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The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional–translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis. [ABSTRACT FROM AUTHOR]

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. [ABSTRACT FROM AUTHOR]

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs. [ABSTRACT FROM AUTHOR]

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1 , the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2 , several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s) play important roles in the synthesis of diverse secondary compounds in Arabidopsis (Arabidopsis thaliana). Comparison of four data sets analyzing seedlings harvested over a 2-d period of constant conditions after growth with varying photoperiods and thermocycles recorded a total of 98 P450 loci as circadian regulated for at least one of the four conditions. Here, we further describe the circadian-regulated pathways using, as reporters, individual P450 loci that are likely to be rate limiting in secondary metabolic pathways. Reverse transcription-polymerase chain reaction gel blot analyses have confirmed circadian regulation of P450s in phenylpropanoid, carotenoid, oxylipin, glucosinolate, and brassinosteroid biosyntheses and have shown that both P450 and non-P450 genes in the many branches of the phenylpropanoid pathway have similar circadian patterns of expression. In silico analyses of the subsets of coregulated promoters have identified overrepresented promoter elements in various biosynthetic pathway genes, including MYB and MYB4 elements that are significantly more abundant in promoters for the core and lignin sections of phenyipropanoid metabolism. Interactions with these elements important for circadian regulation do not involve theMYB transcription factor PAP1, as previously proposed, since the expression patterns of circadian-regulated P450s are the same in pap1-D mutant seedlings as in wild-type seedlings. Further analysis of circadian-regulated promoters in other biochemical pathways provides us with the opportunity to identify novel promoter motifs that might be important in P450 circadian regulation. [ABSTRACT FROM AUTHOR]

In plants, light signals caused by the presence of neighbors accelerate stem growth and flowering and induce a more erect position of the leaves, a developmental strategy known as shade-avoidance syndrome. In addition, mutations in the photoreceptors that mediate shade-avoidance responses enhance disease susceptibility in Arabidopsis thaliana. Here, we describe the Arabidopsis constitutive shade-avoidance1 (csa1) mutant, which shows a shade-avoidance phenotype in the absence of shade and enhanced growth of a bacterial pathogen. The csa1 mutant has a T-DNA inserted within the second exon of a Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) gene, which leads to the production of a truncated mRNA. Arabidopsis plants transformed with the truncated TIR-NBS-LRR gene recapitulate the mutant phenotype, indicating that csa1 is a dominant-negative mutation that interferes with phytochrome signaling. TIR-NBS-LRR proteins have been implicated in defense responses in plants. RPS4, the closest homolog of CSA1, confers resistance to Pseudomonas syringae and complements the csa1 mutant phenotype, indicating that responses to pathogens and neighbors share core-signaling components in Arabidopsis. In Drosophila melanogaster and Caenorhabditis elegans, TIR domain proteins are implicated in both development and immunity. Thus, the dual role of the TIR domain is conserved across kingdoms. [ABSTRACT FROM AUTHOR]

Circadian gating of light signaling limits the timing of maximum responsiveness to light to specific times of day. The fhy3 (for far-red elongated hypocotyl3) mutant of Arabidopsis thaliana is involved in independently gating signaling from a group of photoreceptors to an individual response, fhy3 shows an enhanced response to red light during seedling deetiolation. Analysis of two independent fhy3 alleles links enhanced inhibition of hypocotyl elongation in response to red light with an arrhythmic pattern of hypocotyl elongation. Both alleles also show disrupted rhythmicity of central-clock and clock-output gene expression in constant red light, fhy3 exhibits aberrant phase advances under red light pulses during the subjective day. Release-from-light experiments demonstrate clock disruption in fhy3 during the early part of the subjective day in constant red light, suggesting that FHY3 is important in gating red light signaling for clock resetting. The FHY3 gating function appears crucial in the early part of the day for the maintenance of rhythmicity under these conditions. However, unlike previously described Arabidopsis gating mutants that gate all light signaling, gating of direct red light-induced gene expression in fhy3 is unaffected. FHY3 appears to be a novel gating factor, specifically in gating red light signaling to the clock during daytime. [ABSTRACT FROM AUTHOR]

The circadian clock exerts a major influence on transcriptional regulation in plants and other organisms. We have previously identified a motif called the evening element (EE) that is overrepresented in the promoters of evening-phased genes. Here, we demonstrate that multimerized EEs are necessary and sufficient to confer evening-phased circadian regulation. Although flanking sequences are not required for EE function, they can modulate EE activity. One flanking sequence, taken from the PSEUDORESPONSE REGULATOR 9 promoter, itself confers dawn-phased rhythms and has allowed us to define a new clock promoter motif (the morning element [ME]). Scanning mutagenesis reveals that both activators and repressors of gene expression act through the ME and EE. Although our experiments confirm that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are likely to act as repressors via the EE, they also show that they have an unexpected positive effect on EE-mediated gene expression as well. We have identified a clock-regulated activity in plant extracts that binds specifically to the EE and has a phase consistent with it being an activator of expression through the EE. This activity is reduced in CCA1/LHY null plants, suggesting it may itself be part of a circadian feedback loop and perhaps explaining the reduction in EE activity in these double mutant plants. [ABSTRACT FROM AUTHOR]

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F2 individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION I, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays. [ABSTRACT FROM AUTHOR]

Plants respond to the proximity of neighboring vegetation by elongating to prevent shading. Red-depleted light reflected from neighboring vegetation triggers a shade avoidance response leading to a dramatic change in plant architecture. These changes in light quality are detected by the phytochrome family of photoreceptors. We analyzed global changes in gene expression over time in wild-type, phyB mutant, and phyA phyB double mutant seedlings of Arabidopsis in response to simulated shade. Using pattern fitting software, we identified 301 genes as shade responsive with patterns of expression corresponding to one of various physiological response modes. A requirement for a consistent pattern of expression across 12 chips in this way allowed more subtle changes in gene expression to be considered meaningful. A number of previously characterized genes involved in light and hormone signaling were identified as shade responsive, as well as several putative, novel shade-specific signal transduction factors. In addition, changes in expression of genes in a range of pathways associated with elongation growth and stress responses were observed. The majority of shade-responsive genes demonstrated antagonistic regulation by phyA and phyB in response to shade following the pattern of many physiological responses. An analysis of promoter elements of genes regulated in this way identified conserved promoter motifs potentially important in shade regulation. [ABSTRACT FROM AUTHOR]

DET1 is a pleiotropic regulator of Arabidopsis development and controls the expression of many light-regulated genes. To gain a better understanding of the mechanism by which DET1 controls transcription from light-regulated promoters, we identified elements in the chlorophyll a/b-binding protein 2 (CAB2) promoter that are required for DET1-mediated expression. Using a series of reporter constructs in which the luciferase gene is controlled by CAB2 promoter fragments, we defined two DET1-responsive elements in the CAB2 promoter that are essential for proper CAB2 transcription. A 40-bp DET1 dark-response element (DtRE) is required for both dark and root-specific repression of CAB2, whereas the known CAB upstream factor-1 element is required for DET1 activation-associated effects in the light and repression in the roots. HY5, a factor that binds CAB upstream factor-1, is also required for DET1 effects in the light. DtRE binds two distinct activities in Arabidopsis seedling extracts: a novel activity with binding site CAAAACGC that we have named CAB2 DET1-associated factor 1 plus an activity that is likely to be the myb transcription factor Circadian Clock-Associated 1. Both activities are altered in dark-grown det1 extracts as compared with wild type, correlating a change in extractable DNA binding activity with a major change in CAB2 expression. We conclude that DET1 represses the CAB2 promoter in the dark by regulating the binding of two factors, CAB2 DETl-associated factor 1 and Circadian Clock-Associated 1, to the DtRE. [ABSTRACT FROM AUTHOR]

Examines the role of the Timing of Cab Expression1 (TOC1) gene in the Arabidopsis circadian system, using a series of transgenic plants expressing a gradation in TOC1 levels. Comparison between the effects of TOC1 gene dosage and TOC1 overexpression on the plant circadian clock; Role of TOC1 in the red light-dependent control of circadian gene expression; Analysis of the circadian function in the presence of constitute levels of TOC1 using transgenic plants; Significance of TOC1 gene as a component of the plant clock.