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.

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.

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.

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.

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.

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.

Ambient temperature and genotype differentially affect developmental and phenotypic plasticity in Arabidopsis thaliana.

BACKGROUND: Global increase in ambient temperatures constitute a significant challenge to wild and cultivated plant species. Forward genetic analyses of individual temperature-responsive traits have resulted in the identification of several signaling and response components. However, a comprehensive knowledge about temperature sensitivity of different developmental stages and the contribution of natural variation is still scarce and fragmented at best. RESULTS: Here, we systematically analyze thermomorphogenesis throughout a complete life cycle in ten natural Arabidopsis thaliana accessions grown under long day conditions in four different temperatures ranging from 16 to 28 °C. We used Q10, GxE, phenotypic divergence and correlation analyses to assess temperature sensitivity and genotype effects of more than 30 morphometric and developmental traits representing five phenotype classes. We found that genotype and temperature differentially affected plant growth and development with variing strengths. Furthermore, overall correlations among phenotypic temperature responses was relatively low which seems to be caused by differential capacities for temperature adaptations of individual accessions. CONCLUSION: Genotype-specific temperature responses may be attractive targets for future forward genetic approaches and accession-specific thermomorphogenesis maps may aid the assessment of functional relevance of known and novel regulatory components.

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.

Auxin-induced expression divergence between Arabidopsis species may originate within the TIR1/AFB-AUX/IAA-ARF module.

Auxin is an essential regulator of plant growth and development, and auxin signaling components are conserved among land plants. Yet, a remarkable degree of natural variation in physiological and transcriptional auxin responses has been described among Arabidopsis thaliana accessions. As intraspecies comparisons offer only limited genetic variation, we here inspect the variation of auxin responses between A. thaliana and A. lyrata. This approach allowed the identification of conserved auxin response genes including novel genes with potential relevance for auxin biology. Furthermore, promoter divergences were analyzed for putative sources of variation. De novo motif discovery identified novel and variants of known elements with potential relevance for auxin responses, emphasizing the complex, and yet elusive, code of element combinations accounting for the diversity in transcriptional auxin responses. Furthermore, network analysis revealed correlations of interspecies differences in the expression of AUX/IAA gene clusters and classic auxin-related genes. We conclude that variation in general transcriptional and physiological auxin responses may originate substantially from functional or transcriptional variations in the TIR1/AFB, AUX/IAA, and ARF signaling network. In that respect, AUX/IAA gene expression divergence potentially reflects differences in the manner in which different species transduce identical auxin signals into gene expression responses.

Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1.

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing RxopQ as candidate for targeted crop improvement.

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.

Natural variants of ELF3 affect thermomorphogenesis by transcriptionally modulating PIF4-dependent auxin response genes.

BACKGROUND: Perception and transduction of temperature changes result in altered growth enabling plants to adapt to increased ambient temperature. While PHYTOCHROME-INTERACTING FACTOR4 (PIF4) has been identified as a major ambient temperature signaling hub, its upstream regulation seems complex and is poorly understood. Here, we exploited natural variation for thermo-responsive growth in Arabidopsis thaliana using quantitative trait locus (QTL) analysis. RESULTS: We identified GIRAFFE2.1, a major QTL explaining ~18 % of the phenotypic variation for temperature-induced hypocotyl elongation in the Bay-0 x Sha recombinant inbred line population. Transgenic complementation demonstrated that allelic variation in the circadian clock regulator EARLY FLOWERING3 (ELF3) is underlying this QTL. The source of variation could be allocated to a single nucleotide polymorphism in the ELF3 coding region, resulting in differential expression of PIF4 and its target genes, likely causing the observed natural variation in thermo-responsive growth. CONCLUSIONS: In combination with other recent studies, this work establishes the role of ELF3 in the ambient temperature signaling network. Natural variation of ELF3-mediated gating of PIF4 expression during nightly growing periods seems to be affected by a coding sequence quantitative trait nucleotide that confers a selective advantage in certain environments. In addition, natural ELF3 alleles seem to differentially integrate temperature and photoperiod information to induce architectural changes. Thus, ELF3 emerges as an essential coordinator of growth and development in response to diverse environmental cues and implicates ELF3 as an important target of adaptation.

The DET1-COP1-HY5 pathway constitutes a multipurpose signaling module regulating plant photomorphogenesis and thermomorphogenesis.

Developmental plasticity enables plants to respond to elevated ambient temperatures by adapting their shoot architecture. On the cellular level, the basic-helix-loop-helix (bHLH) transcription factor phytochrome interacting factor 4 (PIF4) coordinates this response by activating hormonal modules that in turn regulate growth. In addition to an unknown temperature-sensing mechanism, it is currently not understood how temperature regulates PIF4 activity. Using a forward genetic approach in Arabidopsis thaliana, we present extensive genetic evidence demonstrating that the de-etiolated 1 (DET1)-constitutive photomorphogenic 1 (COP1)-elongated hypocotyl 5 (HY5)-dependent photomorphogenesis pathway transcriptionally regulates PIF4 to coordinate seedling growth in response to elevated temperature. Our findings demonstrate that two of the most prevalent environmental cues, light and temperature, share a much larger set of signaling components than previously assumed. Similar to the toolbox concept in animal embryonic patterning, multipurpose signaling modules might have evolved in plants to translate various environmental stimuli into adaptational growth processes.

Optimized probe masking for comparative transcriptomics of closely related species.

Microarrays are commonly applied to study the transcriptome of specific species. However, many available microarrays are restricted to model organisms, and the design of custom microarrays for other species is often not feasible. Hence, transcriptomics approaches of non-model organisms as well as comparative transcriptomics studies among two or more species often make use of cost-intensive RNAseq studies or, alternatively, by hybridizing transcripts of a query species to a microarray of a closely related species. When analyzing these cross-species microarray expression data, differences in the transcriptome of the query species can cause problems, such as the following: (i) lower hybridization accuracy of probes due to mismatches or deletions, (ii) probes binding multiple transcripts of different genes, and (iii) probes binding transcripts of non-orthologous genes. So far, methods for (i) exist, but these neglect (ii) and (iii). Here, we propose an approach for comparative transcriptomics addressing problems (i) to (iii), which retains only transcript-specific probes binding transcripts of orthologous genes. We apply this approach to an Arabidopsis lyrata expression data set measured on a microarray designed for Arabidopsis thaliana, and compare it to two alternative approaches, a sequence-based approach and a genomic DNA hybridization-based approach. We investigate the number of retained probe sets, and we validate the resulting expression responses by qRT-PCR. We find that the proposed approach combines the benefit of sequence-based stringency and accuracy while allowing the expression analysis of much more genes than the alternative sequence-based approach. As an added benefit, the proposed approach requires probes to detect transcripts of orthologous genes only, which provides a superior base for biological interpretation of the measured expression responses.

ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization.

Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.

Expression level polymorphisms: heritable traits shaping natural variation.

Natural accessions of many species harbor a wealth of genetic variation visible in a large array of phenotypes. Although expression level polymorphisms (ELPs) in several genes have been shown to contribute to variation in diverse traits, their general impact on adaptive variation has likely been underestimated. At present, ELPs have predominantly been correlated to quantitative trait loci (eQTLs) that occupy central hubs in signaling networks, which pleiotropically affect numerous traits. To increase the sensitivity of detecting minor effect eQTLs or those that act in a trait-specific manner, we emphasize the need for more systematic approaches. This requires, but is not limited to, refining experimental designs such as reduction of tissue complexity and combinatorial methods including a priori defined networks.

Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway.

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.

Natural variation of transcriptional auxin response networks in Arabidopsis thaliana.

Natural variation has been observed for various traits in Arabidopsis thaliana. Here, we investigated natural variation in the context of physiological and transcriptional responses to the phytohormone auxin, a key regulator of plant development. A survey of the general extent of natural variation to auxin stimuli revealed significant physiological variation among 20 genetically diverse natural accessions. Moreover, we observed dramatic variation on the global transcriptome level after induction of auxin responses in seven accessions. Although we detect isolated cases of major-effect polymorphisms, sequencing of signaling genes revealed sequence conservation, making selective pressures that favor functionally different protein variants among accessions unlikely. However, coexpression analyses of a priori defined auxin signaling networks identified variations in the transcriptional equilibrium of signaling components. In agreement with this, cluster analyses of genome-wide expression profiles followed by analyses of a posteriori defined gene networks revealed accession-specific auxin responses. We hypothesize that quantitative distortions in the ratios of interacting signaling components contribute to the detected transcriptional variation, resulting in physiological variation of auxin responses among accessions.