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Identification and Characterization of the Interaction between ZmSBEIIb and ZmPAD1 in Maize

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Abstract

The starch branching enzymes (SBEs) play critical roles in forming branched structures (α-1,6-glycosidic linkages) of starch molecules in maize (Zea mays L.). Here, we use a yeast two-hybrid system to screen for ZmSBEIIb-binding proteins to identify proteins binding to ZmSBEIIb to facilitate a more thorough characterization of ZmSBEIIb. By screening a cDNA expression library, a physical interactor of ZmSBEIIb was identified, which is highly homologous with the α-subunit of 20S proteasome and thus named as ZmPAD1. In order to obtain more detailed information about this interaction, the ZmPAD1 interacting with ZmSBEIIb and thereby impacting proteolytic processes were further studied. First, we determined the interaction strength between ZmPAD1 and different truncated ZmSBEIIb baits in the yeast two-hybrid system; second, we confirmed this interaction with GST pull-down and co-immunoprecipitation assays in vitro and in vivo; third, we applied MG115 to inhibit the function of the proteasome in maize suspension-cultured cells for changing the ZmSBEIIb protein content. In our opinion, the interaction between ZmPAD1 and ZmSBEIIb may play a role leading to ZmSBEIIb degradation by 20S proteasome. These results provide a valuable ZmSBEIIb-binding protein resource for analyzing ZmSBEIIb function and provide new insights into the potential protein regulatory mechanism that controls the function of ZmSBEIIb.

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ACKNOWLEDGMENTS

We thank our teacher Professor Yingdian Wang for his selfless support and useful discussions.

Funding

This study is supported by grants from the Natural Science Foundation of Guangdong Province (project no. 2017A030313206), and National Innovation Training Project (project no. 201813177001) in China.

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Authors and Affiliations

  1. State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, 519087, Zhuhai, P.R. China

    C. Chen

  2. College of Engineering, Beijing Normal University, 519087, Zhuhai, P.R. China

    J. Zhang, M. Qin & Z. Li

  3. College of Chemistry, Chemical Engineering and Environmental Engineering, Liaoning Shihua University, 113001, Liaoning, P.R. China

    C. Li

  4. Zhangzhou Customs House, 363005, Zhangzhou, P.R. China

    W. Lin

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  1. C. Chen
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  2. J. Zhang
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  4. C. Li
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Correspondence to C. Chen.

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Abbreviations: AAC—apparent amylose content; DAP—days after pollination; DBE—starch debranching enzyme; ESV1—early starvation 1; FLO6—floury endosperm 6; GST—glutathione-S-transferase; HIF-1α—hypoxia-inducible factor-1α; IPTG—isopropyl-β-D-thiogalactopyranoside; ISA1—isoamylase1; LESV1—like ESV1; MG115—carbobenzoxyl-leucinyl-leucinyl-norvalinal-H; NB—nutrient broth; NOD1 nucleotide-binding oligomerization domain-containing protein 1; ONPG—o-nitrophenyl-β-galactopyranoside; PAGE—polyacrylamide gel electrophoresis; PSMA3—proteasome subunit alpha 3; Ppase—ADP-Glc pyrophosphorylase; PTST1—protein targeting to starch; SBE—starch branching enzyme; SS—starch synthase.

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Chen, C., Zhang, J., Qin, M. et al. Identification and Characterization of the Interaction between ZmSBEIIb and ZmPAD1 in Maize. Russ J Plant Physiol 68, 66–73 (2021). https://doi.org/10.1134/S1021443721010052

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