Influence of PTGS on Chalcone Synthase Gene Family in Yellow Soybean Seed Coat

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 yellow 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.

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