Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato

Abstract

Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

Figure 1.

SlREC2 positively regulates cold tolerance in tomato. A) Phylogenetic analysis of REC proteins in S. lycopersicum, A. thaliana, O. sativa, M. lewisii and M. verbenaceus. The percentage at branch represents the posterior probabilities of amino acid sequences. B) Expression of SlREC family genes in tomato plants after exposure to 25 °C or 4 °C for 6 h. C, D) Phenotypes (C) and the accumulation of hydrogen peroxide (DAB staining) and superoxide (NBT staining) in tomato leaves (D) after the exposure of plants to 25 °C or 4 °C for 7 d. The plants (C) and leaves (D) were digitally extracted for comparison, respectively. Bar in (C), 5 cm. Bar in (D), 2 cm. E–H) REL (E), net CO₂ assimilation rate (Pn; F), the chlorophyll a fluorescence transient (OJIP) curves (G), and changes in the maximum photochemical efficiency of PSII (F_v/F_m) (H) in tomato wild-type (pTRV) and SlREC2-silenced plants (pTRV-SlREC2) after exposure to 25 °C or 4 °C for 7 d. The false-color code depicted at the bottom of the image ranges from 0 to 1.0, representing the level of damage in the leaves. Bars in (H), 2 cm. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 2.

SlREC2 is essential for alleviating cold-induced photoinhibition in tomato. A, C) Changes in PSII parameters, including the maximum photochemical efficiency of PSII (F_v/F_m; A), and the effective quantum yield of PSII [Y(II)], the quantum yield of regulated energy dissipation of PSII (NPQ), and the photochemical quenching coefficient (qP; C) in tomato wild-type (pTRV) and SlREC2-silenced plants (pTRV-SlREC2) after exposure to 25 °C or 4 °C for 5 d. B, D) Changes in PSI parameters, including the maximum P700 photooxidation level (P_m; B), and the quantum yield of PSI [Y(I)], the donor limitation of PSI [Y(ND)], the acceptor side limitation of PSI [Y(NA)] (D) in tomato wild-type (pTRV) and SlREC2-silenced plants (pTRV-SlREC2) after exposure to 25 °C or 4 °C for 5 d. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 3.

ABA plays a critical role in SlREC2-regulated cold tolerance in tomato. A), ABA content in tomato wild-type (pTRV) and SlREC2-silenced plants (pTRV-SlREC2) after exposure to 25 °C and 4 °C for 12 h. B–D) REL (B) and F_v/F_m (C, D) of pTRV and pTRV-SlREC2 plants as influenced by foliar application of ABA and NDGA (ABA-inhibitor) under cold-stress conditions (4 °C for 7 d). The false-color code depicted at the bottom of the image ranges from 0 to 1.0, representing the level of damage in the leaves. Bars in (C), 2 cm. E, F) NPQ (E) and OJIP curves (F) of pTRV and pTRV-SlREC2 plants as influenced by foliar application of ABA and NDGA under 4 °C for 5 d. Fifty micromolar ABA or NDGA was applied 12 h prior to exposure to cold conditions at 4 °C. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 4.

SlNCED1 acts downstream of SlREC2 in the cold response. A) SlNCED1 gene expression in tomato wild-type (pTRV) and SlREC2-silenced plants (pTRV-SlREC2) after exposure to 25 °C and 4 °C for 6 h. B–D) REL (B) and F_v/F_m (C, D) in tomato plants when silenced or nonsilenced SlREC2 (pTRV-SlREC2 or pTRV) in wild type and SlNCED1-deficient mutant (not) after exposure to 4 °C for 7 d. The false-color code depicted at the bottom of the image ranges from 0 to 1.0, representing the level of damage in the leaves. Bars in (C), 2 cm. E, F) NPQ (E) and OJIP curves (F) in tomato plants when silenced or nonsilenced SlREC2 (pTRV-SlREC2 or pTRV) in wild-type and SlNCED1-deficient mutant (not) after exposure to 4 °C for 5 d. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 5.

SlREC2 interacts with SlBCH1b. A) Subcellular localization of SlREC2 fused to GFP in N. benthamiana leaf mesophyll cells. GFP, green fluorescent protein; DAPI, a fluorescent dye (4′,6-diamidino-2-phenylindole) used to label normal nuclei; Chl, chlorophyll autofluorescence; Bright, brightfield. The arrowheads point to nuclei. Scale bars: 25 µm. B) Y2H assay showing the interaction of SlREC2 with SlBCH1b. SlREC2 was fused to the DNA activation domain (AD), while SlBCH1a and SlBCH1b were fused to the DNA binding domain (BD) of GAL4. DDO, yeast synthetic medium without Trp/Leu; QDO, yeast synthetic medium without Trp/Leu/His/Ade, but with 40 µg mL⁻¹ of X-α-gal and 100 ng mL⁻¹ aureobasidin A. C) Interaction of SlREC2 and SlBCH1b detected by BiFC analysis. SlBCH1b was fused to the C-terminal fragment of YFP (cYFP) and SlREC2 was fused to the N-terminal fragment of YFP (nYFP). The construct combinations were cotransformed into N. benthamiana leaves and expressed for 48 h. The signal was detected by confocal microscopy. Bar, 25 μm. D) LUC complementation imaging assay showing the interaction of SlREC2 and SlBCH1b in N. benthamiana leaves. SlREC2-nLUC/SlBCH1b-cLUC, SlREC2-nLUC/cLUC, nLUC/SlBCH1b-cLUC, and nLUC/cLUC were cotransformed into N. benthamiana leaves and investigated after 72 h. Similar results were obtained in three independent experiments.

Figure 6.

SlBCH1b positively regulates cold tolerance in tomato. A, B) SlBCH1b gene expression (A) and REL (B) in tomato wild-type (pTRV) and SlBCH1b-silenced plants (pTRV-SlBCH1b) after exposure to 25 °C or 4 °C for 6 h and 7 d, respectively. C–F) Phenotypes (C), F_v/F_m (D, E), and P_m (F) in pTRV and pTRV-SlBCH1b plants after exposure to 25 °C or 4 °C for 7 d. The plants were digitally extracted for comparison in (C). Bar in (C), 5 cm. Bars in (D), 2 cm. The false-color code depicted at the bottom of the image ranges from 0 to 1.0, representing the level of damage in the leaves. G, H) OJIP curves (G) and the accumulation of superoxide (NBT staining) and hydrogen peroxide (DAB staining) in tomato leaves (H) after pTRV and pTRV-SlBCH1b plants exposure to 25 °C or 4 °C for 7 d. The plants were digitally extracted for comparison. Bar in (H), 2 cm. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 7.

SlBCH1b regulates CBF-, ABA-, and xanthophyll cycle-pathway genes expression, ABA accumulation, and NPQ changes in response to cold stress. A, B) Expression of SlCBF1 (A) and SlCBF2 (B) genes in tomato wild-type (pTRV) and SlBCH1b-silenced plants (pTRV-SlBCH1b) after exposure to 25 °C and 4 °C for 6 h. C, D) Expression of SlNCED1 (C) and accumulation of ABA (D) in pTRV and pTRV-SlBCH1b plants after exposure to 25 °C and 4 °C for 6 and 12 h, respectively. E, F) Changes of SlZEP1 gene expression (E) and NPQ (F) in pTRV and pTRV-SlBCH1b plants after exposure to 25 °C and 4 °C for 6 h and 5 d, respectively. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 8.

SlREC2 and SlBCH1b act additively to enhance cold tolerance in tomato plants. A–F) REL (A), F_v/F_m (B, C), NPQ (D), the accumulation of superoxide (NBT staining; E) and hydrogen peroxide (DAB staining; F) in tomato wild-type (pTRV), SlREC2-silenced plants (pTRV-SlREC2), SlBCH1b-silenced plants (pTRV-SlBCH1b), and the co-silenced plants of these two genes (pTRV-SlREC2/SlBCH1b) after exposure to 25 °C or 4 °C for 7 d. Bars in (B), 2 cm. The false-color code depicted at the bottom of the image ranges from 0 to 1.0, representing the level of damage in the leaves. The plants were digitally extracted for comparison in (E) and (F), respectively. Bars in (E) and (F), 2 cm. G, H) Expression of SlCBF1 and SlNCED1 in pTRV, pTRV-SlREC2, pTRV-SlBCH1b, and pTRV-SlREC2/SlBCH1b plants after exposure to 25 °C or 4 °C for 6 h. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey's test.

Figure 9.

A proposed model explaining the regulatory mechanism of SlREC2-mediated cold response in tomato. SlREC2 transcript levels significantly upregulated when tomato plants are exposed to cold stress. SlREC2 rapidly induces the gene expression of SlBCH1b and interacts with SlBCH1b protein during cold stress. Subsequently, SlREC2 and SlBCH1b act synergistically to induce SlNECD1-mediated ABA accumulation most likely through enhancing their protein stability or the transcriptional activity of other unknown transcription factors (X) on the SlNECD1 gene. ABA increases the expression levels of CBF genes and photoprotection, thereby enhancing cold tolerance in tomato.