A sucrose non-fermenting-1-related protein kinase-1 gene, IbSnRK1, improves starch content, composition, granule size, degree of crystallinity and gelatinization in transgenic sweet potato

Abstract

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) is an essential energy-sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up-regulated, and the content of ADP-glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up-regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.

Keywords: IbSnRK1; starch content; starch quality; sweet potato.

Figure 1

Expression analysis of IbSnRK1 in sweet potato. (a) Starch content and IbSnRK1 expression in the storage roots of different cultivars. (b) Expression of IbSnRK1 in different tissues of Lushu 3. L, leaf; P, petiole; S, stem; SR, storage root; FR, fibrous root. (c) Expression of IbSnRK1 in response to 175 mm sucrose treatment. The treatment with H₂O was used as control. (d) Time settings used to examine the circadian rhythm during 16‐h light/8‐h dark photoperiods with light on at 5 am and off at 9 pm. Total RNA was extracted from the in vitro grown plants sampled at 10 pm (P1, P4, P7 and P10), 10 am (P2, P5, P8 and P11) and 4 pm (P3, P6, P9 and P12), respectively. The results are expressed as relative values with respect to P1 (set to 1.0). Data are presented as the mean ± SD (n = 3). * and different lowercase letters indicate a significant difference at P < 0.05; ** and different capital letters indicate a significant difference at P < 0.01 by Student's t‐test.

Figure 2

Expression analysis of IbSnRK1 (a), starch content (b) and amylose proportion (c) in the storage roots of the transgenic sweet potato plants, WT and VC. The sweet potato β‐actin gene was used as an internal control. Data are presented as the mean ± SD (n = 3). * and ** indicate a significant difference compared with WT at P < 0.05 and P < 0.01, respectively, by Student's t‐test.

Figure 3

Morphology and size distribution of starch granules from the storage roots of the transgenic sweet potato plants, WT and VC. (a) Starch granules in amyloplasts. The bar indicates a length of 100 μm. (b) Scanning electron micrographs of the starch granules. The dotted line indicates a length of 50 μm. (c) Size distribution and mean volume diameter (MV) of starch granules. (d) Comparison of the starch granule diameter.

Figure 4

Chain length distribution (CLD) of amylopectin from the transgenic sweet potato plants, WT and VC. (a) CLD of the amylopectin after normalization to the total peak area. (b) Differences of CLD between the transgenic plants and WT were calculated as follow: the normalized CLD value for each transgenic plant and VC minus the value obtained for WT. DP, degree of polymerization.

Figure 5

Wide‐angle X‐ray powder diffraction spectra (a) and differential scanning calorimeter thermograms (b) of starches from the storage roots of the transgenic sweet potato plants, WT and VC.

Figure 6

Expression of the genes involved in starch biosynthesis pathway in the storage roots of the transgenic sweet potato plants, WT and VC. Data are presented as the mean ± SD (n = 3). * and ** indicate a significant difference compared with WT at P < 0.05 and P < 0.01, respectively, by Student's t‐test.

Figure 7

Enzyme activities of acid invertase, neutral invertase, SuSy, AGPase, GBSS, SS and SBE in the storage roots of the transgenic sweet potato plants, WT and VC. Data are presented as the mean ± SD (n = 3). * and ** indicate a significant difference compared with WT at P < 0.05 and P < 0.01, respectively, by Student's t‐test.

Figure 8

The content of components related to starch biosynthesis in the storage roots of the transgenic sweet potato plants, WT and VC. Data are presented as the mean ± SD (n = 3). * and ** indicate a significant difference compared with WT at P < 0.05 and P < 0.01, respectively, by Student's t‐test.

Figure 9

Diagram showing the regulation of starch biosynthesis in the storage roots of the IbSnRK1‐overexpressing sweet potato plants. Biosynthesis pathways are shown with solid arrows and regulatory interactions are shown with broken arrows. Fold changes (the mean of the four transgenic lines L13, L14, L17 and L18) are shown in colour, red boxes, white boxes and blue boxes indicate up‐regulation, no obvious change and down‐regulation of expression of genes encoding these enzymes (proteins), respectively.