald et al., 2009) In rice, the aroma is considered to be a special trait that produces the potent ‘popcorn-like’ or ‘pandan-like’ aroma found in a large variety of cereal products and vegetablederived and animal-derived products (Adams and De Kimpe, 2006) This aroma component is a value-added enhances aromatic rice such as the Jasmine types of Thailand and Basmati rice of India and Pakistan This aroma is economically important because it determines the premium price Similarly, in vegetable soybean, the immature seeds are consumed as a vegetable or snack called ‘Edam- characteristic and can be directly linked to consumer ame’, and the aroma plays an important role influencing ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd 75 76 Siwaret Arikit et al consumer preference and acceptance ‘Chamame’, a special group of ‘Edamame’ that contains a pleasant aroma that is thought to be attributable to the presence of 2AP (Fushimi and Masuda, 2001), is preferred by consumers and can command higher prices In Japan, the price of this end product, 2AP, by an unknown pathway (Bradbury et al., 2008) Plant AMADHs belong to the aldehyde dehydrogenase (ALDH) superfamily, which represents a group of NAD(P)+dependent enzymes that oxidize various aldehydes (Peroz- variety can be double that of nonaromatic varieties (Statistics Department, Ministry of Agriculture, Forestry and Fisheries, 2009) The aroma characteristic also contributes to the acceptance of the product by the unfamiliar con- ich et al., 1999) Based on the amino acid sequences, AMADHs are homologous to BADHs and contain similar primary structures (Brauner et al., 2003) However, there are some differences between the two enzymes with respect to substrate specificities (Sˇebela et al., 2000; sumer Hence, the aromatic varieties were targeted as the first priority in vegetable soybean breeding programmes that were introduced to 107 countries all over the world (Shanmugasundaram and Yan, 2004; Takahashi et al., Livingstone et al., 2002) AMADH probably plays a role in physiological processes connected to polyamine degradation, converting 4-aminobutanal to GABA (Petrˇivalsky´ 2006) 2AP has been reported to be biosynthesized in various organisms, including plants, such as pandan (Pandanus amaryllifolius Roxb.) (Buttery et al., 1983), certain varieties et al., 2007) Because natural substrates of AMADH are reactive metabolites that show considerable toxicity, this enzyme was thought to serve as a detoxification enzyme (Tylichova´ et al., 2007) The pathway of GABA biosynthesis of rice (Oryza sativa L.) (Buttery et al., 1982; Widjaja et al., 1996; Yoshihashi, 2002), bread flowers (Vallaris glabra Ktze) (Wongpornchai et al., 2003) and certain varieties of soybean (Glycine max L.) (Fushimi and Masuda, 2001; Plonjarean et al., 2007; Wu et al., 2009) Synthesis of this via polyamine catabolism, 4-aminobutanal and AMADH is normally thought of as an alternative (Kakkar et al., 2000), while the major pathway occurs via the decarboxylation of glutamate (Snedden et al., 1995) However, evidence supporting the existence of this alternative pathway in plants compound has also been documented in microorganisms, in particular Bacillus cereus (Adams and De Kimpe, 2007) and bakers’ yeast (Snowdon et al., 2006), and in animals such as tigers (Panthera tigris tigris) (Brahmachary et al., has previously been reported in soybean (Xing et al., 2007) and pea (Petrˇivalsky´ et al., 2007) At present, however, it has only been reported in rice that AMADH deficiency likely results in 2AP biosynthesis 1990) 2AP can also be formed in food products, such as roasted popcorn and bread crust, by thermal generation during heating (Schieberle and Wener, 1991) So far, the complete biosynthetic pathway for 2AP in There is no evidence so far as to whether the same mechanism or biochemical pathway for 2AP biosynthesis is shared among other plants In this study, we aimed to verify the association between AMADH deficiency and 2AP plants has not been fully elucidated Recently, a key pathway for 2AP biosynthesis in rice was proposed (Bradbury et al., 2008), and a single gene responsible for the aroma trait has been identified by different research groups biosynthesis in the soybean to examine its role in a plant besides rice We predicted that inactivation of AMADH, which induces 2AP biosynthesis, would be conserved among plant species and that the gene responsible for (Bradbury et al., 2005; Vanavichit et al., 2008; Kovach et al., 2009) The identified gene has been given multiple names, including Os2AP (Vanavichit et al., 2008), BAD2 (Bradbury et al., 2005, 2008) and BADH2 (Niu et al., 2008) Its protein product was identified as amino alde- AMADH could be orthologous or homologous to the rice Os2AP We chose to study the above hypotheses in the soybean for the following reasons: first, aromatic and nonaromatic varieties of this plant are available, so the natural variation in the gene that controls this trait could hyde dehydrogenase (AMADH), which shares high sequence similarity with betaine aldehyde dehydrogenase (BADH) (Bradbury et al., 2008) The utilization of 4-aminobutanal by the AMADH enzyme is likely to be the key step be observed; second, the complete genome sequence of the soybean has been released, allowing us to search for homologous genes throughout its genome; and finally, soybean is a dicot species distantly related to rice, which that affects the biosynthesis of 2AP in rice Nonaromatic rice contains the functional AMADH enzyme, which converts 4-aminobutanal to 4-aminobutyrate (GABA), and consequently, 2AP is not formed In aromatic rice, which strengthens the validity of our hypotheses We characterized the AMADH candidate gene in soybean and inactivated it by RNAi to disrupt the synthesis of AMADH and observe its effect on 2AP biosynthesis The results pre- does not have a functional AMADH enzyme, 4-aminobutanal is converted first into precursor(s) and then into the sented here support the idea that inactivation of AMADH is a general key factor that determines 2AP biosynthesis in ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 Inactivation of GmAMADH2 enhances 2-acetyl-1-pyrroline formation in soybeans 77 plants These results could be applied to the manipulation of the target gene in other plants to enhance 2AP biosynthesis Results Determination of the aroma component and assessment of AMADH activity in aromatic and nonaromatic soybeans Ten varieties of aromatic and nonaromatic soybeans were selected to assay AMADH enzymatic activity in immature seeds The aroma characteristic of the ten varieties was ten soybean varieties The enzyme activity staining on the PAGE gel revealed four activity bands for AMADH with 4aminobutanal, but the bands could not utilize betaine aldehyde as a substrate (Figure 1) Among these four activity bands, one band (band c, Figure 1) was present in all varieties of nonaromatic soybean but absent from all aromatic soybean varieties This band is considered to be the band of AMADH associated with 2AP production Characterization of rice Os2AP homologues in soybeans We hypothesized that the gene encoding the AMADH confirmed by measuring the 2AP content in the seeds using headspace gas chromatography (HSGC) 2AP was detected in all five aromatic varieties but not in the nonaromatic varieties (Table 1) This result shows that 2AP is that is associated with 2AP in soybeans could be the homologue of rice Os2AP We then identified the possible homologues in soybeans using the protein sequence of Os2AP (Vanavichit et al., 2008) to perform a homology the determinant of the aromatic phenotype in soybeans The AMADH enzymatic activity of the fractionated extracts from immature seeds was assayed by activity staining after native polyacrylamide gel electrophoresis (PAGE) 4-Aminobutanal and betaine aldehyde were compared as search against the NCBI protein database using the Basic Local Alignment Search Tool for protein searching (BLASTP) Two soybean proteins, accession numbers BAG09376 and BAG09377, were retrieved The amino acid sequence identities of BAG09376 and BAG09377 in substrates for AMADH enzymatic activity The extracts from nonaromatic rice (c.v Nipponbare) calli were also loaded in the same PAGE gel The activity staining test revealed that AMADH from soybean extracts utilized comparison with rice Os2AP were 75% and 74%, respectively Both BAG09376 and BAG09377 were annotated as peroxisomal BADH proteins that were identified in peroxisomes purified from etiolated soybean cotyledons 4-aminobutanal but could not utilize betaine aldehyde as a substrate On the contrary, AMADH from rice extracts, which was used as a positive control, utilized both 4-aminobutanal and betaine aldehyde (Supplementary (Arai et al., 2008) To obtain the full-length genomic sequences of the genes, the two coding sequences (CDSs), AB333793 and AB333794, corresponding to BAG09376 and BAG09377 Figure S1) Thus, 4-aminobutanal was used as a substrate for the analysis of AMADH enzymatic in extracts from the were used to perform a nucleotide search (BLASTN) against a recently released (assembly Glyma1) shotgun genome sequence database for soybean (Phytozome, http://www.phytozome.net/soybean) Two gene models, Table Contents of 2AP in soybean seeds determined by automated headspace gas chromatography with a nitrogen–phosphorus detector (HSGC-NPD) Glyma05g01770 and Glyma06g19820, annotated as BADHs, were retrieved corresponding to AB333793 and 2AP content (ppb) Variety (mean ± SD) Phenotype Okuhara wase n.d Nonaromatic Oishi Edamame n.d Nonaromatic Shirono Mai n.d Nonaromatic Chiang Mai 60 n.d Nonaromatic Jack n.d Nonaromatic Chamame 579.5 ± 28.8 Aromatic Kouri 583.7 ± 36.8 Aromatic Kaori hime 1160.0 ± 50.4 Aromatic Fukunari 609.4 ± 43.1 Aromatic Yuagari musume 1008.5 ± 56.8 Aromatic n.d., not detected Figure AMADH activity assay on a native PAGE gel of crude extracts from soybean seeds from ten varieties The arrows indicate the four bands of enzymes that metabolize 4-aminobutanal Nonaromatic varieties are Okuhara wase in lane 1, Oishi Edamame in lane 2, Shirono Mai in lane 3, Chiang Mai 60 in lane and Jack in lane Aromatic varieties are Chamame in lane 6, Kouri in lane 7, Kaori hime in lane 8, Fukunari in lane and Yuagari musume in lane 10 ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 78 Siwaret Arikit et al AB333794, respectively We renamed the two genes GmAMADH1 and GmAMADH2 in this study and considered both of them to be candidate genes for AMADH Both GmAMADH1 and GmAMADH2 contain 15 exons, similar to other previously reported plant BADHs (Legaria of GmAMADH1 and GmAMADH2 from the Phytozome soybean genome database were identical to those reported previously (Arai et al., 2008) GmAMADH1 and GmAMADH2 are 85% identical and 92% similar at the amino acid level and 85% identical at the nucleotide level et al., 1998; Vanavichit et al., 2008) The exons in the two genes were relatively similar in size, but several introns of GmAMADH1 were longer than those of GmAMADH2 (Figure 2a) The CDSs and deduced amino acid sequences The essential aldehyde dehydrogenase conserved domain and glutamic acid (Glu) and cysteine (Cys) active site residues were found in both GmAMADH1 and GmAMADH2 (data not shown) (a) (b) Figure (a) The gene structures of GmAMADH1 and GmAMADH2 The sequence variation, a TT deletion in exon 10 of GmAMADH2 at position 928, is shown (b) Deduced amino acid sequence of the GmAMADH2 coding sequence translated from the start codon (ATG) The end of translation and the location of the premature stop codon (TAG) are indicated with asterisks (*) ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 Inactivation of GmAMADH2 enhances 2-acetyl-1-pyrroline formation in soybeans 79 Expression analysis of GmAMADH1 and GmAMADH2 The expression of the two genes, GmAMADH1 and GmAMADH2, was analysed in leaves, young seeds and calli, and these results were compared among the ten aromatic and nonaromatic soybean varieties The results of reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that the expression patterns of the two genes differed among these soybean varieties The expression levels of GmAMADH2 were much lower in all tissues of the five aromatic varieties than in the nonaromatic varieties By contrast, the expression levels of GmAMADH1 did not differ in all ten soybean varieties (Figure 3) This result suggested that the lower expression of GmAMADH2 in the aromatic varieties was associated with the presence of 2AP Sequence variation in the CDS of GmAMADH2 Because the expression of GmAMADH2 was lower in all of the aromatic soybean varieties compared with the nonaromatic varieties, the factors that regulate the expression of this gene were investigated The full-length GmAMADH2 cDNA was amplified by RT-PCR from two representative soybean varieties, Chamame (aromatic) and CM60 (nonaromatic), and it was subsequently sequenced Pairwise alignment of the cDNA sequences between the two varieties revealed that the CDSs were almost identical, except for a region in exon 10 at nucleotides 928–932 downstream from the first ATG, where two thymines (T) were absent from the aromatic variety Chamame (Figure 2a) The TT deletion in Chamame caused a frameshift and a premature stop codon, TAG, three bases downstream from the deletion (Figure 2b) Consequently, the deduced amino acid sequence from the CDS of Chamame was truncated and comprised only 311 amino acids By contrast, the nonaromatic variety, CM60, contained the complete 503 amino acid sequence, and this sequence was identical to that of BAG09376 The presence of the 2-bp deletion in exon 10 in the other four aromatic varieties was confirmed by sequencing exon 10 of the genomic DNA (Figure 2a) This result indicated that the lower amount of the GmAMADH2 transcript observed in all aromatic varieties could be caused by the premature stop codon that induced non-sense-mediated decay (NMD) (Isshiki et al., 2001) RNAi-mediated gene suppression of GmAMADH2 To verify that the AMADH associated with 2AP biosynthesis (band c, Figure 1) was encoded by GmAMADH2 and that the level of 2AP would increase when the AMADH was inactivated, we suppressed the expression of GmAMADH2 in nonaromatic soybeans by RNAi-mediated gene suppression The GmAMADH2-RNAi was introduced into two nonaromatic soybean varieties, CM60 and Jack, using Agrobacterium transformation The pANGmAMADH2 construct contained genomic DNA fragments of GmAMADH2 in the sense and antisense directions that spanned 441 bp covering exon 1, intron and part of exon (Figure 4a) To select for the GmAMADH2-RNAi-containing calli, all hygromycin-resistant calli were tested by PCR to detect the GUS linker fragment that served as the loop in the transcribed RNAi construct In this study, we investigated the level of gene suppression, 2AP content and enzyme activity at the callus stage The expression levels of GmAMADH2 in pANGmAMADH2-transformed calli of both varieties were highly suppressed compared with the levels of the wild MADH1 was RNAi vector GmAMADH1 type (Figure 4b) The expression of GmAalso analysed to verify the specificity of the The results showed that the expression of was not suppressed in the two RNAi-trans- formed lines, indicating that the pANGmAMADH2 RNAi vector was specific for GmAMADH2 without co-suppression of GmAMADH1 The accumulation of the siRNA generated by the RNAi mechanism was observed by RNA gel Figure RT-PCR analysis of GmAMADH1 and GmAMADH2 expression in leaves, young seeds and calli Lectin was used as a control Nonaromatic varieties are Okuhara wase in lane 1, Oishi Edamame in lane 2, Shirono Mai in lane 3, Chaing Mai 60 in lane and Jack in lane Aromatic varieties are Chamame in lane 6, Kouri in lane 7, Kaori hime in lane 8, Fukunari in lane and Yuagari musume in lane 10 ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 80 Siwaret Arikit et al (a) (b) Figure (a) The structure of the pANGmAMADH2 RNAi vector (b) RT-PCR analysis of endogenous GmAMADH2, the transgene and GmAMADH1, and RNA gel blot of the siRNA specific to GmAMADH2 among the Jack and CM60 wild-type varieties and the corresponding RNAi lines, Jack-RNAi and CM60-RNAi, respectively Lectin was used as a control for RT-PCR analysis rRNA was stained with 2% methylene blue as a loading control DNA oligomers of 22 and 24 nt were used as size markers for siRNA blot analysis of pANGmAMADH2-transformed calli of both varieties (Figure 4b) In addition, the analysis of 2AP in the seeds and calli were different A second isozyme band (band b, Figure 5) was also completely absent from the pANGmAMADH2-transformed calli compared with wild type indicated that 2AP was synthesized in the RNAi-transformed calli (Table 2) An enzymatic assay of AMADH activity in PAGE gels aromatic soybean calli in agreement with the pANGmAMADH2-transformed calli, although this isozyme normally appeared in both aromatic and nonaromatic seeds Another isozyme band (band a, Figure 5) that was highly was performed to verify whether the product of GmAMADH2 was inhibited by RNAi The result clearly showed that the candidate AMADH isozyme, which was thought to be encoded by GmAMADH2, disappeared from the extracts of calli from both RNAi lines The absence of the stained in young seeds of both aromatic and nonaromatic soybeans was faintly stained in the callus samples of all varieties AMADH band from the RNAi lines was similar to that in the aromatic varieties, Chamame and Kaori hime, which were used as controls (Figure 5) However, it is worth noting that the band patterns of AMADH isozymes in young Plant BADH ⁄ AMADH family BADH ⁄ AMADH homologous protein sequences were retrieved from 11 flowering plant (Angiosperm) genomes, Table Contents of 2AP in soybean calli identified in wild type and RNAi transgenic lines 2AP content (ppb; as FW) Variety (mean ± SD) Phenotype Wild type Yuagari musume 457.3 ± 32.3 Aromatic Kaori hime 325.3 ± 48.3 Aromatic Okuhara wase n.d Nonaromatic Chiang Mai 60, CM60 n.d Nonaromatic Jack n.d Nonaromatic CM60-RNAi (5-1) 324.2 ± 45.2 Aromatic Jack-RNAi (2) 343.2 ± 50.2 Aromatic Transgenic lines n.d., not detected Figure AMADH gel activity assay of the crude extracts from calli of the Jack and CM 60 wild-type varieties and the corresponding RNAi lines, Jack RNAi and CM 60 RNAi, respectively The varieties Chamame and Kaori hime were used as aromatic line controls for the enzyme activity in callus tissues The varieties Jack and Kaori hime were used as nonaromatic and aromatic controls, respectively, for the enzyme activity in seeds The candidate AMADH activity band is indicated by an arrow (c) ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 Inactivation of GmAMADH2 enhances 2-acetyl-1-pyrroline formation in soybeans 81 six dicot species and five monocot species, a lycophyte genome, a chlorophyte genome and a bryophyte genome In all flowering plants, two homologues of BADH ⁄ AMADHs were identified from each species, whereas the three primitive genomes contained only one BADH ⁄ AMADH each dicot species were likely to be clustered within the same species However, in some closely related species, such as pea and soybean, orthologous groups were also clustered For Arabidopsis, one homologue was outgrouped from the other dicots The phylogenetic analysis showed that, among the flowering plant genomes, two distinct groups were clearly defined as dicot and monocot groups (Figure 6a) Among the monocots, two complete orthologous subgroups con- According to an in silico analysis of the NAD-dependent aldehyde dehydrogenase protein domain, an aldehyde dehydrogenase cysteine active site with the PROSITE regular expression [FYLVA] - x - {GVEP} - {DILV} - G - [QE] - taining all five monocot species were clearly identified By contrast, an orthologous group was not found among the dicot species The two BADH ⁄ AMADH homologues in {LPYG} - C - [LIVMGSTANC] - [AGCN] - {HE} - [GSTADNEKR] was detected in all BADH ⁄ AMADH homologues The whole set of BADH ⁄ AMADH homologous proteins (a) Figure (a) Phylogenetic tree of BADH ⁄ AMADH homologues among the higher plants Pisum sativum (CAC48392.2_PISSA and CAC48393.1_PISSA), Zoysia tenuifolia (BAD34953.1_ZOYTE and BAD34949.1_ZOYTE), Oryza sativa (Os04g0464200_ORYSA and Os08g0424500_ORYSA), Zea mays (ACF87737.1_ZEAMA and NP_001105781.1_ZEAMA), Sorghum bicolor (Sb07g020650.1_SORBI and Sb06g019200.1_Sb06g019210.1_SORBI), Arabidopsis thaliana (At3g48170.1_ARATH and At1g74920.1_ARATH), Populus trichocarpa (661953_POPTR and 666405_POPTR), Glycine max (BAG09376.1_GLYMA: GmAMADH2 and BAG09377.1_GLYMA: GmAMADH1), Amaranthus hypochondriacus (AAB58165.1_AMAHY and AAB70010.1_AMAHY), Atriplex hortensis (ABF72123.1_ATRHO and P42757.1_ATRHO) and Hordeum vulgare (BAB62846.1_HORVU and BAB62847.1_HORVU); a lycophyte (Selaginella moellendorffii; 24394_CHLRE); a chlorophyte (Chlamydomonas reinhardtii; 174224_SELMO); and a bryophyte (Physcomitrella patens; EDQ78577.1_PHYPA) The numbers indicated for each node are the bootstrap values (b) Consensus sequences of the aldehyde dehydrogenase cysteine active site domains in BADH ⁄ AMADH homologous proteins The BADH ⁄ AMADH homologues were clustered as monocot, dicot and primitive groups The two subgroups of the monocot class are separated by a line The cysteine active site in each sequence is highlighted with a grey box The distinct amino acids in the two subgroups of monocot homologues are underlined The PROSITE regular pattern of the aldehyde dehydrogenase cysteine active site domain is provided (b) ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 82 Siwaret Arikit et al could be separated into two major groups according to two consensus domain patterns, FANAGQVCSATS and FWTNGQICSATS The former was found only in a monocot subgroup The latter was found in another monocot subgroup as well as in most of the dicot BADH ⁄ AMADHs, from soybean seeds and calli Because the activity staining method used in this study was able to detect both BADH and AMADH activities, this result could suggest that there is no BADH activity in soybean seed and callus extracts This may also imply that both GmAMADH1 and GmA- except for soybean and pea, for which one homologue contained FFTNGQICSATS In the primitive species that have only one BADH ⁄ AMADH protein, the BADH ⁄ AMADHs contained the second type of protein domain MADH2 encode enzymes that only function as AMADHs The lack of BADH activity has also been reported in pea (Pisum sativum) as the pea AMADH did not oxidize betaine aldehyde and elementary aldehydes (Sˇebela et al., (Figure 6b) 2000) On the contrary, in some monocots, such as rice (O sativa), barley (Hordeum vulgare) and oat (A Sativa), the Os2AP (BADH2) orthologues show very low BADH activity on betaine aldehyde, whereas proteins that are Discussion 2AP was detected by HSGC in all varieties of aromatic soybean, confirming that it could be the potent aroma com- orthologous to BADH1 show moderate to high activity towards this substrate (Livingstone et al., 2002, 2003; Bradbury et al., 2008; Takashi et al., 2008) However, both paralogues, BADH1 and BADH2, in those monocots ponent in soybeans as reported previously (Fushimi and Masuda, 2001) Currently, the role of 2AP biosynthesis in plants is unclear; however, 2AP could be formed to detoxify the 4-aminobutanal, which is a reactive amino-carbonyl compound that accumulates when AMADH is inactivated show a broad affinity for a range of amino aldehydes This might support our finding in this study that the BADH1 orthologues, which contain the monocot-specific consensus domain (Figure 6b), have been duplicated from the AMADH ancestor and then evolved the BADH function Loss of AMADH from aromatic soybeans The accumulation of 4-aminobutanal has even been shown in an Escherichia coli AMADH-deficient mutant (Samsonova et al., 2005) According to the enzymatic assay in this study, it is noteworthy that four enzymatic GmAMADH2 is the key gene responsible for 2AP biosynthesis in soybeans activity bands were detected when 4-aminobutanal was utilized in the AMADH enzymatic staining assay using (NH4)2SO4-fractionated extracts from soybean seeds (Figure 1) This could suggest the existence of putative Both GmAMADH1 and GmAMADH2 could be thought of as candidate genes for the aroma trait in soybeans owing to their high similarities to rice Os2AP In this study, we clearly demonstrated that GmAMADH2 is the key gene AMADH isoforms or nonspecific aldehyde dehydrogenases (ALDHs) that can metabolize 4-aminobutanal (Sˇebela et al., 2001) The patterns of the AMADH activity bands in the native PAGE assays for the extracts from young seeds associated with 2AP biosynthesis Both natural aromatic varieties, which contain an inactive form of GmAMADH2, and the GmAMADH2-RNAi knock-down lines lacked AMADH enzymatic activity, which probably resulted in the and calli of soybeans were not similar The reason underlying this phenomenon remains unclear It is possible that some of the AMADH isoforms were not present in calli or that different sets of genes are expressed in calli and seeds Nevertheless, the candidate AMADH enzymatic synthesis of 2AP Previous reports on the genetic control of the aroma trait in vegetable soybean have shown segregation of aromatic and nonaromatic seeds in an F2 population to be : This suggests that a single recessive gene could control the trait (AVRDC, 2003) It is possible band was consistently distinguishable between the aromatic and nonaromatic soybeans in assays of both seed and callus samples that the gene regulating the trait is GmAMADH2 Hence, a functional marker for molecular breeding for aroma in soybean could be designed based on the sequence variation in GmAMADH2 Although GmAMADH2 is considered In this study, we tested the two possible substrates, beta- to be the key player, a role for GmAMADH1 in 2AP biosynthesis could not be ruled out Because GmAMADH1 and GmAMADH2 are highly similar to each other at both the nucleotide and protein levels, and because they are ine aldehyde and 4-aminobutanal, and found that only 4-aminobutanal was utilized by the enzymes extracted almost identical in the conserved domain for NAD-dependent aldehyde dehydrogenases, the similarity in enzymatic GmAMADH1 and GmAMADH2 might have only AMADH and not BADH function ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 Inactivation of GmAMADH2 enhances 2-acetyl-1-pyrroline formation in soybeans 83 function could be expected As multiple AMADH activity bands are shown in Figures and 5, one might think that one of those bands is GmAMADH1 However, inactivation of GmAMADH1 still needs to be performed to verify this prediction In rice, another homologue of Os2AP, BADH1, high similarity of the two BADH ⁄ AMADHs in species of dicots indicates that the two genes might have been independently duplicated in each species after the divergence of the dicot species The difference between the two subgroups of BADH ⁄ AMADH among the monocots revealed has been suggested to also play a role in 2AP biosynthesis (Bradbury et al., 2008), but clear evidence has not yet been reported by the two consensus sequences of the protein domain might explain the differences in the enzymatic function of the two BADH ⁄ AMADH homologues in monocots, as previously reported (Bradbury et al., 2008) According to the GmAMADH2 is independently mutated in soybean, and its sequence variation is not related to those found in rice Os2AP phylogenetic tree in this study, the true orthologues of the two BADH ⁄ AMADH homologues can be distinguished among the monocots Therefore, proteins in the same orthologous group as rice Os2AP might be involved in Although the molecular mechanism that regulates 2AP biosynthesis in soybeans might resemble that in rice, the sequence variation in the CDS of GmAMADH2 that leads to the loss of function in all aromatic soybean varieties is 2AP biosynthesis However, orthology among dicots or comparison across dicots and monocots is not possible Therefore, both homologues of BADH ⁄ AMADH in this gene family need to be analysed in dicots to identify the not related to sequence variations previously reported in rice Os2AP The sequence variations in Os2AP (Kovach et al., 2009) and GmAMADH2 (this study) might have evolved independently, but they appear to have the same effect of inactivation of AMADH In rice, the major allele candidate gene for the aroma In conclusion, aromatic soybeans share a similar mechanism with aromatic rice for 2AP biosynthesis, which involves the inactivation of AMADH Because this molecular mechanism is conserved in distantly evolutionarily of Os2AP in aromatic rice varieties is an 8-bp deletion in exon (Kovach et al., 2009) However, several aromatic rice varieties contain other types of sequence variation in Os2AP that cause the same effect Therefore, it could be related species such as rice and soybean, we suggest that this regulation might also be shared in other plants These data can be used to engineer a metabolic pathway for 2AP production through inactivation of AMADH in other possible that this gene mutates easily This assumption is possibly true in other plants and might explain why 2AP is found in many plants Currently, only one type of sequence variation, the TT deletion, has been identified plants that not accumulate 2AP among the aromatic soybean varieties, as presented in this study However, it is possible that other novel sequence variations could exist BADH ⁄ AMADHs in dicots may have been recently duplicated after the divergence of dicots and monocots As a result of the high similarity in the protein sequences, BADH and AMADH are likely to have a common ancestor Because only one BADH ⁄ AMADH is found in primitive species, the two homologues in flowering plants seem to have evolved through a duplication of the ancestral gene, which might have taken place after the divergence of monocots and dicots Because two orthologous groups can be clearly identified among the monocots, we predict that the two BADH ⁄ AMADHs in each monocot species were duplicated in the monocot common ancestor before the divergence of the monocot species By contrast, the Experimental procedures Plant materials Ten varieties of aromatic and nonaromatic vegetable soybean (G max L.) were used in this experiment The five aromatic soybean varieties (Chamame, Kouri, Kaori hime, Yuagari musume and Fukunari) and the three nonaromatic soybean varieties (Okuhara Wase, Oishi Edamame and Shirono Mai) were collected from a market in Japan Another nonaromatic soybean, Jack, was provided by Professor Randall L Nelson, USDA, Agricultural Research Service, IL, USA The nonaromatic variety, Chiang Mai 60 (CM60), was provided by the Chiang Mai Field Crop Research Center, Department of Agriculture, Thailand Mature seeds of each variety were grown in pots under open-air conditions at Kasetsart University, Nakhon Pathom, Thailand Fresh pods from each plant were harvested between October and November 2008 2AP analysis in soybean seeds Automated HSGC using an Agilent Technologies (Wilmington, DE, USA) model 6890N gas chromatograph, an Agilent Technologies model G1888 headspace autosampler and a nitrogen–phosphorus ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 84 Siwaret Arikit et al detector (NPD) was performed for headspace volatile extraction and quantitative analysis of 2AP in the soybean samples The HSGC conditions were the same as those reported by Sriseadka et al., 2006 with some modifications Chromatographic separation was performed on a fused silica capillary column, phase HP-5MS (60 m · 0.32 mm i.d · 1.0 lm) (Agilent Technologies) Purified helium gas at a flow rate of mL ⁄ was used as the GC carrier gas 2AP contents in soybean samples were determined using an internal standardization method with 2,4-dimethylpyridine (2,4DMP) as the internal standard Identification of 2AP in the soybean headspace was accomplished using an HP 5973 mass-selective detector (Agilent Technologies, Palo Alto, CA, USA) equipped with the Wiley 7N Mass Spectral Library and the NIST 05 Mass Spectral Library, both purchased from Agilent Technologies Additionally, structural confirmation by mass spectral comparison with 2AP was performed The mass spectrometer was operated in the electron impact mode with an electron energy of 70 eV, ion source temperature of 230 °C, quadrupole temperature of 150 °C, mass range m ⁄ z of 29–550, scan rate of 0.68 s ⁄ scan and EM voltage of 1423 V The GC–MS transfer line was set to 280 °C The system operation, as well as data acquisition, collection and evaluation, was accomplished using Agilent ChemStation software version A.01.04 and B.01.03 (Agilent Technologies, Waldbronn, Germany) Reverse transcriptase polymerase chain reaction Total RNA was isolated from immature seeds, leaves and calli of the ten varieties using an RNeasy Plant mini kit (Qiagen, Valencia, CA, USA) The DNAase-treated RNA was reverse-transcribed and PCR-amplified using Titan One Tube RT-PCR kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions The reverse transcription and PCR thermal cycle conditions were performed as follows: reverse transcription at 50 °C for 30 min, then PCR with initial heating at 95 °C for followed by 27–37 cycles of 95 °C, 30 s; 55 °C, 30 s; 68 °C, and a final extension at 68 °C for The number of cycles was adjusted to avoid over-cycling, and all RT-PCR assays were carried out in triplicate GUS linker was used to determine the levels of transcription from the RNAi construct, and Lectin was used as control to determine the mRNA amount The primers for GmAMADH1 were 5¢-TGAAGCTGGTGCTCCTTTGT-3¢ and 5¢AAGATGGTCCATTCAGCAGT-3¢ The primers for GmAMADH2 were 5¢-TGAAGCGGGTGCTCCTTTAG-3¢ and 5¢-AATATGGTCCATTCAGCAGC-3¢ The primers for GUS linker were 5¢-CATGAAGATGCGGACTTACG-3¢ and 5¢-ATCCACGCCGTATTCGG-3¢ The primers for Lectin were 5¢-TCAACGAAAACGAGTCTGGTG-3¢ and 5¢-GGTGGAGGCATCATAGGTAAT-3¢ Polymerase chain reaction (PCR) Crude and fractionated extract preparation All extraction procedures were performed at °C Young soybean seeds and callus tissue were ground into fine powder, and then crude enzymes were extracted with cold extraction buffer (100 mM KPi (pH 7.5), 10% (v ⁄ v) glycerol, 2% (w ⁄ v) PVPP, mM EDTA, mM NAD and mM DTT, at a ratio of 400 mg fresh weight per mL) The homogenate was centrifuged at 20 800 g for 15 The supernatant was fractionated with solid (NH4)2SO4, and the fraction containing 55%–75% saturation was collected The fraction was resuspended in 500 lL of 100 mM KPi (pH 7.5) buffer and desalted through a NAP-5 column (GE Healthcare Biosciences, Uppsala, Sweden) A solution of mM NAD, mM EDTA and mM DTT was added to obtain desalted solutions The crude extracts were assayed immediately AMADH activity gel staining assay A nondenaturing polyacrylamide mini-gel system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used in this study to assay the AMADH activity Total enzyme extracts (20 lg in 16 lL) were mixed with lL of gel-loading buffer containing 50% (v ⁄ v) glycerol and 0.05% (w ⁄ v) bromphenol blue The samples were then separated on 10% PAGE gels, Tris–HCl, pH 8.8 The electrode buffer contained 25 mM Tris–HCl and 192 mM glycine Separation was performed with constant current at 20 mA ⁄ gel at °C Staining was performed at 37 °C in a solution containing 100 mM glycine–NaOH buffer (pH 9.5), mM NAD+, mM 4-aminobutanal or betaine aldehyde, mM (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 0.15 mM 1-methoxy-phenazine methosulphate until the bands were visible The reaction was terminated by adding 10% acetic acid and then distilled water Genomic DNA was extracted from young leaves using the DNeasy Plant mini kit (Qiagen) PCR was performed in 25 lL reaction mixtures containing 50 ng of genomic DNA template, 0.1 mM of dNTPs, 0.25 mM of each forward and reverse primer, 0.25 units of Taq DNA polymerase, 2.0 mM MgCl2 and 1· Thermophilic DNA Polymerase buffer (Promega, Madison, WI, USA) After being preheated at 94 °C for min, the PCR was carried out for 30 cycles under the following conditions: 94 °C denaturation for 30 s, 55 °C annealing for 30 s and a 72 °C extension for min, with a final extension at 72 °C for Primers used for PCR are listed in Supplementary Table I DNA sequencing and sequence assembly The amplified PCR fragments were purified and cloned into the pGEM-T Easy Vector (Promega) The templates were sequenced in both directions with an automatic sequencer using the ABI PRISMÔ Big DyeÔ Terminator Cycle (Applied Biosystem ⁄ PerkinElmer, San Jose, CA, USA) Sequences were assembled and viewed using the phred ⁄ phrap ⁄ consed software (http:// www.phrap.org) Construction of the RNAi vector The GmAMADH2 fragment was amplified from the genomic DNA of the Jack variety by PCR using the forward primer 5¢-CACCATGAGCATCCCAATTCCCCA-3¢ and the reverse primer 5¢-TTCGAGTTTTGCTAGTTCAGG-3¢ The amplified PCR fragment was cloned into the Gateway pENTR ⁄ D-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA), which carries two recombination sites (attL1 and attL2) for the LR Clonase reaction Subsequently, the target ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 Inactivation of GmAMADH2 enhances 2-acetyl-1-pyrroline formation in soybeans 85 fragment was transferred into the pANDA destination vector (Miki and Shimamoto, 2004) by recombinase reactions The resulting pANGmAMADH2 RNAi vector comprised a 35S promoter, 441-bp sense and 441-bp antisense GmAMADH2 fragments that were interrupted by a 1-kb GUS linker fragment (Figure 4a) Callus induction and Agrobacterium transformation Soybean seeds were disinfected with 0.1% HgCl2 and a drop of Tween-20 for 6–7 min, followed by 3–5 washes in distilled water The sterilized seeds were placed on callus induction medium (CC) (Potrykus et al., 1979) with some modifications and allowed to induce calli for month at 25 °C under dark conditions Induced calli were excised from explant ⁄ callus complexes and proliferated in liquid Finer and Nagasawa Lite (FNL) medium (FNL macrosalts, MS micro salts, B5 vitamins, 1% sucrose, g ⁄ L asparagine and mg ⁄ L 2,4-D, pH 5.8), where FNL macrosalts consist of 2830 mg ⁄ L KNO3, 463 mg ⁄ L · (NH4)2SO4, 370 mg ⁄ L MgSO4Ỉ7H2O, 185 mg ⁄ L KH2PO4 and 300 mg ⁄ L CaCl2Ỉ2H2O (Samoylov et al., 1998) Flasks were placed on a shaker at 125 rpm under dim light (5–10 lE ⁄ m2 ⁄ s) at 25 °C The medium was replaced at weekly intervals by pipetting the spent medium out of the flask and replacing it with fresh medium pANGmAMADH2 was mobilized into the Agrobacterium tumefaciens strain AGL1 by electroporation Agrobacterium cultures harbouring the RNAi vector were grown on a plate of YEB medium containing 50 mg ⁄ L kanamycin and 50 mg ⁄ L hygromycin B at 28 °C until colony formation Then, 50 mL of liquid YEB medium containing 50 mg ⁄ L Kanamycin and 50 mg ⁄ L hygromycin B was inoculated with a single colony and shaken at 28 °C and 180 rpm to an OD650 of 0.6–0.8 Agrobacterium cultures were pelleted by centrifugation at 1160 g for 10 and resuspended in liquid cocultivation medium (FNL medium) to OD650 of 0.2 for the inoculations The wounded calli were soaked in the Agrobacterium inoculum suspension for 30 The inoculated calli were randomly placed on co-cultivation medium (MSD20-ASG medium), which consisted of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962), Gamborg’s B5 vitamins (Gamborg et al., 1968) 3% sucrose, 20 mg of l-1 2,4-D, 200 lM acetosyringone and 0.3% Phytagel (pH 5.8) Plates were incubated in the dark for days at 25 °C Following co-cultivation, transgenic calli were selected in selection medium (FNL, 30 mg ⁄ L hygromycin B and 300 mg ⁄ L Cefotaxime) The medium was replaced every week for six additional weeks, and then transgenic calli were placed on solid selective medium (CC, 30 mg ⁄ L hygromycin B) 2AP analysis in soybean callus Portions of callus samples (0.1 g) were homogenized (Ika UltraTurrax T8 homogeniser, Staufen, Germany) with 0.5 mL of ethanol containing 200 ppb of 2-acetyl-(13C-methyl)-1-pyrroline (Yoshihashi et al., 2002) They were extracted at room temperature for h After centrifugation, lL of supernatant was injected onto a DB-Wax extr, 60 m · 0.25 mm i.d · 0.25 lm thickness fused silica capillary column (Agilent Technologies, Wilmington, DE, USA) installed in a Shimadzu GCMS-QP2010 GCMS system (Kyoto, Japan) with helium at a carrier velocity of 41.2 cm ⁄ s The injector and interface temperatures were set at 150 and 250 °C, respectively The oven programme was as follows: column temperature was isothermally maintained at 40 °C for min, programmed first at a rate of 10 °C ⁄ to 100 °C, then °C ⁄ to 140 °C and then 20 °C ⁄ to 250 °C; the column temperature was maintained isothermally at 250 °C for 10 The mass spectrometer was used in the electron ionization mode with the ion source temperature set at 250 °C, the analyser temperature set at 100 °C and ionization energy at 70 eV Single ion monitoring was set up to monitor m ⁄ z 111 for 2AP and m ⁄ z 112 for carbon-13 labelled 2AP Under these conditions, the retention times of 2AP and carbon-13 labelled 2AP were found to be 12.46 Quantification was performed by measuring the area ratios between ions at m ⁄ z 111 and 112, corresponding to 2AP and carbon-13 labelled 2AP Each extract was analysed three times to obtain an average peak area RNA gel blot analysis Total RNA was isolated from callus tissue of the varieties Jack, CM60, the RNAi-transformed Jack (Jack-RNAi) and the RNAi-transformed CM60 (CM60-RNAi), using TRIZOLÒ (Invitrogen) The RNA samples were visualized with 1% agarose gel electrophoresis and quantified by spectrophotometry (OD260) The total RNA samples were dissolved in one volume of 100% formamide and mixed well Twenty microgram of total RNA was resolved on a PAGE gel (15% polyacrylamide gel containing M of urea) and then transferred onto a Hybond-NX membrane (GE Healthcare Biosciences, Uppsala, Sweden) by electro-blotting The hybridization was performed with a DIG-labelled DNA probe complementary to the CDS sequence of GmAMADH2 from exon to exon The detection of signals was performed as previously described (Goto et al., 2003) Phylogenetic and protein domain analyses A set of BADH ⁄ AMADH homologous proteins in eleven higher plant (Angiosperms) genomes (P sativum, Zoysia tenuifolia, O sativa, Zea mays, Sorghum bicolor, Arabidopsis thaliana, Populus trichocarpa, G max, Amaranthus hypochondriacus, Atriplex hortensis and H vulgare), a lycophyte (Selaginella moellendorffii), a chlorophyte genome (Chlamydomonas reinhardtii) and a bryophyte genome (Physcomitrella patens) was obtained using the rice Os2AP protein sequence as query to perform BLASTP searches against the NCBI and Phytozome databases Multiple sequence alignments and phylogenetic trees were analysed using a robust phylogenetic tree analysis web service (http://www.phylogeny.fr/ version2_cgi/index.cgi) with the ‘One click’ mode This mode contained multiple sequences aligned using the MUSCLE program, the aligned sequences were curated using GBLOCK, the phylogeny was identified using PhyML and trees were rendered using TreeDyn (Dereeper et al., 2008) The protein domains were predicted by the PROSITE program (http://www.expasy.org/prosite/) with the default parameters Acknowledgements We gratefully acknowledge the financial support of BIOTEC and Royal Golden Jubilee (RGJ)-PhD program ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87 86 Siwaret Arikit et al Grant No 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Merr roots Plant Physiol Biochem 45, 560–566 Yoshihashi, T (2002) Quantitative analysis on 2-acetyl-1-pyrroline of an aromatic rice by stable isotope dilution method and model studies on its formation during cooking J Food Sci 67, 619–622 Yoshihashi, T., Huong, N.T.T and Inatomi, H (2002) Precursors of 2-acetyl-1-pyrroline, a potent flavor compound of an aromatic rice variety J Agric Food Chem 50, 2001–2004 Supporting information Additional Supporting information may be found in the online version of this article: Figure S1 Substrate-specific test with 4-aminobutanal and betaine aldehyde of enzyme extracts from soybean and rice calli The positive control is the non-aromatic rice c.v Nipponbare Chamame and Kaori hime represent aromatic varieties and CM60 represents a non-aromatic variety of soybean Table S1 List of PCR primers used for amplification of GmAMADH2 Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article ª 2010 The Authors Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 75–87