Plant architecture is optimized for the local light environment. In response to foliar shade or neighbor proximity (low red to far-red light), some plant species exhibit shade-avoiding phenotypes, including increased stem and hypocotyl growth, which increases the likelihood of outgrowing competitor plants. If shade persists, early flowering and the reallocation of growth resources to stem elongation ultimately affect the yield of harvestable tissues in crop species. Previous studies have shown that hypocotyl growth in low red to far-red shade is largely dependent on the photoreceptor phytochrome B and the phytohormone auxin. However, where shade is perceived in the plant and how auxin regulates growth spatially are less well understood. Using the oilseed and vegetable crop species Brassica rapa, we show that the perception of low red to far-red shade by the cotyledons triggers hypocotyl cell elongation and auxin target gene expression. Furthermore, we find that following shade perception, elevated auxin levels occur in a basipetal gradient away from the cotyledons and that this is coincident with a gradient of auxin target gene induction. These results show that cotyledon-generated auxin regulates hypocotyl elongation. In addition, we find in mature B. rapa plants that simulated shade does not affect seed oil composition but may affect seed yield. This suggests that in field settings where mutual shading between plants may occur, a balance between plant density and seed yield per plant needs to be achieved for maximum oil yield, while oil composition might remain constant.

Phytochromes are red/far-red light receptors that function in photomorphogenesis of plants. Photoisomerization of phytochrome by red light leads to its translocation to the nucleus, where it regulates gene expression. We examined whether phytochrome is phosphorylated in response to light, and we report that phytochrome B (phyB)'s N terminus contains a region with a number of phosphoserines, threonines, and tyrosines. The light-dependent phosphorylation of tyrosine 104 (Y104) appears to play a negative role in phyB's activity, because a phosphomimic mutant, phyBY104E, is unable to complement any phyB-related phenotype, is defective in binding to its signaling partner PIF3, and fails to form stable nuclear bodies even though it retains normal photochemistry in vitro. In contrast, plants stably expressing a nonphosphorylatable mutant, phyBY104F, are hypersensitive to light. The proper response to changes in the light environment is crucial for plant survival, and our study brings tyrosine phosphorylation to the forefront of light-signaling mechanisms.

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Strigolactones (SLs) are a diverse class of butenolide-bearing plant hormones associated with several processes of major agricultural concern. SLs initiate symbiosis between plants and arbuscular mycorrhizal fungi, cause germination of crop-devastating parasitic plants, and inhibit shoot branching in vascular plants. SLs are perceived by dual receptor-hydrolase proteins, and capturing the intact ligand inside the receptor remains a key challenge for structural biologists. In addition, many discovered SLs are hard to obtain and too unstable to work with. In a computer-based approach, we investigated the interaction of 20 different SL molecules with nine crystal structures of SL receptors. Our results suggest an important role of the active site for ligand binding and orientation, and that the parasitic plant Striga hermonthica has developed both promiscuous and type-specific SL receptors as part of its host recognition strategy.

Like other complex multicellular organisms, plants are composed of different cell types with specialized shapes and functions. For example, most laminar leaves consist of multiple photosynthetic cell types. These cell types include the palisade mesophyll, which typically forms one or more cell layers on the adaxial side of the leaf. Despite their importance for photosynthesis, we know little about how palisade cells differ at the molecular level from other photosynthetic cell types. To this end, we have used a combination of cell-specific profiling using fluorescence-activated cell sorting and single-cell RNA-sequencing methods to generate a transcriptional blueprint of the palisade mesophyll in Arabidopsis thaliana leaves. We find that despite their unique morphology, palisade cells are otherwise transcriptionally similar to other photosynthetic cell types. Nevertheless, we show that some genes in the phenylpropanoid biosynthesis pathway have both palisade-enriched expression and are light-regulated. Phenylpropanoid gene activity in the palisade was required for production of the ultraviolet (UV)-B protectant sinapoylmalate, which may protect the palisade and/or other leaf cells against damaging UV light. These findings improve our understanding of how different photosynthetic cell types in the leaf can function uniquely to optimize leaf performance, despite their transcriptional similarities.

Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants.

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

Phytochrome A (phyA) is crucial to initiate the early steps of the transition between skoto- and photomorphogenesis upon light exposure and to complete this process under far-red light (typical of dense vegetation canopies). However, under prolonged red or white light, phyA mutants are hyper-photomorphogenic in many respects. To investigate this issue, we analyzed the late response of the transcriptome of the phyA mutant to red light. Compared to the wild-type (WT), hyper-responsive genes outnumbered the genes showing reduced response to red light in phyA. A network analysis revealed the co-expression of PHYTOCHROME INTERACTING FACTOR 1 (PIF1) with those genes showing hyper-promotion by red light in phyA. The enhanced responses of gene expression, cotyledon unfolding, hypocotyl growth, and greening observed in the phyA mutant compared to the WT were absent in the phyA pif1 double mutant compared to pif1, indicating that the hyper-photomorphogenic phenotype of phyA requires PIF1. PIF1 directly binds to gene promoters that displayed PIF1-mediated enhanced response to red light. Expression of mutant PIF1 deficient in interactions with phyA and phyB enhanced the long-term growth response to red light but reduced the expression of selected genes in response to red light. We propose that phytochrome-mediated degradation of PIF1 prevents over-activation of photomorphogenesis during early seedling development.

DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. Thus, we have identified a signal that leads to the targeted removal of ROS-overproducing chloroplasts.

Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.

Even when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL) mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs).

The central regulator of the ethylene (ET) signaling pathway, which controls a plethora of developmental programs and responses to environmental cues in plants, is ETHYLENE-INSENSITIVE2 (EIN2). Here we identify a chromatin-dependent regulatory mechanism at EIN2 requiring two genes: ETHYLENE-INSENSITIVE6 (EIN6), which is a H3K27me3 demethylase also known as RELATIVE OF EARLY FLOWERING6 (REF6), and EIN6 ENHANCER (EEN), the Arabidopsis homolog of the yeast INO80 chromatin remodeling complex subunit IES6 (INO EIGHTY SUBUNIT). Strikingly, EIN6 (REF6) and the INO80 complex redundantly control the level and the localization of the repressive histone modification H3K27me3 and the histone variant H2A.Z at the 5' untranslated region (5'UTR) intron of EIN2. Concomitant loss of EIN6 (REF6) and the INO80 complex shifts the chromatin landscape at EIN2 to a repressive state causing a dramatic reduction of EIN2 expression. These results uncover a unique type of chromatin regulation which safeguards the expression of an essential multifunctional plant stress regulator.

Iron (Fe) is essential for life, but in excess can cause oxidative cytotoxicity through the generation of Fe-catalyzed reactive oxygen species. It is yet unknown which genes and mechanisms can provide Fe-toxicity tolerance. Here, we identify S-nitrosoglutathione-reductase (GSNOR) variants underlying a major quantitative locus for root tolerance to Fe-toxicity in Arabidopsis using genome-wide association studies and allelic complementation. These variants act largely through transcript level regulation. We further show that the elevated nitric oxide is essential for Fe-dependent redox toxicity. GSNOR maintains root meristem activity and prevents cell death via inhibiting Fe-dependent nitrosative and oxidative cytotoxicity. GSNOR is also required for root tolerance to Fe-toxicity throughout higher plants such as legumes and monocots, which exposes an opportunity to address crop production under high-Fe conditions using natural GSNOR variants. Overall, this study shows that genetic or chemical modulation of the nitric oxide pathway can broadly modify Fe-toxicity tolerance.

In response to touch, some carnivorous plants such as the Venus flytrap have evolved spectacular movements to capture animals for nutrient acquisition. However, the molecules that confer this sensitivity remain unknown. We used comparative transcriptomics to show that expression of three genes encoding homologs of the MscS-Like (MSL) and OSCA/TMEM63 family of mechanosensitive ion channels are localized to touch-sensitive trigger hairs of Venus flytrap. We focus here on the candidate with the most enriched expression in trigger hairs, the MSL homolog FLYCATCHER1 (FLYC1). We show that FLYC1 transcripts are localized to mechanosensory cells within the trigger hair, transfecting FLYC1 induces chloride-permeable stretch-activated currents in naïve cells, and transcripts coding for FLYC1 homologs are expressed in touch-sensing cells of Cape sundew, a related carnivorous plant of the Droseraceae family. Our data suggest that the mechanism of prey recognition in carnivorous Droseraceae evolved by co-opting ancestral mechanosensitive ion channels to sense touch.

The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. Arabidopsis thaliana contains a single TSPO/PBR-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

Tissue growth as the result of cell division is an essential part of embryonic development. Previous studies have shown that STIMPY (STIP)/WOX9, a homeodomain transcription factor of the Arabidopsis thaliana WOX family, is required for maintaining cell division and preventing premature differentiation in emerging seedlings. Here we present evidence that STIP performs similar functions during embryogenesis. Complete loss of STIP activity results in early embryonic arrest, most likely due to a failure in cell division. STIMPY-LIKE (STPL)/WOX8, a close homolog of STIP in Arabidopsis, also positively regulates early embryonic growth and can replace STIP function when expressed under the STIP promoter. STPL shares redundant functions with a more distantly related member of the WOX family, WOX2, in regulating embryonic apical patterning. These findings show that combinatorial action of WOX transcription factors is essential for Arabidopsis embryonic development.

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.