Bicolor pattern formation in saddle soybean: Quercetin regulates gene expression through RNA interference

A research group led by Doctoral Student Riho Yamanashi (at the time of the research) of the United Graduate School of Agricultural Science, along with Professor Toshiyuki Fukuhara and Professor Hiromitsu Moriyama from the Division of Bioregulation and Biointeraction, Institute of Agriculture, Professor Hiroshi Tsugawa from the Division of Biotechnology and Life Science, Institute of Engineering at Tokyo University of Agriculture and Technology, and Professor Hisashi Koiwa of Texas A&M University (concurrently Specially Appointed Professor at the Institute of Global Innovation Research, Tokyo University of Agriculture and Technology), have elucidated the mechanism by which the bicolor pattern of saddle soybeans is formed. In this process, they demonstrated for the first time in the world that flavonoids (which are secondary metabolites of plants), especially quercetin, regulate gene expression control through RNA interference. Applications are expected in the regulation of the color of flowers and fruits, as well as controlling the flavonoid and anthocyanin content in vegetables and fruits. The research was published in The Plant Journal.

Domesticated soybean varieties can be broadly classified into three major groups based on seed coat color: yellow (colorless), bicolored yellow-black (saddle), and black. Soybeans used for tofu, miso, natto, and other products are yellow varieties, with Enrei being a representative cultivar. The representative black soybean variety is Tamba-kuro, known as Tamba black beans. "Saddle" soybeans have a bicolored seed coat with a black area around the "hilum" and a yellow peripheral region. They are called this because the black portion resembles a saddle placed on a horse's back.

The beans regarded as the wild, ancestral species of soybeans have black seed coats. As a result, black varieties are considered the wild type with regard to seed coat color, while yellow varieties are considered mutants.

In the seed coats of yellow varieties, RNA interference causes the degradation of mRNA transcribed from the CHS (chalcone synthase) gene, suppressing gene expression (PTGS: post-transcriptional gene silencing), which inhibits the biosynthesis of chalcone, an early intermediate in flavonoid biosynthesis. This prevents the biosynthesis of the anthocyanins that make the seed coat black, resulting in a yellow seed coat. When RNA interference is inhibited, portions of the seed coat alone become black.

In saddle varieties, it has been reported that anthocyanin biosynthesis is inhibited by RNA interference in the yellow portions, but no-one had elucidated the reason that RNA interference is limited to the peripheral region of the seed coat.

The research group focused on the enzyme Dicer, which is essential for inducing RNA interference, and flavonoids, which previous research had shown could inhibit its enzyme activity, and conducted research based on the hypothesis that the enzyme activity of Dicer (DCL4) is specifically inhibited in the region around the hilum of immature seeds. As a result, in the immature seed coat of Shinano-kurakake, Dicer (DCL4) activity was detected only in the peripheral region that becomes yellow (colorless) after maturation and was not detected in the region around the hilum that becomes black after maturation.

On the other hand, in the immature seed coat, phenolic compounds including flavonoids accumulated specifically in the region around the hilum that becomes black. Furthermore, when the researchers compared the Dicer activity inhibition ability of epicatechin, quercetin, and quercetin glycoside, which accumulated in large amounts among these flavonoids, it was found that quercetin inhibits Dicer activity. Furthermore, flavonoids accumulated abundantly in the funiculus that connects the maternal tissue and immature seeds in Shinano-kurakake and Tamba-kuro but did not accumulate in Enrei.

These results indicate that flavonoid aglycons such as quercetin, transported from maternal tissues via the funiculus, accumulate around the hilum of the immature seed coat and inhibit the enzyme activity of DCL4 (double-stranded RNA cleavage activity), thereby inhibiting RNA interference, which leads to anthocyanin accumulation and black coloration.

Moreover, once RNA interference is inhibited, flavonoid biosynthesis resumes, more flavonoid aglycons accumulate, and the inhibition of RNA interference (anthocyanin biosynthesis) is maintained.

On the other hand, the peripheral region of the immature seed coat is distant from the funiculus, with fewer flavonoid aglycons transported from the maternal tissues, maintaining DCL4 activity (RNA interference), and anthocyanins do not accumulate, resulting in a yellow (colorless) appearance. It is believed that the bicolor pattern of saddle beans is formed through this feedforward control mechanism by Dicer DCL4 and flavonoid aglycons (quercetin).

This study demonstrated for the first time in the world that flavonoids (particularly quercetin), which are secondary metabolites of plants, regulate gene expression control through RNA interference. It also became clear that this mechanism gave rise to the useful agricultural trait of the saddle pattern in seeds.

In yellow soybean varieties, the fact that stresses such as viral infection and low temperature inhibit RNA interference in the seed coat, causing partial anthocyanin accumulation that leads to decreased soybean quality, is a major agricultural problem, and this achievement is expected to contribute to maintaining stable soybean quality. Furthermore, it will lead to applications such as regulating the color of flowers and fruits and controlling the flavonoid and anthocyanin content in vegetables and fruits.

Journal Information
Publication: The Plant Journal
Title: Inhibition of DCL4 activity by maternally supplied flavonoid aglycons induces a bicolor pattern in the saddle soybean seed coat
DOI: 10.1111/tpj.70522