In silico analysis of the structural diversity and interactions between invertases and invertase inhibitors from potato (Solanum tuberosum L.)

Abstract

We performed sequence diversity, phylogenetic profiling, 3D structure modelling and in silico interactions between invertases (cell wall/apoplastic and vacuolar) and invertase inhibitors (cell wall/apoplastic and vacuolar) from potato. Cloning and sequencing of invertase inhibitors was performed from different potato cultivars. The comparison of the protein sequences of the different isoforms of invertases and invertase inhibitors exhibited insertions and deletions as well as the variation in terms of amino acid residues. Furthermore, the phylogenetic tree analysis displayed two groups of invertase inhibitors corresponding to the cell wall/apoplast and vacuole. Using Phyre² protein homology recognition engine, it revealed that the structure of invertase inhibitors was predominantly α-helical and that of invertase was α helices and β strands. Results of the Ramachandran plots for each structure showed that the percentage of amino acid residues in favoured region and in allowed region. Also, the Z score and QMEAN score indicated overall good, acceptable and reliable models. In silico interactions between different isoforms of invertase and invertase inhibitors suggested that cell wall/ apoplastic invertase inhibitor exhibited stronger interaction with vacuolar invertase compared to the vacuolar invertase inhibitor. In silico interactions provides valuable information in selecting the appropriate combinations of invertase and invertase inhibitor. Therefore, a better understanding of the interactions between specific invertase and invertase inhibitor alleles will be helpful for an intelligent manipulation of the cold-induced sweetening process of potato tubers.

Keywords: In silico; Invertase; Invertase inhibitor; Phylogenetic; Solanum tuberosum.

Fig. 1

Alignment of the cell wall/apoplastic invertase inhibitors and the vacuolar invertase inhibitors from planta. Amino acid sequences of the cell wall and vacuolar invertase inhibitors from potato were aligned with that of previously published sequences from planta using multiple sequence alignment in MEGA 6.0 using ClustalW (https://www.genome.jp/tools-bin/clustalw (Thompson et al. 1994) using default parameters. The amino acids are represented by a single letter code. The protein sequences listed in the diagram are; StVIF—Solanum tuberosum vacuolar invertase inhibitor (AYV96512), StCIF—Solanum tuberosum cell wall/apoplastic invertase inhibitor (AFI47459), SlVIF—Solanum lycopersicum vacuolar invertase inhibitor (NP_001316149), NtVIF—Nicotiana tabacum vacuolar invertase inhibitor (AAN60076), IbVIF/CIF—Ipomea batata vacuolar/ cell wall invertase inhibitor (AAM94391), SlCIF/VIF—Solanum lycopersicum cell wall invertase inhibitor (NP_001234791), NtCIF—Nicotiana tabacum cell wall invertase inhibitor (CAA73333), Br CIF/VIF—Brassica rapa cell wall/vacuolar invertase inhibitor (XP_009148010), BnCIF/VIF—Brassica napus cell wall/vacuolar invertase inhibitor (XP_013641163), Bo CIF/VIF—Brassica oleracea cell wall/vacuolar invertase inhibitor (XP_013589892), AtVIF—Arabidopsis thaliana vacuolar invertase inhibitor (NP_001320618), GhCIF/VIF—Gossypium hirsutum cell wall/vacuolar invertase inhibitor (XP_016676243) and TcCIF/VIF—Theobroma cacao cell wall/vacuolar invertase inhibitor (XP_017974170). The secondary structure was predicted using PSIPRED v3.3 and compared with that of previously published information from N. tabacum—Nt-INH1/ NtVIF (Greiner et al. , ; Hothorn et al. , ; Reca et al. 2008). The symbols used for the secondary structure are α1-α7 helix. The invertase inhibitor peptide encodes 162 to 192 amino acids. Asterisks at the bottom of the alignment indicate identical residues. Amino acid numbers are listed on right of the alignment. Numbers 1 and 2 at the bottom of the alignment (C residues shaded in grey) denote disulfide bridges connecting the conserved two pairs of cysteine residues. Highly conserved PKF (Proline, Glycine and Phenylalanine shaded in orange) motif in the α6 helix in the known invertase inhibitors (Hothorn et al. 2010) is boxed

Fig. 2

Phylogenetic tree of the cell wall/apoplastic invertase inhibitors and the vacuolar invertase inhibitors from planta. Phylogenetic tree was constructed in MEGA 7 program (Tamura et al. 2011) by neighbour-joining 1000 bootstrap analysis based on the cell wall/apoplastic invertase inhibitor and the vacuolar invertase inhibitor protein sequences from planta. Scale bar 0.20 marks 0.2 amino acid substitution per site. Bootstrap values (%) are indicated at each branch point. Two groups identified. Group A consists of the vacuolar invertase inhibitors from Solanum tuberosum (StVIF—AYV96512), Solanum lycopersicum (SlVIF-NP_001316149), N. tabacum (NtVIF-XP_016474343), Ipomea batata (IbCIF/VIF- AAM94391), Theobroma cacao (TcCIF/VIF-XP_017974170), Gossypium hirsutem (GhCIF/VIF- XP_016676243), Arabidopsis thaliana (AtVIF1-NP_001320618), Brassica oleracea (BoCIF/VIF- XP_013589892), Brassica napus (BnCIF/VIF- XP_013641163) and Brassica rapa (BrCIF/VIF- XP_009148010). Group B consists of the cell wall/apoplastic invertase inhibitors from Solanum tuberosum (StCIF- AFI47459), Solanum lycopersicum (SlCIF- NP_001234791) and Nicotiana tabacum (NtCIF-CAA73333). StKunitz is set as an outgroup which is Solanum tuberosum Kunitz type invertase inhibitor. The protein sequences listed in the diagram are obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/)

Fig. 3

Amino acid residues of invertases (cell wall/apoplastic and vacuolar) interacting with invertase inibitors. Amino acid sequence alignment of the invertases (cell wall/apoplastic and vacuolar) from potato was conducted using multiple sequence alignment in MEGA 6.0 using ClustalW (https://www.genome.jp/tools-bin/clustalw (Thompson et al. 1994) using default parameters. The amino acids are represented by a single letter code. The amino acid residues interacting with invertase inhibitors are highlighted in red. The sequences included in the alignment are: CWINV1-like—beta-fructofuranosidase (XP_006342970), Pain-1—vacuolar invertase (ADM47340), Inv_ap-a pCD141 (AEV46339), Inv_ap-a pCD111 (ADN06440), Inv_ap-b InvGE (AEV46300) and Inv_ap-b InvGF (AEV46318) are cell wall/apoplastic invertases from potato. The protein sequences listed in the diagram are obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/). Asterisks at the bottom of the alignment indicate the identical residues and – indicates deletion. The acid invertases are glycoproteins and have three conserved sequence motifs: β-fructofuranosidase motif (NDPNG(A)), RDP and WECP(V)D motifs are red boxed (Lammens et al. ; Roitsch & Gonzalez . Amino acid numbers are listed on right of the alignment

Fig. 4

Amino acid residues of invertase inhibitors (cell wall/apoplastic and vacuolar) interacting with invertases. Alignment of INH1—cell wall/apoplastic invertase inhibitor and INH2—vacuolar invertase inhibitor from potato was performed using multiple sequence alignment in MEGA 6.0 using ClustalW (https://www.genome.jp/tools-bin/clustalw (Thompson et al. 1994) using default parameters. The secondary structure was predicted using PSIPRED v3.3 and compared with that of previously published information from N. tabacum—Nt-INH1/ NtVIF (Greiner et al. , ; Hothorn et al. , ; Reca et al. 2008). The amino acids are represented by a single letter code. Amino acid residues which are interacting with invertases are highlighted in red. The symbols used for the secondary structure are α1-α7 helix. Four conserved cysteine residues are highlighted in yellow and the numbers 1 and 2 at the bottom of the alignment denote disulfide bridges connecting the conserved two pairs of cysteine residues. Asterisks at the bottom indicate identical residues. Amino acid numbers are listed on right of the alignment. Highly conserved PKF (Proline, Glycine and Phenylalanine shaded in orange) motif in the α6 helix in the known invertase inhibitors (Hothorn et al. 2010) is boxed. The protein sequences listed in the diagram are obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/)