The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism

Abstract

Plants have evolved robust mechanisms to respond and adapt to unfavorable environmental conditions, such as low temperature. The C-repeat/drought-responsive element binding factor CBF1/DREB1b gene encodes a transcriptional activator transiently induced by cold that controls the expression of a set of genes responding to low temperature (the CBF regulon). Constitutive expression of CBF1 confers freezing tolerance but also slows growth. Here, we propose that low temperature-induced CBF1 expression restrains growth at least in part by allowing the accumulation of DELLAs, a family of nuclear growth-repressing proteins, the degradation of which is stimulated by gibberellin (GA). We show that cold/CBF1 enhances the accumulation of a green fluorescent protein (GFP)-tagged DELLA protein (GFP-RGA) by reducing GA content through stimulating expression of GA-inactivating GA 2-oxidase genes. Accordingly, transgenic plants that constitutively express CBF1 accumulate less bioactive GA and as a consequence exhibit dwarfism and late flowering. Both phenotypes are suppressed when CBF1 is expressed in a line lacking two DELLA proteins, GA-INSENSITIVE and REPRESSOR OF GA1-3. In addition, we show that DELLAs contribute significantly to CBF1-induced cold acclimation and freezing tolerance by a mechanism that is distinct from the CBF regulon. We conclude that DELLAs are components of the CBF1-mediated cold stress response.

Figure 1.

Low Temperature Slows Root Growth via a DELLA-Dependent Mechanism. Mean (±se) relative growth of primary roots of 8-day-old wild-type (white), gai-t6 rga-24 (light gray), and the quadruple-DELLA mutant (gai-t6 rga-t2 rgl1-1 rgl2-1; dark gray) seedlings were grown for 5 d in alternating cold (4°C)/warm (22°C) temperatures as indicated (expressed as percentage of non-cold-treated control for each genotype). An asterisk indicates a significant difference between the wild type and gai-t6 rga24/quadruple-DELLA mutant (P < 0.05).

Figure 2.

Cold Enhances CBF1 Transcript and GFP-RGA Protein Levels. (A) Immunodetection of GFP-RGA (with an antibody to GFP) in 7-d-old pRGA:GFP-RGA seedlings that had been subjected to cold (4°C) for the time (2, 4, 8, and 24 h) indicated (and controls, M). PSTAIRE serves as sample loading control. Numbers below the gels represent the fold increase in GFP-RGA protein levels relative to PSTAIRE levels. (B) Expression of CBF1, RD29a, and RGA genes and the GFP-RGA transgene upon cold for the time as in (A) determined by RNA gel blot analysis. Each lane was loaded with 15 μg of total RNA prepared from 7-d-old pRGA:GFP-RGA seedlings. (C) Levels of GAI, RGA, RGL1, RGL2, and RGL3 gene transcripts determined by a quantitative RT-PCR experiment in 7-d-old wild-type seedlings that had been subjected to cold for 4 and 12 h as indicated (and controls, M). ELF1α transcripts provide loading control.

Figure 3.

CBF1 Represses Growth and Delays Floral Transition through the GA-Signaling Pathway. (A) Expression of CBF1 gene in wild-type, CBF1-ox, and CBF1-ox gai-t6 rga-24 plants. Each lane was loaded with 15 μg of total RNA prepared from 2-week-old wild-type and transgenic plants. The RNA gel blot was hybridized with a specific probe for CBF1. (B) Representative 4-week-old wild-type, CBF1-ox, and CBF1-ox gai-t6 rga-24 plants treated with GA (+GA) or control (–GA). (C) and (D) Mean (±se) plant height (cm) (C), and mean (±se) rosette leaf number (D) of 4-week-old wild-type, CBF1-ox, and CBF1-ox gai-t6 rga-24 plants GA-treated (violet) or control (blue).

Figure 4.

CBF1 Represses Plant Growth by Enhancing DELLA Accumulation. (A) GFP fluorescence (viewed by fluorescence confocal microscopy) in nuclear cells of the root tip (RT) and elongating zone (EZ) of 7-d-old seedlings of the pRGA:GFP-RGA (wild-type) and CBF1-ox pRGA:GFP-RGA lines. Images were taken with identical parameters to allow for comparison of fluorescence intensities. (B) Immunodetection of GFP-RGA (with an antibody to GFP) in 7-d-old seedlings of pRGA:GFP-RGA (wild-type) and CBF1-ox pRGA:GFP-RGA lines. PSTAIRE serves as sample loading control. (C) Levels of GAI, RGA, RGL1, RGL2, and RGL3 gene transcripts determined by quantitative RT-PCR experiment in 7-d-old wild-type and CBF1-ox seedlings. ELF1α transcripts provide loading control.

Figure 5.

Cold and CBF1-ox Upregulate GA 2-oxidase Transcript Levels. (A) Relative levels of GA biosynthesis GA20ox1, GA20ox2, GA20ox3, GA3ox1, and GA3ox2 and GA deactivation of GA2ox1, GA2ox2, GA2ox3, GA2ox4, and GA2ox6 gene transcripts (determined by real-time RT-PCR) in 2-week-old wild-type seedlings that had been subjected to cold for 4 h (light gray) and 12 h (dark gray) and control non-cold-treated (Mock, black). Data are means ± se. (B) Relative levels of GA metabolism gene transcripts determined by real-time RT-PCR (as in [A]) in 2-week-old wild-type (black), CBF1-ox (light gray), and CBF1-ox gai-t6 rga-24 (dark gray) plants.

Figure 6.

DELLAs Contribute to CBF1-Induced Freezing Tolerance and Cold Acclimation. Freezing tolerance (expressed as rate of survival [%]; three replicates; n = 24) of 2-week-old wild-type, CBF1-ox, CBF1-ox gai-t6 rga-24, gai, and gai-t6 rga-24 plants nonacclimated (A) or cold-acclimated (4°C, 3 d) (B). Plants were exposed for 24 h at −10°C, and survival was scored after 4 d of recovery under controlled growth conditions. The asterisks indicate no significant difference (P < 0.05) between wild-type and CBF1-ox gai-t6 rga-24 cold-acclimated plants.

Figure 7.

CBF1 Enhances Both CBF Regulon and Sugar Levels in a DELLA-Independent Fashion. (A) Relative levels of RD29a, COR15a, COR47, and P5CS2 gene transcripts (determined by real-time RT-PCR) in 2-week-old wild-type, CBF1-ox, and CBF1-ox gai-t6 rga-24 plants. Data are means ± se. (B) Mean (±se) total soluble sugars (glucose, fructose, and sucrose) were determined from leaf tissue of 2-week-old wild-type, CBF1-ox, CBF1-ox gai-t6 rga-24, gai, and gai-t6 rga-24 plants GA-treated (dark gray) or not (light gray). The levels of individual sugars were determined by enzymatic assays.

Figure 8.

Scheme Showing Contribution of DELLAs to CBF1-Mediated Growth Regulation and Freezing Tolerance. Cold-induced or constitutive expression of CBF1 reduces bioactive GA levels (by increasing of GA2ox3 and GA2ox6 transcript abundance), thus promoting the accumulation in DELLAs by change in their protein stability. In addition, cold/CBF1 specifically increases RGL3 transcript levels. Low temperature also enhances GA2ox1 expression levels via a CBF1-independent mechanism. DELLA accumulation in turn slows plant growth. CBF1 also enhances freezing tolerance through the synergistic DELLA-dependent and COR-dependent pathways. Cold also restrains growth and promotes freezing tolerance by a DELLA-independent and CBF-independent cold response pathway.