No abstract

Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the phase or period of circadian rhythms. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement. We conclude that circadian rhythms of ethylene production are not critical for rhythmic growth.Since the discovery of ethylene production in plants in the 1930s, researchers have tried to elucidate mechanisms governing ethylene formation. A major breakthrough was the completion of the enzymatic pathway for ethylene biosynthesis 50 years later (for review, see Yang and Hoffman, 1984). Shortly thereafter, the first genes encoding ethylene biosynthetic enzymes were cloned (Sato and Theologis, 1989;Van Der Straeten et al., 1990;Hamilton et al., 1991;Spanu et al., 1991). With the use of tomato (Lycopersicon esculentum) and especially Arabidopsis (Arabidopsis thaliana) as model plants, molecular biological and genetic analysis has shed light on many physiological processes involving ethylene (Abeles et al., 1992;Somerville and Meyerowitz, 2002). In higher plants, the enzymes for ethylene biosynthesis are encoded by gene families. The members of these families are differentially responsive to various ethylene-inducing factors, including wounding, fruit ripening, pathogen infections, auxins, and cytokinins (for review, see Fluhr and Mattoo, 1996).In Arabidopsis, there are 12 genes in the family of enzymes that produces the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid (ACC), one of which, ACC SYNTHASE 3 (ACS3), is a pseudogene (Yamagami et al., 2003;Tsuchisaka and Theologis, 2004). ACS1 is not functional as an ACS (Liang et al., 1992). ACS10 and ACS12 do not function as ACSs either, but as aminotransferases (Yamagami et al., 2003). Many of the ACS genes are regulated on the transcriptional level. ACS2 transcription in leaves is switched off when tissues mature (Rodrigues-Pousada et al., 1993; in this paper the gene was designated ACS1). ACS4 can be induced by auxins (Abel et al., 1995). ACS5 is regulated by cytokinins that cause stabilization of the protein (Chae et al., 2003). ACS6 is induce...

Higher plants contain a spatial array of autonomous circadian clocks that regulate gene expression without a localised pacemaker. Circadian timing in plants might be less accurate but more flexible than the vertebrate circadian system.

The expression of CHALCONE SYNTHASE (CHS) expression is an important control step in the biosynthesis of flavonoids, which are major photoprotectants in plants. CHS transcription is regulated by endogenous programs and in response to environmental signals. Luciferase reporter gene fusions showed that the CHS promoter is controlled by the circadian clock both in roots and in aerial organs of transgenic Arabidopsis plants. The period of rhythmic CHS expression differs from the previously described rhythm of chlorophyll a/b-binding protein (CAB) gene expression, indicating that CHS is controlled by a distinct circadian clock. The difference in period is maintained in the wild-type Arabidopsis accessions tested and in the de-etiolated 1 and timing of CAB expression 1 mutants. These clock-affecting mutations alter the rhythms of both CAB and CHS markers, indicating that a similar (if not identical) circadian clock mechanism controls these rhythms. The distinct tissue distribution of CAB and CHS expression suggests that the properties of the circadian clock differ among plant tissues. Several animal organs also exhibit heterogeneous circadian properties in culture but are believed to be synchronized in vivo. The fact that differing periods are manifest in intact plants supports our proposal that spatially separated copies of the plant circadian clock are at most weakly coupled, if not functionally independent. This autonomy has apparently permitted tissue-specific specialization of circadian timing.Light is a key environmental signal for plants, regulating gene expression and development (Neff et al., 2000). Changes in fluence rate and light quality can occur unpredictably and rapidly during the day but have an underlying day-night cycle. Plants have evolved a circadian timing system that allows the anticipation of this predictable rhythm. When plants are deprived of environmental time cues and placed in constant ("free running") environmental conditions, circadian rhythms persist with a period of around 24 h, often for many days (Millar, 1999; McClung, 2000;Murtas and Millar, 2000;Johnson, 2001). Within the circadian system of the whole organism, the term "circadian oscillator" has been used to denote the parts of the system responsible for rhythm generation. Light-dark signals entrain the oscillator via input phototransduction pathways, synchronizing its phase with the environmental light-dark cycle and also affecting its period. Rhythmic output from the oscillator controls a large number of physiological processes in plants ( Lumsden and Millar, 1998). The abundance of 2% to 6% of RNA transcripts in Arabidopsis plants was scored as circadian-regulated in two recent microarray analyses, for example (Harmer et al., 2000;Schaffer et al., 2001).The rhythmic expression of chlorophyll a/bbinding protein (CAB or Light-Harvesting Complex [LHCB]) genes has often been used as a marker for circadian regulation in plants (for review, see Fejes and Nagy, 1998), especially using firefly (Photinus pyralis) luciferase (LUC) repor...

No abstract

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red͞far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB::LUC reporter genes containing the promoter and 5 untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB::Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.Arabidopsis ͉ tobacco ͉ oscillation ͉ luciferase ͉ transgenic plants

No abstract

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.