Arabidopsis Response Regulators Research Articles

Arabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance.

Immune responses in plants can be triggered by damage/microbe-associated molecular patterns (DAMPs/MAMPs) upon recognition by plant pattern recognition receptors (PRRs). DAMPs are signaling molecules synthesized by plants or released from host cellular structures (e.g., plant cell walls) upon pathogen infection or wounding. Despite the hypothesized important role of plant cell wall-derived DAMPs in plant-pathogen interactions, a very limited number of these DAMPs are well characterized. Recent work demonstrated that pectin-enriched cell wall fractions extracted from the cell wall mutant impaired in Arabidopsis Response Regulator 6 (arr6), that showed altered disease resistance to several pathogens, triggered more intense immune responses than those activated by similar cell wall fractions from wild-type plants. It was hypothesized that arr6 cell wall fractions could be differentially enriched in DAMPs. In this work, we describe the characterization of the previous immune-active fractions of arr6 showing the highest triggering capacities upon further fractionation by chromatographic means. These analyses pointed to a role of pentose-based oligosaccharides triggering plant immune responses. The characterization of several pentose-based oligosaccharide structures revealed that β-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance.

Transcriptome Analysis Suggests Cytokinin and Gibberellin Signaling May Account for Differences Between Spring and Winter Canola (Brassica napus L.) Root Development

Spring-type canola produces significantly smaller and less vigorous roots than winter type at later growth stages. To identify the gene-regulatory networks and cross talks among phytohormones that may be responsible for the difference between the root system of the two growth types, we analyzed transcriptomes from the root samples collected at two time points, 30 and 60 days. Out of 169,646 transcripts that were analyzed, 582 and 555 transcripts were found to be significantly differentially expressed between spring and winter types at 30 and 60 days, respectively. Several auxin-responsive genes were significantly differentially expressed between spring and winter types at both time points, but no distinct pattern of differential regulation was observed in either of the growth types. Type-A and type-B ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, and gene sets involved in cytokinin signaling pathway, were upregulated in the spring types at 60 days. This would be consistent with higher cytokinin activity, which is known to inhibit root growth, in spring type root system at the later growth stage. In addition, several gibberellin-responsive and gibberellin-signaling gene sets were also upregulated only in spring types at 60 days. We observed an elongated root system with fewer root branches in spring type compared to winter type at 60 days in this study, which is consistent with a role of gibberellin in root growth and development. Extensive gene expression data generated in this research will further assist in understanding the natural variation of root system in canola growth habits.

To grow or not to grow ….

To grow or not to grow ….

The ASHR3 SET-Domain Protein is a Pivotal Upstream Coordinator for Wound-Induced Callus Formation in Arabidopsis

Plants can induce callus formation at damaged tissue to replenish cellular damages and facilitate tissue regeneration. Physical wounding triggers a transcriptional signaling cascade to induce cell division for callus formation. Several transcriptional regulators involved in wound-induced callus formation have been identified, but how they are coordinated remains elusive. Here, we report that a SET-domain-containing protein, ASH1-RELATED 3 (ASHR3), acts as an upstream regulator of ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1), PLETHORA 3 (PLT3), and WOUND-INDUCED DEDIFFERENTIATION 3 (WIND3) and promotes wound-induced callus formation. Expression of ASHR3 was rapidly induced by wounding, and the ASHR3 protein possibly catalyzed H3K36me3 deposition at the ARR1, PLT3, and WIND3 loci to activate gene expression. In support, loss-of-function mutants of ASHR3 displayed reduced callus formation after wounding on hormone-free medium. Collectively, our study indicates that wounding activates a chromatin modifier, which establishes chromatin landscapes optimizing cell proliferation and reprogramming.

A single gene underlies the dynamic evolution of poplar sex determination.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis.

Exogenous cytokinin is critical for in vitro shoot regeneration. Proteins involved in the cytokinin signal transduction pathway, including type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), participate in shoot regeneration in Arabidopsis (Arabidopsis thaliana). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating WUSCHEL (WUS) expression; however, it is unclear how type-B ARRs inhibit shoot regeneration. Here, we show that ARR12 is a central enhancer of callus formation and shoot regeneration, whereas ARR1 is a strong inhibitor of this process that counteracts the positive effect of ARR12. ARR1 indirectly represses CLAVATA3 (CLV3) expression in an ARR12-dependent manner via competing with ARR12 for binding to the CLV3 promoter, which contributes to its ARR12-dependent inhibitory effect on callus formation and shoot regeneration. In parallel, ARR1 inhibits shoot regeneration through transcriptional activation of INDOLE-3-ACETIC ACID INDUCIBLE17, an auxin response repressor gene, and the consequent indirect repression of WUS expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the CLV3 regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.

Arabidopsis Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease Resistance

The cytokinin signaling pathway, which is mediated by Arabidopsis response regulator (ARR) proteins, has been involved in the modulation of some disease-resistance responses. Here, we describe novel functions of ARR6 in the control of plant disease-resistance and cell-wall composition. Plants impaired in ARR6 function (arr6) were more resistant and susceptible, respectively, to the necrotrophic fungus Plectosphaerella cucumerina and to the vascular bacterium Ralstonia solanacearum, whereas Arabidopsis plants that overexpress ARR6 showed the opposite phenotypes, which further support a role of ARR6 in the modulation of disease-resistance responses against these pathogens. Transcriptomics and metabolomics analyses revealed that, in arr6 plants, canonical disease-resistance pathways, like those activated by defensive phytohormones, were not altered, whereas immune responses triggered by microbe-associated molecular patterns were slightly enhanced. Cell-wall composition of arr6 plants was found to be severely altered compared with that of wild-type plants. Remarkably, pectin-enriched cell-wall fractions extracted from arr6 walls triggered more intense immune responses than those activated by similar wall fractions from wild-type plants, suggesting that arr6 pectin fraction is enriched in wall-related damage-associated molecular patterns, which trigger immune responses. This work supports a novel function of ARR6 in the control of cell-wall composition and disease resistance and reinforces the role of the plant cell wall in the modulation of specific immune responses.

Ethylene mediates salicylic-acid-induced stomatal closure by controlling reactive oxygen species and nitric oxide production in Arabidopsis

Both salicylic acid (SA) and ethylene induce stomatal closure and positively regulate stomatal immunity, but their interactions in guard cell signaling are unclear. Here, we observed that SA induced the expression of ethylene biosynthetic genes; the production of ethylene, reactive oxygen species (ROS) and nitric oxide (NO); and stomatal closure in Arabidopsis thaliana. However, SA-induced stomatal closure was inhibited by an ethylene biosynthetic inhibitor and mutations in ethylene biosynthetic genes, ethylene-signaling genes [RESPONSE TO ANTAGONIST 1 (RAN1), ETHYLENE RESPONSE 1 (ETR1), ETHYLENE INSENSITIVE 2 (EIN2), EIN3 and ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2)], NADPH oxidase genes [ATRBOHD and ATRBOHF], and nitrate reductase genes (NIA1 and NIA2). Furthermore, SA-triggered ROS production in guard cells was impaired in ran1, etr1, AtrbohD and AtrbohF, but not in ein2, ein3 or arr2. SA-triggered NO production was impaired in all ethylene-signaling mutants tested and in nia1 and nia2. The stomata of mutants for CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) showed constitutive ROS and NO production and closure. These results indicate that ethylene mediates SA-induced stomatal closure by activating ATRBOHD/F-mediated ROS synthesis in an RAN1-, ETR1- and CTR1-dependent manner. This in turn induces NIA1/2-mediated NO production and subsequent stomatal closure via the ETR1, EIN2, EIN3 and ARR2-dependent pathway(s).

Role of the Cytokinin-Activated Type-B Response Regulators in Hormone Crosstalk.

Cytokinin is an important phytohormone that employs a multistep phosphorelay to transduce the signal from receptors to the nucleus, culminating in activation of type-B response regulators which function as transcription factors. Recent chromatin immunoprecipitation-sequencing (ChIP-seq) studies have identified targets of type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs) and integrated these into the cytokinin-activated transcriptional network. Primary targets of the type-B ARRs are enriched for genes involved in hormonal regulation, emphasizing the extensive crosstalk that can occur between cytokinin, auxin, abscisic acid, brassinosteroids, gibberellic acid, ethylene, jasmonic acid, and salicylic acid. Examination of hormone-related targets reveals multiple regulatory points including biosynthesis, degradation/inactivation, transport, and signal transduction. Here, we consider this early response to cytokinin in terms of the hormones involved, points of regulatory crosstalk, and physiological significance.

Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen.

Objectives: This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance. Methods: A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated. Results: Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3. Conclusions: Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

MYC2 regulates ARR16, a component of cytokinin signaling pathways, in Arabidopsis seedling development.

MYC2 is a basic helix‐loop‐helix transcription factor that acts as a repressor of blue light‐mediated photomorphogenic growth; however, it promotes lateral root formation. MYC2 also regulates different phytohormone‐signaling pathways in crucial manner. Arabidopsis response regulator 16 (ARR16) is a negative regulator of cytokinin signaling pathways. Here, we show that MYC2 directly binds to the E‐box of ARR16 minimal promoter and negatively regulates its expression in a cytokinin‐dependent manner. While ARR16 and MYC2 influence jasmonic acid and cytokinin signaling, the expression of ARR16 is regulated by cry1, GBF1, and HYH, the components of light signaling pathways. The transgenic studies show that the expression of ARR16 is regulated by MYC2 at various stages of development. The mutational studies reveal that ARR16 positively regulates the hypocotyl growth in blue light, and phenotypic analysis of atmyc2 arr16 double mutant further reveals that arr16 can suppress the short hypocotyl phenotype of atmyc2. Altogether, this work highlights MYC2‐mediated transcriptional repression of ARR16 in Arabidopsis seedling development.

MiR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

Sex-specific polymorphism of MET1 and ARR17 genes in Populus × sibirica

The genus Populus is an effective model in tree genetics. This genus includes dioecious species and, recently, whole genome resequencing of P. trichocarpa and P. balsamifera enabled the identification of sex-linked regions and sex-associated single nucleotide polymorphisms (SNPs). These results created new opportunities to study sex determination in poplars. In the present work, we performed deep sequencing of genes encoding METHYLTRANSFERASE1 (MET1) and homolog of ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17), which are localized in a sex-linked region of Populus genome and contain a number of sex-associated SNPs. Amplicon libraries for 38 samples of P. × sibirica (19 males and 19 females) were sequenced on MiSeq Illumina (300 nt paired-end reads) and approximately 4000× coverage was obtained for each sample. In total, from 80 to 179 SNPs were detected in poplar individuals for MET1, and from 16 to 49 SNPs were detected for ARR17. We identified 17 sex-specific SNPs (11 in MET1 and 6 in ARR17) – they were present in all males but absent in all females. For identified sex-specific SNP sites, females were homozygous, while males were heterozygous. Moreover, colocation of sex-specific SNPs confirming the XY sex-determination system of poplars was revealed: in one allelic variant, males had the same nucleotides as females, while in the other, sex-specific SNPs were present. Based on the data obtained, we developed and successfully applied a high-resolution melting-based approach for sex identification in poplars. The developed molecular markers are useful for distinguishing between male and female poplars in scientific research and can also be applied to select male-only genotypes for use in city landscaping and production of paper, pulp, and biofuel.

The Lateral Root Cap Acts as an Auxin Sink that Controls Meristem Size

Plant developmental plasticity relies on the activities of meristems, regions where stem cells continuously produce new cells [1]. The lateral root cap (LRC) is the outermost tissue of the root meristem [1], and it is known to play an important role during root development [2-6]. In particular, it has been shown that mechanical or genetic ablation of LRC cells affect meristem size [7, 8]; however, the molecular mechanisms involved are unknown. Root meristem size and, consequently, root growth depend on the position of the transition zone (TZ), a boundary that separates dividing from differentiating cells [9, 10]. The interaction of two phytohormones, cytokinin and auxin, is fundamental in controlling the position of the TZ [9, 10].Cytokinin via the ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) control auxin distribution within the meristem, generating an instructive auxin minimum that positions the TZ [10]. We identify a cytokinin-dependent molecular mechanism that acts in the LRC to control the position of the TZ and meristem size. We show that auxin levels within the LRC cells depends on PIN-FORMED 5 (PIN5), a cytokinin-activated intracellular transporter that pumps auxin from the cytoplasm into the endoplasmic reticulum, and on irreversible auxin conjugation mediated by the IAA-amino synthase GRETCHEN HAGEN 3.17 (GH3.17). By titrating auxin in the LRC, the PIN5 and the GH3.17 genes control auxin levels in the entire root meristem. Overall, our results indicate that theLRC serves as an auxin sink that, under the control of cytokinin, regulates meristem size and root growth.

ARR22 overexpression can suppress plant Two-Component Regulatory Systems.

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

ABA inhibits root cell elongation through repressing the cytokinin signaling

ABSTRACTCell elongation, which plays an important role in root penetration into the soil, responds to a variety of environmental factors. A previous study demonstrated that abscisic acid, a phytohormone involved in stress responses, inhibits root growth by delaying the onset of cell elongation. In contrast, we recently reported that cytokinins promote elongation of root cells by enhancing actin bundling. However, the control of root cell elongation through the interaction between abscisic acid and cytokinin signaling has not yet been uncovered. Here, we show that abscisic acid-induced delay in cell elongation requires inhibition of cytokinin signaling; further, stress is signaled to cell elongation by the pathway mediated by B-type ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), which retards root growth.

Transcriptome profiling and phytohormone responses of Arabidopsis roots to different ambient temperatures

ABSTRACT Ambient temperatures influence plant growth and development, however very little is known about changes in root growth in response to ambient temperature change. Here, we performed transcriptome profiling and compared the differences in gene expression at lower and higher temperatures compared with normal plant growth temperatures. Our analysis of the biological processes and molecular functions regulated by differentially expressed genes revealed that low temperature upregulated carbohydrate metabolism and transmembrane transport, and downregulated signal transduction and defense. High temperature upregulated metabolic processes, transport, and auxin biosynthesis, and downregulated catabolic processes. We found that increased temperature specifically affected the levels of Arabidopsis response regulators, ARR1 and ARR12, to decrease cytokinin signaling, altered the level of the brassinosteroid receptor BRI1 to downregulate brassinosteroid signaling, and changed the level of the gibberellin receptor DELLA to upregulate gibberellin signaling and mediate root elongation. These data contribute to our knowledge of how root growth adapts to elevated ambient temperature under climate warming.

Cytokinin modulates context-dependent chromatin accessibility through the type-B response regulators.

The phytohormone cytokinin regulates diverse aspects of plant growth and development, probably through context-dependent transcriptional regulation that relies on a dynamic interplay between regulatory proteins and chromatin. We employed the assay for transposase accessible chromatin with sequencing to profile changes in the chromatin landscape of Arabidopsis roots and shoots in response to cytokinin. Our results reveal differentially accessible chromatin regions indicative of dynamic regulation in response to cytokinin. These changes in chromatin occur preferentially upstream of cytokinin-regulated genes. The changes also largely overlap with binding sites for the type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs), transcription factors that mediate the primary response to cytokinin. Furthermore, the type-B ARRs were found to be necessary for the changes in chromatin state in response to cytokinin. Last, we identified context-dependent responses by comparing root and shoot profiles. This study provides new insight into the dynamics between cytokinin and chromatin with regard to directing transcriptional programmes and how cytokinin mediates its pleiotropic effects.

Ethylene-independent promotion of photomorphogenesis in the dark by cytokinin requires COP1 and the CDD complex

The transition of skotomorphogenesis to photomorphogenesis is induced by the perception of light, and is characterized by the inhibition of hypocotyl elongation and opening of cotyledons. Although it is known that the plant hormone cytokinin inhibits hypocotyl elongation in dark-grown Arabidopsis plants when applied in high concentrations, it is unclear to what extent this response is the result of cytokinin alone or cytokinin-induced ethylene production. Here, we show that cytokinin-induced inhibition of hypocotyl elongation is largely independent of ethylene and suggest a close connection between the cytokinin two-component system and the light-signaling networks. We show that this cytokinin signal is mainly mediated through the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE3 and the ARABIDOPSIS RESPONSE REGULATOR1 in combination with ARR12. Interestingly, mutation of CONSTITUTIVELY PHOTOMORPOGENIC1 (COP1), DE-ETIOLATED1, and CYTOKININ INSENSITIVE4/COP10 renders plants insensitive to cytokinin, and these factors are indispensable for the transcriptional response during cytokinin-induced de-etiolation, indicating that a functional light-signaling pathway is essential for this cytokinin response. In addition, the effect of cytokinin on hypocotyl elongation is strongly dependent on the light conditions, with higher light intensities causing a switch in the response to cytokinin from an inhibitor to a promoter of hypocotyl elongation.

Modulation of Asymmetric Division Diversity through Cytokinin and SPEECHLESS Regulatory Interactions in the Arabidopsis Stomatal Lineage

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types-pavement cells and guard cells-in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the primeregulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomatal lineage divisions and forms a regulatory circuit withSPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition.