Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage

Takao Ohashi; Yurika Mabira; Yutaro Mitsuyoshi; Hiroyuki Kajiura; Ryo Misaki; Takeshi Ishimizu; Kazuhito Fujiyama

doi:10.1016/j.jbiosc.2024.03.006

Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage

J Biosci Bioeng. 2024 Apr 19:S1389-1723(24)00107-5. doi: 10.1016/j.jbiosc.2024.03.006. Online ahead of print.

Authors

Takao Ohashi¹, Yurika Mabira², Yutaro Mitsuyoshi², Hiroyuki Kajiura², Ryo Misaki², Takeshi Ishimizu³, Kazuhito Fujiyama²

Affiliations

¹ International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan; Department of Life Science, Faculty of Science and Engineering, Setsunan University, Neyagawa, Osaka 572-8508, Japan. Electronic address: takao.ohashi@lif.setsunan.ac.jp.
² International Center for Biotechnology, Osaka University, Suita, Osaka 565-0871, Japan.
³ College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.

PMID: 38643032
DOI: 10.1016/j.jbiosc.2024.03.006

Abstract

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

Keywords: Arabidopsis thaliana; Aspergillus aculeatus; Mucilage; Pectin; Pyridylamination; Rhamnogalacturonan I; Rhamnogalacturonase.