Evolutionary alterations in gene expression and enzymatic activities of gibberellin 3-oxidase 1 in Oryza

Kyosuke Kawai, Sayaka Takehara, Toru Kashio, Minami Morii, Akihiko Sugihara, Hisako Yoshimura, Aya Ito, Masako Hattori, Yosuke Toda, Mikiko Kojima, Yumiko Takebayashi, Hiroyasu Furuumi, Ken-ichi Nonomura, Bunzo Mikami, Takashi Akagi, Hitoshi Sakakibara, Hidemi Kitano, Makoto Matsuoka & Miyako Ueguchi-Tanaka

Communications Biology (2022) 5, 67 DOI:10.1038/s42003-022-03008-5

The plant hormone gibberellin (GA) plays important roles in various developmental events, such as stem elongation, induction of seed germination and flowering. Although GA is indispensable for anther and pollen development, our knowledge of GA functions during plant reproduction has been limited to date.

This paper reports the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA₇ synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability.

In this paper, we used the wild strains of genus Oryza that NIG has conserved with the support of National Bioresource Project (NBRP) Rice, Ministry of Education, Culture, Sports, Science and Texhnology (MEXT), Japan.

The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans.

Negishi T#, Kitagawa S#, Horii N, Tanaka Y, Haruta N, Sugimoto A, Sawa H, Hayashi KI, Harata M*, Kanemaki MT*.
# These authors contributed equally * Co-corresponding authors

Genetics (2022) 220, iyab218 DOI:10.1093/genetics/iyab218

To analyze the protein function of C. elegans, it is useful to study the phenotype by suppressing the protein function. For this purpose, nematode individuals with mutated genes and the RNA interference method have been used. However, genes that play an essential role in nematode development may cause a developmental defect due to the loss of the gene, making further analysis difficult. In addition, due to a large amount of maternally-derived mRNA and proteins in the eggs, the functions of proteins normally involved in early development may not be expressed as a phenotype in early development using existing techniques. The auxin-inducible degron (AID) system that we have established rapidly degrades and removes target proteins at any given time and allows us to observe phenotypes that cannot be seen using existing techniques. The AID method has already been applied to C. elegans by a group in the U.S. and is now being used in various studies. However, the conventional AID method has some problems, such as leaky target degradation in the absence of auxin and the effects of high concentrations of auxin.

Therefore, we applied an improved method, AID2, which we developed last year using budding yeast, cultured cells, and mice, to C. elegans to overcome these problems (Figure 1). As a result, we found that AID2 completely suppressed the leaky degradation in C. elegans and rapidly induced target degradation with 1/1300 of the ligand concentration (Figure 2). Furthermore, by degrading the histone H2A.Z protein, we succeeded in observing a developmental defect that had not been reported previously. Furthermore, to induce degradation in the embryo in the egg, we developed a modified ligand suitable for eggshell permeabilization, enabling rapid proteolysis in the embryo (Figure 3).

This research was led by Assistant Professor Takefumi Negishi and Professor Masato Kanemaki at the National Institute of Genetics, and a graduate student Saho Kitagawa and Professor Masahiko Harada at Tohoku University, in collaboration with Professor Kenichiro Hayashi at Okayama University of Science, Professor Hitoshi Sawa at the National Institute of Genetics, and Professor Asako Sugimoto at Tohoku University.