Phytochrome-interacting factors regulate seedling growth through ABA signaling

https://doi.org/10.1016/j.bbrc.2020.04.011Get rights and content

Highlights

  • pif mutants are insensitive to ABA in seedling growth.

  • Overexpression of PIF3 and PIF5 enhances sensitivity to ABA in seedling growth.

  • PIFs positively regulate ABA signaling in post-germination seedling growth.

  • DAP-seq exhibits PIF3- and PIF5-binding sites genome-wide.

Abstract

There is a growing body of evidence that abscisic acid (ABA) and the phytochrome-interacting factor (PIF) family of transcription factors interact in light signaling, the regulation of plant growth development, and adaptation to environmental stimuli. In this study, we investigate the role that PIFs play in the regulation of ABA signaling in Arabidopsis thaliana seedlings grown under long-day conditions. We showed that PIFs positively regulate ABA signaling in post-germination seedling growth. We analyzed the DNA-binding sites for PIF3 and PIF5 by DNA-affinity purification sequencing (DAP-seq) genome-wide. The DAP-seq data showed that G-box motif is the direct binding site of PIF3 and PIF5, and a number of ABA responsive genes are potential targets of PIFs, including PYL3, PYL6, PYL12, SnRK2.2, CPK4, CPK6, ABI5, ABF3, and KIN1. Our results provide a basis for understanding the mechanism for PIFs in regulating ABA signal transduction.

Introduction

Phytochrome-interacting factors (PIFs) are present in a variety of plant lineages from bryophytes to angiosperms [1], and act as key factors in the transition from skotomorphogenesis to photomorphogenesis [2]. PIFs are encoded by a subset of the basic helix–loop–helix (bHLH) superfamily of transcription factors [2]. PIFs negatively regulate light responses by repressing photomorphogenesis and maintaining the skotomorphogenic state of the etiolated seedlings in darkness [2,3]. PIFs accumulate in young dark-grown seedlings, and upon exposure to light the light-activated phytochromes interact with PIFs via rapid phosphorylation, ubiquitination, and proteasome-mediated degradation, which then inhibit PIF function to induce transcriptional reprogramming, resulting in photomorphogenic development [4]. PIFs have been found to bind sequence-specifically to a core DNA G-box motif (CACGTG), suggesting a direct signaling pathway to regulate the expression of their target genes [[5], [6], [7], [8], [9], [10], [11], [12]]. A number of proteins interact directly with PIFs to enhance or inhibit the DNA-binding activity [13]. PIFs can form homodimers or heterodimers to enhance the DNA binding [14], and they can also interact with other transcription factors. PIF3 and PIF4 interact with BZR1 to regulate genes involved in the light and brassinosteroid (BR) pathways [15]. PIF1 and PIF3 interact with HY5 and bind to DNA as the PIF1/PIF3-HY5 complex; in this case, HY5 promotes the DNA-binding activity of PIF1 and PIF3 and regulates genes involved in anthocyanin biosynthesis and reactive oxygen response pathways [16]. Conversely, HY5 also functions antagonistically by binding to the same binding sites targeted by PIF1/PIF3 and inhibits PIF1/PIF3 transcriptional function in regulating chlorophyll and flavonoid biosynthetic pathways [16]. HFR1 and HEC1/HEC2 interact with PIF1 and inhibit seed germination in response to red (R) and far-red (FR) light [17,18], HFR1, PAR1, and PAR2 interact with PIF4/PIF5 and inhibit shade-avoidance responses [19,20]. Light-activated phytochromes interact with PIFs, induce their degradation, and sequester them from binding to DNA [21]. DELLA proteins interact with PIF3 and PIF4 and block the DNA-binding activity of these PIFs [7,22]. DELLA proteins also induce PIFs degradation to regulate target gene expression [23]. Based on these regulating networks, PIFs act as central components of a regulatory node that integrates multiple internal and external signals to optimize plant development [3].

Abscisic acid (ABA) is a phytohormone that plays crucial roles in regulation of plant responses to developmental and environmental cues [24,25]. ABA signal transduction in plant cells has been widely studied, and numerous ABA signaling components, including receptors mediating primary signaling events, have been identified [25]. Members of the PYR/PYL/RCAR family of proteins (part of the START domain) are the best characterized cytosolic ABA receptors [[26], [27], [28]]. A group of type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, are directly downstream components of PYR/PYL/RCARs, and negatively regulate ABA responses. In contrast, a subfamily of ABA-activated SNF1-related kinase 2 (SnRK2 kinases) are positive regulators of ABA signaling [[26], [27], [28]]. In the absence of ABA, PYR/PYL proteins are not bound to PP2Cs, and therefore, PP2C activity is high; this prevents phosphorylation and activation of SnRK2s and downstream factors. In the presence of ABA, PYR/PYLs bind and inhibit PP2Cs, leading to release of SnRKs. Finally, this activates an ABF/AREB/ABI5 clade of bZIP-domain transcription factors via a protein phosphorylation progress to induce physiological ABA responses [26,27]. Thus, reversible protein phosphorylation, mediated by SnRKs and PP2Cs, plays an essential role in the PYR/PYL/RCAR-mediated ABA signaling [29].

PIFs are emerging as integrators of signals from different hormone pathways during growth and development [3]. PIF1 promotes the expression of ABA-biosynthetic genes ABA1, NCED6 and NCED9, and represses the expression of the ABA catabolic gene CYP707A2, resulting in high ABA levels [30]. Low levels of NaCl in soil strongly impair the ability of plants to respond to shade. This block is dependent upon the ABA signaling pathway. Low ratios of R:FR light enhance BR signaling through brassinosteroid signaling kinase 5 (BSK5) and leads to the activation of BRI1-ems-suppressor 1 (BES1). ABA inhibits BSK5 up-regulation and interferes with glycogen synthase kinase 3 (GSK3)-like kinase inactivation by the BR pathway, thus leading to a suppression of the BES1:PIF1 function [31]. PIFs positively regulate the ABA signaling pathway in darkness by binding directly to the G-box motif in the ABI5 promoter. Moreover, the ABA receptors PYL8 and PYL9 interact with PIFs and regulate the protein abundance and transcriptional activity of PIF4. Thus, PIFs function as dark-specific ABA signaling components by linking ABA receptors with ABI5 expression [32]. The aim of the present study was to investigate the role that PIFs play in the regulation of ABA signaling in Arabidopsis thaliana seedlings growth under long-day conditions.

Section snippets

Materials and growth condition

Arabidopsis thaliana ecotype Col-0 was used as the wild-type control. The T-DNA insertion mutants, pif1 (CS66041), pif3 (CS66042), pif4 (CS66043), pif5 (CS66043), pif3/4/5 (CS66048), pif1/3/4/5 (CS66049), and pif1/3/4 (CS66500) were obtained from the Arabidopsis Biological Resource Center (ABRC), and have been described previously [[33], [34], [35], [36]]. Plant were grown in a growth chamber at 22 °C on Murashige & Skoog (MS) medium at 80 μmol photons m−2 s−1 or in compost soil at 120 μmol

PIFs are ubiquitously expressed and response to ABA

RT-qPCR results showed that PIF1, PIF3, PIF4, and PIF5 are expressed in various plant organs and development stages (Fig. 1A). Of note, the four PIFs were mainly expressed in seeds, stems, leaves, flowers, and siliques, but they were rarely expressed in roots (Fig. 1A). PIF1 exhibited a high level of expression, whereas the expression of PIF5 was low (Fig. 1A). We further tested whether the PIFs are stimulated by ABA. The mRNA levels of PIF1, PIF3, and PIF4 were significantly increased by ABA

Discussion

PIF proteins have been shown to play a central role in light signaling [3,48], and ABA inhibits growth and regulates plant stress responses [24]. Increasing evidence has shed light on the interaction of PIFs with ABA signaling involved in the regulation of plant growth development and stress tolerance. NaCl and ABA could act to suppress PIF4/PIF5 function, and thus inhibit hypocotyl elongation under + FR light [31]. In a previous study, pifq mutant Arabidopsis thaliana seedlings were nearly

Funding

This work was supported by National Natural Science Foundation of China (Grant Nos. 31801605 and 31770643), Beijing Natural Science Foundation (Grant Nos. 6182030 and 6172024), Fundamental Research Funds for the Central Universities (Grant No. 2019ZY25).

Declaration of competing interest

The authors declare no conflict of interests.

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