Identification of Arabidopsis phospholipase Ds involved in circadian clock alterations using CRISPR/Cas9-based multiplex editing.

Reciprocal regulation between circadian clock and lipid metabolism is emerging, but its mechanistic details remain elusive in plants. We previously reported that a lipid metabolite phosphatidic acid (PA) bound to the core clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and chemical suppression of phospholipase D (PLD)-catalyzed PA formation perturbed the clock in Arabidopsis. Here, we investigated to identify, among 12 isoforms, specific PLD(s) important to the regulation of clock function. We generated a library of Arabidopsis plants bearing PLD s randomly mutated by multiplex CRISPR/Cas9 system. PCR and gel-based analyses showed that all PLD s, except for ß2 , were effectively edited and the mutations were heritable. Screening of T2 plants identified some with an altered rhythmic expression of CCA1 and this trait was observed in many of their progenies. Genotyping by DNA sequencing revealed that at least two of six PLD s ( α1, α3 , γ1 , δ , ε and ζ2 ) were mutated in the clock-altered T3 plants. This study identifies different combinations of two or more PLDs involved in altering clock outputs, and also suggests a functional redundancy of the six PLDs for the regulation of plant circadian clock.

An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

Circadian clock factors regulate the first condensation reaction of fatty acid synthesis in Arabidopsis.

The circadian clock regulates temporal metabolic activities, but how it affects lipid metabolism is poorly understood. Here, we show that the central clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) regulate the initial step of fatty acid (FA) biosynthesis in Arabidopsis. Triacylglycerol (TAG) accumulation in seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants. Metabolic tracking of lipids in developing seeds indicated that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding ß-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data show a regulatory mechanism for plant lipid biosynthesis by the molecular clock.

Phytochromes transmit photoperiod information via the evening complex in Brachypodium.

BACKGROUND: Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. RESULTS: We find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night. CONCLUSIONS: We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.

COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

Assembly and operation of an imaging system for long-term monitoring of bioluminescent and fluorescent reporters in plants.

BACKGROUND: Non-invasive reporter systems are powerful tools to query physiological and transcriptional responses in organisms. For example, fluorescent and bioluminescent reporters have revolutionized cellular and organismal assays and have been used to study plant responses to abiotic and biotic stressors. Integrated, cooled charge-coupled device (CCD) camera systems have been developed to image bioluminescent and fluorescent signals in a variety of organisms; however, these integrated long-term imaging systems are expensive. RESULTS: We have developed self-assembled systems for both growing and monitoring plant fluorescence and bioluminescence for long-term experiments under controlled environmental conditions. This system combines environmental growth chambers with high-sensitivity CCD cameras, multi-wavelength LEDs, open-source software, and several options for coordinating lights with imaging. This easy-to-assemble system can be used for short and long-term imaging of bioluminescent reporters, acute light-response, circadian rhythms, delayed fluorescence, and fluorescent-protein-based assays in vivo. CONCLUSIONS: We have developed two self-assembled imaging systems that will be useful to researchers interested in continuously monitoring in vivo reporter systems in various plant species.

A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules.

Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally in plants and involve a hormone receptor that binds a hormone, triggering a conformational change in the receptor that enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are characterized well by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio of the hormone receptor to the binding protein. Thus, the same molecular ratchet can have a pico- or micromolar EC50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of the receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including enzyme-linked immunosorbent assay-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation.

Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds.

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense-response applications.

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis.

Identification of protein-protein interactions is an effective method of elucidating new roles for circadian clock-associated proteins that can expand beyond the information collected from transcriptional studies and genetic screens. Tandem affinity purification coupled with liquid chromatography mass spectrometry (APMS) utilizes epitope-tagged versions of your protein of interest to co-precipitate direct and indirect protein partners. Here, we provide a protocol and suggestions for proper design of 6x-His-3x-FLAG-tagged clock proteins and isolation of protein-protein interactions using two immunoprecipitation steps for increased specificity.

Optimisation of root traits to provide enhanced ecosystem services in agricultural systems: A focus on cover crops.

Roots are the interface between the plant and the soil and play a central role in multiple ecosystem processes. With intensification of agricultural practices, rhizosphere processes are being disrupted and are causing degradation of the physical, chemical and biotic properties of soil. However, cover crops, a group of plants that provide ecosystem services, can be utilised during fallow periods or used as an intercrop to restore soil health. The effectiveness of ecosystem services provided by cover crops varies widely as very little breeding has occurred in these species. Improvement of ecosystem service performance is rarely considered as a breeding trait due to the complexities and challenges of belowground evaluation. Advancements in root phenotyping and genetic tools are critical in accelerating ecosystem service improvement in cover crops. In this study, we provide an overview of the range of belowground ecosystem services provided by cover crop roots: (1) soil structural remediation, (2) capture of soil resources and (3) maintenance of the rhizosphere and building of organic matter content. Based on the ecosystem services described, we outline current and promising phenotyping technologies and breeding strategies in cover crops that can enhance agricultural sustainability through improvement of root traits.

High light and temperature reduce photosynthetic efficiency through different mechanisms in the C4 model Setaria viridis.

C4 plants frequently experience high light and high temperature conditions in the field, which reduce growth and yield. However, the mechanisms underlying these stress responses in C4 plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C4 model plant Setaria viridis responded to a four-hour high light or high temperature treatment at photosynthetic, transcriptomic, and ultrastructural levels. Although we observed a comparable reduction of photosynthetic efficiency in high light or high temperature treated leaves, detailed analysis of multi-level responses revealed important differences in key pathways and M/BS specificity responding to high light and high temperature. We provide a systematic analysis of high light and high temperature responses in S. viridis, reveal different acclimation strategies to these two stresses in C4 plants, discover unique light/temperature responses in C4 plants in comparison to C3 plants, and identify potential targets to improve abiotic stress tolerance in C4 crops.

Out of the Dark and Into the Light: A New View of Phytochrome Photobodies.

Light is a critical environmental stimulus for plants, serving as an energy source via photosynthesis and a signal for developmental programming. Plants perceive light through various light-responsive proteins, termed photoreceptors. Phytochromes are red-light photoreceptors that are highly conserved across kingdoms. In the model plant Arabidopsis thaliana, phytochrome B serves as a light and thermal sensor, mediating physiological processes such as seedling germination and establishment, hypocotyl growth, chlorophyll biogenesis, and flowering. In response to red light, phytochromes convert to a biologically active form, translocating from the cytoplasm into the nucleus and further compartmentalizes into subnuclear compartments termed photobodies. PhyB photobodies regulate phytochrome-mediated signaling and physiological outputs. However, photobody function, composition, and biogenesis remain undefined since their discovery. Based on photobody cellular dynamics and the properties of internal components, photobodies have been suggested to undergo liquid-liquid phase separation, a process by which some membraneless compartments form. Here, we explore photobodies as environmental sensors, examine the role of their protein constituents, and outline the biophysical perspective that photobodies may be undergoing liquid-liquid phase separation. Understanding the molecular, cellular, and biophysical processes that shape how plants perceive light will help in engineering improved sunlight capture and fitness of important crops.

Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis.

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

Time Will Tell: Intercellular Communication in the Plant Clock.

Multicellular organisms have evolved local and long-distance signaling mechanisms to synchronize development and response to stimuli among a complex network of cells, tissues, and organs. Biological timekeeping is one such activity that is suggested to be coordinated within an organism to anticipate and respond to daily and seasonal patterns in the environment. New research into the plant clock suggests circadian rhythms are communicated between cells and across long distances. However, further clarity is required on the nature of the signaling molecules and the mechanisms underlying signal translocation. Here we summarize the roles and properties of tissue-specific circadian rhythms, discuss the evidence for local and long-distance clock communication, and evaluate the potential signaling molecules and transport mechanisms involved in this system.

A genome resource for green millet Setaria viridis enables discovery of agronomically valuable loci.

Wild and weedy relatives of domesticated crops harbor genetic variants that can advance agricultural biotechnology. Here we provide a genome resource for the wild plant green millet (Setaria viridis), a model species for studies of C4 grasses, and use the resource to probe domestication genes in the close crop relative foxtail millet (Setaria italica). We produced a platinum-quality genome assembly of S. viridis and de novo assemblies for 598 wild accessions and exploited these assemblies to identify loci underlying three traits: response to climate, a 'loss of shattering' trait that permits mechanical harvest and leaf angle, a predictor of yield in many grass crops. With CRISPR-Cas9 genome editing, we validated Less Shattering1 (SvLes1) as a gene whose product controls seed shattering. In S. italica, this gene was rendered nonfunctional by a retrotransposon insertion in the domesticated loss-of-shattering allele SiLes1-TE (transposable element). This resource will enhance the utility of S. viridis for dissection of complex traits and biotechnological improvement of panicoid crops.

The evening complex is central to the difference between the circadian clocks of Arabidopsis thaliana shoots and roots.

The circadian clock regulates the timing of many aspects of plant physiology, and this requires entrainment of the clock to the prevailing day:night cycle. Different plant cells and tissues can oscillate with different free-running periods, so coordination of timing across the plant is crucial. Previous work showed that a major difference between the clock in mature shoots and roots involves light inputs. The objective of this work was to define, in Arabidopsis thaliana, the operation of the root clock in more detail, and in particular how it responds to light quality. Luciferase imaging was used to study the shoot and root clocks in several null mutants of clock components and in lines with aberrant expression of phytochromes. Mutations in each of the components of the evening complex (EARLY FLOWERING 3 and 4, and LUX ARRHYTHMO) were found to have specific effects on roots, by affecting either rhythmicity or period and its response to light quality. The data suggest that the evening complex is a key part of the light input mechanism that differs between shoots and roots and show that roots sense red light via phytochrome B.

A mobile ELF4 delivers circadian temperature information from shoots to roots.

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Explaining how the plant circadian clock responds to temperature oscillations is crucial to understanding plant responsiveness to the environment. Here, we found a prevalent temperature-dependent function of the Arabidopsis clock component EARLY FLOWERING 4 (ELF4) in the root clock. Although the clocks in roots are able to run in the absence of shoots, micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information, but trafficking is essential for controlling the period of the root clock in a temperature-dependent manner. Low temperatures favour ELF4 mobility, resulting in a slow-paced root clock, whereas high temperatures decrease movement, leading to a faster clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.

Transgenic overexpression of endogenous FLOWERING LOCUS T-like gene MeFT1 produces early flowering in cassava.

Endogenous FLOWERING LOCUS T homolog MeFT1 was transgenically overexpressed under control of a strong constitutive promoter in cassava cultivar 60444 to determine its role in regulation of flowering and as a potential tool to accelerate cassava breeding. Early profuse flowering was recorded in-vitro in all ten transgenic plant lines recovered, causing eight lines to die within 21 days of culture. The two surviving transgenic plant lines flowered early and profusely commencing as soon as 14 days after establishment in soil in the greenhouse. Both transgenic lines sustained early flowering across the vegetative propagation cycle, with first flowering recorded 30-50 days after planting stakes compared to 90 days for non-transgenic controls. Transgenic plant lines completed five flowering cycles within 200 days in the greenhouse as opposed to twice flowering event in the controls. Constitutive overexpression of MeFT1 generated fully mature male and female flowers and produced a bushy phenotype due to significantly increased flowering-induced branching. Flower induction by MeFT1 overexpression was not graft-transmissible and negatively affected storage root development. Accelerated flowering in transgenic plants was associated with significantly increased mRNA levels of MeFT1 and the three floral meristem identity genes MeAP1, MeLFY and MeSOC1 in shoot apical tissues. These findings imply that MeFT1 encodes flower induction and triggers flowering by recruiting downstream floral meristem identity genes.

PCH1 regulates light, temperature, and circadian signaling as a structural component of phytochrome B-photobodies in Arabidopsis.

The members of the phytochrome (phy) family of bilin-containing photoreceptors are major regulators of plant photomorphogenesis through their unique ability to photointerconvert between a biologically inactive red light-absorbing Pr state and an active far-red light-absorbing Pfr state. While the initial steps in Pfr signaling are unclear, an early event for the phyB isoform after photoconversion is its redistribution from the cytoplasm into subnuclear foci known as photobodies (PBs), which dissipate after Pfr reverts back to Pr by far-red irradiation or by temperature-dependent nonphotochemical reversion. Here we present evidence that PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) functions both as an essential structural component of phyB-containing PBs and as a direct regulator of thermal reversion that is sufficient to stabilize phyB as Pfr in vitro. By examining the genetic interaction between a constitutively active phyBY276H-YFP allele (YHB-YFP) and PCH1, we show that the loss of PCH1 prevents YHB from coalescing into PBs without affecting its nuclear localization, whereas overexpression of PCH1 dramatically increases PB levels. Loss of PCH1, presumably by impacting phyB-PB assembly, compromises a number of events elicited in YHB-YFP plants, including their constitutive photomorphogenic phenotype, red light-regulated thermomorphogenesis, and input of phyB into the circadian clock. Conversely, elevated levels of both phyB and PCH1 generate stable, yet far-red light-reversible PBs that persisted for days. Collectively, our data demonstrate that the assembly of PCH1-containing PBs is critical for phyB signaling to multiple outputs and suggest that altering PB dynamics could be exploited to modulate plant responses to light and temperature.