Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways

Abstract

Cold-induced sweetening (CIS) in potato is detrimental to the quality of processed products. Conversion of starch to reducing sugars (RS) by amylases is considered one of the main pathways in CIS but is not well studied. The amylase genes StAmy23, StBAM1, and StBAM9 were studied for their functions in potato CIS. StAmy23 is localized in the cytoplasm, whereas StBAM1 and StBAM9 are targeted to the plastid stroma and starch granules, respectively. Genetic transformation of these amylases in potatoes by RNA interference showed that β-amylase activity could be decreased in cold-stored tubers by silencing of StBAM1 and collective silencing of StBAM1 and StBAM9. However, StBAM9 silencing did not decrease β-amylase activity. Silencing StBAM1 and StBAM9 caused starch accumulation and lower RS, which was more evident in simultaneously silenced lines, suggesting functional redundancy. Soluble starch content increased in RNAi-StBAM1 lines but decreased in RNAi-StBAM9 lines, suggesting that StBAM1 may regulate CIS by hydrolysing soluble starch and StBAM9 by directly acting on starch granules. Moreover, StBAM9 interacted with StBAM1 on the starch granules. StAmy23 silencing resulted in higher phytoglycogen and lower RS accumulation in cold-stored tubers, implying that StAmy23 regulates CIS by degrading cytosolic phytoglycogen. Our findings suggest that StAmy23, StBAM1, and StBAM9 function in potato CIS with varying levels of impact.

Keywords: cold-induced sweetening; potato; reducing sugar; starch degradation; tuber.; α-Amylase; β-amylase.

Fig. 1.

Phylogenetic analysis of β-amylases and gene structures of StBAM1–9. (A) A phylogram of 56 β-amylases from eight plant species. The glucosyl hydrolase domains of 56 β-amylases were aligned by MUSCLE and were used to construct a maximum-likelihood tree. The robustness of the tree is derived from 100 bootstrap replicates. The scale bar refers to 0.1 amino acid substitution per site. The phylogram displayed with Tree Of Life software (Letunic and Bork, 2016) showed that plant β-amylases were clustered into four subfamilies. Potato proteins are shown in red, tomato proteins in purple, Arabidopsis proteins in blue, rice proteins in rose red, P. trifoliate proteins in black, poplar proteins in dark green, soybean proteins in orange, and barley proteins in grey. (B) Gene structures for potato StBAMs based on the number and position of CDS (yellow), intron (solid lines) and untranslated region (blue).

Fig. 2.

The phylogenetic relations of 38 α-amylases from eight plant species. Thirty-eight α-amylases were aligned by MUSCLE with default settings and used to generate a neighbour-joining tree. The scale bar refers to amino acid substitution per site. The phylogram showed that plant α-amylases were clustered into three subfamilies. α-Amylases of tomato, rice, poplar, and soybean were named in terms of the corresponding locus names, and the names of α-amylase for potato, Arabidopsis, barley, and apple were derived from the reports.

Fig. 3.

Alignment of the conserved glucosyl hydrolase domains from potato StBAMs, soybean GmBMY1, and Arabidopsis AtBAM1, AtBAM4, and AtBAM9. Most and same colour shading indicates identical residues and conservative substitutions for each column, whereas the less conserved residues are presented as a different colour. An overall picture of sequence conservation is displayed in the bar graph, together with tall yellow bars representing high sequence conservation and short brown bars representing low sequence conservation. Substrate binding sites and two catalytic residues are marked with black and larger red arrowheads, respectively. The deletion and substitution of the flexible loop and inner loop in StBAM9, AtBAM9, and AtBAM4 were indicated with red lines.

Fig. 4.

Subcellular localizations of StAmy23, StBAM1, and StBAM9 in Nicotiana benthamiana leaves. (A) StAmy23–GFP was coexpressed with cytosol marker RFP (A1–A4). (B) StBAM9–GFP was coexpressed with starch granule marker StGBSS–RFP (B1–B7). (C) StBAM1–GFP was coexpressed with StGBSS–RFP (C1–C7). The first column indicates GFP fluorescence (A1, B1, and C1), the second column indicates RFP fluorescence (A2, B5, and C5) or chlorophyll autofluorescence (B2 and C2), the third column shows merged images, and the last column shows ×4 enlargement of merged images (A4, B4, B7, C4 and C7). Bars: 10 μm. (D, E) Western blots probed with GFP antibody (E) and RFP antibody (F) showing stable protein fusions of potato StAmy23–GFP, StBAM1–GFP, StBAM9–GFP, StGBSS–RFP, and free GFP and RFP with expected size.

Fig. 5.

Transcripts of StAmy23, StBAM1, and StBAM9 in transgenic tubers stored at 4 °C for 0, 15 and 30 d. (A) The relative expression of StAmy23 in RNAi-StAmy23 tubers. (B) The relative expression of StBAM1 in RNAi-StBAM1 tubers. (C) The relative expression of StBAM9 in RNAi-StBAM9 tubers. (D) The relative expression of StBAM1 in RNAi-(StBAM1+StBAM9) tubers. (E) The relative expression of StBAM9 in RNAi-(StBAM1+StBAM9) tubers. The columns represent the mean values of three biological replicates and the bars indicate the standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 6.

The sugar content and crisp colour of RNAi-StAmy23, RNAi-StBAM1, RNAi-StBAM9 and RNAi-(StBAM1+StBAM9) tubers stored at 4 °C for 0, 15 and 30 d. (A–D) Reducing sugar (RS) content. (E–H) Glucose content. (I–L) Fructose content. (M–P) Sucrose content. (Q) Colour of potato crisps from RNAi-StAmy23 tubers stored at 4 °C for 0 and 15 d. (R) Colour of potato crisps from RNAi-StBAM1, RNAi-StBAM9 and RNAi-(StBAM1+StBAM9) tubers stored at 4 °C for 0 and 30 d. The columns represent the mean values of three biological replicates and the bars indicate the standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 7.

The amylase activity of RNAi-StAmy23, RNAi-StBAM1, RNAi-StBAM9 and RNAi-(StBAM1+StBAM9) tubers stored at 4 °C for 0 and 30 d. (A) α-Amylase activity of RNAi-StAmy23 tubers. (B) β-Amylase activity of RNAi-StBAM1 tubers. (C) β-Amylase activity of RNAi-StBAM9 tubers. (D) β-Amylase activity of RNAi-(StBAM1+StBAM9) tubers. The columns represent the mean values of three biological replicates and the bars indicate the standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 8.

The starch content of RNAi-StAmy23, RNAi-StBAM1, RNAi-StBAM9 and RNAi-(StBAM1+StBAM9) tubers stored at 4 °C for 0, 15 and 30 d. (A–D) Starch content. (E–H) Soluble starch content. (I–L) Phytoglycogen content. (M–P) Malto-oligosaccharide content. The columns represent the mean values of three biological replicates and the bars indicate the standard deviation. *P < 0.05, **P < 0.01 by Student’s t test.

Fig. 9.

The interaction between StBAM9 and StBAM1. (A–C) StBAM9 and StBAM1 interact on the starch granules in a BiFC assay. (A) Transient coexpression of StBAM9–YFP^N and StBAM1–YFP^C in N. benthamiana. (B) As a negative control, StBAM1–YFP^C and YFP^N were coexpressed. (C) StBAM9–YFP^N and YFP^C were coexpressed. Bars: 10 μm. (D) StBAM9 and StBAM1 interact in a yeast two-hybrid assay. Yeast coexpressing StBAM9 and StBAM9-P (StBAM9 without transit peptide) with StBAM1 grows on the double dropout (–Leu–Trp) medium by addition of X-α-Gal and triple dropout (–Leu–Trp–His) medium, respectively. The colour of the clones on SD/–Leu–Trp/X-a-Gal and the survival on SD/–Leu–Trp–His selecting plates represent the interaction.