Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

25 Years of thermomorphogenesis research: milestones and perspectives.

In 1998, Bill Gray and colleagues showed that warm temperatures trigger arabidopsis hypocotyl elongation in an auxin-dependent manner. This laid the foundation for a vibrant research discipline. With several active members of the 'thermomorphogenesis' community, we here reflect on 25 years of elevated ambient temperature research and look to the future.

Novel exotic alleles of EARLY FLOWERING 3 determine plant development in barley.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

Auxin-dependent regulation of cell division rates governs root thermomorphogenesis.

Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.

An exotic allele of barley EARLY FLOWERING 3 contributes to developmental plasticity at elevated temperatures.

Increase in ambient temperatures caused by climate change affects various morphological and developmental traits of plants, threatening crop yield stability. In the model plant Arabidopsis thaliana, EARLY FLOWERING 3 (ELF3) plays prominent roles in temperature sensing and thermomorphogenesis signal transduction. However, how crop species respond to elevated temperatures is poorly understood. Here, we show that the barley ortholog of AtELF3 interacts with high temperature to control growth and development. We used heterogeneous inbred family (HIF) pairs generated from a segregating mapping population and systematically studied the role of exotic ELF3 variants in barley temperature responses. An exotic ELF3 allele of Syrian origin promoted elongation growth in barley at elevated temperatures, whereas plant area and estimated biomass were drastically reduced, resulting in an open canopy architecture. The same allele accelerated inflorescence development at high temperature, which correlated with early transcriptional induction of MADS-box floral identity genes BM3 and BM8. Consequently, barley plants carrying the exotic ELF3 allele displayed stable total grain number at elevated temperatures. Our findings therefore demonstrate that exotic ELF3 variants can contribute to phenotypic and developmental acclimation to elevated temperatures, providing a stimulus for breeding of climate-resilient crops.

Recent advances in understanding thermomorphogenesis signaling.

Plants show remarkable phenotypic plasticity and are able to adjust their morphology and development to diverse environmental stimuli. Morphological acclimation responses to elevated ambient temperatures are collectively termed thermomorphogenesis. In Arabidopsis thaliana, morphological changes are coordinated to a large extent by the transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which in turn is regulated by several thermosensing mechanisms and modulators. Here, we review recent advances in the identification of factors that regulate thermomorphogenesis of Arabidopsis seedlings by affecting PIF4 expression and PIF4 activity. We summarize newly identified thermosensing mechanisms and highlight work on the emerging topic of organ- and tissue-specificity in the regulation of thermomorphogenesis.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development.

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 µM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

High temperature perception in leaves promotes vascular regeneration and graft formation in distant tissues.

Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism. This article has an associated 'The people behind the papers' interview.

Evolution and Functions of Plant U-Box Proteins: From Protein Quality Control to Signaling.

Posttranslational modifications add complexity and diversity to cellular proteomes. One of the most prevalent modifications across eukaryotes is ubiquitination, which is orchestrated by E3 ubiquitin ligases. U-box-containing E3 ligases have massively expanded in the plant kingdom and have diversified into plant U-box proteins (PUBs). PUBs likely originated from two or three ancestral forms, fusing with diverse functional subdomains that resulted in neofunctionalization. Their emergence and diversification may reflect adaptations to stress during plant evolution, reflecting changes in the needs of plant proteomes to maintain cellular homeostasis. Through their close association with protein kinases, they are physically linked to cell signaling hubs and activate feedback loops by dynamically pairing with E2-ubiquitin-conjugating enzymes to generate distinct ubiquitin polymers that themselves act as signals. Here, we complement current knowledgewith comparative genomics to gain a deeper understanding of PUB function, focusing on their evolution and structural adaptations of key U-box residues, as well as their various roles in plant cells.

Arabidopsis EARLY FLOWERING 3 controls temperature responsiveness of the circadian clock independently of the evening complex.

Daily changes in light and temperature are major entrainment cues that enable the circadian clock to generate internal biological rhythms that are synchronized with the external environment. With the average global temperature predicted to keep increasing, the intricate light-temperature coordination that is necessary for clock functionality is expected to be seriously affected. Hence, understanding how temperature signals are perceived by the circadian clock has become an important issue. In Arabidopsis, the clock component EARLY FLOWERING 3 (ELF3) not only serves as a light Zeitnehmer, but also functions as a thermosensor participating in thermomorphogenesis. However, the role of ELF3 in temperature entrainment of the circadian clock is not fully understood. Here, we report that ELF3 is essential for delivering temperature input to the clock. We demonstrate that in the absence of ELF3, the oscillator is unable to respond to temperature changes, resulting in an impaired gating of thermoresponses. Consequently, clock-controlled physiological processes such as rhythmic growth and cotyledon movement were disturbed. Genetic analyses suggest that the evening complex is not required for ELF3-controlled thermoresponsiveness. Together, our results reveal that ELF3 is an essential Zeitnehmer for temperature sensing of the oscillator, and thereby for coordinating the rhythmic control of thermoresponsive physiological outputs.

How to Detect QTLs in the Plant Circadian Clock.

One of the most powerful methods to identify loci controlling complex quantitative traits has been the quantitative trait locus (QTL) mapping. The QTL mapping approach has proven immensely useful to improve our understanding of key pathways such as flowering time, growth, and disease resistance. Since major circadian clock parameters such as period, phase, and amplitude are quantitative in nature, the QTL mapping approach could also be used to study the complex genetic architecture of the circadian clock. Here, we describe a simple QTL mapping method to identify components controlling clock parameters in natural populations of Arabidopsis thaliana.

On the evolution of plant thermomorphogenesis.

Plants have a remarkable capacity to acclimate to their environment. Acclimation is enabled to a large degree by phenotypic plasticity, the extent of which confers a selective advantage, especially in natural habitats. Certain key events in evolution triggered adaptive bursts necessary to cope with drastic environmental changes. One such event was the colonization of land 400-500 mya. Compared to most aquatic habitats, fluctuations in abiotic parameters became more pronounced, generating significant selection pressure. To endure these harsh conditions, plants needed to adapt their physiology and morphology and to increase the range of phenotypic plasticity. In addition to drought stress and high light, high temperatures and fluctuation thereof were among the biggest challenges faced by terrestrial plants. Thermomorphogenesis research has emerged as a new sub-discipline of the plant sciences and aims to understand how plants acclimate to elevated ambient temperatures through changes in architecture. While we have begun to understand how angiosperms sense and respond to elevated ambient temperature, very little is known about thermomorphogenesis in plant lineages with less complex body plans. It is unclear when thermomorphogenesis initially evolved and how this depended on morphological complexity. In this review, we take an evolutionary-physiological perspective and generate hypotheses about the emergence of thermomorphogenesis.

Plant thermotropism: an underexplored thermal engagement and avoidance strategy.

Various strategies evolved in plants to adjust the position of organs relative to the prevailing temperature condition, which allows optimal plant growth and performance. Such responses are classically separated into nastic and tropic responses. During plant thermotropic responses, organs move towards (engage) or away (avoid) from a directional temperature cue. Despite thermotropism being a classic botanical concept, the underlying ecological function and molecular and biophysical mechanisms remain poorly understood to this day. This contrasts to the relatively well-studied thermonastic movements (hyponasty) of e.g., rosette leaves. In this review, we provide an update on the current knowledge on plant thermotropisms and propose directions for future research and application.

Cut and paste: temperature-enhanced cotyledon micrografting for Arabidopsis thaliana seedlings.

BACKGROUND: Cotyledon micrografting represents a useful tool for studying the central role of cotyledons during early plant development, especially their interplay with other plant organs with regard to long distance transport. While hypocotyl micrografting methods are well-established, cotyledon micrografting is still inefficient. By optimizing cotyledon micrografting, we aim for higher success rates and increased throughput in the model species Arabidopsis thaliana. RESULTS: We established a cut and paste cotyledon surgery procedure on a flat and solid but moist surface which improved handling of small seedlings. By applying a specific cutting and joining pattern, throughput was increased up to 40 seedlings per hour. The combination of short-day photoperiods and low light intensities for germination and long days plus high light intensities, elevated temperature and vertical plate positioning after grafting significantly increased 'ligation' efficiency. In particular high temperatures affected success rates favorably. Altogether, we achieved up to 92% grafting success in A. thaliana. Reconnection of vasculature was demonstrated by transport of a vasculature-specific dye across the grafting site. Phloem and xylem reconnection were completed 3-4 and 4-6 days after grafting, respectively, in a temperature-dependent manner. We observed that plants with grafted cotyledons match plants with intact cotyledons in biomass production and rosette development. CONCLUSIONS: This cut and paste cotyledon-to-petiole micrografting protocol simplifies the handling of plant seedlings in surgery, increases the number of grafted plants per hour and greatly improves success rates for A. thaliana seedlings. The developed cotyledon micrografting method is also suitable for other plant species of comparable size.

Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA.

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development are completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes.

Development of Wild and Cultivated Plants under Global Warming Conditions.

Global warming is one of the most detrimental aspects of climate change, affecting plant growth and development across the entire life cycle. This Review explores how different stages of development are influenced by elevated temperature in both wild plants and crops. Starting from seed development and germination, global warming will influence morphological adjustments, termed thermomorphogenesis, and photosynthesis primarily during the vegetative phase, as well as flowering and reproductive development. Where applicable, we distinguish between moderately elevated temperatures that affect all stages of plant development and heat waves that often occur during the reproductive phase when they can have devastating consequences for fruit development. The parallel occurrence of elevated temperature with other abiotic and biotic stressors, particularly the combination of global warming and drought or increased pathogen pressure, will potentiate the challenges for both wild and cultivated plant species. The key components of the molecular networks underlying the physiological processes involved in thermal responses in the model plant Arabidopsis thaliana are highlighted. In crops, temperature-sensitive traits relevant for yield are illustrated for winter wheat (Triticum aestivum L.) and soybean (Glycine max L.), representing cultivated species adapted to temperate vs. warm climate zones, respectively. While the fate of wild plants depends on political agendas, plant breeding approaches informed by mechanistic understanding originating in basic science can enable the generation of climate change-resilient crops.

A Mobile Auxin Signal Connects Temperature Sensing in Cotyledons with Growth Responses in Hypocotyls.

Plants have a remarkable capacity to adjust their growth and development to elevated ambient temperatures. Increased elongation growth of roots, hypocotyls, and petioles in warm temperatures are hallmarks of seedling thermomorphogenesis. In the last decade, significant progress has been made to identify the molecular signaling components regulating these growth responses. Increased ambient temperature utilizes diverse components of the light sensing and signal transduction network to trigger growth adjustments. However, it remains unknown whether temperature sensing and responses are universal processes that occur uniformly in all plant organs. Alternatively, temperature sensing may be confined to specific tissues or organs, which would require a systemic signal that mediates responses in distal parts of the plant. Here, we show that Arabidopsis (Arabidopsis thaliana) seedlings show organ-specific transcriptome responses to elevated temperatures and that thermomorphogenesis involves both autonomous and organ-interdependent temperature sensing and signaling. Seedling roots can sense and respond to temperature in a shoot-independent manner, whereas shoot temperature responses require both local and systemic processes. The induction of cell elongation in hypocotyls requires temperature sensing in cotyledons, followed by the generation of a mobile auxin signal. Subsequently, auxin travels to the hypocotyl, where it triggers local brassinosteroid-induced cell elongation in seedling stems, which depends upon a distinct, permissive temperature sensor in the hypocotyl.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

myTAI: evolutionary transcriptomics with R.

Motivation: Next Generation Sequencing (NGS) technologies generate a large amount of high quality transcriptome datasets enabling the investigation of molecular processes on a genomic and metagenomic scale. These transcriptomics studies aim to quantify and compare the molecular phenotypes of the biological processes at hand. Despite the vast increase of available transcriptome datasets, little is known about the evolutionary conservation of those characterized transcriptomes. Results: The myTAI package implements exploratory analysis functions to infer transcriptome conservation patterns in any transcriptome dataset. Comprehensive documentation of myTAI functions and tutorial vignettes provide step-by-step instructions on how to use the package in an exploratory and computationally reproducible manner. Availability and implementation: The open source myTAI package is available at https://github.com/HajkD/myTAI and https://cran.r-project.org/web/packages/myTAI/index.html. Contact: hgd23@cam.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1.

Thermomorphogenesis is defined as the suite of morphological changes that together are likely to contribute to adaptive growth acclimation to usually elevated ambient temperature [1, 2]. While many details of warmth-induced signal transduction are still elusive, parallels to light signaling recently became obvious (reviewed in [3]). It involves photoreceptors that can also sense changes in ambient temperature [3-5] and act, for example, by repressing protein activity of the central integrator of temperature information PHYTOCHROME-INTERACTING FACTOR 4 (PIF4 [6]). In addition, PIF4 transcript accumulation is tightly controlled by the evening complex member EARLY FLOWERING 3 [7, 8]. According to the current understanding, PIF4 activates growth-promoting genes directly but also via inducing auxin biosynthesis and signaling, resulting in cell elongation. Based on a mutagenesis screen in the model plant Arabidopsis thaliana for mutants with defects in temperature-induced hypocotyl elongation, we show here that both PIF4 and auxin function depend on brassinosteroids. Genetic and pharmacological analyses place brassinosteroids downstream of PIF4 and auxin. We found that brassinosteroids act via the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), which accumulates in the nucleus at high temperature, where it induces expression of growth-promoting genes. Furthermore, we show that at elevated temperature BZR1 binds to the promoter of PIF4, inducing its expression. These findings suggest that BZR1 functions in an amplifying feedforward loop involved in PIF4 activation. Although numerous negative regulators of PIF4 have been described, we identify BZR1 here as a true temperature-dependent positive regulator of PIF4, acting as a major growth coordinator.