Arabidopsis thaliana ecotype Columbia (Col-0) plants were used as the wild type. The mutant of pif7-1 (Sail_622_G02) were obtained from the Arabidopsis Biological Resource Center. Seeds of each genotype were surface sterilized and sown on Murashige and Skoog medium consisting of 0.8% agar, stratified at 4°C in the dark for 3 days, and transferred to a growth chamber at 23°C under constant light. After 10 days, the seedlings were transferred to soil (3:1 [v/v] potting soil:vermiculite) and grown in a greenhouse under a 16-h white light (100–120 μmol m^–2 s^–1)/8-h dark cycle. Seeds used for measurement of hypocotyl length analysis were simultaneously harvested from plants grown in the same batches at 22°C under long-day (16 h light/8 h dark) conditions. Dry seeds were stored at room temperature for 2–5 months before germination analysis.

The seed was cold stratified at 4°C for 3 days and then sowed on 1/2MS medium for germination in the grow camber under a 16-h white light (100–120 μmol m^–2 s^–1)/8-h dark cycle. For hypocotyl elongation measurements, seeds were sown on 1/2MS on the growth media. Seedlings were grown on vertical plates in an incubator for 3 d at 22°C. High temperature treatment (28°C) started on day 4 for 3 days.

To generate transgenic plants overexpressing PIF7, the full-length coding sequence of this gene was amplified using the primers listed in Supplemental Table 1. The amplified fragments were cloned into the Nde/EcoRI (New England Biolabs) site of the pRI101-6Flag vector [6 × Flag tag inserted in the EcoRI/SacI site of the pRI101-AN vector (3262; Takara)] to generate the PIF7-Flash construct, using an In-fusion HD Cloning Kit (638911, Clontech). These constructs were transformed into Arabidopsis plants using the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998).

To check the interaction of AFP2 and PIF7 in yeast, sequences encoding AFP2 and PIF7 were amplified using the primers listed in Supplemental Table 1. The PCR products were cloned into the prey vector pGADT7 (630442, Clontech) and bait vector pGBKT7 (630443, Clontech) to generate pGBKT7-AFP2 (BD-AFP2) and pGADT7-PIF7 (AD-PIF7), respectively, using In-fusion Cloning Technology (Clontech). Yeast strains Y187 and AH109 were transformed with the prey and bait vectors, respectively, by polyethylene glycol-mediated yeast transformation as described by the manufacturer. After screening on minus Trp medium (630413, Clontech) or minus Leu medium (630414, Clontech), three independent clones were mated and grown on minus Trp/Leu medium (630417, Clontech) for 3 d to confirm mating success, and the corresponding clones were transferred to minus TPR/Leu/His/Ade medium (630428, Clontech) to measure their growth status.

The transgenic plants AFP2-FLAG and PIF7-Flash/AFP2-FLAG were used in Co-IP assays to detect the interaction between AFP2 and PIF7 in planta. Seedlings were grown in a grown cabinet under long-day (16 h light/8 h dark) conditions at 22°C for 7 days. The samples were harvested, ground to a fine powder in liquid nitrogen, and homogenized in IP buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA [pH 8.0], 1% Triton X-100, 0.6 mM PMSF, 20 μM MG132, Roche protease inhibitor cocktail). The extracts were centrifuged at 14,000 g for 20 min. The supernatant was mixed with 20 μl anti-MYC resin (ChromoTek) for 4 h at 4°C. The beads were washed three times with washing buffer (50 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl, 5 mM EDTA, 0.1% [v/v] Triton X-100, 20 μM MG132, and a protease inhibitor tablet). After brief centrifugation, the immunoprecipitates were separated on a 12% (w/v) SDS-polyacrylamide gel and detected by immunoblot analysis with anti-Myc (Clontech) or anti-Flag (Sigma-Aldrich) antibodies, and the immunoblot signals were detected using an ECL Kit (Invitrogen).

BiFC experiments were performed using the leaves of Nicotiana benthamiana plants grown in a greenhouse (16 h light/8 h dark) for 2 weeks as described previously [22]. In brief, full-length or truncated AFP2 and PIF7 were recombined into the binary BiFC vector pSPYNE and pSPYCE, respectively. The constructs were transformed into competent A. tumefaciens strain LBA4404 cells (9115, Clontech), and the cultures were incubated in a rotator at 250 rpm for 12 h at 28°C. After culture, Agrobacteria harboring nYFP or cYFP were mixed together and centrifuged at 12000 g for 5 min at 4°C, and the pellets were dissolved in injection solution (10 mM Tri-HCl buffer, 25 mM MgCl₂, pH 5.6) to an OD₆₀₀ of 0.1. The Agrobacterium solution was injected into N. benthamiana leaves, and YFP fluorescence was observed 3 d after injection under a confocal microscope (LSM710; Zeiss).

Total RNA was extracted from the seedling samples using a Plant Total RNA extraction kit (NEB) and reverse-transcribed to cDNA using the reverse transcriptase kit (Takara Bio) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio) on a LightCycler 96 (Roche). The gene expression results were normalized to the expression of PP2A as the internal control. All experiments were performed three biological replicates and technical repeats [23,24]. Primers are listed in Supplemental Tab. 1.

The transient transactivation assay was performed in Arabidopsis protoplasts as described previously. For transient transcription activity assays using specific promoters, the YUC8 and IAA19 promoter sequence was amplified from Col-0 genomic DNA and cloned into the pGreenII-0800-LUC vector as a reporter and the REN gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter in the pGreenII 0800-LUC vector as the internal control. The coding sequences of AFP2 and PIF7 were cloned into pGREEN-62-SK under the control of the 35S promoter and used as effectors. Reporters and effectors were transformed into Arabidopsis protoplasts in different combinations and incubated in the dark for 18 h. LUC and REN activities were separately determined at 18 h post-transformation using the Dual-Luciferase Reporter Assay System (Promega, E1910).

For immunoblot analysis, freshly harvested or after-ripened seeds were rapidly frozen in liquid nitrogen and ground in extraction buffer (50 mM sodium phosphate, pH 7.0, 100 mM NaCl, 5 mM EDTA, 0.1% [v/v] Triton X-100, 0.1% [w/v] sodium deoxycholate, and protease inhibitor tablet; Roche). Following centrifugation at 14,000 g for 5 min at 4°C, the supernatant was collected. Approximately 10 mg of total protein was separated on a 12% (w/v) SDS–PAGE gel and transferred to a nitrocellulose membrane, which was then probed with the appropriate primary antibody (anti-Myc, anti-GST, 1:3000, Clontech; anti-Flag, 1:3000, Sigma-Aldrich) and horseradish-conjugated goat anti-mouse secondary antibody (1:3000, Promega). Signals were detected using an ECL Kit (Invitrogen).

The coding sequence of PIF7 or AFP2 was cloned into pRI101-NGFP, pRI101-m Cherry, or pRI101-CFP. The constructs were transformed into Agrobacterium strain GV3101. The Agrobacterium cultures were infiltrated into N. benthamiana leaves, and fluorescent signals from GFP, CFP, or mCherry fluorescent protein (YFP) were observed at 72 h post-infiltration.

It is known that PIF4 plays the vital role in hypocotyl elongation during thermomorphogenesis [13]. Here we accessed the function of its homology, PIF7, during hypocotyl elongation. At first we generated the transgenic line expressing GUS marker under the control of the native promoter of PIF7 (pPIF7:GUS). GUS staining showed the expression of PIF7 in the cotyledon and the hypocotyl of the young seedling. High temperature treatment partially increased the GUS staining in cotyledon and hypocotyl (Fig. 1A) suggesting the potential function of PIF7 during heat stress. We then obtained the loss-of-function mutant, SAIL_622_G02, from ABRC, in which a T-DNA was inserted into its third and fourth exon through flanking primers analysis, such insertion also abolished the functional transcripts of PIF7 (Fig. 1B), thus such mutant was named as pif7-1. We also generated the transgenic line overexpressing PIF7-Flash fusion under the control of the 35S promoter (35S:PIF7-Flash, abbreviated as PIF7-Flash), the expression of PIF7-Flash was also confirmed by western blotting analysis using anti-MYC antibody (Fig. 1C). Under the normal condition at 22°C, the hypocotyl length of PIF7-Flash was longer than wild-type Col and pif7-1 mutant, ambient HTs treatment promoted the hypocotyl elongation, but HT did not obviously promote the hypocotyl elongation in pif7-1 mutant, and PIF7-Flash seedling still showed longer hypocotyl independent of ambient environment temperature (Figs. 1C and 1D). Thus, these data suggest that PIF7 is associated with the hypocotyl elongation during thermomorphogenesis.

The auxin-biosynthesis related genes including YUC8 and IAA19 is dramatically induced by ambient HT, and mediates PIF4-dependent hypocotyl elongation during thermomorphogenesis [1]. Here we also compared their expression in pif7-1 mutant or PIF7-Flash seedlings, and found HT indeed induced the expression of YUC8 and IAA19, such induction was weak in the pfi7-1 seedlings, but stronger in the PIF7-Flash seedlings (Fig. 2). Similarly, PIF7 also regulated the expression of IAA19 and SAUR24, Thus, this genetic analysis suggests that PIF7 promote hypocotyl elongation through auxin-biosynthesis related gene such as YUC8, etc.

The wild-type Col seedling was treated with 28°C for indicated time, and the total RNA was extracted for RT-qPCR analysis. The PP2A was used as the internal control. The values are shown as means SD of triplicate experiments. Bars with different letters are significantly different at p < 0.05 (Tukey’s test).

To better understand the mechanism of which PIF7 control hypocotyl elongation during HTs, we used PIF7 as the bait and perform yeast two-hybrid approach to screen the Arabidopsis normalized cDNA library to look for PIF7-interaction protein. After two-round of screening, we obtained several positive clones. One clone encoding APF2 was selected for further study as AFP2 is also reported to particulate plant response to HTs stress. We first confirmed the interaction of PIF7 and AFP2 in yeast cell as the yeast co-expressing BD-PIF7 and AD-AFP2 grow well on the selective medium, but not for the BD-PIF7 or AD-AFP2 with their corresponding empty vector (Fig. 3A). We then generated the construct containing PIF7-Flash fusion under the control of 35S promoter (35S:PIF7-Flash, abbreviated as PIF7-Flash), or the AFP2-FLAG fusion under the control of 35S promoter (35S:AFP2-FLAG, abbreviated as AFP2-FLAG), and co-expressed them in tobacco leaves, and then checked their interaction between AFP2 and PIF7 (Figs. 3C and 3D). As shown in Fig. 3B, we found AFP2-FLAG could be co-immunoreacted by PIF7-Flash, but not by anti-MYC alone, suggesting the interaction between PIF7 and AFP2 in planta.

As we mentioned above, PIF7 modulates the hypocotyl elongation under HTs, we then analyzed the function of AFP2 during thermomorphogenesis. We used the afp2-1 mutant and the transgenic line overexpressing AFP2-FLAG that is used before, to compare the hypocotyl length before or after HT stress. Under normal condition, all of the wild-type Col, afp2-1 and AFP2-FLAG transgenic line presented the similar length, however, HTs treatment increased the hypocotyl length, but HTs-induced hypocotyl elongation was attenuated in the AFP2-FLAG seedlings, but more obvious for afp2-1 mutant seedling (Figs. 1C and 1D), suggesting that AFP2 as the negative regulator controls hypocotyl elongation during seedling thermomorphogenesis process. We then checked the YUC8 and IAA19 expressing among Col, afp2-1 and AFP2-FLAG seedlings. As shown in Fig. 2, expressions of YUC8 and IAA19 were lower in the AFP2-FLAG lines, but higher in the afp2-1 line, compared with Col seedlings. These data suggest that AFP2 control hypocotyl elongation through auxin biosynthesis pathway.

We further dissect the genetic relationship between AFP2 and PIF7 during thermomorphogenesis by reciprocally crossing AFP2-FLAG or afp2 with PIF7-Flash or pif7 mutant, and then checked their hypocotyl elongation after HTs stress. As shown in Figs. 1C and 1D, HTs promoted the hypocotyl elongation for wild-type Col and PIF7-Flash seedling, but such induction was not effective in AFP2-FLAG and pif7-1 mutant seedlings. However, overexpressing AFP2-FALG in PIF7-Flash seedlings (AFP2-FLAG/PIF7-Flash) attenuated the hypocotyl elongation in contrast to PIF7-Flash seedling. In agreement with it, the expressions of YUC8 and IAA19 in AFP2-FLAG/PIF7-Flash was lower than that in PIF7-Flash background alone (Fig. 2). These data suggest that AFP2 antagonizes the function of PIF7 through influencing the auxin biosynthesis.

Furthermore, we checked the effects of AFP2 and PIF7 on the transcriptional level of YUC8 or IAA19 by transient protoplast analysis. As shown in Figs. 4A and 4B, overexpressing PIF7 alone obviously activated the transcriptional level of YUC8 or IAA19, however, coexpressing AFP2 with PIF7 attenuated the activation effect of PIF7 on the transcriptional level of YUC8 or IAA19, confirming the antagonistic effect between AFP2 or PIF7 on YUC8 or IAA19 expressions.

We previously study showed that AFP2 present circadian expression during daytime and nighttime. Here we further explore the expressing profile of AFP2 during seedling thermomorphogenesis. HTs treatment did not obviously affect the transcriptional level of AFP2. We then generated the transgenic seedling expressing the AFP2-FLAG fusion under the control of its native promoter (pAFP2:AFP2-FLAG), and checked the endogenous AFP2 protein level after HT treatment. As show in Fig. 5A, HT gradually decreased the protein abundance of AFP2 at the first 24 h treatment and sustained the lower level of AFP2 during the following 48 h treatment, suggesting that HT induced the degradation of AFP2. To further test the effect of HT on the stability of AFP2, we expressed GST-AFP2 in bacteria and used the extracted GST-AFP2 protein to test the effect of HT on AFP2 stability by cell-free analysis. As shown in Fig. 5B upper panel, we incubated GST-AFP2 with the crude protein extraction from the seedling subjected with 48 h of HT treatment, observed the gradually degradation of GST-AFP2. As the control, the crude protein extract from the seedling after HT treatment with additional proteasome inhibitor MG132 did not affect the stability of GST-APF2 (Fig. 5C, bottom panel). These data suggest that HT induced the degradation of AFP2 through ubiquitin-dependent degradation pathway.