An interplay between bZIP16, bZIP68, and GBF1 regulates nuclear photosynthetic genes during photomorphogenesis in Arabidopsis.

The development of a seedling into a photosynthetically active plant is a crucial process. Despite its importance, we do not fully understand the regulatory mechanisms behind the establishment of functional chloroplasts. We herein provide new insight into the early light response by identifying the function of three basic region/leucine zipper (bZIP) transcription factors: bZIP16, bZIP68, and GBF1. These proteins are involved in the regulation of key components required for the establishment of photosynthetically active chloroplasts. The activity of these bZIPs is dependent on the redox status of a conserved cysteine residue, which provides a mechanism to finetune light-responsive gene expression. The blue light cryptochrome (CRY) photoreceptors provide one of the major light-signaling pathways, and bZIP target genes overlap with one-third of CRY-regulated genes with an enrichment for photosynthesis/chloroplast-associated genes. bZIP16, bZIP68, and GBF1 were demonstrated as novel interaction partners of CRY1. The interaction between CRY1 and bZIP16 was stimulated by blue light. Furthermore, we demonstrate a genetic link between the bZIP proteins and cryptochromes as the cry1cry2 mutant is epistatic to the cry1cry2bzip16bzip68gbf1 mutant. bZIP16, bZIP68, and GBF1 regulate a subset of photosynthesis associated genes in response to blue light critical for a proper greening process in Arabidopsis.

Light and temperature regulate m6A-RNA modification to regulate growth in plants.

N6-methyladenosine is a highly dynamic, abundant mRNA modification which is an excellent potential mechanism for fine tuning gene expression. Plants adapt to their surrounding light and temperature environment using complex gene regulatory networks. The role of m6A in controlling gene expression in response to variable environmental conditions has so far been unexplored. Here, we map the transcriptome-wide m6A landscape under various light and temperature environments. Identified m6A-modifications show a highly specific spatial distribution along transcripts with enrichment occurring in 5'UTR regions and around transcriptional end sites. We show that the position of m6A modifications on transcripts might influence cellular transcript localization and the presence of m6A-modifications is associated with alternative polyadenylation, a process which results in multiple RNA isoforms with varying 3'UTR lengths. RNA with m6A-modifications exhibit a higher preference for shorter 3'UTRs. These shorter 3'UTR regions might directly influence transcript abundance and localization by including or excluding cis-regulatory elements. We propose that environmental stimuli might change the m6A landscape of plants as one possible way of fine tuning gene regulation through alternative polyadenylation and transcript localization.

Cryptochromes and UBP12/13 deubiquitinases antagonistically regulate DNA damage response in Arabidopsis.

Cryptochromes (CRYs) are evolutionarily conserved blue-light receptors that evolved from bacterial photolyases that repair damaged DNA. Today, CRYs have lost their ability to repair damaged DNA; however, prior reports suggest that human CRYs can respond to DNA damage. Currently, the role of CRYs in the DNA damage response (DDR) is lacking, especially in plants. Therefore, we evaluated the role of plant CRYs in DDR along with UBP12/13 deubiquitinases, which interact with and regulate the CRY2 protein. We found that cry1cry2 was hypersensitive, while ubp12ubp13 was hyposensitive to UVC-induced DNA damage. Elevated UV-induced cyclobutane pyrimidine dimers (CPDs) and the lack of DNA repair protein RAD51 accumulation in cry1cry2 plants indicate that CRYs are required for DNA repair. On the contrary, CPD levels diminished and RAD51 protein levels elevated in plants lacking UBP12 and UBP13, indicating their role in DDR repression. Temporal transcriptomic analysis revealed that DDR-induced transcriptional responses were subdued in cry1cry2, but elevated in ubp12ubp13 compared to WT. Through transcriptional modeling of the time-course transcriptome, we found that genes quickly induced by UVC (15 min) are targets of CAMTA 1-3 transcription factors, which we found are required for DDR. This transcriptional regulation seems, however, diminished in the cry1cry2 mutant, indicating that CAMTAs are required for CRY2-mediated DDR. Furthermore, we observed enhanced CRY2-UBP13 interaction and formation of CRY2 nuclear speckles under UVC, suggesting that UVC activates CRY2 similarly to blue light. Together, our data reveal the temporal dynamics of the transcriptional events underlying UVC-induced genotoxicity and expand our knowledge of the role of CRY and UBP12/13 in DDR.

Oligodendrocyte precursor cells engulf synapses during circuit remodeling in mice.

Oligodendrocyte precursor cells (OPCs) give rise to myelinating oligodendrocytes throughout life, but the functions of OPCs are not limited to oligodendrogenesis. Here we show that OPCs contribute to thalamocortical presynapse elimination in the developing and adult mouse visual cortex. OPC-mediated synapse engulfment increases in response to sensory experience during neural circuit refinement. Our data suggest that OPCs may regulate synaptic connectivity in the brain independently of oligodendrogenesis.

Systematic histone H4 replacement in Arabidopsis thaliana reveals a role for H4R17 in regulating flowering time.

Despite the broad array of roles for epigenetic mechanisms on regulating diverse processes in eukaryotes, no experimental system is currently available in plants for the direct assessment of histone function. In this work, we present the development of a genetic strategy in Arabidopsis (Arabidopsis thaliana) whereby modified histone H4 transgenes can completely replace the expression of endogenous histone H4 genes. Accordingly, we established a collection of plants expressing different H4 point mutants targeting residues that may be post-translationally modified in vivo. To demonstrate its utility, we screened this new H4 mutant collection to uncover substitutions in H4 that alter flowering time. We identified different mutations in the H4 tail (H4R17A) and the H4 globular domain (H4R36A, H4R39K, H4R39A, and H4K44A) that strongly accelerate the floral transition. Furthermore, we identified a conserved regulatory relationship between H4R17 and the ISWI chromatin remodeling complex in plants: As with other biological systems, H4R17 regulates nucleosome spacing via ISWI. Overall, this work provides a large set of H4 mutants to the plant epigenetics community that can be used to systematically assess histone H4 function in Arabidopsis and a roadmap to replicate this strategy for studying other histone proteins in plants.

UBP12 and UBP13 deubiquitinases destabilize the CRY2 blue light receptor to regulate Arabidopsis growth.

Light is a crucial exogenous signal sensed by cryptochrome (CRY) blue light receptors to modulate growth and the circadian clock in plants and animals. However, how CRYs interpret light quantity to regulate growth in plants remains poorly understood. Furthermore, CRY2 protein levels and activity are tightly regulated in light to fine-tune hypocotyl growth; however, details of the mechanisms that explain precise control of CRY2 levels are not fully understood. We show that in Arabidopsis, UBP12 and UBP13 deubiquitinases physically interact with CRY2 in light. UBP12/13 negatively regulates CRY2 by promoting its ubiquitination and turnover to modulate hypocotyl growth. Growth and development were explicitly affected in blue light when UBP12/13 were disrupted or overexpressed, indicating their role alongside CRY2. UBP12/13 also interacted with and stabilized COP1, which is partially required for CRY2 turnover. Our combined genetic and molecular data support a mechanistic model in which UBP12/13 interact with CRY2 and COP1, leading to the stabilization of COP1. Stabilized COP1 then promotes the ubiquitination and degradation of CRY2 under blue light. Despite decades of studies on deubiquitinases, the knowledge of how their activity is regulated is limited. Our study provides insight into how exogenous signals and ligands, along with their receptors, regulate deubiquitinase activity by protein-protein interaction. Collectively, our results provide a framework of cryptochromes and deubiquitinases to detect and interpret light signals to control plant growth at the most appropriate time.

WRKY transcription factors and ethylene signaling modify root growth during the shade-avoidance response.

Shade-intolerant plants rapidly elongate their stems, branches, and leaf stalks to compete with neighboring vegetation, maximizing sunlight capture for photosynthesis. This rapid growth adaptation, known as the shade-avoidance response (SAR), comes at a cost: reduced biomass, crop yield, and root growth. Significant progress has been made on the mechanistic understanding of hypocotyl elongation during SAR; however, the molecular interpretation of root growth repression is not well understood. Here, we explore the mechanisms by which SAR induced by low red:far-red light restricts primary and lateral root (LR) growth. By analyzing the whole-genome transcriptome, we identified a core set of shade-induced genes in roots of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) seedlings grown in the shade. Abiotic and biotic stressors also induce many of these shade-induced genes and are predominantly regulated by WRKY transcription factors. Correspondingly, a majority of WRKY genes were among the shade-induced genes. Functional analysis using transgenics of these shade-induced WRKYs revealed that their role is essentially to restrict primary root and LR growth in the shade; captivatingly, they did not affect hypocotyl elongation. Similarly, we also found that ethylene hormone signaling is necessary for limiting root growth in the shade. We propose that during SAR, shade-induced WRKY26, 45, and 75, and ethylene reprogram gene expression in the root to restrict its growth and development.

Chimeric Activators and Repressors Define HY5 Activity and Reveal a Light-Regulated Feedback Mechanism.

The first exposure to light marks a crucial transition in plant development. This transition relies on the transcription factor HY5 controlling a complex downstream growth program. Despite its importance, its function in transcription remains unclear. Previous studies have generated lists of thousands of potential target genes and competing models of HY5 transcription regulation. In this work, we carry out detailed phenotypic and molecular analysis of constitutive activator and repressor HY5 fusion proteins. Using this strategy, we were able to filter out large numbers of genes that are unlikely to be direct targets, allowing us to eliminate several proposed models of HY5's mechanism of action. We demonstrate that the primary activity of HY5 is promoting transcription and that this function relies on other, likely light-regulated, factors. In addition, this approach reveals a molecular feedback loop via the COP1/SPA E3 ubiquitin ligase complex, suggesting a mechanism that maintains low HY5 in the dark, primed for rapid accumulation to reprogram growth upon light exposure. Our strategy is broadly adaptable to the study of transcription factor activity. Lastly, we show that modulating this feedback loop can generate significant phenotypic diversity in both Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum).

A Statistical Description of Plant Shoot Architecture.

Plant architectures can be characterized statistically by their spatial density function, which specifies the probability of finding a branch at each location in the territory occupied by a plant. Using high-precision 3D scanning, we analyzed 557 plant shoot architectures, representing three species, grown across three to five environmental conditions, and through 20-30 developmental time points. We found two elegant properties in the spatial density functions of these architectures: all functions could be nearly modified in one direction without affecting the density in orthogonal directions (called "separability"), and all functions shared the same underlying shape, aside from stretching and compression (called "self-similarity"). Surprisingly, despite their striking visual diversity, we discovered that all architectures could be described as variations on a single underlying function: a Gaussian density function truncated at roughly two SDs. We also observed systematic variation in the spatial density functions across species, growth conditions, and time, which suggests functional specialization despite following the same general design form.

High-Resolution Laser Scanning Reveals Plant Architectures that Reflect Universal Network Design Principles.

Transport networks serve critical functions in biological and engineered systems, and yet their design requires trade-offs between competing objectives. Due to their sessile lifestyle, plants need to optimize their architecture to efficiently acquire and distribute resources while also minimizing costs in building infrastructure. To understand how plants resolve this design trade-off, we used high-precision three-dimensional laser scanning to map the architectures of tomato, tobacco, or sorghum plants grown in several environmental conditions and through multiple developmental time points, scanning in total 505 architectures from 37 plants. Using a graph-theoretic algorithm that we developed to evaluate design strategies, we find that plant architectures lie along the Pareto front between two simple length-based objectives-minimizing total branch length and minimizing nutrient transport distance-thereby conferring a selective fitness advantage for plant transport processes. The location along the Pareto front can distinguish among species and conditions, suggesting that during evolution, natural selection may employ common network design principles despite different optimization trade-offs.

Cryptochromes Interact Directly with PIFs to Control Plant Growth in Limiting Blue Light.

Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome wide and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.

Integration of Light and Photoperiodic Signaling in Transcriptional Nuclear Foci.

Light regulates major plant developmental transitions by orchestrating a series of nuclear events. This study uncovers the molecular function of the natural variant, TZP (Tandem Zinc-finger-Plus3), as a signal integrator of light and photoperiodic pathways in transcriptional nuclear foci. We report that TZP acts as a positive regulator of photoperiodic flowering via physical interactions with the red-light receptor phytochrome B (phyB). We demonstrate that TZP localizes in dynamic nuclear domains regulated by light quality and photoperiod. This study shows that phyB is indispensable not only for localizing TZP to transcriptionally active nuclear photobodies, but also for recruiting TZP on the promoter of the floral inducer FLOWERING LOCUS T (FT). Our findings signify a unique transcriptional regulatory role to the highly enigmatic plant nuclear photobodies, where TZP directly activates FT gene expression and promotes flowering.

Linking photoreceptor excitation to changes in plant architecture.

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.

Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3).

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3(NPH3), with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.

Cryptochrome 1 and phytochrome B control shade-avoidance responses in Arabidopsis via partially independent hormonal cascades.

Plants respond to a reduction in the red/far-red ratio (R:FR) of light, caused by the proximity of other plants, by initiating morphological changes that improve light capture. In Arabidopsis, this response (shade avoidance syndrome, SAS) is controlled by phytochromes (particularly phyB), and is dependent on the TAA1 pathway of auxin biosynthesis. However, when grown in real canopies, we found that phyB mutants and mutants deficient in TAAI (sav3) still display robust SAS responses to increased planting density and leaf shading. The SAS morphology (leaf hyponasty and reduced lamina/petiole ratio) could be phenocopied by exposing plants to blue light attenuation. These responses to blue light attenuation required the UV-A/blue light photoreceptor cry1. Moreover, they were mediated through mechanisms that showed only limited overlap with the pathways recruited by phyB inactivation. In particular, pathways for polar auxin transport, auxin biosynthesis and gibberellin signaling that are involved in SAS responses to low R:FR were not required for the SAS responses to blue light depletion. By contrast, the brassinosteroid response appeared to be required for the full expression of the SAS phenotype under low blue light. The phyB and cry1 inactivation pathways appeared to converge in their requirement for the basic/helix-loop-helix (bHLH) transcription factors PHYTOCHROME INTERACTING FACTORs 4 and 5 (PIF4 and PIF5) to elicit the SAS phenotype. Our results suggest that blue light is an important control of SAS responses, and that PIF4 and PIF5 are critical hubs for a diverse array of signaling routes that control plant architecture in canopies.

Phototropism: mechanism and outcomes.

Plants have evolved a wide variety of responses that allow them to adapt to the variable environmental conditions in which they find themselves growing. One such response is the phototropic response - the bending of a plant organ toward (stems and leaves) or away from (roots) a directional blue light source. Phototropism is one of several photoresponses of plants that afford mechanisms to alter their growth and development to changes in light intensity, quality and direction. Over recent decades much has been learned about the genetic, molecular and cell biological components involved in sensing and responding to phototropic stimuli. Many of these advances have been made through the utilization of Arabidopsis as a model for phototropic studies. Here we discuss such advances, as well as studies in other plant species where appropriate to the discussion of work in Arabidopsis.

Regulation of phototropic signaling in Arabidopsis via phosphorylation state changes in the phototropin 1-interacting protein NPH3.

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.