A Guide to Quantify Arabidopsis Seedling Thermomorphogenesis at Single Timepoints and by Interval Monitoring.

Temperature-induced elongation of hypocotyls, petioles, and roots, together with hyponastic leaf responses, constitute key model phenotypes that can be used to assess a plant's capacity for thermomorphogenesis. Phenotypic responses are often quantified at a single time point during seedling development at different temperatures. However, to capture growth dynamics, several time points need to be assessed, and ideally continuous measurements are taken. Here we describe a general experimental setup and technical solutions for recording and measuring seedling phenotypes at single and multiple time points. Furthermore, we present an R-package called "rootdetectR," which allows easy processing of hypocotyl, root or petiole length, and growth rate data and provides different options of data presentation.

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.

Plasticity of maternal environment-dependent expression-QTLs of tomato seeds.

Seeds are essential for plant reproduction, survival, and dispersal. Germination ability and successful establishment of young seedlings strongly depend on seed quality and on environmental factors such as nutrient availability. In tomato (Solanum lycopersicum) and many other species, seed quality and seedling establishment characteristics are determined by genetic variation, as well as the maternal environment in which the seeds develop and mature. The genetic contribution to variation in seed and seedling quality traits and environmental responsiveness can be estimated at transcriptome level in the dry seed by mapping genomic loci that affect gene expression (expression QTLs) in contrasting maternal environments. In this study, we applied RNA-sequencing to construct a linkage map and measure gene expression of seeds of a tomato recombinant inbred line (RIL) population derived from a cross between S. lycopersicum (cv. Moneymaker) and S. pimpinellifolium (G1.1554). The seeds matured on plants cultivated under different nutritional environments, i.e., on high phosphorus or low nitrogen. The obtained single-nucleotide polymorphisms (SNPs) were subsequently used to construct a genetic map. We show how the genetic landscape of plasticity in gene regulation in dry seeds is affected by the maternal nutrient environment. The combined information on natural genetic variation mediating (variation in) responsiveness to the environment may contribute to knowledge-based breeding programs aiming to develop crop cultivars that are resilient to stressful environments.

The Mediator complex subunit MED25 interacts with HDA9 and PIF4 to regulate thermomorphogenesis.

Thermomorphogenesis is, among other traits, characterized by enhanced hypocotyl elongation due to the induction of auxin biosynthesis genes like YUCCA8 by transcription factors, most notably PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Efficient binding of PIF4 to the YUCCA8 locus under warmth depends on HISTONE DEACETYLASE 9 (HDA9) activity, which mediates histone H2A.Z depletion at the YUCCA8 locus. However, HDA9 lacks intrinsic DNA-binding capacity, and how HDA9 is recruited to YUCCA8, and possibly other PIF4-target sites, is currently not well understood. The Mediator complex functions as a bridge between transcription factors bound to specific promoter sequences and the basal transcription machinery containing RNA polymerase II. Mutants of Mediator component Mediator25 (MED25) exhibit reduced hypocotyl elongation and reduced expression of YUCCA8 at 27°C. In line with a proposed role for MED25 in thermomorphogenesis in Arabidopsis (Arabidopsis thaliana), we demonstrated an enhanced association of MED25 to the YUCCA8 locus under warmth and interaction of MED25 with both PIF4 and HDA9. Genetic analysis confirmed that MED25 and HDA9 operate in the same pathway. Intriguingly, we also showed that MED25 destabilizes HDA9 protein. Based on our findings, we propose that MED25 recruits HDA9 to the YUCCA8 locus by binding to both PIF4 and HDA9.

Arabidopsis latent virus 1, a comovirus widely spread in Arabidopsis thaliana collections.

Transcriptome studies of Illumina RNA-Seq datasets of different Arabidopsis thaliana natural accessions and T-DNA mutants revealed the presence of two virus-like RNA sequences which showed the typical two-segmented genome characteristics of a comovirus. This comovirus did not induce any visible symptoms in infected A. thaliana plants cultivated under standard laboratory conditions. Hence it was named Arabidopsis latent virus 1 (ArLV1). Virus infectivity in A. thaliana plants was confirmed by quantitative reverse transcription polymerase chain reaction, transmission electron microscopy and mechanical inoculation. Arabidopsis latent virus 1 can also mechanically infect Nicotiana benthamiana, causing distinct mosaic symptoms. A bioinformatics investigation of A. thaliana RNA-Seq repositories, including nearly 6500 Sequence Read Archives (SRAs) in the NCBI SRA database, revealed the presence of ArLV1 in 25% of all archived natural A. thaliana accessions and in 8.5% of all analyzed SRAs. Arabidopsis latent virus 1 could also be detected in A. thaliana plants collected from the wild. Arabidopsis latent virus 1 is highly seed-transmissible with up to 40% incidence on the progeny derived from infected A. thaliana plants. This has probably led to a worldwide distribution in the model plant A. thaliana with as yet unknown effects on plant performance in a substantial number of studies.

Effects of sublethal single, simultaneous and sequential abiotic stresses on phenotypic traits of Arabidopsis thaliana.

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four Arabidopsis thaliana accessions. Combined sublethal stresses were applied either simultaneously (high temperature and drought) or sequentially (flooding followed by drought). In total, we analysed the phenotypic responses of 1782 individuals across these stresses and different developmental stages. Overall, abiotic stresses and their combinations resulted in distinct patterns of effects across the traits analysed, with both quantitative and qualitative differences across accessions. Stress combinations had additive effects on some traits, whereas clear positive and negative interactions were observed for other traits: 9 out of 12 traits for high temperature and drought, 6 out of 12 traits for post-submergence and drought showed significant interactions. In many cases where the stresses interacted, the strength of interactions varied across accessions. Hence, our results indicated a general pattern of response in most phenotypic traits to the different stresses and stress combinations, but it also indicated a natural genetic variation in the strength of these responses. This includes novel results regarding the lack of a response to drought after submergence and a decoupling between leaf number and flowering time after submergence. Overall, our study provides a rich characterization of trait responses of Arabidopsis plants to sublethal abiotic stresses at the phenotypic level and can serve as starting point for further in-depth physiological research and plant modelling efforts.

Wandering between hot and cold: temperature dose-dependent responses.

Plants in most natural habitats are exposed to a continuously changing environment, including fluctuating temperatures. Temperature variations can trigger acclimation or tolerance responses, depending on the severity of the signal. To guarantee food security under a changing climate, we need to fully understand how temperature response and tolerance are triggered and regulated. Here, we put forward the concept that responsiveness to temperature should be viewed in the context of dose-dependency. We discuss physiological, developmental, and molecular examples, predominantly from the model plant Arabidopsis thaliana, illustrating monophasic signaling responses across the physiological temperature gradient.

Epigenetic regulation of thermomorphogenesis and heat stress tolerance.

Many environmental conditions fluctuate and organisms need to respond effectively. This is especially true for temperature cues that can change in minutes to seasons and often follow a diurnal rhythm. Plants cannot migrate and most cannot regulate their temperature. Therefore, a broad array of responses have evolved to deal with temperature cues from freezing to heat stress. A particular response to mildly elevated temperatures is called thermomorphogenesis, a suite of morphological adaptations that includes thermonasty, formation of thin leaves and elongation growth of petioles and hypocotyl. Thermomorphogenesis allows for optimal performance in suboptimal temperature conditions by enhancing the cooling capacity. When temperatures rise further, heat stress tolerance mechanisms can be induced that enable the plant to survive the stressful temperature, which typically comprises cellular protection mechanisms and memory thereof. Induction of thermomorphogenesis, heat stress tolerance and stress memory depend on gene expression regulation, governed by diverse epigenetic processes. In this Tansley review we update on the current knowledge of epigenetic regulation of heat stress tolerance and elevated temperature signalling and response, with a focus on thermomorphogenesis regulation and heat stress memory. In particular we highlight the emerging role of H3K4 methylation marks in diverse temperature signalling pathways.

2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions.

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.

Protein kinase and phosphatase control of plant temperature responses.

Plants must cope with ever-changing temperature conditions in their environment. Suboptimal high and low temperatures, and stressful extreme temperatures, induce adaptive mechanisms that allow optimal performance and survival, respectively. These processes have been extensively studied at the physiological, transcriptional and (epi)genetic level. Cellular temperature signalling cascades and tolerance mechanisms also involve post-translational modifications (PTMs), particularly protein phosphorylation. Many protein kinases are known to be involved in cold acclimation and heat stress responsiveness but research on the role and importance of kinases and phosphatases in triggering responses to mild changes in temperature such as thermomorphogenesis is inadequately understood. In this review, we summarize the current knowledge on the roles of kinases and phosphatases in plant temperature responses. We discuss how kinases can function over a range of temperatures in different signalling pathways and provide an outlook to the application of PTM-modifying factors for the development of thermotolerant crops.

The membrane-localized protein kinase MAP4K4/TOT3 regulates thermomorphogenesis.

Plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity, in a process called thermomorphogenesis. To this date, the regulation of thermomorphogenesis has been exclusively shown to intersect with light signalling pathways. To identify regulators of thermomorphogenesis that are conserved in flowering plants, we map changes in protein phosphorylation in both dicots and monocots exposed to warm temperature. We identify MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)/TARGET OF TEMPERATURE3 (TOT3) as a regulator of thermomorphogenesis that impinges on brassinosteroid signalling in Arabidopsis thaliana. In addition, we show that TOT3 plays a role in thermal response in wheat, a monocot crop. Altogether, the conserved thermal regulation by TOT3 expands our knowledge of thermomorphogenesis beyond the well-studied pathways and can contribute to ensuring food security under a changing climate.

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

The diverse and unanticipated roles of histone deacetylase 9 in coordinating plant development and environmental acclimation.

Plants tightly control gene transcription to adapt to environmental conditions and steer growth and development. Different types of epigenetic modifications are instrumental in these processes. In recent years, an important role for the chromatin-modifying RPD3/HDA1 class I HDAC HISTONE DEACETYLASE 9 (HDA9) emerged in the regulation of a multitude of plant traits and responses. HDACs are widely considered transcriptional repressors and are typically part of multiprotein complexes containing co-repressors, DNA, and histone-binding proteins. By catalyzing the removal of acetyl groups from lysine residues of histone protein tails, HDA9 negatively controls gene expression in many cases, in concert with interacting proteins such as POWERDRESS (PWR), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15), WRKY53, ELONGATED HYPOCOTYL 5 (HY5), ABA INSENSITIVE 4 (ABI4), and EARLY FLOWERING 3 (ELF3). However, HDA9 activity has also been directly linked to transcriptional activation. In addition, following the recent breakthrough discovery of mutual negative feedback regulation between HDA9 and its interacting WRKY-domain transcription factor WRKY53, swift progress in gaining understanding of the biology of HDA9 is expected. In this review, we summarize knowledge on this intriguing versatile-and long under-rated-protein and propose novel leads to further unravel HDA9-governed molecular networks underlying plant development and environmental biology.

HISTONE DEACETYLASE 9 stimulates auxin-dependent thermomorphogenesis in Arabidopsis thaliana by mediating H2A.Z depletion.

Many plant species respond to unfavorable high ambient temperatures by adjusting their vegetative body plan to facilitate cooling. This process is known as thermomorphogenesis and is induced by the phytohormone auxin. Here, we demonstrate that the chromatin-modifying enzyme HISTONE DEACETYLASE 9 (HDA9) mediates thermomorphogenesis but does not interfere with hypocotyl elongation during shade avoidance. HDA9 is stabilized in response to high temperature and mediates histone deacetylation at the YUCCA8 locus, a rate-limiting enzyme in auxin biosynthesis, at warm temperatures. We show that HDA9 permits net eviction of the H2A.Z histone variant from nucleosomes associated with YUCCA8, allowing binding and transcriptional activation by PHYTOCHROME INTERACTING FACTOR 4, followed by auxin accumulation and thermomorphogenesis.

Root Tropisms: Investigations on Earth and in Space to Unravel Plant Growth Direction.

Root tropisms are important responses of plants, allowing them to adapt their growth direction. Research on plant tropisms is indispensable for future space programs that envisage plant-based life support systems for long-term missions and planet colonization. Root tropisms encompass responses toward or away from different environmental stimuli, with an underexplored level of mechanistic divergence. Research into signaling events that coordinate tropistic responses is complicated by the consistent coincidence of various environmental stimuli, often interacting via shared signaling mechanisms. On Earth the major determinant of root growth direction is the gravitational vector, acting through gravitropism and overruling most other tropistic responses to environmental stimuli. Critical advancements in the understanding of root tropisms have been achieved nullifying the gravitropic dominance with experiments performed in the microgravity environment. In this review, we summarize current knowledge on root tropisms to different environmental stimuli. We highlight that the term tropism must be used with care, because it can be easily confused with a change in root growth direction due to asymmetrical damage to the root, as can occur in apparent chemotropism, electrotropism, and magnetotropism. Clearly, the use of Arabidopsis thaliana as a model for tropism research contributed much to our understanding of the underlying regulatory processes and signaling events. However, pronounced differences in tropisms exist among species, and we argue that these should be further investigated to get a more comprehensive view of the signaling pathways and sensors. Finally, we point out that the Cholodny-Went theory of asymmetric auxin distribution remains to be the central and unifying tropistic mechanism after 100 years. Nevertheless, it becomes increasingly clear that the theory is not applicable to all root tropistic responses, and we propose further research to unravel commonalities and differences in the molecular and physiological processes orchestrating root tropisms.

POWERDRESS-mediated histone deacetylation is essential for thermomorphogenesis in Arabidopsis thaliana.

Ambient temperature affects plant growth and even minor changes can substantially impact crop yields. The underlying mechanisms of temperature perception and response are just beginning to emerge. Chromatin remodeling, via the eviction of the histone variant H2A.Z containing nucleosomes, is a critical component of thermal response in plants. However, the role of histone modifications remains unknown. Here, through a forward genetic screen, we identify POWERDRESS (PWR), a SANT-domain containing protein known to interact with HISTONE DEACETYLASE 9 (HDA9), as a novel factor required for thermomorphogenesis in Arabidopsis thaliana. We show that mutations in PWR impede thermomorphogenesis, exemplified by attenuated warm temperature-induced hypocotyl/petiole elongation and early flowering. We show that inhibitors of histone deacetylases diminish temperature-induced hypocotyl elongation, which demonstrates a requirement for histone deacetylation in thermomorphogenesis. We also show that elevated temperature is associated with deacetylation of H3K9 at the +1 nucleosomes of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and YUCCA8 (YUC8), and that PWR is required for this response. There is global misregulation of genes in pwr mutants at elevated temperatures. Meta-analysis revealed that genes that are misregulated in pwr mutants display a significant overlap with genes that are H2A.Z-enriched in their gene bodies, and with genes that are differentially expressed in mutants of the components of the SWR1 complex that deposits H2A.Z. Our findings thus uncover a role for PWR in facilitating thermomorphogenesis and suggest a potential link between histone deacetylation and H2A.Z nucleosome dynamics in plants.

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in Arabidopsis thaliana.

Microscopically visible chromatin is partitioned into two major components in Arabidopsis thaliana nuclei. On one hand, chromocenters are conspicuous foci of highly condensed "heterochromatic" domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich "euchromatin" emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes Arabidopsis a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, i.e., the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.

Thermosensing Enlightened.

Understanding the molecular networks driving plant responses to high ambient temperatures is crucial for developing crop cultivars resistant to global warming. Although several factors involved in temperature signalling are known, a thermosensing mechanism had remained elusive. However, two recent publications demonstrate that the photoreceptor phytochrome B (phyB) also acts as a thermosensor.

Molecular and genetic control of plant thermomorphogenesis.

Temperature is a major factor governing the distribution and seasonal behaviour of plants. Being sessile, plants are highly responsive to small differences in temperature and adjust their growth and development accordingly. The suite of morphological and architectural changes induced by high ambient temperatures, below the heat-stress range, is collectively called thermomorphogenesis. Understanding the molecular genetic circuitries underlying thermomorphogenesis is particularly relevant in the context of climate change, as this knowledge will be key to rational breeding for thermo-tolerant crop varieties. Until recently, the fundamental mechanisms of temperature perception and signalling remained unknown. Our understanding of temperature signalling is now progressing, mainly by exploiting the model plant Arabidopsis thaliana. The transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) has emerged as a critical player in regulating phytohormone levels and their activity. To control thermomorphogenesis, multiple regulatory circuits are in place to modulate PIF4 levels, activity and downstream mechanisms. Thermomorphogenesis is integrally governed by various light signalling pathways, the circadian clock, epigenetic mechanisms and chromatin-level regulation. In this Review, we summarize recent progress in the field and discuss how the emerging knowledge in Arabidopsis may be transferred to relevant crop systems.