Arabidopsis CBF3 and DELLAs positively regulate each other in response to low temperature

Abstract

The C-repeat binding factor (CBF) is crucial for regulation of cold response in higher plants. In Arabidopsis, the mechanism of CBF3-caused growth retardation is still unclear. Our present work shows that CBF3 shares the similar repression of bioactive gibberellin (GA) as well as upregulation of DELLA proteins with CBF1 and -2. Genetic analysis reveals that DELLAs play an essential role in growth reduction mediated by CBF1, -2, -3 genes. The in vivo and in vitro evidences demonstrate that GA2-oxidase 7 gene is a novel CBF3 regulon. Meanwhile, DELLAs contribute to cold induction of CBF1, -2, -3 genes through interaction with jasmonate (JA) signaling. We conclude that CBF3 promotes DELLAs accumulation through repressing GA biosynthesis and DELLAs positively regulate CBF3 involving JA signaling. CBFs and DELLAs collaborate to retard plant growth in response to low temperature.

Figure 1. CBF3 suppresses plant growth through negative regulation of bioactive GA level and GA reduction in low temperature is mediated by CBF3.

(a) Representative phenotypes of 4-week-old CBF1-ox, CBF2-ox and CBF3-ox plants with or without GA₃ application. Dwarfism caused by CBF1, -2, -3 overexpression can be partially rescued by 10⁻⁵ M GA₃ application. Phenotypes including (b) the areas of fifth rosette leaves, (c) the final heights, (d) the rosette leaf numbers and (e) GA₁₊₃ contents are shown. In cbf3 mutant cold induced growth repression and GA reduction are weakened according to (f–h) growth phenotypes and (i) GA₁₊₃ contents. (SE, n = 20, *P < 0.05, **P < 0.01).

Figure 2. CBF3 enhances DELLA accumulation in low temperature and do not affect GA-mediated DELLA degradation.

(a) GFP fluorescence in the root tip (first row) and elongating zone (second row) of 8-day-old of pRGA::GFP:RGA, CBF1-ox pRGA::GFP:RGA, CBF2-ox pRGA::GFP:RGA and CBF3-ox pRGA::GFP:RGA seedlings. Images are taken with identical parameters for comparison of fluorescence levels. (b) Immunoblot analysis of GFP:RGA levels in leaves from 4-week-old plants indicated. (c) GFP:RGA levels in 8-d-old seedlings treated at 12 °C for 4 h. The β-tubulin is used as loading control.

Figure 3. The della-global mutation weakens growth retardation caused by low temperature and CBF1, -2, -3 overexpression.

(a–c) Comparison of Ler and della-global plants. Up or down arrows represent increase or decrease relative to wild type, respectively. (d–f) Comparison between Ws and CBF1-ox, CBF2-ox and CBF3-ox plants as well as comparison between della-global and CBF1-ox della-global plants, CBF2-ox della-global plants, CBF3-ox della-global plants. (SE, n = 20, *P < 0.05, **P < 0.01).

Figure 4. CBF3 increases transcript levels of RGL3, GA2ox3 and GA2ox7.

(a) Relative expression levels of GA metabolism and signaling genes in Ws and CBF1-ox, CBF2-ox and CBF3-ox plants. (b) Relative expression levels of RGL3, GA2ox3 and GA2ox7 in Ws plants under 12 °C treatment. (c) Relative expression levels of RGL3, GA2ox3 and GA2ox7 in Col and cbf3 plants under 12 °C treatment. Data are means ± SE.

Figure 5. GA2ox7 is a CBF3 regulon.

(a) Schematic diagram of three CRT/DRE-like elements in the promoter region of GA2ox7. Core sequences of L1, L2 and L3 are shown. (b) ChIP qRT-PCR analysis of CBF3 binding to the three CRT/DRE-like elements of GA2ox7. The −0.2 kb to −0.35 kb promoter region of RD29a containing three CRT/DRE-like elements serves as positive control and one area without CRT/DRE-like elements in the CBF-noninduced gene GAI is used as negative control. Data are means ± SE. (c) Oligonucleotides of L2 and L2-m (mutated version) elements within the GA2ox7 promoter used in the EMSA. Underlined letters are core sequences of CRT/DRE. Three nucleotides are substituted in L2-m. (d) CBF3 binds to L2 element of GA2ox7 promoter in vitro. Unlabeled L2 and L2-m elements fragment are used as competitors. (e) Dual-LUC Assays using transient expression system in tobacco leaves. CBF3 driven by 35S promoter was served as the effector and LUC under control of GA2ox7 promoter truncations as indicated were reporters. The relative activity (LUC/REN) were shown. Reporters co-transformed with the blank pC1304 vector were used as controls. Data are means ± SE.

Figure 6. DELLAs contribute to cold induction of CBF1, -2, -3 through interaction with JA signaling.

(a–c) Altered cold induction levels of CBF1, -2, -3 in Ler and della-global plants under GA₃, MeJA, GA₃ together with MeJA or 0.1% ethanol treatments. (d) Expression level of CBF1, -2, -3 in Ler and della-global plants under GA₃ or MeJA treatments at 22 °C. (e) CBF3 expression level under 12 °C and 4 °C in seedlings indicated. Data are means ± SE.

Figure 7. Model for positive regulation between CBF3 and DELLAs in response to low temperature.

In warm temperature, DELLAs interact with JAZs to prevent JAZs binding to ICE1. Meanwhile, DELLAs are degraded through GA mediated signaling. In cold temperature, ICE1 is modified to gain the function for activation of CBF3 transcription. CBF3 activates GA2ox7 to decrease the bioactive GA level and subsequently promotes the accumulation of DELLAs. Increased DELLAs release more ICE1 to enhance next round of CBF3 cold induction.