Identification of TIMING OF CAB EXPRESSION 1 as a temperature-sensitive negative regulator of tuberization in potato

Abstract

For many potato cultivars, tuber yield is optimal at average daytime temperatures in the range 14-22 °C. Above this range, tuber yield is reduced for most cultivars. We previously reported that moderately elevated temperature increases steady-state expression of the core circadian clock gene TIMING OF CAB EXPRESSION 1 (StTOC1) in developing tubers, whereas expression of the StSP6A tuberization signal is reduced, along with tuber yield. In this study we provide evidence that StTOC1 links environmental signalling with potato tuberization by suppressing StSP6A autoactivation in the stolons. We show that transgenic lines silenced in StTOC1 expression exhibit enhanced StSP6A transcript levels and changes in gene expression in developing tubers that are indicative of an elevated sink strength. Nodal cuttings of StTOC1 antisense lines displayed increased tuber yields at moderately elevated temperatures, whereas tuber yield and StSP6A expression were reduced in StTOC1 overexpressor lines. Here we identify a number of StTOC1 binding partners and demonstrate that suppression of StSP6A expression is independent of StTOC1 complex formation with the potato homolog StPIF3. Down-regulation of StTOC1 thus provides a strategy to mitigate the effects of elevated temperature on tuber yield.

Keywords: Circadian clock; StSP6A; TIMING OF CAB EXPRESSION 1; environment; heat stress; potato; tuberization.

Fig. 1.

(A) Quantitative RT–PCR of primary transgenic tissue culture leaves to assess the level of StTOC1 expression. RQ, relative quantitation. Error bars represent the SE of three biological replicates. StTOC1 expression was significantly lower than in wild-type (WT) controls as determined by Student’s t-test (P<0.05). (B) Gene expression analysis as determined by quantitative RT–PCR for tubers of selected Desiree StTOC1 antisense transgenic lines (TOCAS32, TOCAS44, and TOCAS67) compared with the WT control. Error bars represent the SE of three biological replicates. StTOC1 expression was significantly lower than in WT controls as determined by Student’s t-test (P<0.05).

Fig. 2.

Gene expression levels of StTOC1, StSP6A, and StSP5G as determined by quantitative RT–PCR of swelling stolons from line TOCAS44 compared with the wild type (WT) control. Plants were grown at normal (22 °C) and elevated (30 °C) temperature. RQ, relative quantitation. Error bars represent the SE of three biological replicates. Significant differences in expression in the transgenic lines as estimated using Student’s t-test are indicated by asterisks (P<0.05). Values labelled with the same letter were not significantly different based on one-way ANOVA with Fisher’s least significant difference test.

Fig. 3.

Comparison of tuber yields at normal (18 °C) and elevated (24, 26 °C) temperature from nodal cuttings of TOCAS32 and TOCAS44 lines compared with the wild type (WT) control. Error bars represent the SE of difference from ANOVA of the interaction term (line and temperature).

Fig. 4.

(A) Comparison of tuber yields at normal (14–18 °C) and elevated (20–26 °C) temperature from nodal cuttings of Desiree StTOC1 sense lines TOCSNS8, TOCSNS12, and TOCSNS14 compared with the wild type (WT). Error bars represent the SE of difference from ANOVA of the interaction term (line and temperature). (B) Comparison of tuber yield in stem node cuttings from WT plants and lines TOCHA3 and TOCHA11 overexpressing the tagged StTOC1-HA protein. Plants were grown at a daytime maximum temperature of 18 °C. Significant differences between transgenic lines and the WT, as estimated using Student’s t-test, are indicated by asterisks (P<0.01). (C, D) Comparison of gene expression levels of StTOC1 (C) and StSP6A (D) as determined by quantitative RT–PCR of leaves from lines TOCHA3 and TOCHA11 compared with WT. Plants were grown at a daytime maximum temperature of 18 °C. RQ, relative quantitation. Error bars represent the SE of three biological replicates. Significant differences between transgenic lines, as estimated using Student’s t-test, are indicated by asterisks (P<0.01).

Fig. 5.

Relative luminescence units (RLU) from transient expression studies of the 2.64 kb StSP6A promoter fused to the LUC reporter gene (p6A::LUC) co-transformed with the 35S::StTOC1, 35S::StPIF3, and 35S::StSP6A effector constructs. Agrobacterium tumefaciens cells containing these constructs were co-infiltrated into N. benthamiana leaves and luciferase activity was measured in excised leaf discs at intervals of 1 h for 14 h. Error bars represent the SD of n=12 leaf discs. Studies were repeated three times, with similar results.

Fig. 6.

In planta protein–protein interactions between StTOC1, StPIF3, and StSP6A. Representative confocal microscopy images showing the reconstituted BiFC fluorescence in epidermal N. benthamiana cells. YFP fluorescence generated by protein–protein interaction was detected 2 days after infiltration. The interactions tested are indicated in panels A–I. (C) and (F) show the StSP6A-YFN43 and StTOC1-YFN43 fusions infiltrated with the empty YFC43 vector, used as negative controls. Weak fluorescence in the cytosol and nucleus was observed for the StSP6A-YFN43 negative control (C). Strong fluorescence by the reconstituted YFP protein (A) denotes that StSP6A and StTOC1 interact in the nucleus. BiFC studies confirmed a nuclear interaction of the StPIF3 and StTOC1 proteins (E), and the dimerization of StTOC1 (D). No YFP fluorescence was observed in leaves infiltrated with the StSP6A-YFN43 and StPIF3-YFC43 constructs (B), indicating that these proteins do not interact. (G), (H), and (I) are higher magnifications of (A), (D), and (E), respectively.

Fig. 7.

Hierarchical tree graph showing the over-represented AgriGO v2 GO terms for the genes differentially expressed in TOCAS swelling stolons, The Phureja DM1-3 PGSC protein ID for the entire genome was used as background, and the expression data were analysed using the Fisher test method, Yekutieli false discovery rate, at a P-value≤0.05, with a minimum of 10 mapping entries. Boxes represent GO terms labelled by their GO ID. Box colours indicate significance (e) levels: lower e-values are red, through orange, to yellow for higher e-values; non-significant terms are shown in white boxes.