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. 2019 Nov 12:10:1444.
doi: 10.3389/fpls.2019.01444. eCollection 2019.

Upregulated Lipid Biosynthesis at the Expense of Starch Production in Potato (Solanum tuberosum) Vegetative Tissues via Simultaneous Downregulation of ADP-Glucose Pyrophosphorylase and Sugar Dependent1 Expressions

Xiaoyu Xu  1   2 , Thomas Vanhercke  1 , Pushkar Shrestha  1 , Jixun Luo  1 , Sehrish Akbar  1 , Christine Konik-Rose  1 , Lauren Venugoban  1 , Dawar Hussain  1 , Lijun Tian  1 , Surinder Singh  1 , Zhongyi Li  1 , Peter J Sharp  2 , Qing Liu  1
Affiliations

Upregulated Lipid Biosynthesis at the Expense of Starch Production in Potato (Solanum tuberosum) Vegetative Tissues via Simultaneous Downregulation of ADP-Glucose Pyrophosphorylase and Sugar Dependent1 Expressions

Xiaoyu Xu et al. Front Plant Sci. .

Abstract

Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco (Nicotiana tabacum) leaf and potato (Solanum tuberosum) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways.

Keywords: ADP-glucose pyrophosphorylase; RNA interference; Solanum tuberosum; potato; sugar dependent1; triacylglycerol.

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Figures

Figure 1
Figure 1
Contents of total fatty acids (TFA) and major carbohydrate in the mature potato tubers of WT and the eleven selected T0 generation plants. (A) TFA content; (B) Total soluble sugars content; (C) Total starch content; (D) Regression analysis among TFA, total soluble sugars (triangles) and starch (circles) in WT (open symbols) and transgenic lines (black symbols). The relationship between TFA and starch is negative (y = -12.753x + 43.179, R² = 0.7662), while the relationship between TFA and the total soluble sugars is positive (y = 5.0997x + 0.3807, R² = 0.784). The data represent the mean values ± standard deviation (SD) of three biological replicates. Letters (a, b, c, etc.) above the bars are all based on the least significant difference (LSD). Different letters between lines are statistically significantly different at P < 0.05.
Figure 2
Figure 2
Gene expression analysis and total carbon allocation in the potato tubers of WT (open bars) and the two selected WT-derived lines, WT-L5 (hatched bars) and WT-L10 (black bars) at two developmental stages. (A) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) result at the flowering stage; (B) Real-time qRT-PCR result at the mature stage; (C) Total carbon allocation at the flowering stage; (D) Total carbon allocation at the mature stage. The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on LSD, bars marked with different letters are statistically significantly different at P < 0.05.
Figure 3
Figure 3
Contents of neutral and polar lipids in the potato tubers of WT (open bars) and the two selected WT-derived lines, WT-L5 (hatched bars) and WT-L10 (black bars) at two developmental stages. (A) Neutral lipids contents at the flowering stage; (B) Neutral lipids contents at the mature stage; (C) Polar lipids contents at the flowering stage; (D) Polar lipids contents at the mature stage. The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on LSD, bars marked with different letters are statistically significantly different at P < 0.05.
Figure 4
Figure 4
Analysis of the starch properties in the mature potato tubers. (A) Amylose content; (B) Chain length distribution (CLD) variation in debranched starch samples of WT-L5 relative to WT; (C) CLD variation in debranched starch samples of WT-L10 relative to WT; The CLD was reflected as the degree of polymerization (DP), each mark above the horizontal axis corresponds to the difference of a chain length in mole percentage. The error bars represent the standard errors, and the difference value was obtained by subtracting the CLD of WT from the two selected RNAi lines, respectively. (D) Swelling power of potato flour; (E) SDS-PAGE of potato starch granule bound proteins (GBPs). The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on least significant difference (LSD), bars marked with different letters are statistically significantly different at P < 0.05.
Figure 5
Figure 5
Gene expression analysis and total carbon allocation in the potato tubers of the HO69 (open bars) and three super-transformed lines, 69-L1 (bar with upward trend), 69-L2 (hatched bars) and 69-L3 (black bars) at two developmental stages. (A) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) result at the flowering stage; (B) Real-time qRT-PCR result at the mature stage; (C) Total carbon allocation at the flowering stage; (D) Total carbon allocation at the mature stage. The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on LSD, bars marked with different letters are statistically significantly different at P < 0.05.
Figure 6
Figure 6
Contents of neutral and polar lipids in the potato tubers of the HO69 (open bars) and three super-transformed lines, 69-L1 (bar with upward trend), 69-L2 (hatched bars) and 69-L3 (black bars) at two developmental stages. (A) Neutral lipids contents at the flowering stage; (B) Neutral lipids contents at the mature stage; (C) Polar lipids contents at the flowering stage; (D) Polar lipids contents at the mature stage. The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on LSD, bars marked with different letters are statistically significantly different at P < 0.05.
Figure 7
Figure 7
Analysis of the starch properties in the mature potato tubers. (A) Amylose ratio; (B) Difference of chain length distribution (CLD) between debranched starch of 69 and WT; (C) Difference of CLD between debranched starch of 69-L1 and 69; (D) Difference of CLD between debranched starch of 69-L2 and 69; (E) Difference of CLD between debranched starch of 69-L3 and 69; The CLD was reflected as DP. Each mark above the horizontal axis corresponds to the difference of a chain length in mole percentage. The error bars are standard errors. The difference value was obtained by subtracting the CLD of 69 from the three super-transformed lines, respectively. (F) Swelling power of potato flour; (G) SDS-PAGE of potato starch GBPs. The two obscure tracks between 69-L3 and 69-L2 are misplaced samples which are irrelevant with other samples. The data represent the mean values ± SD of three biological replicates. Letters (a, b, c) above the bars are based on LSD, bars marked with different letters are statistically significantly different at P < 0.05.
Figure 8
Figure 8
Confocal scanning microscopy analysis of the LDs distribution in mature potato tubers. The LDs were stained with BODIPY (yellow), bright-field images of the unstained starch granules and cell wall structures formed the major background contrasted to the LDs. (A, B) Visualization of LDs in WT. (C, D) Visualization of LDs in WT-L5. (E, F) Visualization of LDs in HO69. (G, H) Visualization of LDs in 69-L3. The scale bars are located in the lower left corner for each photograph, sizes of the scale bars are all normalized to 20 µm.
Figure 9
Figure 9
Microscopic observation of starch granules in mature potato tubers. (A) scanning electron microscopy (SEM) image of WT; (B) SEM image of WT-L5; (C) SEM image of WT-L10; (D) SEM image of HO69; (E) SEM image of 69-L3; (F) Polarized light image of WT; (G) Polarized light image of WT-L5; (H) Polarized light image of WT-L10; (I) Polarized light image of HO69; (J) Polarized light image of 69-L3. The starch granules were observed under a view of 300× magnification in the SEM, with scale bars located in the lower left corner for each photograph. Sizes of the scale bars are all normalized to 20 µm. For the images taken by light microscopy, the scale bars are located in the lower middle position of each photograph. Size of the scale bars are normalized to 50 µm.
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