CRISPR/Cas9 editing of CBP80 enhances drought tolerance in potato (Solanum tuberosum)

Abstract

Developing drought-tolerant potato varieties is increasingly important due to climate change and water scarcity, as potatoes are highly sensitive to water deficits that can significantly reduce yield and tuber quality. The cap-binding protein CBP80, involved in the abscisic acid (ABA) signalling pathway, has emerged as a promising target for improving drought tolerance in plants. In this study, we used CRISPR/Cas9 to edit the StCBP80 gene in the tetraploid potato cultivar Spunta. Given the complexity of editing all four alleles in a tetraploid genome, eight independent partially edited lines (two or three alleles edited) were obtained. Two of these lines were selected for detailed molecular and phenotypic characterization. Under restricted water conditions, the selected lines exhibited reduced transpiration rates and improved leaf area index compared to non-edited controls. Gene expression analysis by quantitative real-time PCR showed differential expression of drought-responsive genes (P5CS, PDH, and MYB33), supporting a role for StCBP80 in stress response modulation. Moreover, the edited lines showed lower yield penalties, both in biomass and tuber production, under drought stress. This work represents one of the first applications of genome editing to enhance drought tolerance in a commercial potato cultivar, and highlights CBP80 as a promising target for crop improvement. These findings provide valuable insights for the development of stress-resilient potato varieties using genome editing approaches.

Keywords: abiotic stress; abscisic acid; cap binding proteins; climate change; genome edited plants.

Figure 1

Schematic representation of the StCBP80 gene of Solanum tuberosum (potato), which is composed of 19 exons interspersed with intronic regions. In this simplified representation, each “Exon” is depicted as a box, and each “Intron” is shown as a line separating the exons. The lengths of the boxes and lines are not proportional to the actual sequences but are designed to illustrate the gene’s structural organization. A zoomed-in view of the first four exons is shown, highlighting the design site of two sgRNAs: sgRNAG9 (exon 1), and sgRNAG104 (exon 2). These sgRNAs were strategically positioned for CRISPR/Cas9-mediated editing to disrupt the StCBP80 gene function. The information on the gene structure and sgRNA locations was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/gene/102588913).

Figure 2

Sequences highlighted in blue indicate the nucleotide sequences of the non-edited control StCBP80 gene and the edited lines CBP80-39 and CBP80-32 at the sgRNA-G9 target site. Mutations detected in the edited lines include a single-base insertion (A) in one allele and two independent single-base deletions (G) in the CBP80-39 line. In the CBP80-32 line, a single-base deletion (G) and a single-base insertion (A) were detected in two distinct alleles.

Figure 3

Relative expression levels of StCBP80 (A), PDH (B), P5CS (C), and MYB33 (D) in the non-edited control and the edited lines (CBP80-32 and CBP80-39) under well-watered (WW) and water-deficit (WD) conditions. Statistical analyses were conducted using unpaired, two-tailed Student’s t-tests with Welch’s correction for unequal variances, applying a significance threshold (alpha) of 0.05. The comparisons presented are: non-edited control WD versus CBP80-39 WD, and non-edited control WD versus CBP80-32 WD. A single asterisk (*) denotes statistically significant differences (p < 0.05), while a double asterisk (**) indicates highly statistically significant differences (p < 0.01). Five biological samples were used for each genotype and condition, with three technical replicates per sample.

Figure 4

(A) Temporal variation of canopy cover for edited lines (CBP80-32 and CBP80-39) and the non-edited control under two water regimes: well-watered (WW) and water-deficit (WD). The water-deficit phase occurred between days 50 and 80, followed by rewatering from day 81 onwards. Data represent mean ± standard error for each treatment (n= 5 plants per genotype and water condition). (B) Temporal variation of canopy cover for edited lines (CBP80-32 and CBP80-39) and the non-edited control during the assay.

Figure 5

Transpiration rate as a function of (i) genotype (edited lines and non-edited control, upper panel) and of (ii) plant cover (%, lower panel) for well irrigated plants measured over the experimental period. 32: CBP80-32, 39: CBP80-39 and N-Ed: non-edited control. Different letters indicate significant differences statistically significant differences (p < 0.05). (n= 5 plants per genotype and water condition).

Figure 6

Transpiration rate as a function of genotype for water-limited plants during the drying period (orange bars) and after rewatering (blue bars). Different letters indicate significant differences among genotypes (p<0.05) in each day of measurement. Well-irrigated plants throughout the experiment are included as a reference (grey bars). 32: CBP80-32, 39: CBP80-39 and N-Ed: non-edited control. (n= 5 plants per genotype and water condition). No error bars are shown at days 7 and 13 for water-limited plants because transpiration measurements began only after the soil water content dropped to 30% of field capacity.

Figure 7

(A) Stomatal density in StCBP80-edited lines and non-edited control. The graph represents stomatal density (stomata mm^-2) on the abaxial leaf surface of the StCBP80-edited lines (CBP80-32 and CBP80-39) and the non-edited control. Error bars indicate standard deviation. *Above the bars indicate statistically significant differences (p < 0.05). 32: CBP80-32, 39: CBP80-39 and N-Ed: non-edited control. (B) Representative microscopic images of stomata impressions.

Figure 8

Normalized transpiration rate (NTR) as a function of the fraction of transpirable soil water (FTSW) for lines 32 and 39, and the non-edited control. The threshold of FTSW for the onset of NTR sharp decline (FTSWt) and the slope of NTR decline above FTSWt are indicated in each panel, with confidence intervals shown in brackets. 32: CBP80-32, 39: CBP80-32 and SP: non-edited control.

Figure 9

Tuber yield per plant (g) in the edited lines (CBP80-32 and CBP80-39) and the non-edited control under both well-watered (WW) and water-deficit (WD) conditions. * Above the bars indicate statistically significant differences (p < 0.05) and ns indicate non statistically significant differences (p ≥ 0.05), (n= 5 plants per genotype and water condition).

Figure 10

Yield per plant (g plant^-1) as a function of cumulative transpiration per plant (L plant^-1) in the non-edited control (yellow symbols) and the edited lines CBP80-32 (blue symbols) and CBP80-39 (orange symbols) across different water regimes.