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Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava

Abstract

Key message

Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure.

Abstract

Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6–13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

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Acknowledgements

We would like to thank Editage (https://www.editage.com/) for English language editing.

Funding

This work was supported by the following funding: Strategic Funds for the Promotion of Science and Technology and SICORP Grant Number JPMJSC16C4 of Japan Science and Technology Agency (JST), the Science and Technology Research Partnership for Sustainable Development (SATREPS) Grant Number JPMJSA1508 in a collaboration between the JST and the Japan International Cooperation Agency (JICA), and RIKEN Center for Sustainable Resource Science (CSRS).

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Contributions

YU and MS supervised the experiments, and YU, MS and YN wrote the manuscript. YU, CU, YO and MT prepared the vector construction, transgenic lines and plant management. YU and ST prepared the phylogenic tree and alignment analysis. YU and CU performed the SBE active staining and measurement of amylose content and resistant starch content. MO and YN determined the chain length distribution. YN gave critical suggestions.

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Correspondence to Yoshinori Utsumi or Motoaki Seki.

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Supplementary material 1 (XLSX 10.5 kb)

Supplementary Figure 1 Vector construction for suppression of SBE1, SBE2c and both (TIF 679.9 kb)

11103_2021_1209_MOESM3_ESM.tif

Supplementary Figure 2 Selection of transgenic lines. The detection of SBE enzyme activities on zymograms in leaves of transgenic plants and 60444 (WT). SBE activities in 10 µg of soluble protein from leaves (TIF 15519.9 kb)

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Utsumi, Y., Utsumi, C., Tanaka, M. et al. Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava. Plant Mol Biol 108, 413–427 (2022). https://doi.org/10.1007/s11103-021-01209-w

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Keywords

  • Cassava
  • High-amylose starch
  • Starch branching enzyme
  • Amylopectin