A Gas-and-Brake Mechanism of bHLH Proteins Modulates Shade Avoidance

Abstract

Plants detect proximity of competitors through reduction in the ratio between red and far-red light that triggers the shade avoidance syndrome, inducing responses such as accelerated shoot elongation and early flowering. Shade avoidance is regulated by PHYTOCHROME INTERACTING FACTORs, a group of basic helix-loop-helix (bHLH) transcription factors. Another (b)HLH protein, KIDARI (KDR), which is non-DNA-binding, was identified in de-etiolation studies and proposed to interact with LONG HYPOCOTYL IN FAR-RED1 (HFR1), a (b)HLH protein that inhibits shade avoidance. Here, we established roles of KDR in regulating shade avoidance in Arabidopsis (Arabidopsis thaliana) and investigated how KDR regulates the shade avoidance network. We showed that KDR is a positive regulator of shade avoidance and interacts with several negative growth regulators. We identified KDR interactors using a combination of yeast two-hybrid screening and dedicated confirmations with bimolecular fluorescence complementation. We demonstrated that KDR is translocated primarily to the nucleus when coexpressed with these interactors. A genetic approach confirmed that several of these interactions play a functional role in shade avoidance; however, we propose that KDR does not interact with HFR1 to regulate shade avoidance. Based on these observations, we propose that shade avoidance is regulated by a three-layered gas-and-brake mechanism of bHLH protein interactions, adding a layer of complexity to what was previously known.

Figure 1.

Arabidopsis kdr mutants exhibit a deviating low R:FR response. A, Relative expression of KDR determined by RT-qPCR in wild-type Col-0 shoots grown in control light conditions (R:FR = 2) and low R:FR (R:FR = 0.2) for 90 min. Data represent means ± se, n = 4. Asterisks indicate statistically significant difference by Student’s t test (***P < 0.001). B, Hypocotyl length of seedlings of Arabidopsis wild type (Col-0), knockout line (kdr-1), or activation-tagged line (kdr-D) grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 d of light treatment. Data represent means ± se, n = 76. C, Change in petiole length (Δ Petiole length) of Arabidopsis wild-type (Col-0) rosette plants, knockout line (kdr-1), or activation-tagged line (kdr-D) grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 24 h of light treatment. Plants were grown in short (left)- or long-day (right) conditions. Data represent means ± se, n = 10. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05).

Figure 2.

Heterologous overexpression of KDR leads to long hypocotyl. A, Hypocotyl length of seedlings of Arabidopsis wild type (Col-0), activation-tagged line (kdr-D), and independent homozygous transgenic lines overexpressing KDR in Col-0 background grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 d of light treatment. Data represent means ± se, n = 38. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05). B, Relative expression level of KDR determined by RT-qPCR in wild-type Col-0, knockout line (kdr-1), activation-tagged line (kdr-D), and independent homozygous transgenic lines overexpressing KDR in Col-0 background grown in white light. Data represent means ± se, n = 3. C, Positive correlation between hypocotyl elongation and expression level of KDR measured in Col-0, kdr-D and in the transgenic lines overexpressing KDR using seedlings grown in control light conditions. The correlation was determined by one-phase association curve fitting, equation: y = y0 + (plateau − y0) (1 − e^−kx). Parameters, y0 = 1.115; plateau = 3.117; k = 0.05431. D, Representative seedlings of hypocotyl length experiment in A, for each genotype the growth is shown in control light (left) and low R:FR (right). Bar = 1 cm.

Figure 3.

Overexpression of KDR leads to a constitutive early flowering. A, Number of days to bolting (left), number of rosette leaves to bolting (middle), and total number of leaves to bolting (right) of Arabidopsis wild type (Col-0) and two independent transgenic lines overexpressing KDR in Col-0 background grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2). Data represent means ± se, n = 21. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05). B, Representative rosette plants of experiment in A grown for 20 d in pots containing soil.

Figure 4.

Y2H protein–protein interaction assays confirm the interactions found with screening of two prey libraries of Arabidopsis against the bait KDR. In the GAL4 Y2H assay, the GAL4-DNA binding domain (BD) fused to KDR was coexpressed with the GAL4-activation domain (AD) fused to the full-length CDSs of HFR1, PAR1, PAR2 (left) and AIF2, AIF4, IBH1, IBL1 (right). Yeast cells coexpressing the indicated combinations of constructs were grown on nonselective (−LT) or selective (−LTH + 2 or 5 mm 3-AT and −LTA) media. The strength of interaction is shown by the capability of the yeast to grow on stronger selection media, as indicated by the arrow. L, Leu; T, Trp; H, His; A, adenine.

Figure 5.

Y2H protein–protein interaction studies and subcellular localization of KDR and its targets in planta. A, In the GAL4 Y2H assay, the GAL4 DNA-binding domain (BD) fused to KDR (left), HFR1 (middle), and PAR1 (right) was coexpressed with the GAL4-activation domain (AD) fused to PIF4, PIF5, and PIF7. The mating of the yeast was confirmed through the growth on nonselective medium (−LT). The assay showed that whereas the bait KDR does not interact with the preys PIF4, PIF5, and PIF7, the bait HFR1 does interact with PIF4, PIF5, and PIF7. PAR1 interacts most strongly with PIF4 and PIF5. B, PIF7 can interact with other PIFs in yeast. When used as bait, PIF7 interacts with the prey HFR1 but not with PAR1 and PAR2. C, KDR fused to CFP and the interactors HFR1, PAR1, PAR2, AIF2, AIF4, IBH1, and IBL1 fused to YFP were transiently expressed in epidermal leaf cells of N. benthamiana using A. tumefaciens. Images were taken 2 d after agroinfiltration. Scale bar = 20 µm. L, Leu; T, Trp; H, His; A, adenine.

Figure 6.

Colocalization of KDR and its interactors in planta. KDR and its interactors were transiently coexpressed in epidermal leaf cells of N. benthamiana. KDR was fused to CFP, whereas HFR1, PAR1, PAR2, AIF2, AIF4, IBH1, and IBL1 were fused to YFP. Colocalization observations were made on individual cells, representing individual transformation events (n = 92 [KDR-CFP + HFR1-YFP], 133 [KDR-CFP + PAR1-YFP], 60 [KDR-CFP + PAR2-YFP], 32 [KDR-CFP + AIF4-YFP; KDR-CFP + IBL1-YFP], 36 [KDR-CFP + AIF2-YFP], 39 [KDR-CFP + IBH1-YFP]). Representative images are shown; all cells showed similar localization patterns of the two fluorescently tagged proteins. Images were taken 2 d after infiltration with A. tumefaciens. Scale bar = 20 µm.

Figure 7.

BiFC experiments confirm the interactions found with the Y2H assay. BiFC experiments performed by A. tumefaciens transient transformation of N. benthamiana leaf epidermis. The interaction of KDR with PAR1, PAR2, AIF2, AIF4, IBH1, and IBL1 was visualized as the reconstituted YFP signal in different nucleus compartments based on the type of interaction. No interaction was found between HFR1 and KDR and the negative controls using PAR1L66E. The autofluorescence of the chloroplasts is shown in red and the BiFC signal of Venus (YFP) in green. Images were taken 2 d after agroinfiltration. Scale bars = 10 μm.

Figure 8.

KDR interactors are differentially expressed, and their misexpression affects phenotypic responses to low R:FR. A, Relative expression of HFR1, PAR1, PAR2, AIF2, AIF4, IBH1, and IBL1 in control white light or low R:FR light for 90 min in wild-type Col-0 shoots. Data represent means ± se, n = 4. Asterisks indicate statistically significant difference by Student’s t test (***P < 0.001 and **P < 0.01). B, C, and D, Hypocotyl length of seedlings of Arabidopsis wild type (Col-0), overexpressing lines (35S:HFR1-GFP, 35S:PAR1-GFP, and 35S:PAR2), and mutants (hfr1-201, hfr1-5, PAR RNAi #9, pif4 pif5 pif7, par2-1, aif2-1, aif2-2, aif4, and ibl1) grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 d of light treatment. Data represent means ± se, n = 38. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05)

Figure 9.

Overexpression of KDR rescues hypocotyl length in response to low R:FR in PAR2 but not HFR1 overexpressor. A, Hypocotyl length of seedlings of Arabidopsis wild type (Col-0), 35S:KDR #9; 35S:HFR1-GFP, and 35S:PAR2 overexpression lines and the double overexpression lines 35S:KDR #9 × 35S:HFR1-GFP and 35S:KDR #9 × 35S:PAR2 grown in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 d of light treatment. Data represent means ± se, n = 38. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05) B, Representative seedlings as in experiment (A), for each genotype the growth is shown in control light (left) and low R:FR (right). The arrows indicate the hypocotyl-root transition. Scale bar = 1 cm.

Figure 10.

Overexpression of KDR in different pif knockout lines. Hypocotyl length of seedlings of Arabidopsis wild type (Col-0), knockout lines (pif7, pif4 pif5, and pif4 pif5 pif7), KDR-overexpressing line (35S:KDR #9), and independent lines of pif7, pif4 pif5, and pif4 pif5 pif7-overexpressing KDR (pif7 35S:KDR, pif4 pif5 35S:KDR, and pif4 pif5 pif7 35S:KDR) in control light conditions (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 d of light treatment. Data represent means ± se, n = 38. Different letters indicate statistically significant differences by two-way ANOVA with post-hoc Tukey test (P < 0.05).

Figure 11.

Proposed network regulating cell elongation in low R:FR. The HHbH module is composed of atypical (b)HLH and typical bHLH members, which can be positive or negative growth regulators, and they interact in an antagonistic and redundant manner to regulate cell elongation adequately and rapidly.