Breeding Science 54 : 355-360 (2004) Influence of PTGS on Chalcone Synthase Gene Family in Yellow Soybean Seed Coat

Atsushi Kasai1), Mutsumi Watarai2), Setsuzo Yumoto3,4), Shinji Akada1), Ryuji Ishikawa2), Takeo Harada2), Minoru Niizeki2) and Mineo Senda*1)

1) Gene Research Center, Hirosaki University, 3 Bunkyo, Hirosaki, Aomori 036-8561, Japan 2) Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo, Hirosaki, Aomori 036-8561, Japan 3) Tokachi Agricultural Experiment Station, S9-2, Shinsei, Memuro, Kasai, Hokkaido 082-0071, Japan 4) Present address: National Agricultural Research Center for Tohoku Region, Kariwano, Nishi-senboku, Akita 019-2112, Japan

Most commercial soybean varieties have yellow seeds duced in the pigmented seed coats of soybeans carrying the i due to loss of pigmentation in the seed coat. We previ- allele (Wang et al. 1994, Todd and Vodkin 1996, Senda et al. ously showed that inhibition of seed coat pigmentation 2002b). Consequently, CHS activity in the non-pigmented in yellow soybeans is controlled by post-transcriptional seed coat with the I allele is significantly lower than that in gene silencing (PTGS) of chalcone synthase (CHS) the pigmented seed coat with the i allele (Wang et al. 1994). genes. Soybean CHS genes are composed of at least Since CHS is an important enzyme in the biosynthesis of eight members, CHS1–CHS8. In this study, we com- anthocyanin and proanthocyanidin pigments, the reduction of pared the steady state mRNA level of each CHS mem- CHS mRNA by the I allele is likely to be the basis for the in- ber in the seed coat between a yellow soybean cultivar hibition of seed coat pigmentation (Wang et al. 1994, Todd displaying CHS PTGS and its pigmented seed coat mu- and Vodkin 1996, Senda et al. 2002a, 2002b). A number of tant in which the CHS PTGS is abrogated. Northern hy- spontaneous mutants with alteration from the I allele to the i bridization analysis suggested that in the pigmented allele have been isolated in yellow soybeans, and seed coats seed coat, the transcripts of CHS7 and CHS8 were of these mutants (i/i genotype) are fully pigmented (Todd abundant among the total CHS transcripts, whereas and Vodkin 1996, Senda et al. 2004). those of other CHS members (CHS1–CHS6) were less In the soybean genome, CHS is encoded by a multigene abundant. Reverse transcription and subsequent real family composed of at least eight members, CHS1 to CHS8 time PCRs using member-specific primers revealed re- (Akada and Dube 1995, DDBJ/Genbank/EMBL accession duction of the amount of mRNA of most CHS members number AY237728), and CHS mRNA is derived from in the non-pigmented seed coat of the yellow soybean as mRNAs of these CHS members (Senda et al. 2002b). Recent- compared to the pigmented seed coat of the mutant, in- ly, it was shown that inhibition of seed coat pigmentation in dicating the influence of PTGS on the CHS gene family. yellow soybeans is controlled by post-transcriptional gene silencing (PTGS) of CHS genes, and that the steady state level Key Words: yellow soybean, non-pigmented seed coat, of CHS mRNA is increased significantly in the pigmented chalcone synthase, gene family, CHS mem- seed coat of the mutant (i/i) due to abrogation of CHS PTGS ber, post-transcriptional gene silencing. (Senda et al. 2004). However, it remained unknown which CHS member-derived transcript(s) exist in the pigmented seed coat of the mutant, and to what extent PTGS influences each CHS member in the non-pigmented seed coat of the Introduction yellow soybean. In this study, we investigated the influence of CHS PTGS on each CHS member in the non-pigmented Yellow soybeans have non-pigmented seed coats. Seed seed coat of a yellow soybean cultivar (cv. Toyohomare) by coat pigmentation in soybeans is determined by the I (in- comparing the amount of mRNA with that in a pigmented hibitor) locus. The dominant I allele inhibits pigmentation seed coat mutant in which CHS PTGS is abrogated. throughout the entire seed coat, resulting in uniformly yel- low colored mature harvested seeds, whereas the recessive i Materials and Methods allele leads to a completely pigmented seed coat. It has been shown that the steady state level of mRNA of the chalcone Plant materials synthase (CHS) gene is specifically reduced in the non- Japanese soybean cultivar Toyohomare has yellow pigmented seed coat with the I allele, whereas it is not re- seeds determined by a dominant allele of the I locus, and a pigmented seed coat mutant of Toyohomare with mutation Communicated by Y. Takahata of I to i was isolated spontaneously as a pigmented seed. Received March 25, 2004. Accepted June 21, 2004. Toyohomare (I/I) and its mutant line (i/i) are abbreviated *Corresponding author (e-mail: [email protected]) here as TH and THM, respectively. TH and THM were 356 Kasai, Watarai, Yumoto, Akada, Ishikawa, Harada, Niizeki and Senda provided by the Tokachi Agricultural Experimental Station, Results Japan. It has been already shown that CHS PTGS occurs in the non-pigmented seed coat of TH, but not in the pigmented Reduction of the steady state CHS mRNA level in the seed seed coat of THM (Senda et al. 2004). coat of yellow soybean already occurs in the very early stage of seed development RNA extraction from seed coats It was shown previously that post-transcriptional gene Seed coat RNAs were extracted essentially according silencing (PTGS) of CHS genes is involved in the inhibition to the protocol of Wang and Vodkin (1994). of seed coat pigmentation in yellow soybeans (Senda et al. 2004). The soybean cultivar Toyohomare (TH) has yellow PCR amplification and sequencing seeds because of the loss of seed coat pigmentation due to Soybean genomic DNA was extracted from young CHS PTGS, whereas a spontaneous mutant line of TH with leaves according to the protocol of Ausubel et al. (1987). a change from I to i at the I locus (THM) (i/i genotype) has PCR amplification with AmpliTaq Gold DNA polymerase pigmented seeds due to the abrogation of CHS PTGS (Senda (Applied Biosystems, USA) was conducted according to the et al. 2004). In order to investigate when CHS PTGS occurs manufacturer’s recommendations. DNA sequences of PCR- in the seed development of TH, comparative northern hy- amplified fragments were determined using a BigDye bridization analysis between TH and THM using a CHS Terminator Cycle Sequencing Kit and an ABI PRISM 310 probe was carried out with RNAs isolated from seed coats at Genetic Analyzer (Applied Biosystems, USA). various stages of seed development. Since the CHS probe used is a mixture of portions of exon 2 from all CHS mem- Southern and northern hybridization analyses bers (described as a non-specific CHS probe in Senda et al. Southern and northern hybridization analyses, includ- 2002b), the hybridized band reflects the total CHS tran- ing the preparation of the soybean CHS probe, were per- scripts. As shown in Figure 1, the steady state level of CHS formed as described previously (Senda et al. 2002b). transcripts was reduced in TH compared to THM in seeds with less than 25-mg to at least 600-mg fresh weight, sug- Reverse transcription and quantitative real time PCR gesting that CHS PTGS already occurs in seeds at a very ear- Residual genomic DNA was eliminated by treating ly stage and is maintained throughout seed development. For seed coat RNA with RNase-free DNase I (Promega, USA) northern hybridization and RT-PCR analyses presented be- according to the manufacturer’s recommendations. Reverse low, we used RNAs isolated from seed coats in the earliest transcription (RT) of DNase-treated seed coat RNA was stage (less than 25-mg fresh weight, Fig. 1) when CHS PTGS carried out using the SuperScript III First Strand Synthesis already occurs in TH. It should be noted that as reported by System (Invitrogen Corp., USA), and with the resultant Senda et al. (2004), even though CHS PTGS occurs, a small cDNA as a template, quantitative real time PCR was per- amount of CHS mRNA remains in the non-pigmented seed formed using a SYBR Green qPCR Kit (Finnzymes, coat of TH (Fig. 1). Finland) with a DNA Engine Opticon 2 continuous fluores- cence detection system (MJ Research Inc., USA). PCR prod- Transcripts of CHS7 and CHS8 constitute most of the CHS uct melting curves confirmed the specificity of single-target transcripts in the seed coat of the mutant amplification, and fold expression of each CHS member in Soybean CHS genes consist of at least eight members, the non-pigmented seed coat of the yellow soybean relative CHS1 to CHS8 (Akada and Dube 1995, DDBJ/Genbank/ to that in the pigmented seed coat of the mutant was deter- EMBL accession number AY237728). Specific fragments of mined in triplicate. The soybean actin gene (SAc3, Shah et each CHS member from CHS1 to CHS8 were amplified by al. 1982) was used as a control for the total RNA transcripts. member-specific forward primers designed based on the se- The sequence of the reverse primer used for RT from seed quences of CHS1–CHS7, as described previously (Shimizu coat RNA was 5′-GAGGTGGTGAACATGTATCC-3′, for et al. 1999). For CHS8, the specific forward primer was which the annealing site is located in exon 3 of SAc3. The se- 5′-GCACACCTTCATTTCAACCTC-3′. All eight forward quences of the forward and reverse primers used for real- primers were derived from the 5′-untranslated region (UTR) time PCR of SAc3 were 5′-CGACAATGGAACTGGAATG of individual CHS members. Only the CHS3-specific for- G-3′, which is located in exon 1, and 5′-ATGTCATCCCAG ward primer was difficult to be designed in the 5′-UTR, and TTGCTGAC-3′, which is located in the complementary se- a common forward primer of CHS3 and CHS4 (abbreviated quence of exon 2, respectively. Quantified mRNA levels of as CHS3/4) was used to quantify both CHS3 and CHS4 tran- CHS members were divided by those of SAc3 for normaliza- scripts (Shimizu et al. 1999). The sequence of the reverse tion. primer used for amplification of each fragment from CHS1– CHS6 was 5′-CAGGATAGGTACTCTGATC-3′, as de- scribed in Shimizu et al. (1999). The sequences of the re- verse primers used for CHS7 and CHS8 were 5′-CAGGATA GGTGCTCTGATC-3′ and 5′-CAGGATAGGTGCTCTGA GC-3′, respectively (the nucleotide differences compared PTGS influence on CHS gene family in yellow soybean seed coat 357

Fig. 1. Northern hybridization analysis of the seed coat RNAs isolated from different stages of seed development in TH and THM. Total RNAs were electrophoresed on formaldehyde gels, blotted onto nylon membranes, and hybridized with the CHS probe. Equal amounts of total seed coat RNA were analyzed, as indicated by the equal loading of 18S rRNA. with the sequence of the reverse primer for CHS1–CHS6 are CHS7–CHS8 groups, respectively. RNA gel blot compari- underlined). These reverse primers were derived from com- son between TH and THM using each probe was carried out. plementary sequences of a region conserved in exon 1 of all In THM, the hybridization signal was weak with the CHS1 CHS members. Using these forward and reverse primers, probe, while an intense band was detected with the CHS7 member-specific fragments were amplified from the geno- probe, indicating that transcripts of CHS7 and CHS8 consti- mic DNA of TH by PCR. Each fragment was electrophoresed tute most of the CHS transcripts in the pigmented seed coat in a 2 % agarose gel and blotted onto a nylon membrane. The of the mutant in which CHS PTGS doesn’t occur (Fig. 2C). nucleotide sequences of the nine product fragments, all with In TH, compared to THM, the signal intensities were sig- different sizes (164 bp–235 bp), were determined and the nificantly reduced using either the CHS1 or the CHS7 probe, percentage of nucleotide identity between any two of the suggesting that CHS PTGS occurs in both the CHS1–CHS6 nine fragments from the 3′-end to the 5′-upstream sequence and CHS7–CHS8 groups (Fig. 2C). With respect to the de- was calculated simply with no gaps, leading to classification gree of reduction of the amounts of mRNAs in the TH seed of these genes into two major groups, CHS1–CHS6 and coat, the reduction of the CHS7–CHS8 group was signifi- CHS7–CHS8 (Table 1). When the sequence comparison was cantly stronger than that of the CHS1–CHS6 group. limited to the coding region, the difference between the two groups was more prominent (Fig. 2A). The amplified frag- Influence of PTGS on each CHS member in the yellow soy- ment of CHS1 when used as a probe hybridized to the frag- bean seed coat ment of each member in the CHS1–CHS6 group, but did not Reverse transcription (RT) of the seed coat RNA iso- hybridize to the CHS7–CHS8 group, supporting these re- lated from each of TH and THM, and quantitative real-time sults (Fig. 2B). Conversely, the amplified fragment of CHS7 PCR of the reverse transcription products, were performed when used as a probe hybridized to the fragment of each to compare the amount of the mRNA from each CHS mem- member in the CHS7–CHS8 group, but did not hybridize to ber in TH versus THM. The sequence of the reverse primer the CHS1–CHS6 group (Fig. 2B). Thus, the CHS1 and used for RT from seed coat RNA was 5′-CC(A/G)GGAACA CHS7 probes specifically detected the CHS1–CHS6 and TCCTTGAGGAG-3′. This reverse primer was designed

Table 1. Sizes and percent nucleotide identity of CHS member-specific PCR fragments CHS3 4 CHS3 4 CHS1 CHS2 / / CHS4 CHS5 CHS6 CHS7 CHS8 (CHS3)1) (CHS4)1) CHS1 196 bp 75.6 % 74.3 %74.2 %83.5 %71.4 % 66.3 %57.7 %56.6 % CHS2 172 bp 77.9 %78.5 %79.3 %76.7 % 70.3 %59.3 %61.0 % CHS3/4 (CHS3)1) 187 bp 80.2 %88.4 %68.4 % 66.3 %57.2 %57.2 % CHS3/4 (CHS4)1) 194 bp 100 %71.6 % 65.5 %53.6 %49.5 % CHS4 164 bp 77.4 % 72.0 %57.9 %56.7 % CHS5 235 bp 67.2 %50.2 %55.1 % CHS6 232 bp 54.1 %53.2 % CHS7 207 bp 63.3 % CHS8 216 bp 1) Using a common forward primer of CHS3 and CHS4 (abbreviated as CHS3/4), two fragments derived from CHS3 and CHS4 are amplified. CHS3/4 (CHS3) and CHS3/4 (CHS4) indicate CHS3- and CHS4-derived PCR fragments, respectively. 358 Kasai, Watarai, Yumoto, Akada, Ishikawa, Harada, Niizeki and Senda

Fig. 2. (A) Sequence alignment of nucleotide sequences of the coding regions included in the member-specific amplified fragments. Identical nucleotides are shown white on a black background. A line under the sequences indicates the annealing site of the reverse primer used for PCR in Figure 2B. The start codon is double-underlined. (B) Amplification of the member-specific fragments shown in an EtBr-stained agarose gel. In the case of CHS3/4, although two fragments derived from CHS3 and CHS4 are amplified, these were not separated each other in an agarose gel and therefore these were detected as a single band. Each number corresponds to that of a CHS member. These fragments were blotted onto nylon membranes, and hybridized with the CHS1 or CHS7 probe. (C) Northern hybridization analysis of the seed coat RNAs in TH and THM. Total RNAs were electrophoresed on formaldehyde gels, blotted to nylon membranes, and hybridized with the CHS1 or CHS7 probe. The probe specific to the gene encoding another enzyme, dihydroflavonol reductase (DFR) of the antho- cyanin biosynthetic pathway was also used. Equal amounts of total seed coat RNA were analyzed, as indicated by the equal loading of 18S rRNA. based on the consensus sequence derived from a conserved control for mRNA quantification, the soybean actin gene region in exon 2 of all CHS members. For the quantitative (SAc3) was used, and the quantified mRNA level of each real-time PCRs for each CHS member, the same set of prim- CHS member was normalized by that of SAc3. The influence ers as described above was used except in the case of CHS8, of PTGS on each CHS member was determined by the ratio because analysis of the RT-PCR product melting curves and of the normalized quantified mRNA level in TH to that in agarose gel electrophoresis of the RT-PCR products for THM. The results are summarized in Table 2. For both CHS5 CHS8 showed that there was an additional minor-fragment and CHS6, RT-PCR products were not obtained even in that interfered with the quantification. We therefore THM, and even using another primer set designed based on searched for another primer set for mRNA quantification of a searched sequence with Primer3 for PCR subsequent to CHS8 using Primer3 (http://frodo.wi.mit.edu/cgi-bin/ RT, clear amplified fragments with the expected sizes were primer3/primer3_www.cgi), and based on that search only not detected (data not shown), indicating that few or no tran- the reverse primer was re-designed as 5′-ATGTGCTCACT scripts are derived from CHS5 and CHS6 regardless of GTTGGTGATTC-3′, for which the annealing site was 13- whether or not CHS PTGS occurs. For CHS2, CHS3/4 and bp downstream from that of the former reverse primer. As a CHS4, the amount of mRNA in TH was about one-third of

Table 2. Relative fold expression of each CHS member in TH as compared to THM CHS1 CHS2 CHS3/4 CHS4 CHS5 CHS6 CHS7 CHS8 2.1 ± 0.291) 0.36 ± 0.051) 0.31 ± 0.021) 0.30 ± 0.081) N.D.2) N.D.2) 0.14 ± 0.051) 0.09 ± 0.021) 1) Value was calculated by dividing the quantified mRNA level in TH by that in THM. Results are the means ± SD of at least three inde- pendent experiments. 2) N.D.: not determined. PTGS influence on CHS gene family in yellow soybean seed coat 359 that in THM, while in CHS7 and CHS8, there was a consid- (CHS7 and CHS8). Taken together with the data that in erable decrease (about 10-fold) in TH. Only CHS1 showed THM, the transcripts of CHS7 and CHS8 were abundant the opposite effect, namely an increase (about 2-fold) in the among the total CHS transcripts and those of other CHS amount of mRNA in the TH seed coat. These data strongly members were less abundant, it was suggested that in each suggest that CHS PTGS influences most CHS members in CHS member, the smaller the amount of mRNA exists in the non-pigmented seed coat of yellow soybeans. THM, the less the influence of CHS PTGS in TH is. How- ever, for CHS1, the result was contrary to our expectations; Discussion the amount of the mRNA was 2-fold higher in the non- pigmented seed coat of the yellow soybean despite the CHS Plants possess an antiviral defense mechanism that tar- PTGS. As described above, a small amount of CHS mRNA gets viral RNAs for degradation in a sequence-specific man- remains even in yellow soybeans because of the incomplete ner (Vance and Vaucheret 2001, Voinnet 2001, Waterhouse degradation by CHS PTGS. One possibility is that the et al. 2001). Expression of plant transgenes can also be af- amount of CHS1 mRNA was too small to be influenced by fected by a defense mechanism whereby transgene mRNA, CHS PTGS in the non-pigmented seed coat of the yellow and in some cases homologous endogenous mRNA, is de- soybean. In fact, RT-PCR analysis indicated that the level of stroyed post-transcriptionally (Vaucheret et al. 1998). This CHS1 mRNA was very low in the non-pigmented seed coat phenomenon is called post-transcriptional gene silencing of the yellow soybean (Senda et al. 2002a). In the genome of (PTGS). Recently, it was reported that naturally occurring yellow soybeans with the I/I genotype, an extra CHS1 exists CHS PTGS is involved in the inhibition of soybean seed coat and in this CHS1 region, different sequence deletions are pigmentation (Senda et al. 2004). This CHS PTGS is in- present specific to seed coat pigmented mutants whose duced by the I allele in the genome of yellow soybeans, not genotype was changed from I to i (Todd and Vodkin 1996, by viruses, and does not result in the complete degradation Senda et al. 2002a, 2002b). We previously isolated this of CHS mRNA; instead, a small amount of CHS mRNA re- extra CHS1 and designated it as ICHS1 to distinguish it from mains in the non-pigmented seed coat of yellow soybeans. A another CHS1 (Senda et al. 2002a). In THM, as well as some reasonable explanation for these findings is that a certain seed coat pigmented mutants with the i/i genotype, at least threshold of CHS activity is required to make the seeds pig- the promoter region of ICHS1 was deleted (Todd and mented and below this threshold, the seeds are not pigment- Vodkin 1996, Senda et al. 2002a, 2002b, 2004). If the amount ed even though CHS mRNA is present (Senda et al. 2004). of mRNA derived from a CHS1 member is not influenced by The soybean CHS gene family is composed of at least eight CHS PTGS in the TH seed coat, it is decreased in THM as members and CHS mRNA consists of the mRNAs of these compared to that in TH, because of ICHS1 deletion. This CHS members. The influence of CHS PTGS on each CHS agrees well with the result shown in Table 2 (obtained by the member in the yellow soybean seed coat has remained un- relative quantification of the amount of CHS1 mRNA) that known. In this study, first, we showed that CHS7 and CHS8 the level of CHS1 mRNA is higher in TH than in THM. transcripts were abundant among the total CHS transcripts in Seed color is one of the most important characters in the pigmented seed coat of the mutant with the i/i genotype, soybean breeding because it determines the high quality of in which CHS PTGS was abrogated. In the promoter regions yellow soybeans. Undesirable pigmentation on the non- of many CHS genes, conserved regions known as the H-box pigmented seed coats of yellow soybeans such as (1) the pig- and G-box have been found (Oberholzer et al. 2000). In mented mutation of the I allele described above, (2) mottling Phaseolus, the H-box and G-box were both essential for ex- by certain plant viruses (Bernard and Weiss 1973) and (3) pression in the pigmented flower parts (Faktor et al. 1996). the pigmentation induced by low temperatures (Takahashi A remarkable feature is that in the soybean CHS members 1997), affects the seed quality. Recently, mottling formation analyzed so far, all CHS members except CHS2 have a com- is related with suppression of CHS PTGS by a viral silencing plete H-box (CCTACC), and only CHS7 and CHS8 possess suppressor protein (Senda et al. 2004). Although it remains a complete G-box (CACGTG) as well as an H-box (Shimizu to be investigated whether the low-temperature-induced pig- et al. 1999, DDBJ/Genbank/EMBL accession number mentation is related to the suppression of CHS PTGS, the I AY237728). It is unknown whether a set of an H-box and allele has been suggested to be associated with this pigmen- G-box is required for expression in the pigmented seed coat tation (Takahashi 1997). The results of our study showed of soybean. Further investigations are needed to elucidate that CHS7 and CHS8 play major roles in determining the the relationships between the transcription of CHS members seed coat pigmentation. If the expression of these two mem- and their promoter sequences. bers can be inhibited completely in the seed coat regardless Second, RT and quantitative real-time PCR analyses of whether or not the mutation of the I allele or suppression revealed that in the non-pigmented seed coat of the yellow of CHS PTGS occurs, it might prevent undesirable pigmenta- soybean, CHS PTGS influenced the most CHS members tion on the non-pigmented seed coats of yellow soybeans, whose transcripts existed in the pigmented seed coat of the leading to the maintenance of high quality in seed production. mutant. Three members (CHS2, CHS3/4 and CHS4) showed less reduction in the amount of mRNA than two members 360 Kasai, Watarai, Yumoto, Akada, Ishikawa, Harada, Niizeki and Senda

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