Exploring Plant α-Amylase Inhibitors: Mechanisms and Potential Application for Insect Pest Control

Abstract

α-Amylases are found in microbes, plants, and animals, including insect pests. They play crucial roles in catalyzing the hydrolysis of α-1,4-glucan bonds within starch, glycogen, and related carbohydrates, forming shorter oligomers. In green plants, these enzymes are pivotal for starch degradation during photosynthesis and seed germination, whereas in phytophagous insect pests, they predominantly facilitate seed parasitism by degrading raw starch granules. Amylase inhibitors in plants appear to function as part of their defense against pests and pathogens. In the context of insect pests, some of these amylase inhibitors can target α-amylases in the digestive system of certain insects. Both mono- and dicotyledonous plants harbor multiple genes encoding proteinaceous α-amylase inhibitors. Previous studies have demonstrated that α-amylase inhibitors, whether produced in vitro or overexpressed in transgenic plants, can exhibit entomotoxic activity against certain insect pests. Field trials involving transgenic plants that overexpress α-amylase inhibitors have been conducted, laying the foundation for the potential commercialization of crops engineered with these genes. Herein, this review explores the molecular interactions between plant α-amylase inhibitors and insect α-amylases, shedding light on the underlying mechanisms of action, structural diversity, and assessing the broader biotechnological applications of this promising strategy.

Keywords: crop protection; insecticidal protein; starch catabolism; transgenic plants; α‐amylase.

FIGURE 1

In silico analyses of the five more representative sequences of plant‐ and actinobacteria‐derived α‐amylase inhibitor proteins belonging to the seven families: knottin (or inhibitor cystine knot; ICK), γ‐thionin (or defensin), CM‐protein (or cereal‐type), kunitz‐type, thaumatin‐like, lectin‐like, and actinobacteria (Streptomyces sp.) (Table 1; Supporting Information Files S1 to S8), retrieved from UniProt database Release 2023_05 [111]. (A) The evolutionary tree was constructed from amino acid protein sequences (Supporting Information File S8) with the maximum likelihood method, Approximate Likelihood‐Ratio Test (aLRT) SH‐like branch support, and WAG substitution model using the Phylogeny.fr tool webserver [138]. (B) Conserved motifs in amino acid sequences (Supporting Information File S9) were identified using the MEME suite web server with default parameters [139]. (C) Pairwise identity matrix was generated from amino acid sequences (Supporting Information File S8) using the Sequence Demarcation Tool version 1.2 with default parameters [140].

FIGURE 2

Representative three‐dimensional (3D) structures of different α‐amylases and plant α‐amylase inhibitors performed with AlphaFold2 and AlphaFold2‐multimer [112]. (A) 3D structure of a plant α‐amylase (Phaseolus vulgaris, V7CTK1_PHAVU). (B) 3D structure of an insect pest α‐amylase (Callosobruchus chinensis α‐amylase, A0A168VCK5_CALCS). (C) 3D structure of a human α‐amylase (Human pancreatic α‐amylase, P04746 AMYP_HUMAN). (D) 3D structure of an animal α‐amylase (Porcine pancreatic α‐amylase, P00690 AMYP_PIG). (E) 3D structure of a proteinaceous α‐amylase inhibitor (P. vulgaris α‐amylase inhibitor isoform 1, αAI‐1, P02873 LEA1_PHAVU). (F) 3D structure of a proteinaceous α‐amylase inhibitor (P. vulgaris α‐amylase inhibitor isoform 2, αAI‐2, Q41114 LEA2_PHAVU). (G) 3D structure of protein‐protein interaction between the plant‐derived proteinaceous αAI‐1 α‐amylase inhibitor (P02873 LEA1_PHAVU) and insect pest α‐amylase (C. chinensis α‐amylase, A0A168VCK5_CALCS). (H) 3D structure of protein‐protein interaction between the plant‐derived proteinaceous αAI‐2 α‐amylase inhibitor (Q41114 LEA2_PHAVU) and insect pest α‐amylase (C. chinensis α‐amylase, A0A168VCK5_CALCS). Importantly, biochemical studies showed that P. vulgaris αAI‐1 inhibits porcine pancreatic α‐amylase (PPA) and α‐amylases of Callosobruchus maculatus and C. chinensis, but does not inhibit α‐amylase of Zabrotes subfasciatus. In contrast, P. vulgaris αAI‐2 does not inhibit PPA and α‐amylases of C. maculatus and C. chinensis, but inhibits α‐amylase of Z. subfasciatus. Protein sequences were retrieved from the Uniprot database Release 2023_05 [111]. Regions of different degrees of confidence are expressed with different colors according to the predicted local distance difference test (plDDT) value.

FIGURE 3

Representative three‐dimensional (3D) structures of different plant α‐amylase inhibitor proteins performed with AlphaFold2 and AlphaFold2‐multimer [112]. (A) 3D structure of a plant α‐amylase inhibitor from knottin/ICK family (Cucumis melo, P32041 ITR3_CUCMC). (B) 3D structure of a plant α‐amylase inhibitor from γ‐thionin/defensin family (Vigna radiata, Q8W434_VIGRA). (C) 3D structure of a plant α‐amylase inhibitor from CM‐protein/cereal‐type family (Triticum aestivum, P16851 IAAC2_WHEAT). (D) 3D structure of a plant α‐amylase inhibitor from the kunitz‐type family (Hordeum vulgare, P07596 IAAS_HORVU). (E) 3D structure of a plant α‐amylase inhibitor from the thaumatin‐like family (Zea mays, P13867 IAAT_MAIZE). (F) 3D structure of a plant α‐amylase inhibitor from the lectin‐like family (Phaseolus vulgaris, P02873 LEA1_PHAVU). (G) 3D structure of an actinobacteria α‐amylase inhibitor from the actinobacteria family (Streptomyces rochei, P07512 IAA_STRRO). Protein sequences were retrieved from the Uniprot database Release 2023_05 [111]. Regions of different degrees of confidence are expressed with different colors according to the predicted local distance difference test (plDDT) value.