Hui Shi

A Quartet of PIF bHLH Factors Provides a Transcriptionally Centered Signaling Hub That Regulates Seedling Morphogenesis through Differential Expression-Patterning of Shared Target Genes in Arabidopsis

Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of Arabidopsis Phytochrome (phy)-Interacting bHLH Factors (PIF1, 3, 4, and 5) are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple pif-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.

EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings.

The ability to switch from skotomorphogenesis to photomorphogenesis is essential for seedling development and plant survival. Recent studies revealed that COP1 and phytochrome-interacting factors (PIFs) are key regulators of this transition by repressing the photomorphogenic responses and/or maintaining the skotomorphogenic state of etiolated seedlings. Here we report that the plant hormone ethylene plays a crucial role in the transition from skotomorphogenesis to photomorphogenesis by facilitating greening of etiolated seedlings upon light irradiation. Activation of EIN3/EIL1 is both necessary and sufficient for ethylene-induced enhancement of seedling greening, as well as repression of the accumulation of protochlorophyllide, a phototoxic intermediate of chlorophyll synthesis. EIN3/EIL1 were found to induce gene expression of two key enzymes in the chlorophyll synthesis pathway, protochlorophyllide oxidoreductase A and B (PORA/B). ChIP and EMSA assays demonstrated that EIN3 directly binds to the specific elements present in the PORA and PORB promoters. Genetic studies revealed that EIN3/EIL1 function in cooperation with PIF1 in preventing photo-oxidative damage and promoting cotyledon greening. Moreover, activation of EIN3 reverses the blockage of greening triggered by cop1 mutation or far-red light irradiation. Consistently, EIN3 acts downstream of COP1 and its protein accumulation is enhanced by COP1 but decreased by light. Taken together, EIN3/EIL1 represent a new class of transcriptional regulators along with PIF1 to optimize de-etiolation of Arabidopsis seedlings. Our study highlights the essential role of ethylene in enhancing seedling development and survival through protecting etiolated seedlings against photo-oxidative damage.

A Molecular Framework of Light-Controlled Phytohormone Action in Arabidopsis

Environmental changes strongly affect plant growth and development. Phytohormones, endogenous plant-made small molecules such as ethylene, regulate a wide range of processes throughout the lifetime of plants. The ability of plants to integrate external signals with endogenous regulatory pathways is vital for their survival. Ethylene has been found to suppress hypocotyl elongation in darkness while promoting it in light. How ethylene regulates hypocotyl elongation in such opposite ways is largely unknown. In particular, how light modulates and even reverses the function of ethylene has yet to be characterized. Here we show that the basic-helix-loop-helix transcription factor phytochrome-interacting factor 3 (PIF3) is directly activated by ETHYLENE-INSENSITIVE 3 (EIN3) and is indispensible for ethylene-induced hypocotyl elongation in light. Ethylene via EIN3 concomitantly activates two contrasting pathways: the PIF3-dependent growth-promoting pathway and an ethylene response factor 1 (ERF1)-mediated growth-inhibiting pathway. In the light, growth-promoting PIFs are limiting due to light-dependent destabilization, and thus ethylene stimulates growth under these conditions. In contrast, ERF1 is destabilized, and thus limiting, under dark conditions, explaining why ethylene inhibits growth in the dark. Our findings provide a mechanistic insight into how light modulates internal hormone-regulated plant growth.

Combinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis

A subfamily of four Phytochrome (phy)-Interacting bHLH transcription Factors (PIFs) collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoactivated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a transition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process. To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChIP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied preferentially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.

Research ArticleCombinatorial Complexity in a Transcriptionally Centered Signaling Hub in Arabidopsis

A subfamily of four Phytochrome (phy)-Interacting bHLH transcription Factors (PIFs) collectively promote skotomorphogenic development in dark-grown seedlings. This activity is reversed upon exposure to light, by photoactivated phy molecules that induce degradation of the PIFs, thereby triggering the transcriptional changes that drive a transition to photomorphogenesis. The PIFs function both redundantly and partially differentially at the morphogenic level in this process. To identify the direct targets of PIF transcriptional regulation genome-wide, we analyzed the DNA-binding sites for all four PIFs by ChIP-seq analysis, and defined the genes transcriptionally regulated by each PIF, using RNA-seq analysis of pif mutants. Despite the absence of detectable differences in DNA-binding-motif recognition between the PIFs, the data show a spectrum of regulatory patterns, ranging from single PIF dominance to equal contributions by all four. Similarly, a broad array of promoter architectures was found, ranging from single PIF-binding sites, containing single sequence motifs, through multiple PIF-binding sites, each containing one or more motifs, with each site occupied preferentially by one to multiple PIFs. Quantitative analysis of the promoter occupancy and expression level induced by each PIF revealed an intriguing pattern. Although there is no robust correlation broadly across the target-gene population, examination of individual genes that are shared targets of multiple PIFs shows a gradation in correlation from strongly positive, through uncorrelated, to negative. This finding suggests a dual-layered mechanism of transcriptional regulation, comprising both a continuum of binding-site occupancy by each PIF and a superimposed layer of local regulation that acts differentially on each PIF, to modulate its intrinsic transcriptional activation capacity at each site, in a quantitative pattern that varies between the individual PIFs from gene to gene. These findings provide a framework for probing the mechanisms by which transcription factors with overlapping direct-target genes integrate and selectively transduce signals to their target networks.

Ethylene-orchestrated circuitry coordinates a seedling’s response to soil cover and etiolated growth

Significance Seedlings’ ability to both adapt to their soil environment and acquire photoautotrophic capacity under various buried conditions is a life-or-death issue for terrestrial flowering plants. By designing and utilizing a standardized real-soil assay, we identify the key features of germinating seedlings’ soil response and deduce that the gaseous phytohormone ethylene acts as the primary regulator of soil-induced plant morphogenetic changes. Moreover, our study illustrates that an EIN3/EIL1-conducted PIF3–ERF1 molecular circuitry enables seedlings to synchronize the rate of protochlorophyllide biosynthesis with upward growth, a mechanism critical to the prevention of photooxidative damage during seedlings’ initial transition from dark to light in natural conditions. The early life of terrestrial seed plants often starts under the soil in subterranean darkness. Over time and through adaptation, plants have evolved an elaborate etiolation process that enables seedlings to emerge from soil and acquire autotrophic ability. This process, however, requires seedlings to be able to sense the soil condition and relay this information accordingly to modulate both the seedlings’ growth and the formation of photosynthetic apparatus. The mechanism by which soil overlay drives morphogenetic changes in plants, however, remains poorly understood, particularly with regard to the means by which the cellular processes of different organs are coordinated in response to disparate soil conditions. Here, we illustrate that the soil overlay quantitatively activates seedlings’ ethylene production, and an EIN3/EIN3-like 1–dependent ethylene-response cascade is required for seedlings to successfully emerge from the soil. Under soil, an ERF1 pathway is activated in the hypocotyl to slow down cell elongation, whereas a PIF3 pathway is activated in the cotyledon to control the preassembly of photosynthetic machinery. Moreover, this latter PIF3 pathway appears to be coupled to the ERF1-regulated upward-growth rate. The coupling of these two pathways facilitates the synchronized progression of etioplast maturation and hypocotyl growth, which, in turn, ultimately enables seedlings to maintain the amount of protochlorophyllide required for rapid acquisition of photoautotrophic capacity without suffering from photooxidative damage during the dark-to-light transition. Our findings illustrate the existence of a genetic signaling pathway driving soil-induced plant morphogenesis and define the specific role of ethylene in orchestrating organ-specific soil responses in Arabidopsis seedlings.

Salt-Induced Stabilization of EIN3/EIL1 Confers Salinity Tolerance by Deterring ROS Accumulation in Arabidopsis

Ethylene has been regarded as a stress hormone to regulate myriad stress responses. Salinity stress is one of the most serious abiotic stresses limiting plant growth and development. But how ethylene signaling is involved in plant response to salt stress is poorly understood. Here we showed that Arabidopsis plants pretreated with ethylene exhibited enhanced tolerance to salt stress. Gain- and loss-of-function studies demonstrated that EIN3 (ETHYLENE INSENSITIVE 3) and EIL1 (EIN3-LIKE 1), two ethylene-activated transcription factors, are necessary and sufficient for the enhanced salt tolerance. High salinity induced the accumulation of EIN3/EIL1 proteins by promoting the proteasomal degradation of two EIN3/EIL1-targeting F-box proteins, EBF1 and EBF2, in an EIN2-independent manner. Whole-genome transcriptome analysis identified a list of SIED (Salt-Induced and EIN3/EIL1-Dependent) genes that participate in salt stress responses, including several genes encoding reactive oxygen species (ROS) scavengers. We performed a genetic screen for ein3 eil1-like salt-hypersensitive mutants and identified 5 EIN3 direct target genes including a previously unknown gene, SIED1 (At5g22270), which encodes a 93-amino acid polypeptide involved in ROS dismissal. We also found that activation of EIN3 increased peroxidase (POD) activity through the direct transcriptional regulation of PODs expression. Accordingly, ethylene pretreatment or EIN3 activation was able to preclude excess ROS accumulation and increased tolerance to salt stress. Taken together, our study provides new insights into the molecular action of ethylene signaling to enhance plant salt tolerance, and elucidates the transcriptional network of EIN3 in salt stress response.

HFR1 Sequesters PIF1 to Govern the Transcriptional Network Underlying Light-Initiated Seed Germination in Arabidopsis

This study identifies the LONG HYPOCOTYL IN FAR-RED1 (HFR1)– PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) pair as the central transcriptional regulatory module for rapid light-initiated transcriptome changes in imbibed seeds. HFR1 interacts with PIF1 to prevent PIF1 from binding to DNA, promoting light-initiated germination. The functionally antagonistic HFR1–PIF1 pair constructs a fail-safe mechanism for fine-tuning seed germination under low-level illumination. Seed germination is the first step for seed plants to initiate a new life cycle. Light plays a predominant role in promoting seed germination, where the initial phase is mediated by photoreceptor phytochrome B (phyB). Previous studies showed that PHYTOCHROME-INTERACTING FACTOR1 (PIF1) represses seed germination downstream of phyB. Here, we identify a positive regulator of phyB-dependent seed germination, LONG HYPOCOTYL IN FAR-RED1 (HFR1). HFR1 blocks PIF1 transcriptional activity by forming a heterodimer with PIF1 that prevents PIF1 from binding to DNA. Our whole-genomic analysis shows that HFR1 and PIF1 oppositely mediate the light-regulated transcriptome in imbibed seeds. Through the HFR1–PIF1 module, light regulates expression of numerous genes involved in cell wall loosening, cell division, and hormone pathways to initiate seed germination. The functionally antagonistic HFR1–PIF1 pair constructs a fail-safe mechanism for fine-tuning seed germination during low-level illumination, ensuring a rapid response to favorable environmental changes. This study identifies the HFR1–PIF1 pair as a central module directing the whole genomic transcriptional network to rapidly initiate light-induced seed germination.

Arabidopsis DET1 degrades HFR1 but stabilizes PIF1 to precisely regulate seed germination.

Significance How organisms respond to environment changes is a fundamental and intriguing question in biology. Light is the energy resource and a crucial environmental cue for plant major developmental switches, such as seed germination. Studying the underlying mechanism is important for us to understand the basic principles of plant development and improve crop productions. Here we identify DET1 as a novel central repressor of seed germination. We further reveal that seeds use a multilevel regulatory circuit of triple feed-forward loops to sensitively and precisely mediate light-regulated germination. This study provides a comprehensive framework of how light regulates seed germination. Seed is an essential propagation organ and a critical strategy adopted by terrestrial flowering plants to colonize the land. The ability of seeds to accurately respond to light is vital for plant survival. However, the underlying mechanism is largely unknown. In this study, we reveal a circuit of triple feed-forward loops adopted by Arabidopsis seeds to exclusively repress germination in dark conditions and precisely initiate germination under diverse light conditions. We identify that de-etiolated 1 (DET1), an evolutionarily conserved protein, is a central repressor of light-induced seed germination. Genetic analysis demonstrates that DET1 functions upstream of long hypocotyl in far-red 1 (HFR1) and phytochrome interacting factor 1 (PIF1), the key positive and negative transcription regulators in seed germination. We further find that DET1 and constitutive photomorphogenic 10 (COP10) target HFR1 for protein degradation by assembling a COP10–DET1–damaged DNA binding protein 1–cullin4 E3 ligase complex. Moreover, DET1 and COP10 directly interact with and promote the protein stability of PIF1. Computational modeling reveals that phytochrome B (phyB)–DET1–HFR1–PIF1 and phyB–DET1–Protease–PIF1 are new signaling pathways, independent of the previously identified phyB-PIF1 pathway, respectively mediating the rapid and time-lapse responses to light irradiation. The model-simulated results are highly consistent with their experimental validations, suggesting that our mathematical model captures the essence of Arabidopsis seed germination networks. Taken together, this study provides a comprehensive molecular framework for light-regulated seed germination, improving our understanding of how plants respond to changeable environments.

The Red Light Receptor Phytochrome B Directly Enhances Substrate-E3 Ligase Interactions to Attenuate Ethylene Responses

Plants germinating under subterranean darkness assume skotomorphogenesis, a developmental program strengthened by ethylene in response to mechanical pressure of soil. Upon reaching the surface, light triggers a dramatic developmental transition termed de-etiolation that requires immediate termination of ethylene responses. Here, we report that light activation of photoreceptor phyB results in rapid degradation of EIN3, the master transcription factor in the ethylene signaling pathway. As a result, light rapidly and efficiently represses ethylene actions. Specifically, phyB directly interacts with EIN3 in a light-dependent manner and also physically associates with F box protein EBFs. The light-activated association of phyB, EIN3, and EBF1/EBF2 proteins stimulates robust EIN3 degradation by SCFEBF1/EBF2 E3 ligases. We reveal that phyB manipulates substrate-E3 ligase interactions in a light-dependent manner, thus directly controlling the stability of EIN3. Our findings illustrate a mechanistic model of how plants transduce light information to immediately turn off ethylene signaling for de-etiolation initiation.