# Threshold-dependent negative autoregulation of PIF4 gene expression optimizes growth and fitness in Arabidopsis

## TL;DR

This study shows that PIF4, a key plant growth regulator, controls its own expression through a threshold-dependent negative feedback mechanism, which helps optimize plant growth and fitness under different environmental conditions.

## Contribution

The study reveals that PIF4 undergoes threshold-dependent negative autoregulation, a novel mechanism for controlling plant growth in response to environmental signals.

## Key findings

- PIF4 promoter activity is higher in pif4 mutants and lower in overexpression lines, indicating negative autoregulation.
- COP1 promotes PIF4 autoinhibition, while phyB inhibits it, showing differential regulation by upstream factors.
- Enhanced PIF4 function in transgenic lines reduces biomass and yield, highlighting the importance of feedback control.

## Abstract

PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is a vital transcription factor that controls plant growth by integrating environmental signals like light and temperature. Recent studies have shown many upstream regulators, such as HEMERA (HMR), HEAT SHOCK TRANSCRIPTION FACTORS (HSFs), TEOSINTE BRANCHED 1/CYCLOIDEA/PCF 5 (TCP5), and the B-BOX (BBX) proteins, play roles in regulating PIF4 transcription. However, the role of PIF4 in controlling its own gene expression is unknown. Here, we demonstrate that the PIF4 undergoes negative autoregulation. We show that PIF4 promoter activity is higher in the pif4 mutant but significantly reduced in PIF4 overexpression transgenic lines. Moreover, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) enhances PIF4 protein stability and promotes PIF4 autoinhibition. However, Phytochrome B (phyB), a photoreceptor that decreases PIF4 stability, inhibits autoinhibition. We further develop a network-based mathematical model incorporating the PIF4 autoinhibition and other key interactions. Our modeling and data analysis reveal that PIF4 autoregulation depends on a threshold of cellular PIF4 concentration. Our model also successfully predicts the hypocotyl growth and PIF4 promoter activity in various light and temperature conditions. Moreover, we show that the transgenic lines with enhanced PIF4 function negatively influence biomass and yield, irrespective of photoperiod and temperature. Together, the negative feedback of PIF4 dampens its own function and restrains unregulated growth. Our study thus elucidates the mechanisms of how the phyB-COP1/DET1-PIF4 module controls PIF4 transcription in tune with the endogenous PIF4 level.

PIF4, a bHLH transcription factor, is a key regulator of growth in response to diverse environmental cues. Specifically, PIF4 promotes thermosensory growth by targeting many downstream growth and hormone signaling genes. However, the regulatory mechanisms of PIF4 gene expression remain elusive. Through experiment and mathematical modelling, we show that PIF4 negatively regulates its own transcription above a threshold PIF4 protein concentration, which is dependent on photoperiod and temperature. Elevated levels of endogenous PIF4 protein are associated with reduced PIF4 promoter activity and lead to increased growth. Additionally, we show that upstream regulators of PIF4, COP1 and phyB, differentially regulate the PIF4 autoinhibition. COP1 promotes PIF4 autoinhibition, while phyB photoreceptor dampens it. Moreover, our study provides evidence that PIF4 autoinhibition affects the expression of downstream genes, regulating biomass and grain yield in response to varying light and temperatures. Together, our study highlights that PIF4-mediated negative feedback is a key regulatory mechanism in controlling growth.