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.

Ethylene-Mediated Regulation of A2-Type CYCLINs Modulates Hyponastic Growth in Arabidopsis.

Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth.

Ethylene promotes hyponastic growth through interaction with ROTUNDIFOLIA3/CYP90C1 in Arabidopsis.

Upward leaf movement, called hyponastic growth, is employed by plants to cope with adverse environmental conditions. Ethylene is a key regulator of this process and, in Arabidopsis thaliana, hyponasty is induced by this phytohormone via promotion of epidermal cell expansion in a proximal zone of the abaxial side of the petiole. ROTUNDIFOLIA3/CYP90C1 encodes an enzyme which was shown to catalyse C-23 hydroxylation of several brassinosteroids (BRs) - phytohormones involved in, for example, organ growth, cell expansion, cell division, and responses to abiotic and biotic stresses. This study tested the interaction between ethylene and BRs in regulating hyponastic growth. A mutant isolated in a forward genetic screen, with reduced hyponastic response to ethylene treatment, was allelic to rot3. The cause of the reduced hyponastic growth in this mutant was examined by studying ethylene-BR interaction during local cell expansion, pharmacological inhibition of BR synthesis and ethylene effects on transcription of BR-related genes. This work demonstrates that rot3 mutants are impaired in local cell expansion driving hyponasty. Moreover, the inhibition of BR biosynthesis reduces ethylene-induced hyponastic growth and ethylene increases sensitivity to BR in promoting cell elongation in Arabidopsis hypocotyls. Together, the results show that ROT3 modulates ethylene-induced petiole movement and that this function is likely BR related.

Shedding light on large-scale chromatin reorganization in Arabidopsis thaliana.

Plants need to respond quickly and appropriately to various types of light signals from the environment to optimize growth and development. The immediate response to shading, reduced photon flux (low light), and changes in spectral quality involves changes in gene regulation. In the case of more persistent shade, the plant shows a dramatic change in the organization of chromatin. Both plant responses are controlled via photoreceptor signaling proteins. Recently, several studies have revealed similar features of chromatin reorganization in response to various abiotic and biotic signals, while others have unveiled intricate molecular networks of light signaling towards gene regulation. This opinion paper briefly describes the chromatin (de)compaction response from a light-signaling perspective to provide a link between chromatin and the molecular network of photoreceptors and E3 ubiquitin ligase complexes.

Modulation of ethylene- and heat-controlled hyponastic leaf movement in Arabidopsis thaliana by the plant defence hormones jasmonate and salicylate.

Upward leaf movement (hyponastic growth) is adopted by several plant species including Arabidopsis thaliana, as a mechanism to escape adverse growth conditions. Among the signals that trigger hyponastic growth are, the gaseous hormone ethylene, low light intensities, and supra-optimal temperatures (heat). Recent studies indicated that the defence-related phytohormones jasmonic acid (JA) and salicylic acid (SA) synthesized by the plant upon biotic infestation repress low light-induced hyponastic growth. The hyponastic growth response induced by high temperature (heat) treatment and upon application of the gaseous hormone ethylene is highly similar to the response induced by low light. To test if these environmental signals induce hyponastic growth via parallel pathways or converge downstream, we studied here the roles of Methyl-JA (MeJA) and SA on ethylene- and heat-induced hyponastic growth. For this, we used a time-lapse camera setup. Our study includes pharmacological application of MeJA and SA and biological infestation using the JA-inducing caterpillar Pieris rapae as well as mutants lacking JA or SA signalling components. The data demonstrate that MeJA is a positive, and SA, a negative regulator of ethylene-induced hyponastic growth and that both hormones repress the response to heat. Taking previous studies into account, we conclude that SA is the first among many tested components which is repressing hyponastic growth under all tested inductive environmental stimuli. However, since MeJA is a positive regulator of ethylene-induced hyponastic growth and is inhibiting low light- and heat-induced leaf movement, we conclude that defence hormones control hyponastic growth by affecting stimulus-specific signalling pathways.

Genetical Genomics Reveals Large Scale Genotype-By-Environment Interactions in Arabidopsis thaliana.

One of the major goals of quantitative genetics is to unravel the complex interactions between molecular genetic factors and the environment. The effects of these genotype-by-environment interactions also affect and cause variation in gene expression. The regulatory loci responsible for this variation can be found by genetical genomics that involves the mapping of quantitative trait loci (QTLs) for gene expression traits also called expression-QTL (eQTLs). Most genetical genomics experiments published so far, are performed in a single environment and hence do not allow investigation of the role of genotype-by-environment interactions. Furthermore, most studies have been done in a steady state environment leading to acclimated expression patterns. However a response to the environment or change therein can be highly plastic and possibly lead to more and larger differences between genotypes. Here we present a genetical genomics study on 120 Arabidopsis thaliana, Landsberg erecta × Cape Verde Islands, recombinant inbred lines (RILs) in active response to the environment by treating them with 3 h of shade. The results of this experiment are compared to a previous study on seedlings of the same RILs from a steady state environment. The combination of two highly different conditions but exactly the same RILs with a fixed genetic variation showed the large role of genotype-by-environment interactions on gene expression levels. We found environment-dependent hotspots of transcript regulation. The major hotspot was confirmed by the expression profile of a near isogenic line. Our combined analysis leads us to propose CSN5A, a COP9 signalosome component, as a candidate regulator for the gene expression response to shade.

Ethylene-induced differential petiole growth in Arabidopsis thaliana involves local microtubule reorientation and cell expansion.

• Hyponastic growth is an upward petiole movement induced by plants in response to various external stimuli. It is caused by unequal growth rates between adaxial and abaxial sides of the petiole, which bring rosette leaves to a more vertical position. The volatile hormone ethylene is a key regulator inducing hyponasty in Arabidopsis thaliana. Here, we studied whether ethylene-mediated hyponasty occurs through local stimulation of cell expansion and whether this involves the reorientation of cortical microtubules (CMTs). • To study cell size differences between the two sides of a petiole in ethylene and control conditions, we analyzed epidermal imprints. We studied the involvement of CMT orientation in epidermal cells using the tubulin marker line as well as genetic and pharmacological means of CMT manipulation. • Our results demonstrate that ethylene induces cell expansion at the abaxial side of the- petiole and that this can account for the observed differential growth. At the abaxial side, ethylene induces CMT reorientation from longitudinal to transverse, whereas, at the adaxial side, it has an opposite effect. The inhibition of CMTs disturbed ethylene-induced hyponastic growth. • This work provides evidence that ethylene stimulates cell expansion in a tissue-specific manner and that it is associated with tissue-specific changes in the arrangement of CMTs along the petiole.

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings.

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.

Petiole hyponasty: an ethylene-driven, adaptive response to changes in the environment.

BACKGROUND: Many plant species can actively reorient their organs in response to dynamic environmental conditions. Organ movement can be an integral part of plant development or can occur in response to unfavourable external circumstances. These active reactions take place with or without a directional stimulus and can be driven either by changes in turgor pressure or by asymmetric growth. Petiole hyponasty is upward movement driven by a higher rate of cell expansion on the lower (abaxial) compared with the upper (adaxial) side. Hyponasty is common among rosette species facing environmental stresses such as flooding, proximity of neighbours or elevated ambient temperature. The complex regulatory mechanism of hyponasty involves activation of pathways at molecular and developmental levels, with ethylene playing a crucial role. SCOPE: We present current knowledge on the mechanisms that promote hyponasty in the context of other organ movements, including tropic and nastic reactions together with circumnutation. We describe major environmental cues resulting in hyponasty and briefly discuss their perception and signal transduction. Since ethylene is a central agent triggering hyponasty, we focus on ethylene in controlling different stages during plant development and summarize current knowledge on the relationship between ethylene and cell growth.

Large-scale chromatin de-compaction induced by low light is not accompanied by nucleosomal displacement.

Arabidopsis thaliana is widely used as model to study chromatin compaction dynamics during development and in response to the environment. Signals such as prolonged heat treatment, low light and pathogen infestation are known to induce large-scale de-condensation of nuclear chromatin. Here we demonstrate that the response to different environments varies at the nucleosomal level. Our results show that in contrast to previous reports on heat and biotic infestation, low light intensity signaling does not alter nucleosomal occupancy, despite the marked effects of low light on global chromatin compaction.

Kinome profiling reveals an interaction between jasmonate, salicylate and light control of hyponastic petiole growth in Arabidopsis thaliana.

Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ∼omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.

Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition.

Plants grow in dense vegetations at the risk of being out-competed by neighbors. To increase their competitive power, plants display adaptive responses, such as rapid shoot elongation (shade avoidance) to consolidate light capture. These responses are induced upon detection of proximate neighbors through perception of the reduced ratio between red (R) and far-red (FR) light that is typical for dense vegetations. The plant hormone auxin is a central regulator of plant development and plasticity, but until now it has been unknown how auxin transport is controlled to regulate shade-avoidance responses. Here, we show that low R:FR detection changes the cellular location of the PIN-FORMED 3 (PIN3) protein, a regulator of auxin efflux, in Arabidopsis seedlings. As a result, auxin levels in the elongating hypocotyls are increased under low R:FR. Seedlings of the pin3-3 mutant lack this low R:FR-induced increase of endogenous auxin in the hypocotyl and, accordingly, have no elongation response to low R:FR. We hypothesize that low R:FR-induced stimulation of auxin biosynthesis drives the regulation of PIN3, thus allowing shade avoidance to occur. The adaptive significance of PIN3-mediated control of shade-avoidance is shown in plant competition studies. It was found that pin3 mutants are outcompeted by wild-type neighbors who suppress fitness of pin3-3 by 40%. We conclude that low R:FR modulates the auxin distribution by a change in the cellular location of PIN3, and that this control can be of great importance for plants growing in dense vegetations.

Photoreceptors CRYTOCHROME2 and phytochrome B control chromatin compaction in Arabidopsis.

Development and acclimation processes to the environment are associated with large-scale changes in chromatin compaction in Arabidopsis (Arabidopsis thaliana). Here, we studied the effects of light signals on chromatin organization. A decrease in light intensity induces a large-scale reduction in chromatin compaction. This low light response is reversible and shows strong natural genetic variation. Moreover, the degree of chromatin compaction is affected by light quality signals relevant for natural canopy shade. The photoreceptor CRYPTOCHROME2 appears a general positive regulator of low light-induced chromatin decompaction. Phytochrome B also controls light-induced chromatin organization, but its effect appears to be dependent on the genetic background. We present a model in which chromatin compaction is regulated by the light environment via CRYPTOCHROME2 protein abundance, which is controlled by phytochrome B action.

Regulatory network identification by genetical genomics: signaling downstream of the Arabidopsis receptor-like kinase ERECTA.

Gene expression differences between individuals within a species can be largely explained by differences in genetic background. The effect of genetic variants (alleles) of genes on expression can be studied in a multifactorial way by the application of genetical genomics or expression quantitative trait locus mapping. In this paper, we present a strategy to construct regulatory networks by the application of genetical genomics in combination with transcript profiling of mutants that are disrupted in single genes. We describe the network identification downstream of the receptor-like kinase ERECTA in Arabidopsis (Arabidopsis thaliana). Extending genetical genomics on the Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population with expression profiling of monogenic mutants enabled the identification of regulatory networks in the so far elusive ERECTA signal transduction cascade. We provide evidence that ERECTA is the causal gene for the major hotspot for transcript regulation in the Arabidopsis Ler/Cvi recombinant inbred population. We further propose additional genetic variation between Ler and Cvi in loci of the signaling pathway downstream of ERECTA and suggest candidate genes underlying these loci. Integration of publicly available microarray expression data of other monogenic mutants allowed us to link ERECTA to a downstream mitogen-activated protein kinase signaling cascade. Our study shows that microarray data of monogenic mutants can be effectively used in combination with genetical genomics data to enhance the identification of genetic regulatory networks.

Endogenous abscisic acid as a key switch for natural variation in flooding-induced shoot elongation.

Elongation of leaves and stem is a key trait for survival of terrestrial plants during shallow but prolonged floods that completely submerge the shoot. However, natural floods at different locations vary strongly in duration and depth, and, therefore, populations from these locations are subjected to different selection pressure, leading to intraspecific variation. Here, we identified the signal transduction component that causes response variation in shoot elongation among two accessions of the wetland plant Rumex palustris. These accessions differed 2-fold in petiole elongation rates upon submergence, with fast elongation found in a population from a river floodplain and slow elongation in plants from a lake bank. Fast petiole elongation under water consumes carbohydrates and depends on the (inter)action of the plant hormones ethylene, abscisic acid, and gibberellic acid. We found that carbohydrate levels and dynamics in shoots did not differ between the fast and slow elongating plants, but that the level of ethylene-regulated abscisic acid in petioles, and hence gibberellic acid responsiveness of these petioles explained the difference in shoot elongation upon submergence. Since this is the exact signal transduction level that also explains the variation in flooding-induced shoot elongation among plant species (namely, R. palustris and Rumex acetosa), we suggest that natural selection results in similar modification of regulatory pathways within and between species.

A kinetic analysis of hyponastic growth and petiole elongation upon ethylene exposure in Rumex palustris.

BACKGROUND AND AIMS: Complete submergence is an important stress factor for many terrestrial plants, and a limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris is one such species and manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment. This study aimed to investigate the kinetics of ethylene-induced leaf hyponasty and petiole elongation. METHODS: Leaf hyponasty and petiole elongation was studied using a computerized digital camera set-up followed by image analyses. Linear variable displacement transducers were used for fine resolution monitoring and measurement of petiole growth rates. KEY RESULTS: We show that submergence-induced hyponastic growth and petiole elongation in R. palustris can be mimicked by exposing plants to ethylene. The petiole elongation response to ethylene is shown to depend on the initial angle of the petiole. When petiole angles were artificially kept at 0 degrees, rather than the natural angle of 35 degrees, ethylene could not induce enhanced petiole elongation. This is very similar to submergence studies and confirms the idea that there are endogenous, angle-dependent signals that influence the petiole elongation response to ethylene. CONCLUSIONS: Our data suggest that submergence and ethylene-induced hyponastic growth and enhanced petiole elongation responses in R. palustris are largely similar. However, there are some differences that may relate to the complexity of the submergence treatment as compared with an ethylene treatment.

Ethylene-induced hyponastic growth in Arabidopsis thaliana is controlled by ERECTA.

Plants can respond quickly and profoundly to detrimental changes in their environment. For example, Arabidopsis thaliana can induce an upward leaf movement response through differential petiole growth (hyponastic growth) to outgrow complete submergence. This response is induced by accumulation of the phytohormone ethylene in the plant. Currently, only limited information is available on how this response is molecularly controlled. In this study, we utilized quantitative trait loci (QTL) analysis of natural genetic variation among Arabidopsis accessions to isolate novel factors controlling constitutive petiole angles and ethylene-induced hyponastic growth. Analysis of mutants in various backgrounds and complementation analysis of naturally occurring mutant accessions provided evidence that the leucin-rich repeat receptor-like Ser/Thr kinase gene, ERECTA, controls ethylene-induced hyponastic growth. Moreover, ERECTA controls leaf positioning in the absence of ethylene treatment. Our data demonstrate that this is not due to altered ethylene production or sensitivity.

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana.

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.

Auxin perception and polar auxin transport are not always a prerequisite for differential growth.

Using time-lapse photography, we studied the response kinetics of low light intensity-induced upward leaf-movement, called hyponastic growth, in Arabidopsis thaliana. This response is one of the traits of shade avoidance and directs plant organs to more favorable light conditions. Based on mutant- and pharmacological data we demonstrated that among other factors, functional auxin perception and polar auxin transport (PAT) are required for the amplitude of hyponastic growth and for maintenance of the high leaf angle, upon low light treatment. Here, we present additional data suggesting that auxin and PAT antagonize the hyponastic growth response induced by ethylene treatment. We conclude that ethylene- and low light-induced hyponastic growth occurs at least partly via separate signaling routes, despite their strong similarities in response kinetics.