Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation

Abstract

Shade caused by the proximity of neighboring vegetation triggers a set of acclimation responses to either avoid or tolerate shade. Comparative analyses between the shade-avoider Arabidopsis thaliana and the shade-tolerant Cardamine hirsuta revealed a role for the atypical basic-helix-loop-helix LONG HYPOCOTYL IN FR 1 (HFR1) in maintaining the shade tolerance in C. hirsuta, inhibiting hypocotyl elongation in shade and constraining expression profile of shade-induced genes. We showed that C. hirsuta HFR1 protein is more stable than its A. thaliana counterpart, likely due to its lower binding affinity to CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), contributing to enhance its biological activity. The enhanced HFR1 total activity is accompanied by an attenuated PHYTOCHROME INTERACTING FACTOR (PIF) activity in C. hirsuta. As a result, the PIF-HFR1 module is differently balanced, causing a reduced PIF activity and attenuating other PIF-mediated responses such as warm temperature-induced hypocotyl elongation (thermomorphogenesis) and dark-induced senescence. By this mechanism and that of the already-known of phytochrome A photoreceptor, plants might ensure to properly adapt and thrive in habitats with disparate light amounts.

Keywords: Cardamine hirsuta; HFR1; PIFs; shade avoidance; shade tolerance.

Figure EV1. Characterization of RNAi‐HFR1 and chfr1 mutants in Cardamine hirsuta

1. A, B

   Relative expression levels of ChHFR1 gene, normalized to EF1α in Ch^WT, (A) two RNAi‐HFR1 lines (#01 and #21) and (B) the two chfr1 mutants of C. hirsuta. Seedlings were grown for 7 days in W. Expression values are the mean ± SE of three independent biological replicates relative to Ch^WT. Asterisks mark significant differences (Student t‐test: **P‐value < 0.01) relative to Ch^WT.

2. C

   The two identified chfr1‐1 and chfr1‐2 mutants have one nucleotide insertion at position 420 of the ChHFR1 ORF, which leads to a frame shift and a premature stop codon.

Figure 1. Hypocotyls of Cardamine hirsuta seedlings with reduced levels of ChHFR1 strongly elongate in response to simulated shade

1. A, B

   Hypocotyl length of Ch^WT, (A) RNAi‐ChHFR1 transgenic, and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR > 1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W + FR) for 4 more days, producing various R:FR. Aspect of representative 7‐day‐old Ch^WT, RNAi‐HFR1 and chfr1‐1 seedlings grown in W or W + FR (R:FR, 0.02), as indicated, is shown in lower panel.

2. C, D

   Effect of W + FR exposure on the expression of PIL1, YUC8, and XTR7 genes in seedlings of Ch^WT, (C) RNAi‐HFR1, and (D) chfr1 mutant lines. Expression was analyzed in 7‐day‐old W‐grown seedlings transferred to W + FR (R:FR, 0.02) for 0, 1, 4, 8, and 12 h. Transcript abundance is normalized to EF1α levels.

Data information: Values are the means ± SE of three independent biological replicates relative to Ch^WT value at 0 h. Asterisks mark significant differences (Student's t‐test: **P‐value < 0.01; *P‐value < 0.05) relative to Ch^WT value at the same time point.

Figure EV2. Alignments of HFR1, PIF4, PIF5, and PIF7 partial DNA sequences in Arabidopsis thaliana and Cardamine hirsuta

1. Location of shared primers and amplicons used for comparison of expression levels by RT‐qPCR between species.

2. Transcript abundance of PIF4 and PIF5, normalized to YLS8, SPC25, and EF1α in Ch^WT and At^WT grown as in Fig 2. Expression values are the means ± SE of three independent biological replicates relative to the data of At^WT grown in continuous W at day 3. Asterisks mark significant differences (2‐way ANOVA: **P‐value < 0.01, ***P‐value < 0.001) between Ch^WT and At^WT when grown under W (black asterisks) or W + FR (red asterisks).

Figure EV3. ChHFR1 and AtHFR1 complement the Arabidopsis thaliana hfr1‐5 mutant long hypocotyl phenotype

1. Cartoon of HFR1 promoters from A. thaliana (pAtHFR1) and C. hirsuta (pChHFR1). These promoters cover 2,000 bp from the beginning of the translation start of the two HFR1 genes. The positions of G‐boxes (CACGTG) are indicated with arrows.

2. GUS staining of representative A. thaliana seedlings expressing GUS under the pAtHFR1 (line #03). Seven‐day‐old W‐grown seedlings were treated with W + FR for the indicated amount of time.

3. Correlation between Hyp_W+FR‐Hyp_W (means ± SE of at least four biological replicates, data shown in Fig 3C) and relative levels of ChHFR1 or AtHFR1 expression (means ± SE of three biological replicates, data shown in Fig 3B). The estimated regression equations and the R ² values are shown for each plot.

Figure 2. Levels of HFR1 transcript are higher in Cardamine hirsuta than Arabidopsis thaliana seedlings

Seedlings of Ch^WT and At^WT were grown for 3 days in W then either kept under the same conditions or transferred to W + FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of At^WT grown in continuous W at day 3. Asterisks mark significant differences (2‐way ANOVA: **P‐value < 0.01, ***P‐value < 0.001) between Ch^WT and At^WT when grown under W (black asterisks) or W + FR (red asterisks).

Figure 3. The activity of ChHFR1 is higher than that of AtHFR1 in Arabidopsis thaliana seedlings

1. Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 promoter of Arabidopsis thaliana (pAtHFR1) used to complement hfr1‐5 mutant of A. thaliana (At hfr1‐5).

2. Relative expression of HFR1 in seedlings of At^WT, At hfr1‐5, hfr1>pAt:ChHFR1 (in blue), and hfr1>pAt:AtHFR1 (in red) lines grown under W + FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old At^WT. Transcript abundance is normalized to UBQ10 levels.

3. Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W + FR (R:FR, 0.02). The mean hypocotyl length in W (Hyp_W) and W + FR (Hyp_W+FR) of at least four biological replicates was used to calculate Hyp_W+FR‐Hyp_W. Error bars represent SE.

4. Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~ 20 µmol/m²·s¹) after which they were incubated for 3 h in high W (~ 100 µmol/m²·s¹) and transferred to W + FR (R:FR, 0.06) for 3 h.

Data information: Different letters denote significant differences (one‐way ANOVA with the Tukey test, P‐value < 0.05) among means. Source data are available online for this figure.

Figure EV4. ChHFR1 protein accumulates in high W

1. Alignment of AtHFR1 and ChHFR1 protein sequences. Putative COP1 interacting motifs, defined in AtHFR1, are highlighted with a light gray box. VP motifs are highlighted with blue letters. Amino acid sequences inside the blue line rectangles correspond to the synthetic AtHFR1, ChHFR1, and At/ChHFR1 VP peptides used in the microscale thermophoresis assays (Appendix Table S3).

2. Cartoon representing the light treatments given to seedlings to estimate relative HFR1‐3xHA levels. Seedlings grown for 7 days in low W (~ 20 µmol/m²·s¹, R:FR ≈ 6.4) were first moved to high W (~ 100 µmol/m²·s¹, R:FR ≈ 3.9) for 3 h and then either transferred to high W (control) or high W + FR (R:FR ≈ 0.06) for 3 h. Seedling samples were collected at the time points indicated with asterisks.

3. Relative HFR1‐3xHA protein levels of hfr1>35S:ChHFR1 seedlings (line #16) grown as indicated in B, with a representative immunoblot in a lower panel. Relative protein levels are the mean ± SE of three independent biological replicates relative to the data point of 0 h in high W (0 h W). Asterisks mark significant differences in protein levels (Student t‐test: **P‐value < 0.01; *P‐value < 0.05) relative to the 0 h W value.

Source data are available online for this figure.

Figure 4. ChHFR1 and AtHFR1 proteins show different stability in shade

1. Expression of HFR1 and protein levels of HFR1‐3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~ 20 µmol/m²·s¹) after which they were incubated for 3 h in high W (~ 100 µmol/m²·s¹) and then either kept at high W or transferred to W + FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W + FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.

2. Cartoon of constructs containing ChHFR1 or AtHFR1 under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves.

3. Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In (A) and (C), asterisks mark significant differences (Student's t‐test: *P‐value < 0.05, **P‐value < 0.01) between the indicated pairs.

4. Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf disks treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~ 200 µmol/m²·s¹) for 3 days after agroinfiltration and then leaf circles were treated with W + FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2‐way ANOVA: *P‐value < 0.05) between ChHFR1 and AtHFR1 at the same time point.

Source data are available online for this figure.

Figure 5. AtHFR1 interacts more strongly than ChHFR1 with the WD40 domain of COP1

1. Overview of the COP1 WD40‐AtHFR1 complex (PDB ID 6QTV). The COP1 WD40 domain and the AtHFR1 VP peptide are shown in surface representation and colored in blue and orange, respectively. The N‐terminus of HFR1 VP peptide, the amino acid of which differs between AtHFR1 and ChHFR1, is highlighted in magenta.

2. Table summaries of the microscale thermophoresis binding assay (see Fig EV5). The sequence of the respective synthetic peptides is indicated.

3. Cartoon of constructs containing ChHFR1, AtHFR1, ChHFR1* and AtHFR1* derivatives under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves.

4. Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates. Asterisks mark significant differences (Student t‐test: *P‐value < 0.05, **P‐value < 0.01) between the indicated pairs.

Source data are available online for this figure.

Figure EV5. Microscale thermophoresis (MST) experimental traces and analysis

1. A–F

   Raw MST traces and analysis of AtCOP1 WD40 with different peptides in triplicates (duplicates for At/ChHFR1 VP). The concentration of AtCOP1 WD40 is fixed at 0.15 μM mixed with 16 serially diluted peptide concentrations at 1:1 ratio. Panels A, C, and E show the normalized MST traces. The blue box area illustrates the fluorescence before activation of the infrared (IR) laser and red box area illustrates average fluorescence after activation of the IR laser. Average values ± SD (error bars) were subsequently used for fluorescence normalization. k_D fit displaying fraction bound as a function of ligand concentration is shown in adjacent right panels B, D, and F. (A) Raw MST traces for AtHFR1 (in blue) and ChHFR1 (in light‐brown) VP peptides. Individual concentrations that showed slight aggregation or precipitation are shown in gray and were excluded from the k_D fit calculation. (B) Fitted data over a concentration range from 0.032 to 500 μM for AtHFR1 VP (blue dots) and 0.032 to 1,000 μM for ChHFR1 VP (light‐brown dots) were used to derive the corresponding dissociation constant k_D. (C) Raw MST traces for At/ChHFR1 VP peptide (in orange). One concentration that showed slight precipitation or aggregation is shown in gray. A concentration range of 0.0154 to 506 μM was used for the At/ChHFR1 VP. (D) No k_D was determined, as no binding between COP1 WD40 and the At/ChHFR1 VP peptide (orange dots) was detected. (E) A concentration range from 0.0076 to 250 μM for HsTRIB1 (in red) and AtCRY1 (in green) peptides was used. Raw MST traces show no aggregation or precipitation effects during this binding. One AtCRY1 VP outlier is shown in gray. (F) The k_D for HsTRIB1 (brown dots) and AtCRY1 (green dots) VP peptides was calculated using the normalized traces.

Figure 6. AtHFR1 interacts with AtPIF7

1. A

   Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The BD‐ and the AD‐derivative constructs used in the assay are shown on the left side of the panel. SD‐LW or SD‐HLW refer to the selective medium (plated as drops in dilutions of 1, 1:10, and 1:100) indicative of transformed cells or interaction between the hybrid proteins, respectively. Truncated forms of murine p53 (BD‐fused) and SV40 large T‐antigen (AD‐fused), known to interact, were used as a positive control. Empty vectors (/) were used as negative controls.

2. B, C

   Hypocotyl length of seedlings of At^WT, (B) pif7‐1, hfr1‐5, pif7‐1 hfr1‐5 (top graph), pif7‐2, hfr1‐5, and pif7‐2 hfr1‐5 (bottom graph) mutants, and (C) transgenic 35S:GFP‐ΔNt‐HFR1 (35S:ΔNt‐HFR1), two lines of 35S:PIF7‐CFP (35S:PIF7 #1 and #2), and 35S:GFP‐ΔNt‐HFR1 35S:PIF7‐CFP double transgenic (35S:ΔNt‐HFR1 × 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W + FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica).

3. D

   Aspect of representative 7‐day‐old W‐grown seedlings shown in C. Scale bar is 1 cm.

Figure 7. Cardamine hirsuta has an attenuated hypocotyl elongation at warm temperature and delayed dark‐induced senescence (DIS)

1. In At^WT, PIFs promote hypocotyl elongation as a response to warm temperature (28ºC). High ChHFR1 activity is expected to inhibit this response by repressing PIFs more effectively in Ch^WT and attenuate hypocotyl elongation at 28°C.

2. Seedlings were grown for 7 days in W at either 22°C, 2 days at 22°C then transferred to 28°C for additional 5 days (22°C > 28°C) or for 7 days at 28°C, as represented in the panel.

3. Hypocotyl length of seedlings of (left) At^WT, pifq, pif7‐2, Ch^WT, (middle) 35S:GFP‐ΔNt‐HFR1 (ΔNtHFR1), hfr1‐5 and (right) chfr1‐1 and chfr1‐2 lines grown at warm temperatures. Hypocotyl lengths are the means ± SE of three biological replicates. Asterisks mark significant differences (Student's t‐test: *P‐value < 0.05, **P‐value < 0.01) relative to the same genotype grown at 22°C (left and right graphs, black asterisks), and between the indicated pairs (middle graph, red asterisks).

4. In At^WT, PIF‐mediated DIS involves a reduction of chlorophyll levels. HFR1 activity might inhibit DIS through repression of PIFs. If PIF activity is attenuated in Ch^WT, DIS would be delayed in this species compared to At^WT.

5. Seedlings were grown for 7 days in W and then transferred to total darkness for several days to induce senescence, as illustrated at the right panel.

6. Relative chlorophylls levels of (left) At^WT, pifq, Ch^WT, (middle) ΔNtHFR1, hfr1‐5 and (right) chfr1‐1, and chfr1‐2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.

7. Aspect of 4‐day‐old dark‐grown seedlings of At^WT, pifq, pif7‐2, hfr1‐5 and ΔNt‐HFR1 (left panel), and At^WT, Ch^WT, and chfr1‐1 (right panel).

Figure 8. Model summarizing how PIF‐HFR1 transcriptional module is differently balanced in Arabidopsis thaliana and Cardamine hirsuta

Shade (low R:FR) displaces phytochrome photoequilibrium toward the inactive form, allowing PIFs to promote the expression of shade avoidance‐related genes, such as HFR1. PIF transcript or/and protein levels are induced in response to warm temperatures, resulting in enhanced expression of growth‐promoting genes. HFR1 abundance is also increased by warm temperature. HFR1 modulates these responses by heterodimerizing with PIFs and inhibiting their DNA‐binding ability. As a result, HFR1 attenuates hypocotyl elongation of A. thaliana seedlings in response to shade or warm temperature. In C. hirsuta, higher HFR1 activity inhibits more effectively PIF action than in A. thaliana. In addition, PIF abundance is attenuated in C. hirsuta. Both changes alter the PIF‐HFR1 balance in C. hirsuta, resulting in lower PIF transcriptional activity. As a consequence, shade‐ and warm temperature‐induced hypocotyl elongation are repressed and DIS is delayed in this species.