Sundaramoorthy Jagadeesh, Park Gyu Tae, Chang Jeong Ho, Lee Jeong-Dong, Kim Jeong Hoe, Seo Hak Soo, Chung Gyuhwa, Song Jong Tae
School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea.
Department of Biology Education, Kyungpook National University, Daegu, Republic of Korea.
PLoS One. 2016 Jul 21;11(7):e0159865. doi: 10.1371/journal.pone.0159865. eCollection 2016.
In soybean, flavonoid 3'5'-hydroxylase (F3'5'H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and two EMS-induced mutants of Glycine max (PE1837 and PE636). F3'5'H expression in CW12700, PE1837, and PE636 was normal, whereas that in CW13381 was aberrant and missing the third exon. Sequence analysis of F3'5'H of CW13381 revealed the presence of an indel (~90-bp AT-repeat) in the second intron. In addition, the F3'5'H of CW12700, PE1837, and PE636 harbored unique single-nucleotide substitutions. The single nucleotide polymorphisms resulted in substitutions of amino acid residues located in or near the SRS4 domain of F3'5'H, which is essential for substrate recognition. 3D structure modeling of F3'5'H indicated that the substitutions could interfere with an interaction between the substrate and heme group and compromise the conformation of the active site of F3'5'H. Recombination analysis revealed a tight correlation between all of the mutant alleles at the W1 locus and white flower color. On the basis of the characterization of the new mutant alleles, we discussed the biological implications of F3'5'H and DFR in the determination of flower colors in soybean.
在大豆中，类黄酮3'5'-羟化酶（F3'5'H）和二氢黄酮醇-4-还原酶（DFR）在花青素色素的产生中起着关键作用。编码前者的W1位点或编码后者的W3和W4功能丧失，总是会产生白花。在本研究中，我们寻找了负责大豆白花产生的新遗传成分，并分离出四个白花突变系，即两个野生大豆种质（CW12700和CW13381）以及两个通过甲基磺酸乙酯（EMS）诱变的栽培大豆突变体（PE1837和PE636）。CW12700、PE1837和PE636中F3'5'H的表达正常，而CW13381中的表达异常且缺失第三个外显子。对CW13381的F3'5'H进行序列分析，发现在第二个内含子中存在一个插入缺失（约90bp的AT重复序列）。此外，CW12700、PE1837和PE636的F3'5'H存在独特的单核苷酸替换。这些单核苷酸多态性导致F3'5'H的底物识别必需的SRS4结构域内或附近的氨基酸残基发生替换。F3'5'H的三维结构建模表明，这些替换可能会干扰底物与血红素基团之间的相互作用，并损害F3'5'H活性位点的构象。重组分析显示，W1位点的所有突变等位基因与白花颜色之间存在紧密关联。基于新突变等位基因的特征，我们讨论了F3'5'H和DFR在大豆花色决定中的生物学意义。
