Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst).
ABSTRACT: From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.
Project description:The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCF(ZTL), TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis.
Project description:ZEITLUPE (ZTL), a photoreceptor with E3 ubiquitin ligase activity, communicates end-of-day light conditions to the plant circadian clock. It still remains unclear how ZTL protein accumulates in the light but does not destabilize target proteins before dusk. Two deubiquitylating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 and 13 (UBP12 and UBP13), which regulate clock period and protein ubiquitylation in a manner opposite to ZTL, associate with the ZTL protein complex. Here we demonstrate that the ZTL interacting partner, GIGANTEA (GI), recruits UBP12 and UBP13 to the ZTL photoreceptor complex. We show that loss of UBP12 and UBP13 reduces ZTL and GI protein levels through a post-transcriptional mechanism. Furthermore, a ZTL target protein is unable to accumulate to normal levels in ubp mutants. This demonstrates that the ZTL photoreceptor complex contains both ubiquitin-conjugating and -deconjugating enzymes, and that these two opposing enzyme types are necessary for circadian clock pacing. This shows that deubiquitylating enzymes are a core element of circadian clocks, conserved from plants to animals.
Project description:Nucleocytoplasmic partitioning of core clock components is essential for the proper operation of the circadian system. Previous work has shown that the F-box protein ZEITLUPE (ZTL) and clock element GIGANTEA (GI) heterodimerize in the cytosol, thereby stabilizing ZTL. Here, we report that ZTL post-translationally and reciprocally regulates protein levels and nucleocytoplasmic distribution of GI in Arabidopsis. We use ectopic expression of the N-terminus of ZTL, which contains the novel, light-absorbing region of ZTL (the LOV domain), transient expression assays and ztl mutants to establish that the levels of ZTL, a cytosolic protein, help govern the abundance and distribution of GI in the cytosol and nucleus. Ectopic expression of the ZTL N-terminus lengthens period, delays flowering time and alters hypocotyl length. We demonstrate that these phenotypes can be explained by the competitive interference of the LOV domain with endogenous GI-ZTL interactions. A complex of the ZTL N-terminus polypeptide with endogenous GI (LOV-GI) blocks normal GI function, causing degradation of endogenous ZTL and inhibition of other GI-related phenotypes. Increased cytosolic retention of GI by the LOV-GI complex additionally inhibits nuclear roles of GI, thereby lengthening flowering time. Hence, we conclude that under endogenous conditions, GI stabilization and cytoplasmic retention occurs naturally through a LOV domain-mediated GI-ZTL interaction, and that ZTL indirectly regulates GI nuclear pools by sequestering GI to the cytosol. As the absence of either GI or ZTL compromises clock function and diminishes the protein abundance of the other, our results highlight how their reciprocal co-stabilization is essential for robust circadian oscillations.
Project description:A LOV (Light, Oxygen, or Voltage) domain containing blue-light photoreceptor ZEITLUPE (ZTL) directs circadian timing by degrading clock proteins in plants. Functions hinge upon allosteric differences coupled to the ZTL photocycle; however, structural and kinetic information was unavailable. Herein, we tune the ZTL photocycle over two orders of magnitude. These variants reveal that ZTL complexes with targets independent of light, but dictates enhanced protein degradation in the dark. In vivo experiments definitively show photocycle kinetics dictate the rate of clock component degradation, thereby impacting circadian period. Structural studies demonstrate that photocycle dependent activation of ZTL depends on an unusual dark-state conformation of ZTL. Crystal structures of ZTL LOV domain confirm delineation of structural and kinetic mechanisms and identify an evolutionarily selected allosteric hinge differentiating modes of PAS/LOV signal transduction. The combined biochemical, genetic and structural studies provide new mechanisms indicating how PAS/LOV proteins integrate environmental variables in complex networks.
Project description:The rhythmic opening/closing and volatile emissions of flowers are known to attract pollinators at specific times. That these rhythms are maintained under constant light or dark conditions suggests a circadian clock involvement. Although a forward and reverse genetic approach has led to the identification of core circadian clock components in Arabidopsis thaliana, the involvement of these clock components in floral rhythms has remained untested, probably because of the weak diurnal rhythms in A. thaliana flowers. Here, we addressed the role of these core clock components in the flowers of the wild tobacco Nicotiana attenuata, whose flowers open at night, emit benzyl acetone (BA) scents and move vertically through a 140° arc. We first measured N. attenuata floral rhythms under constant light conditions. The results suggest that the circadian clock controls flower opening, BA emission and pedicel movement, but not flower closing. We generated transgenic N. attenuata lines silenced in the homologous genes of Arabidopsis LATE ELONGATED HYPOCOTYL (LHY) and ZEITLUPE (ZTL), which are known to be core clock components. Silencing NaLHY and NaZTL strongly altered floral rhythms in different ways, indicating that conserved clock components in N. attenuata coordinate these floral rhythms.
Project description:The circadian clock is the endogenous timer that coordinates physiological processes with daily and seasonal environmental changes. In Arabidopsis thaliana, establishment of the circadian period relies on targeted degradation of TIMING OF CAB EXPRESSION 1 (TOC1) by the 26S proteasome. ZEITLUPE (ZTL) is the F-box protein that associates with the SCF (Skp/Cullin/F-box) E3 ubiquitin ligase that is responsible for marking TOC1 for turnover. CULLIN1 (CUL1) is a core component of SCF complexes and is involved in multiple signaling pathways. To assess the contribution of CUL1-containing SCF complexes to signaling within the plant oscillator, circadian rhythms were examined in the recessive, temperature-sensitive CUL1 allele axr6-3. The activity of CUL1 in this mutant declines progressively with increasing ambient temperature, resulting in more severe defects in CUL1-dependent activities at elevated temperature. Examination of circadian rhythms in axr6-3 revealed circadian phenotypes comparable to those observed in ztl null mutants; namely, lengthened circadian period, altered expression of core oscillator genes, and limited degradation of TOC1. In addition, treatment of seedlings with exogenous auxin did not alter TOC1 stability. These results demonstrate that CUL1 is required for TOC1 degradation and further suggest that this protein is the functional cullin for the SCF(ZTL) complex.
Project description:BACKGROUND: A plant's endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata. RESULTS: Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed. CONCLUSIONS: We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.
Project description:BACKGROUND:Bamboo is an important member of the family Poaceae and has many inflorescence and flowering features rarely observed in other plant groups. It retains an unusual form of perennialism by having a long vegetative phase that can extend up to 120 years, followed by flowering and death of the plants. In contrast to a large number of studies conducted on the annual, reference plants Arabidopsis thaliana and rice, molecular studies to characterize flowering pathways in perennial bamboo are lacking. Since photoperiod plays a crucial role in flower induction in most plants, important genes involved in this pathway have been studied in the field grown Bambusa tulda, which flowers after 40-50 years. RESULTS:We identified several genes from B. tulda, including four related to the circadian clock [LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), ZEITLUPE (ZTL) and GIGANTEA (GI)], two circadian clock response integrators [CONSTANS A (COA), CONSTANS B (COB)] and four floral pathway integrators [FLOWERING LOCUS T1, 2, 3, 4 (FT1, 2, 3, 4)]. These genes were amplified from either gDNA and/or cDNA using degenerate as well as gene specific primers based on homologous sequences obtained from related monocot species. The sequence identity and phylogenetic comparisons revealed their close relationships to homologs identified in the temperate bamboo Phyllostachys edulis. While the four BtFT homologs were highly similar to each other, BtCOA possessed a full-length B-box domain that was truncated in BtCOB. Analysis of the spatial expression of these genes in selected flowering and non-flowering tissue stages indicated their possible involvement in flowering. The diurnal expression patterns of the clock genes were comparable to their homologs in rice, except for BtZTL. Among multiple BtCO and BtFT homologs, the diurnal pattern of only BtCOA and BtFT3, 4 were synchronized in the flower inductive tissue, but not in the non-flowering tissues. CONCLUSION:This study elucidates the photoperiodic regulation of bamboo homologs of important flowering genes. The finding also identifies copy number expansion and gene expression divergence of CO and FT in bamboo. Further studies are required to understand their functional role in bamboo flowering.
Project description:Plants sense photoperiod signals to confirm the optimal flowering time. Previous studies have shown that Cryptochrome2 (CRY2) functions to promote floral transition in the long-day plant (LDP) Arabidopsis; however, the function and molecular mechanism by which CRY2 regulates floral transition in short-day plants (SDPs) is still unclear. In this study, we identified a CRY2 homologous gene, ClCRY2, from Chrysanthemum lavandulifolium, a typical SDP. The morphological changes in the C. lavandulifolium shoot apex and ClFTs expression analysis under SD conditions showed that adult C. lavandulifolium completed the developmental transition from vegetative growth to reproductive growth after eight SDs. Meanwhile, ClCRY2 mRNA exhibited an increasing trend from 0 to 8 d of SD treatment. ClCRY2 overexpression in wild-type (WT) Arabidopsis and C. lavandulifolium resulted in early flowering. The transcript levels of the CONSTANS-like (COL) genes ClCOL1, ClCOL4, and ClCOL5, and FLOWERING LOCUS T (FT) homologous gene ClFT1 were upregulated in ClCRY2 overexpression (ClCRY2-OE) C. lavandulifolium under SD conditions. The transcript levels of some circadian clock-related genes, including PSEUDO-REPONSE REGULATOR 5 (PRR5), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and GIGANTEA (GI-1 and GI-2), were upregulated in ClCRY2-OE C. lavandulifolium, while the expression levels of other circadian clock-related genes, such as EARLY FLOWERING 3 (ELF3), ELF4, LATE ELONGATED HYPOCOTYL (LHY), PRR73, and REVEILLE8 (RVE8), were downregulated in ClCRY2-OE C. lavandulifolium under SD conditions. Taken together, the results suggest that ClCRY2 promotes floral transition by fine-tuning the expression of circadian clock-related gene, ClCOLs and ClFT1 in C. lavandulifolium under SD conditions.
Project description:The circadian clock coordinates an organism's growth, development and physiology with environmental factors. One illuminating example is the rhythmic growth of hypocotyls and cotyledons in Arabidopsis thaliana. Such daily oscillations in leaf position are often referred to as sleep movements or nyctinasty. Here, we report that plantlets of the liverwort Marchantia polymorpha show analogous rhythmic movements of thallus lobes, and that the circadian clock controls this rhythm, with auxin a likely output pathway affecting these movements. The mechanisms of this circadian clock are partly conserved as compared to angiosperms, with homologs to the core clock genes PRR, RVE and TOC1 forming a core transcriptional feedback loop also in M. polymorpha.