Down-Regulation of Cytokinin Receptor Gene SlHK2 Improves Plant Tolerance to Drought, Heat, and Combined Stresses in Tomato

Abstract

Environmental stresses negatively affect the growth and development of plants. Several previous studies have elucidated the response mechanisms of plants to drought and heat applied separately; however, these two abiotic stresses often coincide in environmental conditions. The global climate change pattern has projected that combined drought and heat stresses will tend to increase in the near future. In this study, we down-regulated the expression of a cytokinin receptor gene SlHK2 using RNAi and investigated the role of this gene in regulating plant responses to individual drought, heat, and combined stresses (drought + heat) in tomato. Compared to the wild-type (WT), SlHK2 RNAi plants exhibited fewer stress symptoms in response to individual and combined stress treatments. The enhanced abiotic stress tolerance of SlHK2 RNAi plants can be associated with increased membrane stability, osmoprotectant accumulation, and antioxidant enzyme activities. Furthermore, photosynthesis machinery was also protected in SlHK2 RNAi plants. Collectively, our results show that down-regulation of the cytokinin receptor gene SlHK2, and consequently cytokinin signaling, can improve plant tolerance to drought, heat, and combined stress.

Keywords: abiotic stress; antioxidants; combined stress; cytokinin; drought stress; heat stress; photosynthesis; tomato.

Figure 1

Phenotypes of the WT and SlHK2 RNAi (L1 and L2) lines under well-watered (control) and drought, heat, and combined stress conditions. The 5-week-old SlHK2 RNAi and WT plants grown under well-watered conditions are used for treatment. Drought: 5 days of no watering; heat: 5 days of 32/26 °C (day/night) with watering once every day; combined D + H: 5 days of no watering and 32/26 °C (day/night).

Figure 2

(a) Relative water content %, (b) electrolyte leakage %, transcript levels of (c) SlDREB1, (d) SlNCED, (e) SlHSP 17.4, and (f) SlHSP 21, in leaves of WT and SlHK2 RNAi plants grown under well-watered (C), drought (D), heat (H), and combined stress (D + H) conditions. Values represent mean ± SE (n = 6). The relative expression levels were determined by quantitative real-time PCR using the SlActin gene as reference. Different letters indicate significant differences at p < 0.05 among treatments.

Figure 3

Transcript levels of (a) SlCER3, (b) SlLPT1, (c) SlTTS1, (d) SlKCS1, and (e) cuticular wax accumulation, in leaves of WT and SlHK2 RNAi plants. For the quantitative real-time PCR analysis, the plants were used after five days of well-watered (C), drought (D), heat (H), and combined stress (D + H) conditions. The relative expression levels were determined by quantitative real-time PCR using the SlActin gene as reference. The well-watered SlHK2 RNAi and WT plants were used to determine cuticular waxes. Cuticular waxes were extracted and analyzed using GC-MS. Values represent mean ± SE (n = 6). Different letters indicate significant differences among treatments at p < 0.05.

Figure 4

(a) Photosynthetic rate (A, in µmol m⁻²s⁻¹), (b) stomatal conductance (gs, in µmol H₂O m⁻²s⁻¹), (c) PSII operating efficiency (Fq’/Fm’), (d) electron transport rate (ETR), (e) non-photochemical quenching (NPQ), (f) the fraction of open PSII centers (qL), (g) maximum photochemical yield of photosystem II (Fv/Fm), (h) transcript levels of the photosystem II oxygen-evolving complex protein gene (PsbP), (i) photosystem II oxygen-evolving complex protein gene (PsbQ), and (j) chlorophyll fluorescence imaging (Fv/Fm), in leaves of WT and SlHK2 RNAi lines (L1 and L2). The color range shows the plants’ values from black (minimum) to pink (maximum) in chlorophyll fluorescence imaging. Spots on the leaf circle display areas damaged by these stresses in (j). The relative expression levels were determined by quantitative real-time PCR (using SlActin gene as reference). Different letters show significant differences among treatments at p < 0.05.

Figure 5

(a) Malondialdehyde content (MDA), (b) hydrogen peroxide (H₂O₂), and (c) histochemical staining results of H₂O₂ in WT and SlHK2 RNAi plants after 5 days of drought (D), heat (H), and combined (D + H) stresses. For H₂O₂ staining, six leaves each of WT and SlHK2 RNAi lines (L1 and L2) were used for each treatment. Values represent mean ± SE (n = 6). Different letters indicate significant differences among treatments at p < 0.05.

Figure 6

The activities of (a) glutathione reductase (GR), (b) ascorbate peroxidase APX, (c) superoxide dismutase (SOD), (d) peroxidase (POD), (e) dehydroascorbate reductase (DHAR), (f) catalase (CAT), (g) proline, and (h) osmoprotectants in leaves of the WT and SlHK2 RNAi tomato plants under drought (D), heat (H) and combined (D + H) stresses. Blue represents a higher relative concentration, while green represents the lower relative concentration of the heat map in WT and SlHK2 RNAi lines (L1 and L2). Values represent mean ± SE (n = 6). Different letters indicate significant differences among treatments at p < 0.05.

Figure 7

Transcript levels of (a) SlGR1, (b) SclAPX, (c) SlSOD, (d) SlCAT1, (e) SlSPS, (f) SlP5CS, and (g) SlT6PS genes, in leaves of SlHK2 RNAi and WT tomato plants. Control, normal (hydrated) conditions (C); drought stress, 5 days no watering (D); heat stress, 5 days 32/26 °C day/night (H); combined drought and heat stress, 5 days no watering + 32/26 °C day/night for 5 days (D + HS). The relative expression level is determined by quantitative real-time PCR (using the SlActin gene as reference). Values represent mean ± SE (n = 6). Different letters show significant differences among treatments at p < 0.05.