Identification of Gene Targets for the Sprouting Inhibitor CIPC

Abstract

Sprout suppressants are widely used in industry to ensure year-round availability of potato tubers, significantly decreasing wastage by repressing premature growth of buds on the tuber surface during storage. Despite its ban from 2020 in the EU, isopropyl N-(3-chlorophenyl) carbamate (also known as chlorpropham or CIPC) remains the most widely used suppressant worldwide. However, the mechanism of action of CIPC remains obscure. Here, we report on a combined targeted transcriptomic and genetic approach to identify components in the tuber bud cell-division machinery that might be involved in CIPC's mode of action. This involved RNAseq analysis of dissected, staged tuber buds during in vitro sprouting with and without CIPC to identify lead genes, followed by the development and application of an Arabidopsis root assay to assess cell division response to CIPC in selected mutants. The ease of use of this model plant, coupled with its immense genetic resources, allowed us to test the functionality of lead genes encoding cell-division-associated proteins in the modulation of plant growth response to CIPC. This approach led to the identification of a component of the augmin complex (a core player in mitosis) as a potential target for CIPC.

Keywords: CIPC; cell division; chlorpropham; potato; sprouting.

FIGURE 1

CIPC leads to early changes in bud morphology during sprouting. SEM images of apical buds on the surface of a potato tuber at Day 0 through to Day 9 after sprouting initiation (as indicated) for explants grown without (A,B,D,F) or with (C,E,G) 100‐μM CIPC. Scale bars = 1 mm.

FIGURE 2

Principal component analysis of RNAseq data shows that CIPC‐treated buds display distinct transcriptomes. PCA of RNAseq data obtained from either control (nontreated) (circles) or CIPC‐treated (triangles) buds at time points (as indicated) after initiation of sprouting, with Day 0 data from untreated buds. The first two principal components (PC1 and PC2) are plotted to show distinctions between sample groups, with each sample is plotted as a point. Samples are colored according to time from sectioning and treatment. Clustering of point indicates a shift in the transcriptome of control samples with time that is distinct from the CIPC‐treated samples.

FIGURE 3

K‐means clustering of RNAseq data indicates cell cycle–associated genes show a distinct pattern of expression in CIPC‐treated buds compared with controls within 5 days of sprouting initiation. The top 2000 genes from RNAseq analysis showing the most variable expression (analyzed using SESeq2) were grouped using K‐means clustering to detect patterns of expression. Data have been scaled and plotted as a heatmap, as indicated by the scale. Datasets are defined by age (days after initiation of sprouting) and whether they represent untreated control or CIPC‐treated buds (as indicated). Four clusters were identified: A—abiotic stress response; B—toxic substance response/hydrogen peroxide catabolism; C—cell cycle/mitosis; D—photosynthesis. Expression was calculated by normalizing gene counts with the variance‐stabilizing transform, centering by subtracting mean expression for each gene, and scaling by dividing by gene standard deviation.

FIGURE 4

Comparison of cell cycle‐associated genes identifies 39 whose expression is downregulated within 5 days of CIPC‐treatment compared with control. Comparison of gene sets corresponding to GO‐Cell Cycle term in RNAseq data from control and CIPC‐treated buds at 0, 1, 5, and 9 days after sprouting initiation. (A) Comparison of upregulated cell cycle genes from Day 0 to Day 1. (B) Comparison of downregulated genes from Day 1 to Day 5 in CIPC‐treated samples and Day 5 to Day 9 in both conditions. The cell cycle GO term was not significantly downregulated in the control from Day 1 to Day 5. Values give absolute number of genes, with percentage indicating the relative contribution within the comparison made.

FIGURE 5

An Arabidopsis root growth assay identifies lead cell cycle genes functionally involved in the CIPC‐response. (A) Root lengths of Col‐0 seedlings at 7 days after germination on medium containing a range of CIPC concentration, as indicated. (B–D) Root lengths of T‐DNA mutants for genes identified as potentially involved in CIPC growth suppression. For each set of T‐DNA mutants analyzed, Col‐0 seedlings were assayed in parallel. Seedlings were germinated on either 10 μM (gray bars) or 20 μM (black bars) CIPC and root length measured after 7 days. Two‐way ANOVA was carried out with Dunnet's multiple comparison test for each assay, with samples showing a difference in growth after 7 days at a particular CIPC concentration indicated where p < 0.001. Bars show mean with standard error of the mean. For A–D, n > 34 per sample per treatment.