Timing to perfection: the biology of central and peripheral circadian clocks

The mammalian circadian system, which is comprised of multiple cellular clocks located in the organs and tissues, orchestrates their regulation in a hierarchical manner throughout the 24?hr of the day. At the top of the hierarchy are the suprachiasmatic nuclei, which synchronize subordinate organ and tissue clocks using electrical, endocrine, and metabolic signaling pathways that impact the molecular mechanisms of cellular clocks. The interplay between the central neural and peripheral tissue clocks is not fully understood and remains a major challenge in determining how neurological and metabolic homeostasis is achieved across the sleep-wake cycle. Disturbances in the communication between the plethora of body clocks can desynchronize the circadian system, which is believed to contribute to the development of diseases such as obesity and neuropsychiatric disorders. This review will highlight the relationship between clocks and metabolism, and describe how cues such as light, food, and reward mediate entrainment of the circadian system.     

Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks

Plants synchronize developmental and metabolic processes with the earth's 24-h rotation through the integration of circadian rhythms and responses to light. We characterize the time for coffee (tic) mutant that disrupts circadian gating, photoperiodism, and multiple circadian rhythms, with differential effects among rhythms. TIC is distinct in physiological functions and genetic map position from other rhythm mutants and their homologous loci. Detailed rhythm analysis shows that the chlorophyll a/b-binding protein gene expression rhythm requires TIC function in the mid to late subjective night, when human activity may require coffee, in contrast to the function of EARLY-FLOWERING3 (ELF3) in the late day to early night. tic mutants misexpress genes that are thought to be critical for circadian timing, consistent with our functional analysis. Thus, we identify TIC as a regulator of the clock gene circuit. In contrast to tic and elf3 single mutants, tic elf3 double mutants are completely arrhythmic. Even the robust circadian clock of plants cannot function with defects at two different phases.     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

The right-handed slanting of Arabidopsis thaliana roots is due to the combined effects of positive gravitropism,circumnutation and thigmotropism

When primary Arabidopsis roots grow down a tilted agar plate, they do not elongate following the gravitational vector along a straight line, but instead they slant noticeably to the right-hand. This process is seen mostly in the ecotypes Wassilewskjia and Landsberg, whereas it is attenuated in the ecotype Columbia, and in some mutants is even inverted. The origin of the slanting of Arabidopsis roots, that evidently constitutes a form of chirality, has so far not been sufficiently clarified. In the present paper we describe it as the general result of the cumulative effects of positive gravitropism, circumnutation and a thigmotropic obstacle-avoiding movement, and in particular, as the consequence of the alternating movement of circumnutation of the root to the right and to the left of the gravitational vector. This movement, which does not appear symmetrical in its nature, since the waves made to the right-hand are complete whereas those made to the left-hand are reduced or aborted, appears to be the reason for the observed slanting. In addition, evidence is furnished supporting the hypothesis that the strong left-handed cell-files torsion, seen in right-handed coiling roots, is not the consequence of a primary process, but of an artifact, and is due to the adjustment of the three dimensional root circumnutating helix to the flat two dimensional agar surface.     

The circadian clocks of plants and cyanobacteria

Classical research on the circadian rhythms of plants helped to demonstrate that all living organisms utilize circadian clocks to adapt their day–night cycles and that the clock is the basis for photoperiodic time measurements. Molecular models for the circadian oscillator have now been elucidated in Drosophila, Neurospora, mice and cyanobacteria. All share a similar feedback structure, but key proteins in each of the oscillators are different. A plant clock model has yet to be proposed, but clock mutants of Arabidopsis are expected to reveal key proteins in the mechanism. Here we discuss how a self-sustained oscillation is established in eukaryotic and prokaryotic models, and the polyphyletic evolution of these clock systems.     

Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

Unique ethylene-regulated touch responses of Arabidopsis thaliana roots to physical hardness

Although touch responses of plant roots are an important adaptive behavior, the molecular mechanism remains unclear. We have developed a bioassay for measuring root-bending responses to physical hardness in Arabidopsis thaliana seedlings. Our test requires a two-layer solid medium. Primary roots growing downward through an upper layer of 0.3% phytagel either penetrate the lower layer or bend along an interface between the upper and lower layers with different concentrations (0.2–0.5%, corresponding to 1.57–6.79 gw mm⁻² in hardness). In proportion to increasing hardness of the lower layer, we found that the percentage of bending roots increased and ethylene production decreased, suggesting an inverse relationship between the root-bending response and ethylene production. Studies with ethylene biosynthesis modulators and mutants also suggested that bending and non-bending responses of roots to medium hardness depend, respectively, on decreased and increased ethylene biosynthesis. In addition, the degrees of root-tip softening and differential root-cell growth, both possible factors determining root-bending response, were enhanced and attenuated by decreased and increased amounts of ethylene, respectively—also in bending roots and non-bending roots. Our findings indicate that ethylene regulates root touch responses, probably through a combination of root-tip softening (or hardening) and differential root-cell growth.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.