A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation

Abstract

Traditional functional genetic studies in crops are time consuming, complicated and cannot be readily scaled up. The reason is that mutant or transformed crops need to be generated to study the effect of gene modifications on specific traits of interest. However, many crop species have a complex genome and a long generation time. As a result, it usually takes several months to over a year to obtain desired mutants or transgenic plants, which represents a significant bottleneck in the development of new crop varieties. To overcome this major issue, we are currently establishing a versatile plant genetic screening platform, amenable to high throughput screening in almost any crop species, with a unique workflow. This platform combines protoplast transformation and fluorescence activated cell sorting. Here we show that tobacco protoplasts can accumulate high levels of lipid if transiently transformed with genes involved in lipid biosynthesis and can be sorted based on lipid content. Hence, protoplasts can be used as a predictive tool for plant lipid engineering. Using this newly established strategy, we demonstrate the major role of ABI3 in plant lipid accumulation. We anticipate that this workflow can be applied to numerous highly valuable metabolic traits other than storage lipid accumulation. This new strategy represents a significant step toward screening complex genetic libraries, in a single experiment and in a matter of days, as opposed to years by conventional means.

Keywords: fluorescence activated cell sorting; high throughput screening; lipid accumulation; metabolic engineering; protoplast.

FIGURE 1

Schematic representation of the high throughput screen developed in this work. Protoplasts (green spheres) are isolated from a leaf and transformed with DNA constructs. After 48 h incubation, to allow for transgene expression and metabolite accumulation, protoplasts are stained with a neutral lipid specific fluorescent stain and screened for fluorescence by flow cytometry or fluorescence activated cell sorting.

FIGURE 2

Protoplast lipid quantification results mimic total tissue lipid analysis results. Thin layer chromatography plate separation of total fatty acids (TFA) extracted from leaves or from isolated protoplasts of WT or HO lines and total lipid and triacylglycerol (TAG) quantification of the same samples quantified by gas chromatography. Concentrations are given in percent relative to leaf fresh weight or in micrograms per million cells. Bars represent standard deviation. Stars indicate statistically relevant variation compared to WT (P-values < 0.05). FFA, free fatty acids; DAG, diacylglycerol. See Supplementary Figure S1 for standards run next to samples.

FIGURE 3

Fluorescence activated cell sorting of lipid rich tobacco leaf protoplasts. (A) Microscopy images of protoplasts extracted from WT or HO (line with high leaf oil content) tobacco leaves in bright field (top) and their lipid content (bottom), visualized under fluorescent light after BODIPY^TM 493/503 staining. (B) Cell sorting of the same protoplast samples, with the P1 population defined as the protoplasts with the highest stain fluorescence to chlorophyll auto-fluorescence ratio, and P2 defined as the remainder of the intact cells (based on previous data not show). (C) Average stain fluorescence (left) and lipid content (right) for each sorted population. Average stain fluorescence of each whole sample is also indicated (blue bars). Where N.D. is in brackets, this indicates not determined. Error bars represent the standard deviation. Asterisks represent statistically significant differences (P-value < 0.05) compared with WT (one asterisk) or with both WT and HO P2 population (two asterisks).

FIGURE 4

Lipid accumulation in tobacco leaf transiently transformed protoplasts. Lipid accumulation in WT tobacco leaf protoplasts compared to WT protoplasts transiently transformed with WRI1 or DGAT1 or both, was measured with BODIPY^TM staining and flow cytometry recording of the average particle stain fluorescence for the whole sample. Error bars represent the standard deviation. Asterisks represent statistically significant differences (P-value < 0.05) compared with WT.

FIGURE 5

WRI1 and DGAT1 have a synergistic positive effect on protoplast lipid biosynthesis. (A) Interaction plot of predicted treatment means with 5% Least Significant Difference (LSD) bar on the log10-transformed scale and (B) Plot of the back-transformed predicted means (with back-transformed 95% confidence interval limits) from the analysis of the data previously presented in Figure 4B. WT corresponds to “WRI1−:DGAT1−” and the samples transformed with DGAT1, WRI1 and both, correspond to WRI1−:DGAT1+, “WRI1+:DGAT1−” and “WRI1+:DGAT1+,” respectively.

FIGURE 6

Comparison of the relative effect of various transiently transformed effectors on lipid accumulation in tobacco leaf protoplasts. Lipid accumulation, compared to WT, in tobacco leaf protoplasts transiently transformed with WRI1, DGAT1, ABI3, FUS3, LEC1, or LEC2 and various combinations of these constructs, was measured by flow cytometry and based on the average particle stain fluorescence for the whole sample following BODIPY^TM staining. Bars represent standard deviation. (A) Comparative additional effect of ABI3, FUS3, LEC1, or LEC2 on lipid content in protoplasts co-transformed with WRI1 and DGAT1. (B) Individual effect of WRI1, DGAT1, ABI3, LEC2 and of various combinations of these constructs on lipid content in co-transformed protoplasts. A single star represents statistically significant difference (P-value < 0.05) compared with WT, while the double stars represent additional statistically significant difference (P-value < 0.05) compared with WRI1 and DGAT1 combination (shown for samples with significant increase only).