Atmospheric nitrogen dioxide suppresses the activity of phytochrome interacting factor 4 to suppress hypocotyl elongation

Main conclusion Ambient concentrations of atmospheric nitrogen dioxide (NO2) inhibit the binding of PIF4 to promoter regions of auxin pathway genes to suppress hypocotyl elongation in Arabidopsis. Abstract Ambient concentrations (10–50 ppb) of atmospheric nitrogen dioxide (NO2) positively regulate plant growth to the extent that organ size and shoot biomass can nearly double in various species, including Arabidopsis thaliana (Arabidopsis). However, the precise molecular mechanism underlying NO2-mediated processes in plants, and the involvement of specific molecules in these processes, remain unknown. We measured hypocotyl elongation and the transcript levels of PIF4, encoding a bHLH transcription factor, and its target genes in wild-type (WT) and various pif mutants grown in the presence or absence of 50 ppb NO2. Chromatin immunoprecipitation assays were performed to quantify binding of PIF4 to the promoter regions of its target genes. NO2 suppressed hypocotyl elongation in WT plants, but not in the pifq or pif4 mutants. NO2 suppressed the expression of target genes of PIF4, but did not affect the transcript level of the PIF4 gene itself or the level of PIF4 protein. NO2 inhibited the binding of PIF4 to the promoter regions of two of its target genes, SAUR46 and SAUR67. In conclusion, NO2 inhibits the binding of PIF4 to the promoter regions of genes involved in the auxin pathway to suppress hypocotyl elongation in Arabidopsis. Consequently, PIF4 emerges as a pivotal participant in this regulatory process. This study has further clarified the intricate regulatory mechanisms governing plant responses to environmental pollutants, thereby advancing our understanding of how plants adapt to changing atmospheric conditions. Supplementary Information The online version contains supplementary material available at 10.1007/s00425-024-04468-1.


Introduction
Nitrogen dioxide (NO 2 ) is often considered as a toxic gaseous air pollutant (Wellburn 1990).Upon combustion of fuels, nitrogen (N) in the air is oxidized into nitric oxide (NO), which is then rapidly converted into NO 2 (Wellburn 1990;Wallington and Nielsen 1999).Atmospheric NO 2 can be either detrimental or beneficial to plants depending on the concentration and plant species (Capron and Mansfield 1977;Sandhu and Gupta 1989;Wellburn 1990;Saxe 1994).
We recently reported that atmospheric NO 2 at ambient concentrations (10-50 ppb) positively regulates Arabidopsis plant growth to increase shoot biomass (Takahashi et al. 2005(Takahashi et al. , 2014a) ) and organ size (Takahashi et al. 2014a), and accelerates flowering (Takahashi et al. 2014b).We found that NO 2 increased the leaf size and shoot biomass of Arabidopsis by 2.5-fold (Takahashi et al. 2014a(Takahashi et al. , 2014b)), and these increases were attributable to stimulation of both cell proliferation and cell enlargement by NO 2 (Takahashi et al. 2014a).Nitrogen analyses of gaseous 15 NO 2 -fed Arabidopsis plants have indicated that the contribution of NO 2 to total plant nitrogen is minor (> 5%) and that NO 2 may function as a signal rather than a nutrient (Takahashi et al. 2014a).However, the molecular mechanism underlying the responses of plant cells to NO 2 is unknown.
It has been reported that the mechanism of hypocotyl elongation is distinct from the mechanism of biomass production (Ivakov et al. 2017;Costigliolo-Rojas et al. 2022).Consequently, investigations that focus on the effects of NO 2 on hypocotyl elongation may not provide insights into the mechanisms underlying NO 2 -mediated biomass production.However, by utilizing a range of Arabidopsis mutants related to hypocotyl elongation, research investigating the key molecules involved in NO 2 -mediated processes can provide valuable information on how NO 2 regulates plant cell behavior.
Phytochrome-interacting factor 4 (PIF4) is a basic helix-loop-helix (bHLH) transcription factor that functions as a central regulator in integrating environmental and developmental signals (Leivar and Quail 2011).It regulates the expression of various target genes involved in cell elongation and plant development.Mutants with a defective PIF4 gene (pif4) exhibit a short hypocotyl, whereas PIF-overexpressing lines exhibit a long hypocotyl (Huq and Quail 2002).In the present study, we investigated the effects of NO 2 on hypocotyl elongation of various Arabidopsis pif mutants to elucidate the involvement and functional significance of PIFs in mediating the physiological responses to NO 2 exposure.

Analysis of hypocotyl length
We harvested seedlings of -NO 2 control plants and + NO 2 -treated plants at 3-14 days of age; hypocotyl length was determined according to Fankhauser and Casal (2004).Seedlings were sandwiched between two acetate sheets (Holbein Works, Ltd., Osaka, Japan) and scanned using a flatbed scanner (CanoScan 5600F, Canon, Tokyo, Japan) at a resolution of 600 dpi.Hypocotyl length was analyzed using ImageJ software (National Institute of Health, Bethesda, MA, USA).

Analysis of shoot biomass
Shoots harvested from 4-week-old plants were washed with pure water, lyophilized, and then weighed (Takahashi et al. 2005).

RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis
Shoots of 8-day-old -NO 2 control and + NO 2 -treated plant seedlings were harvested at ZT8, frozen in liquid N, and stored at -80 °C until use (where ZT (zeitgeber) is the number of hours from dawn).Frozen shoots were ground in liquid N using a mortar and pestle, and homogenized.Total RNA was then extracted using the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions.The total RNA (1 μg) was reverse-transcribed into cDNA with ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd., Osaka, Japan) in a 10-µL reaction volume.qRT-PCR was performed using the Kapa SYBR FAST qRT-PCR Kit (Kapa Biosystems, Wilmington, MA, USA) and the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturers' instructions according to Takahashi et al. (2014a).Relative gene transcript levels were calculated using the comparative ΔΔ -CT method (Livak and Schmittgen 2001); the gene encoding protein phosphatase 2A (PP2A) was used as the reference gene.

Protein extraction and immunoblot analysis
Shoots from 9-day-old PIF4::PIF4-HA seedlings were harvested at ZT8, frozen in liquid N, and stored at -80 °C until use.Frozen shoots were ground in liquid N using a mortar and pestle and homogenized with 0.1 mL extraction buffer (per 100 mg tissue) containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 10 mM NaF, 2 mM Na 3 VO 4 , 25 mM glycerol phosphate, 10% glycerol, and 0.1% Nonidet P-40, containing 1 mM phenylmethylsulfonyl fluoride, 1 × cOmplete ULTRA protease inhibitor cocktail (Roche, Mannheim, Germany), and 20 µM MG132.The homogenate was centrifuged twice at 16,000 g for 10 min at 4 °C, and the resulting supernatant was used for immunoblot analysis.The homogenate protein content was determined following the method of Bradford (1976), with bovine serum albumin as the standard.

Chromatin immunoprecipitation (ChIP)-qPCR assay
The ChIP assays were conducted following the method of Kaufmann et al. (2010) with modifications.Briefly, shoots (0.5 g) harvested from 9-day-old Arabidopsis plants harboring PIF4::PIF4-HA or WT (control) plants at ZT8 were fixed in phosphate-buffered saline (PBS) containing 1% (v/v) formaldehyde by vacuum infiltration.Formaldehyde was quenched with 0.1 M glycine under vacuum.After grinding the plant material in liquid N and filtering using Miracloth (Millipore), nuclei were isolated, and isolated chromatin was sheared using a QSonica Q700 cup horn sonicator (QSonica, Newtown, CT, USA) at 70% amplification, with 10 cycles of 30 s sonication followed by 30 s cooling.After sonication, the extract was centrifuged at 21,880 g for 10 min at 4 °C.After chromatin isolation, the chromatin concentration was measured, and immunoprecipitation was performed using Dynabeads (Invitrogen, Carlsbad, CA, USA) coated with rat anti-HA (Roche, Basel, Switzerland).Cross-links were reversed by incubation at 65 °C for 12 h, and DNA was purified using the IPure V2 kit (Diagenode, Liège, Belgium) and eluted in 100 μL Tris-EDTA (pH 8.0).The ChIP-qPCR analyses were conducted using 1 μL eluted DNA solution and specific primers, according to the manufacturer's instructions (Diagenode) (Table S1).The ChIP-qPCR data are presented as relative amounts of immunoprecipitated DNA compared to input or % input.

Statistical analyses
GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses.Student's t-test or Mann-Whitney U test was used to compare two groups.Data were subjected to one-way ANOVA followed by Tukey's post hoc test, or two-way ANOVA (NO 2 treatment, genotype, N × G interaction) followed by Tukey's post hoc test.Differences at P < 0.05 were considered to be statistically significant.Significant differences among groups are indicated by letters in figures.

Results and discussion
NO 2 suppresses hypocotyl elongation in WT but not in pif4 mutants PIF4 encodes a bHLH transcription factor known as a phytochrome-interacting factor, which plays a central role in integrating environmental and developmental signals (Leivar and Quail 2011); it regulates the expression of various target genes involved in cell elongation and plant development.We investigated the effect of NO 2 on the hypocotyl length of a pif4 mutant with defective hypocotyl elongation (Huq and Quail 2002).First, we determined the effect of NO 2 on the hypocotyl length of WT at 3-14 days after sowing (DAS).The hypocotyl length reached a plateau at 11-13 DAS in both + NO 2 and -NO 2 plants, and was 1.7fold higher in -NO 2 plants than in + NO 2 plants (Fig. S1).Although NO 2 inhibited hypocotyl elongation in WT, it had no significant effect on the hypocotyl length of the pif4 mutant (Fig. 1).NO 2 inhibited hypocotyl elongation in other single pif mutants, such as pif1, pif3, and pif5, but did not significantly inhibit hypocotyl elongation in the quadruple mutant pifq.These results indicate that PIF4 uniquely eliminates NO 2 -triggered inhibition of hypocotyl elongation in Arabidopsis.This finding suggests that PIF4 is involved in the mechanism underlying NO 2 -mediated physiological processes in Arabidopsis.The inhibition of hypocotyl elongation by NO 2 was still observed in WT grown under higher light intensity (Fig. S2).This indicated that the effect of NO 2 on hypocotyl elongation is not generally limited by the existing hypocotyl length.This suggests that the pifq and pif4 mutants are indeed responsive to NO 2 .
We speculated that NO 2 may suppress PIF4 activity transcriptionally and/or translationally to suppress Arabidopsis hypocotyl elongation.Notably, hypocotyl length was shortest in the pif4 or pifq mutants among all the lines tested in this study.
The hypocotyl elongation of overexpressors in the presence and absence of NO 2 was very similar to that of WT, except that the inhibitory effect of NO 2 on hypocotyl elongation in PIF1-and PIF4-overexpressors was not statistically significant (Fig. 1b).The fact that the PIF4-overexpressors produced the longest hypocotyls among all the lines tested in this study in the presence or absence of NO 2 is notable, given that PIF4 uniquely regulates NO 2 -induced hypocotyl elongation among PIF genes, as mentioned above.PIF1 has a limited regulatory role in the modulation of hypocotyl elongation; instead, its predominant function lies in the suppression of seed germination (Oh et al. 2004;Leiver and Quail 2011).As shown in Fig. 1b, among all the PIF-overexpressing lines, the PIF1 overexpressor produced the shortest hypocotyls in the presence or absence of NO 2 .
The hypocotyl length of one of the PIF4-overexpressors (35S::PIF4-Myc) was almost the same in the presence or absence of NO 2 .This may have been because of the substantial abundance of the PIF4 protein in the overexpressor, resulting in a diminished impact of NO 2 on hypocotyl elongation.The hypocotyl of the PIF4-overexpressing line in which PIF4 was under the control of its native promoter (PIF4::PIF4:HA) was shorter in the presence of NO 2 than in the absence of NO 2 (Fig. 1c).

NO 2 suppressed the expression of PIF4 target genes
We hypothesized that the suppression of hypocotyl elongation by NO 2 may result from inhibition of the transcription of the target genes of PIF4.To test this hypothesis, we selected 11 genes reported to be target genes of PIF4, namely YUCCA8 (YUC8), IAA19, IAA29, SAUR19, SAUR46, SAUR63, SAUR67, PRE1, PRE2, PRE5, PRE6 (Oh et al. 2012), and tested the effect of NO 2 on their transcript levels in WT Arabidopsis (Col-0) using qRT-PCR (Fig. 2).All of the tested genes were significantly down-regulated by NO 2 (Fig. 2).Thus, NO 2 inhibited the transcriptional expression of a variety of auxin pathway genes under the control of PIF4 to suppress hypocotyl elongation.
We also examined the transcript levels of these 11 genes in WT and the pif4 mutant (Fig. 2).The transcript levels of these 11 genes were lower in the pif4 mutant than in WT in the absence of NO 2 , and NO 2 exposure did not significantly alter their transcriptional responses (Fig. 2).A two-way ANOVA revealed a significant interaction between genotype and NO 2 treatment for almost all the genes.
In this study, NO 2 suppressed the expression of YUC8, IAA19, IAA29, SAUR19, SAUR46, SAUR63, SAUR67, PRE1, PRE2, PRE5, and PRE6 (Fig. 2).The NO 2 -induced down-regulation of auxin-responsive genes (IAA19, IAA29, SAUR19, SAUR46, SAUR63, SAUR67) may result in a reduction of cell elongation, leading to a shortened hypocotyl length.Some of these genes play critical roles in processes that affect cell elongation, such as promoting auxin transport (Chae et al. 2012) and stimulating membrane acidification (Spartz et al. 2014).PRE proteins are a class of HLH proteins that function as a secondary repressor (Buti et al. 2020) and the expression of their encoding genes is activated by auxin (Zheng et al. 2017).YUC8 is involved in the auxin biosynthesis pathway and is regulated by PIF4 in high-temperature-induced hypocotyl elongation (Sun et al. 2012).These results indicated that NO 2 requires a functional PIF4 gene to affect the expression of genes involving the auxin biosynthesis.

NO 2 did not affect PIF4 transcription or PIF4-HA protein accumulation
Our results show that NO 2 inhibited the transcriptional expression of auxin pathway genes.To determine whether this was attributable to down-regulation of PIF4 transcription and/or PIF4 protein accumulation, we performed qRT-PCR and protein immunoblot analyses.The transcript levels of PIF4 did not differ between -NO 2 and + NO 2 plants across the diurnal cycle (Fig. 3a, Fig. S3), demonstrating that the inhibition of the transcriptional expression of auxin pathway genes by NO 2 is not due to down-regulation of PIF4 transcript levels.
Since no anti-PIF4 antibody was available, we used a transgenic line expressing a fusion construct of PIF4 and the HA antigen (PIF4::PIF4-HA) (designated as PIF4-HA) (Yamashino et al. 2013).The amount of PIF4-HA was determined by immunoblot analysis using an anti-HA antibody according to Yamashino et al. (2013) (Fig. 3b, Fig. S4).The PIF4 protein levels did not differ between -NO 2 and + NO 2 plants.Together, these results indicate that NO 2 -induced down-regulation of PIF4-controlled auxin pathway genes was not because of a reduction in PIF4 transcription or PIF4 protein accumulation.

NO 2 inhibited binding of PIF4 to the promoter of target genes
Despite the failure of NO 2 to alter PIF4 transcript or PIF4 protein levels, NO 2 distinctly suppressed the expression of the target genes of PIF4.We, therefore, suspected that NO 2 may alter the binding of PIF4 to the promoter regions of its target genes.To address this question, we performed ChIP assays using proteins extracted from PIF4-overexpressors grown in the presence and absence of NO 2 .The hypocotyl length of the PIF4::PIF4-HA transgenic line was shorter in the presence of NO 2 than in the absence of NO 2 (Fig. 1c), whereas the hypocotyl length elongation of 35S::PIF4-Myc was not inhibited by NO 2 .Therefore, we used the PIF4::PIF4-HA transgenic line for ChIP analysis.Immunoprecipitation was performed using an anti-HA antibody.
The ChIP assays with the SAUR46 and SAUR67 genes clearly indicated distinct decreases in percent input values or relative amounts of immunoprecipitated DNA upon NO 2 treatment (Fig. 3c).Overall, the ChIP assays of PIF4::PIF4-HA plant extracts using anti-HA antibody showed that NO 2 decreased PIF4 binding to the promoter regions of target genes SAUR67 and SAUR46 by about one half.Both SAUR46 and SAUR67 are responsive to auxin (Nemhauser et al. 2006), suggesting that NO 2 may affect gene expression through auxin pathways in Arabidopsis.
These findings indicate that NO 2 may post-translationally modify the PIF4 protein to substantially inhibit its binding to the promoter regions of SAUR67 and SAUR46.
To date, nitration of PIF4 proteins following exposure to NO 2 has not been detected.Alternatively, given that the DELLA protein is a transcriptional co-repressor and DELLA blocks PIF4 transcriptional activity by binding to its DNArecognition domain (de Lucas et al. 2008;Li et al. 2016), it is possible that NO 2 -induced down-regulation of PIF activity is mediated by increasing DELLA protein levels.Whether In this study, the ChIP assay results showed that PIF4 binding to promoters of genes other than SAUR67 and SAUR46 was not affected by NO 2 (Fig. S5).These findings suggest that, beyond PIF4, additional proteins like PIF4interacting proteins such as brassinazole resistant 1 (BZR1) and auxin response factor 6 (ARF6) may play roles in the transcriptional regulation of the 11 genes examined in this study.NO 2 may exert inhibitory effects on these proteins, leading to the suppression of gene expression.

The pif4 mutant did not exhibit a shoot biomass response to NO 2 treatment
Given the involvement of PIF4 in the inhibition of hypocotyl elongation by NO 2 , we investigated the effects of NO 2 on biomass in the pif4 mutant to determine whether PIF4 is also involved in stimulation of shoot biomass by NO 2 .To consolidate this, we evaluated the impact of NO 2 on shoot biomass in both WT and the pif4 mutant.
In the WT plants, NO 2 notably increased shoot biomass at 28 days after sowing.In contrast, there were no significant differences in shoot biomass between -NO 2 and + NO 2 conditions in the pif4 mutant (Fig. 4).The shoot biomass of the pif4 mutant was almost the same as that of the WT in the absence of NO 2 .The results obtained using the pifq mutant were almost the same as those obtained using the pif4 mutant.These results suggested that PIF4 is involved in the response to NO 2 not only in the hypocotyl, but also in the shoot.The opposite effects of NO 2 on the shoot and hypocotyl are indicative of organ-specific response mechanisms to NO 2 .Costigliolo- Rojas et al. (2022) recently reported the organ-specific regulatory mechanisms in the shoot and hypocotyl under shaded and warm conditions.Consistent with the results of earlier studies by Shimizu et al. (2016) and Kim et al. (2020) that demonstrated the tissue-specific function of PIF4, our findings suggest that PIF4 may modulate organ-specific regulatory pathways in response to NO 2 .

NO 2 inhibited hypocotyl elongation through PIF4 signaling
In this study, we investigated the effects of NO 2 on hypocotyl elongation and found that NO 2 suppressed hypocotyl elongation in Arabidopsis WT grown in the light (Fig. 1).
Significant suppression of hypocotyl elongation by NO 2 was observed in WT and the pif1, pif3, and pif5 mutants (Fig. 1a), but not in the pifq or pif4 mutants (Fig. 1a).PIF1, PIF3, PIF4, and PIF5 have been reported to act redundantly in etiolated seedlings (Zhang et al. 2013).In green seedlings, PIF4 and PIF5 are involved in hypocotyl elongation Plants were harvested at ZT6 and total RNA extracted, followed by qPCR analysis to determine PIF4 transcript levels.Transcript levels were normalized to the value in -NO 2 WT plants (set to 1).Arabidopsis PP2A was used as an internal control.Each bar represents mean ± SD of data from four independent biological replicates.b Immunoblot analysis of PIF4-HA proteins in Arabidopsis PIF4::PIF4-HA plants.Plants were grown in the presence and absence of NO 2 for 9 days as described for Fig. 1.Total proteins were extracted, and immunoblot analysis was performed using anti-HA-tag and anti-ACT11 (load control).Intensity of band around 35 kDa in the blot with anti-HA antibody (Fig. S4a) and intensity of band around 40 kDa in the blot with anti-actin antibody (Fig. S4c) were quantified.PIF4-HA protein amount was calculated from PIF4-HA signal normalized to actin (ACT11) signal.Each bar represents mean ± SD of data from four to five independent biological replicates.c Chromatin immunoprecipitation (ChIP) assays for SAUR67 and SAUR46 in PIF4::PIF4-HA Arabidopsis plants.Plants were grown in the presence and absence of NO 2 for 9 days, as described for Fig. 1.Arabidopsis PP2A (load control) was used as an internal control.WT (Col-0) plants were used as a negative control.Plants were harvested at ZT6.Each bar represents mean ± SD of data from three independent technical replicates; *P < 0.05, ***P < 0.001 (two-way ANOVA) (Hornitschek et al. 2012).However, such PIF redundancy is not involved in the effects of NO 2 on hypocotyl elongation.PIF4 is similarly essential in responses to NO 2 and high temperature (Koini et al. 2009).
PIF4 is a bHLH transcription factor involved in the integration of multiple signals for plant growth regulation.It is activated by various environmental factors including light and temperature, and hormonal signals; it regulates target genes involved in cell elongation (Koini et al. 2009).Recently, it has been revealed that PIF4 integrates cues of nitrate responses (Pereyra et al. 2023).Thus, whether alone or in combination with other proteins, PIF4 might regulate the expression of genes involved in auxin pathways in the response to NO 2 exposure.

NO 2 inhibited binding of PIF4 to the promoter regions of target genes
Atmospheric NO 2 did not affect hypocotyl length in the pif4 mutant (Fig. 1a); in fact, pif4 had the shortest hypocotyl length among all the lines tested in this study.Therefore, we presumed that NO 2 suppresses PIF4 gene expression or PIF4 protein activity to suppress hypocotyl elongation.
However, qRT-PCR and immunoblot analyses showed that NO 2 did not affect the PIF4 transcript level or PIF4 protein level (Fig. 3a and b, respectively).However, NO 2 reduced the ability of PIF4 to bind to the promoters of its target genes (Fig. 3c); therefore, the PIF4 protein may be modified posttranslationally by NO 2 .
NO 2 is a potent, non-discriminating nitrating agent as well as a hydrophobic molecule, and cell membranes are not significant barriers to NO 2 transport (Signorelli et al. 2011).In in vivo studies, NO 2 has been shown to be involved in protein tyrosine nitration (Kolbert et al. 2017) and protein nitrosylation (Heo and Campbell 2004).Protein tyrosine nitration is a covalent post-translational protein modification that plays a vital role in cell physiological processes including cellular signaling (Rubbo and Radi 2008;Ischiropoulos 2009).We recently identified nitratable proteins in Arabidopsis and showed that its proteins are selectively nitrated (Takahashi et al. 2015).Protein nitration has been shown to inhibit protein activity and interactions (Álvarez et al. 2011;Lozano-Juste et al. 2011).It can prevent proteins from exercising their normal functions such as phosphorylation or mimic the structural changes that occur after phosphorylation (Lindermayr and Durner 2009).For example, nitration of the β-subunit of F1-ATPase results in reduced ATPase activity (Fujisawa et al. 2009).Previously, we reported the nitration of PsbO1 by NO 2 and showed that this resulted in inhibition of oxygen evolution in Arabidopsis leaves (Takahashi et al. 2017a,b).Nitrated proteins may also be involved in cellular signaling underlying NO 2 -regulated plant growth (Takahashi and Morikawa 2019).
NO 2 is involved in the nitrosylation of plant proteins (Heo and Campbell 2004;Keszler et al. 2010).S-nitrosylation of proteins is a typical redox signaling mechanism (Sanz et al. 2015).Many proteins are nitrosylated in plants fumigated with NO 2 (Heo and Campbell 2004;Kovacs and Lindermayr 2013;Takahashi et al. 2015).Therefore, nitrosylated proteins may play a role in cellular signaling underlying NO 2 -regulated plant growth (Takahashi et al. 2005(Takahashi et al. , 2014a)).NO-mediated up-regulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor promotes TIR1-Aux/IAA, facilitating Aux/IAA degradation and subsequently enhancing activation of gene expression (Terrile et al. 2012).
To integrate a variety of environmental, phytohormonal, and developmental signals, PIF4 cooperatively interacts with other protein factors such as ARF6 and BZR1 (positive regulatory factors) (Oh et al. 2014(Oh et al. , 2016) ) and DELLA (a negative regulatory factor) (Li et al. 2016).NO 2 , which can permeate from the apoplast through the plasma membrane to reach the cytosol and nucleus, may modify these proteins, eventually suppressing binding of PIF4 to the promoters of its target genes.These modifications may inhibit the formation of complexes that include PIF4 and prevent PIF4 from For the 4-week-old WT, pif4, and pifq plants, the shoot biomass is presented as the mean ± SD of the data from 10 independent biological replicates.Statistical significance was assessed using a one-way ANOVA followed by Tukey's post hoc test: *** P < 0.001 binding to the promoter regions of its target genes.Determining the mechanism by which NO 2 modifies these protein factors that play important roles in controlling PIF4 activity is an important and intriguing objective for future studies.

Fig. 2
Fig. 2 Transcript levels of 11 genes under regulation of PIF4 in wildtype control (Col-0) and pif4 mutant plants grown in the absence (-NO 2 ) and presence (+ NO 2 ) of NO 2 as described in the legend of Fig. 1.Transcript levels were normalized to that of PP2A and are displayed relative to that in -NO 2 plants (set to 1) as the control.Each

Fig. 3 a
Fig.3a Transcript levels of PIF4 in Arabidopsis plants grown in the presence and absence of NO 2 .Plants were grown for 8 days as described for Fig.1.Plants were harvested at ZT6 and total RNA extracted, followed by qPCR analysis to determine PIF4 transcript levels.Transcript levels were normalized to the value in -NO 2 WT plants (set to 1).Arabidopsis PP2A was used as an internal control.Each bar represents mean ± SD of data from four independent biological replicates.b Immunoblot analysis of PIF4-HA proteins in Arabidopsis PIF4::PIF4-HA plants.Plants were grown in the presence and absence of NO 2 for 9 days as described for Fig.1.Total proteins were extracted, and immunoblot analysis was performed using anti-HA-tag and anti-ACT11 (load control).Intensity of band around

Fig. 4
Fig.4Shoot biomass of the wild-type control (WT; Col-0) as well as the pif4 and pifq mutant plants grown in the presence and absence of NO 2 as described in the legend of Fig.1.For the 4-week-old WT, pif4, and pifq plants, the shoot biomass is presented as the mean ± SD of the data from 10 independent biological replicates.Statistical significance was assessed using a one-way ANOVA followed by Tukey's post hoc test: *** P < 0.001