PHOSPHATASE 2A dephosphorylates PHYTOCHROME INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. Here, we describe four regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'ß, B''α and B''ß) that interact with PIF3 in yeast two-hybrid, in vitro and in vivo assays. The pp2ab''αß and b''αß/b'αß mutants displayed short hypocotyls, while the overexpression of the B subunits induced longer hypocotyls compared to the wild type under red light. The light-induced degradation of PIF3 was faster in the b''αß/b'αß quadruple mutant compared to in the wild type. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylated PIF3-MYC in vitro. RNA-seq analyses showed that B''α and B''ß alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4 and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

Shining Light on Plant Growth: Recent Insights into Phytochrome Interacting Factors.

Light serves as a pivotal environmental cue regulating various aspects of plant growth and development, including seed germination, seedling de-etiolation, and shade avoidance. Within this regulatory framework, the basic helix-loop-helix transcription factors known as PHYTOCHROME INTERACTING FACTORS (PIFs) play an essential role in orchestrating responses to light stimuli. Phytochromes, acting as red/far-red light receptors, initiate a cascade leading to the degradation of PIFs (except PIF7), thereby triggering transcriptional reprogramming to facilitate photomorphogenesis. Recent research has unveiled multiple post-translational modifications that regulate the abundance and/or activity of PIFs, including phosphorylation, dephosphorylation, ubiquitination, deubiquitination and SUMOylation. Moreover, intriguing findings indicate that PIFs can influence chromatin modifications. These include modulation of Histone 3 Lysine-9 acetylation (H3K9ac), as well as occupancy of histone variants such as H2A.Z (associated with gene repression) and H3.3 (associated with gene activation), thereby intricately regulating downstream gene expression in response to environmental cues. This review summarizes recent advances in understanding PIFs' role in regulating various signaling pathways with a major focus on photomorphogenesis.

ELONGATED HYPOCOTYL 5 interacts with HISTONE DEACETYLASE 9 to suppress glucosinolate biosynthesis in Arabidopsis.

Glucosinolates (GSLs) are defensive secondary metabolites produced by Brassicaceae species in response to abiotic and biotic stresses. The biosynthesis of GSL compounds and the expression of GSL-related genes are highly modulated by endogenous signals (i.e., circadian clocks) and environmental cues, such as temperature, light, and pathogens. However, the detailed mechanism by which light signaling influences GSL metabolism remains poorly understood. In this study, we found that a light-signaling factor, ELONGATED HYPOCOTYL 5 (HY5), was involved in the regulation of GSL content under light conditions in Arabidopsis (Arabidopsis thaliana). In hy5-215 mutants, the transcript levels of GSL pathway genes were substantially upregulated compared with those in wild-type plants. The content of GSL compounds was also substantially increased in hy5-215 mutants, whereas 35S::HY5-GFP/hy5-215 transgenic lines exhibited comparable levels of GSL-related transcripts and GSL content to those in WT plants. HY5 physically interacts with HISTONE DEACETYLASE9 (HDA9) and binds to the proximal promoter region of MYB29 and IMD1 to suppress aliphatic GSL biosynthetic processes. These results demonstrate that HY5 suppresses GSL accumulation during the daytime, thus properly modulating GSL content daily in Arabidopsis plants.

The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.

Light triggers an enhancement of global translation during photomorphogenesis in Arabidopsis, but little is known about the underlying mechanisms. The phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) at a conserved serine residue in the N-terminus has been shown as an important mechanism for the regulation of protein synthesis in mammalian and yeast cells. However, whether the phosphorylation of this residue in plant eIF2α plays a role in regulation of translation remains elusive. Here, we show that the quadruple mutant of SUPPRESSOR OF PHYA-105 family members (SPA1-SPA4) display repressed translation efficiency after light illumination. Moreover, SPA1 directly phosphorylates the eIF2α C-terminus under light conditions. The C-term-phosphorylated eIF2α promotes translation efficiency and photomorphogenesis, whereas the C-term-unphosphorylated eIF2α results in a decreased translation efficiency. We also demonstrate that the phosphorylated eIF2α enhances ternary complex assembly by promoting its affinity to eIF2ß and eIF2γ. This study reveals a unique mechanism by which light promotes translation via SPA1-mediated phosphorylation of the C-terminus of eIF2α in plants.

Characterization of PIF4 Phosphorylation by SPA1.

The PHYTOCHROME INTERACTING FACTORs (PIFs) play pivotal roles in regulating thermo- and photo-morphogenesis in Arabidopsis. One of the main hubs in thermomorphogenesis is PIF4, which regulates plant development under high ambient temperature along with other PIFs. PIF4 enhances its own transcription and PIF4 protein is stabilized under high ambient temperature. However, the mechanisms of thermo-stabilization of PIF4 are less understood. Recently, it was shown that SUPPRESSOR OF PHYA-105 1 (SPA1) can function as a serine/threonine kinase to phosphorylate PIF4 in vitro, and the phosphorylated form of PIF4 is more stable under high ambient temperature conditions. In this chapter, we describe the in vitro kinase assay of PIF4 by SPA1. In principle, this protocol can be applied for other putative substrates and kinases.

Light signaling in plants-a selective history.

In addition to providing the radiant energy that drives photosynthesis, sunlight carries signals that enable plants to grow, develop and adapt optimally to the prevailing environment. Here we trace the path of research that has led to our current understanding of the cellular and molecular mechanisms underlying the plant's capacity to perceive and transduce these signals into appropriate growth and developmental responses. Because a fully comprehensive review was not possible, we have restricted our coverage to the phytochrome and cryptochrome classes of photosensory receptors, while recognizing that the phototropin and UV classes also contribute importantly to the full scope of light-signal monitoring by the plant.

LHP1 and INO80 cooperate with ethylene signaling for warm ambient temperature response by activating specific bivalent genes.

Ethylene signaling has been indicated as a potential positive regulator of plant warm ambient temperature response but its underlying molecular mechanisms are largely unknown. Here, we show that LHP1 and INO80 cooperate with ethylene signaling for warm ambient temperature response by activating specific bivalent genes. We found that the presence of warm ambient temperature activates ethylene signaling through EIN2 and EIN3, leading to an interaction between LHP1 and accumulated EIN2-C to co-regulate a subset of LHP1-bound genes marked by H3K27me3 and H3K4me3 bivalency. Furthermore, we demonstrate that INO80 is recruited to bivalent genes by interacting with EIN2-C and EIN3, promoting H3K4me3 enrichment and facilitating transcriptional activation in response to warm ambient temperature. Together, our findings illustrate a novel mechanism wherein ethylene signaling orchestrates LHP1 and INO80 to regulate warm ambient temperature response through activating specific bivalent genes in Arabidopsis.

COP1 controls light-dependent chromatin remodeling.

Light is a crucial environmental factor that impacts various aspects of plant development. Phytochromes, as light sensors, regulate myriads of downstream genes to mediate developmental reprogramming in response to changes in environmental conditions. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is an E3 ligase for a number of substrates in light signaling, acting as a central repressor of photomorphogenesis. The interplay between phytochrome B (phyB) and COP1 forms an antagonistic regulatory module that triggers extensive gene expression reprogramming when exposed to light. Here, we uncover a role of COP1 in light-dependent chromatin remodeling through the regulation of VIL1 (VIN3-LIKE 1)/VERNALIZATION 5, a Polycomb protein. VIL1 directly interacts with phyB and regulates photomorphogenesis through the formation of repressive chromatin loops at downstream growth-promoting genes in response to light. Furthermore, we reveal that COP1 governs light-dependent formation of chromatin loop and limiting a repressive histone modification to fine-tune expressions of growth-promoting genes during photomorphogenesis through VIL1.

PHYTOCHROME-INTERACTING FACTORS are involved in starch degradation adjustment via inhibition of the carbon metabolic regulator QUA-QUINE STARCH in Arabidopsis.

As sessile organisms, plants encounter dynamic and challenging environments daily, including abiotic/biotic stresses. The regulation of carbon and nitrogen allocations for the synthesis of plant proteins, carbohydrates, and lipids is fundamental for plant growth and adaption to its surroundings. Light, one of the essential environmental signals, exerts a substantial impact on plant metabolism and resource partitioning (i.e., starch). However, it is not fully understood how light signaling affects carbohydrate production and allocation in plant growth and development. An orphan gene unique to Arabidopsis thaliana, named QUA-QUINE STARCH (QQS) is involved in the metabolic processes for partitioning of carbon and nitrogen among proteins and carbohydrates, thus influencing leaf, seed composition, and plant defense in Arabidopsis. In this study, we show that PHYTOCHROME-INTERACTING bHLH TRANSCRIPTION FACTORS (PIFs), including PIF4, are required to suppress QQS during the period at dawn, thus preventing overconsumption of starch reserves. QQS expression is significantly de-repressed in pif4 and pifQ, while repressed by overexpression of PIF4, suggesting that PIF4 and its close homologs (PIF1, PIF3, and PIF5) act as negative regulators of QQS expression. In addition, we show that the evening complex, including ELF3 is required for active expression of QQS, thus playing a positive role in starch catabolism during night-time. Furthermore, QQS is epigenetically suppressed by DNA methylation machinery, whereas histone H3 K4 methyltransferases (e.g., ATX1, ATX2, and ATXR7) and H3 acetyltransferases (e.g., HAC1 and HAC5) are involved in the expression of QQS. This study demonstrates that PIF light signaling factors help plants utilize optimal amounts of starch during the night and prevent overconsumption of starch before its biosynthesis during the upcoming day.

SWAP1-SFPS-RRC1 splicing factor complex modulates pre-mRNA splicing to promote photomorphogenesis in Arabidopsis.

Light signals perceived by a group of photoreceptors have profound effects on the physiology, growth, and development of plants. The red/far-red light-absorbing phytochromes (phys) modulate these aspects by intricately regulating gene expression at multiple levels. Here, we report the identification and functional characterization of an RNA-binding splicing factor, SWAP1 (SUPPRESSOR-OF-WHITE-APRICOT/SURP RNA-BINDING DOMAIN-CONTAINING PROTEIN1). Loss-of-function swap1-1 mutant is hyposensitive to red light and exhibits a day length-independent early flowering phenotype. SWAP1 physically interacts with two other splicing factors, (SFPS) SPLICING FACTOR FOR PHYTOCHROME SIGNALING and (RRC1) REDUCED RED LIGHT RESPONSES IN CRY1CRY2 BACKGROUND 1 in a light-independent manner and forms a ternary complex. In addition, SWAP1 physically interacts with photoactivated phyB and colocalizes with nuclear phyB photobodies. Phenotypic analyses show that the swap1sfps, swap1rrc1, and sfpsrrc1 double mutants display hypocotyl lengths similar to that of the respective single mutants under red light, suggesting that they function in the same genetic pathway. The swap1sfps double and swap1sfpsrrc1 triple mutants display pleiotropic phenotypes, including sterility at the adult stage. Deep RNA sequencing (RNA-seq) analyses show that SWAP1 regulates the gene expression and pre-messenger RNA (mRNA) alternative splicing of a large number of genes, including those involved in plant responses to light signaling. A comparative analysis of alternative splicing among single, double, and triple mutants showed that all three splicing factors coordinately regulate the alternative splicing of a subset of genes. Our study uncovered the function of a splicing factor that modulates light-regulated alternative splicing by interacting with photoactivated phyB and other splicing factors.

Phase separation at the heart of "heat" sensing.

Phytochrome B is known as a receptor for both light and temperature signals. In this issue of Molecular Cell, Chen et al. (2022) show how these two environmental signals are perceived distinctly by a single photoreceptor through liquid-liquid phase separation (LLPS).

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

ABI3- and PIF1-mediated regulation of GIG1 enhances seed germination by detoxification of methylglyoxal in Arabidopsis.

Methylglyoxal (MG) is a toxic by-product of the glycolysis pathway in most living organisms and was previously shown to inhibit seed germination. MG is detoxified by glyoxalase I and II family proteins in plants. MG is abundantly produced during early embryogenesis in Arabidopsis seeds. However, the mechanism that alleviates the toxic effect of MG in maturing seeds is poorly understood. In this study, by T-DNA mutant population screening, we found that mutations in a glyoxalase I gene (named GERMINATION-IMPAIRED GLYOXALASE 1, GIG1) led to significantly impaired germination compared with wild-type seeds. Transformation of full-length GIG1 cDNA under the constitutively active cauliflower mosaic virus 35S promoter in the gig1 background completely recovered the seed germination phenotype. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that GIG1 is uniquely expressed in seeds and is upregulated by abscisic acid (ABA) and downregulated by gibberellic acid (GA) during seed germination. An ABA signaling component, ABI3, directly activated GIG1 in maturing seeds. In addition, PHYTOCHROME INTERACTING FACTOR 1 (PIF1) also plays cooperatively with ABI3 in the regulation of GIG1 expression in the early stage of imbibed seeds. Furthermore, GIG1 expression is stably silenced by epigenetic repressors such as polycomb repressor complexes. Altogether, our results indicate that light and ABA signaling cooperate to enhance seed germination by the upregulation of GIG1 to detoxify MG in maturing seeds.

Phytochrome B triggers light-dependent chromatin remodelling through the PRC2-associated PHD finger protein VIL1.

To compensate for a sessile nature, plants have developed sophisticated mechanisms to sense varying environmental conditions. Phytochromes (phys) are light and temperature sensors that regulate downstream genes to render plants responsive to environmental stimuli1-4. Here, we show that phyB directly triggers the formation of a repressive chromatin loop by physically interacting with VERNALIZATION INSENSITIVE 3-LIKE1/VERNALIZATION 5 (VIL1/VRN5), a component of Polycomb Repressive Complex 2 (PRC2)5,6, in a light-dependent manner. VIL1 and phyB cooperatively contribute to the repression of growth-promoting genes through the enrichment of Histone H3 Lys27 trimethylation (H3K27me3), a repressive histone modification. In addition, phyB and VIL1 mediate the formation of a chromatin loop to facilitate the repression of ATHB2. Our findings show that phyB directly utilizes chromatin remodelling to regulate the expression of target genes in a light-dependent manner.

An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis.

Plant growth and development are acutely sensitive to high ambient temperature caused in part due to climate change. However, the mechanism of high ambient temperature signaling is not well defined. Here, we show that HECATEs (HEC1 and HEC2), two helix-loop-helix transcription factors, inhibit thermomorphogenesis. While the expression of HEC1 and HEC2 is increased and HEC2 protein is stabilized at high ambient temperature, hec1hec2 double mutant showed exaggerated thermomorphogenesis. Analyses of the four PHYTOCHROME INTERACTING FACTOR (PIF1, PIF3, PIF4 and PIF5) mutants and overexpression lines showed that they all contribute to promote thermomorphogenesis. Furthermore, genetic analysis showed that pifQ is epistatic to hec1hec2. HECs and PIFs oppositely control the expression of many genes in response to high ambient temperature. PIFs activate the expression of HECs in response to high ambient temperature. HEC2 in turn interacts with PIF4 both in yeast and in vivo. In the absence of HECs, PIF4 binding to its own promoter as well as the target gene promoters was enhanced, indicating that HECs control PIF4 activity via heterodimerization. Overall, these data suggest that PIF4-HEC forms an autoregulatory composite negative feedback loop that controls growth genes to modulate thermomorphogenesis.

Spatial regulation of thermomorphogenesis by HY5 and PIF4 in Arabidopsis.

Plants respond to high ambient temperature by implementing a suite of morphological changes collectively termed thermomorphogenesis. Here we show that the above and below ground tissue-response to high ambient temperature are mediated by distinct transcription factors. While the central hub transcription factor, PHYTOCHROME INTERCTING FACTOR 4 (PIF4) regulates the above ground tissue response, the below ground root elongation is primarily regulated by ELONGATED HYPOCOTYL 5 (HY5). Plants respond to high temperature by largely expressing distinct sets of genes in a tissue-specific manner. HY5 promotes root thermomorphogenesis via directly controlling the expression of many genes including the auxin and BR pathway genes. Strikingly, the above and below ground thermomorphogenesis is impaired in spaQ. Because SPA1 directly phosphorylates PIF4 and HY5, SPAs might control the stability of PIF4 and HY5 to regulate thermomorphogenesis in both tissues. These data collectively suggest that plants employ distinct combination of SPA-PIF4-HY5 module to regulate tissue-specific thermomorphogenesis.

Light-regulated pre-mRNA splicing in plants.

Light signal perceived by the red/far-red absorbing phytochrome (phy) family of photoreceptors regulates plant growth and development throughout the life cycle. Phytochromes regulate the light-triggered physiological responses by controlling gene expression both at the transcriptional and post-transcriptional levels. Recent large-scale RNA-seq studies have demonstrated the roles of phys in altering the global transcript diversity by modulating the pre-mRNA splicing in response to light. Moreover, several phy-interacting splicing factors/regulators from different species have been identified using forward genetics and protein-protein interaction studies, which modulate the light-regulated pre-mRNA splicing. In this article, we summarize our current understanding of the role of phys in the light-mediated pre-mRNA splicing and how that contributes to the regulation of gene expression to promote photomorphogenesis.

Phytochrome Signaling Networks.

The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle. The holistic regulatory networks orchestrated by phytochromes, including conformational switching, subcellular localization, direct protein-protein interactions, transcriptional and posttranscriptional regulations, and translational and posttranslational controls to promote photomorphogenesis, are highly coordinated and regulated at multiple levels. During the past decade, advances using innovative approaches have substantially broadened our understanding of the sophisticated mechanisms underlying the phytochrome-mediated light signaling pathways. This review discusses and summarizes these discoveries of the role of the modular structure of phytochromes, phytochrome-interacting proteins, and their functions; the reciprocal modulation of both positive and negative regulators in phytochrome signaling; the regulatory roles of phytochromes in transcriptional activities, alternative splicing, and translational regulation; and the kinases and E3 ligases that modulate PHYTOCHROME INTERACTING FACTORs to optimize photomorphogenesis.