Identification of genes encoding granule-bound starch synthase involved in amylose metabolism in banana fruit

Abstract

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

Figure 1. Changes in total starch content (A, B), amylose content (C, D), and GBSS activity (E, F) in banana pulp at different stages of development and storage.

The vertical bars represent standard error (±SE) of three replicates. Three biological experiments were performed, which produced similar results.

Figure 2. SDS-PAGE and western blotting analyses of GBSS proteins in banana pulp at different stages of development and ripening.

M1: Protein marker; A: SDS-PAGE; B: Western blotting; a: Variation of GBSS protein levels at different development stages of banana fruit by SDS-PAGE analysis; b: Variation of GBSS protein levels at different ripening stages of banana fruit by SDS-PAGE analysis; c: Variation of GBSS protein levels at different development stages of banana fruit by western blotting analysis; d: Variation of GBSS protein levels at different ripening stages of banana fruit by western blotting analysis. Each lane contained 20 µL of the GBSS protein extract.

Figure 3. Phylogeny of MaGBSSI and MaGBSSII amino acid sequences.

Austrostipa aristiglumis GBSSI (ABU98330), Microlaena stipoises GBSSI (ABU99332), MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, and MaGBSSI-4 (Musa acuminate L. AAA group cv. Brazilian), Castilleja ambigua GBSSI (ACZ73348), Junellia seriphioides GBSSI (ABQ52190), Malus×domestica GBSSI-2 (ACB97678), Nelumbo nucifera GBSSI (ACM78591), Gossypium hirsutum GBSSI (ACV72639), Malus×domestica GBSSI-1 (ACB97677), Glycine max GBSSI-1 (NP001237971), Vigna unguiculata GBSSI-1 (ABP35818), Lotus japonicus GBSSI-1 (ACB30384), Lotus japonicus GBSSI-2 (ACB30385), Phaseolus vulgaris GBSSI-2 (BAC76613), Vigna radiata GBSSI-2 (ACB30382), Cicer arietinum GBSSII (XP004489397), Pisum sativum GBSSII (Q43093), MaGBSSII-1 and MaGBSSII-2 (Musa acuminate L. AAA group cv. Brazilian), Vitis vinifera GBSSII (XP002278470), Solanum lycopersicum GBSSII (XP004232219), Solanum tuberosum GBSSII (Q43847), Manihot esculenta GBSSII (AAF13168). Numbers presented as percentages indicate the identity of MaGBSSI and MaGBSSII amino acid sequences from different species.

Figure 4. Expression of MaGBSSI and MaGBSSII genes in different banana tissues.

The y-axis represents the relative fold difference in mRNA level, which is calculated using the 2^−ΔΔCt formula with MaActin as internal control. Relative expression levels are presented as fold-changes relative to the expression level obtained in root tissue. The vertical bars represent standard error (±SE) of three replicates. Three biological experiments were performed, which produced similar results.

Figure 5. Expression of MaGBSS genes (A) and scanning electron microscopy (SEM) of starch granules (B) at different development stages in banana fruit.

The y-axis represents the relative fold difference in mRNA level, which is calculated using the 2^−ΔΔCt formula with MaActin as internal control. Relative expression levels are presented as fold-changes relative to the expression level obtained at 0 day of fruit development. The vertical bars represent standard error (±SE) of three replicates (A). Red arrow represents the starch granules. Three biological experiments were performed, which produced similar results.

Figure 6. Expression of MaGBSS genes (A) and scanning electron microscopy (SEM) of starch granules (B) in banana fruit stored for various periods of time.

The y-axis represents the relative fold difference in mRNA level, which is calculated using the 2^−ΔΔCt formula with MaActin as internal control. Relative expression levels are presented as fold-changes relative to the expression level obtained at 0 day of fruit postharvest. The vertical bars represent standard error (±SE) of three replicates (A). Red arrow represents the starch granules. Three biological experiments were performed, which produced similar results.