Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Heterogeneity of the Glyoxylate-Condensing Enzymes.

Wegener, Warner S. (Albert Einstein Medical Center, Philadelphia, Pa.), Henry C. Reeves, and Samuel J. Ajl. Heterogeneity of the glyoxylate-condensing enzymes. J. Bacteriol. 90:594-598. 1965.-Evidence is presented that the enzymatic condensations of glyoxylate with acetyl-CoA (malate synthase), propionyl-CoA (alpha-hydroxyglutarate synthase), butyryl-CoA (beta-ethylmalate synthase), and valeryl-CoA (beta-n-propylmalate synthase) are catalyzed by different enzymes. The possibility that these activities resulted from a single enzyme possessing a broad fatty acid acyl-CoA substrate specificity was ruled out. The latter was suggested by the fact that cells grown on a number of short-chain fatty acids exhibited all the above activities. The conclusion that these reactions are catalyzed by different enzymes is based on the following considerations: (i) the enzymes can be differentially inactivated by heat; (ii) under various growth conditions, where all the condensing enzymes are present, their respective activities do not show a constant ratio, as would be expected if they were catalyzed by a single enzyme; and (iii) under appropriate growth conditions, one or more of these enzymes has been shown to be present to the exclusion of others.     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Procedures for the Generation of Mature Chlamydomonas reinhardtii Zygotes for Molecular and Biochemical Analyses

Zygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas reinhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quantities and a method was developed for the extraction from zygotes of RNA that could be translated in vitro.     

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes [9]. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.     

Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents

Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.Abbreviations DW dry weight - MS Murashige & Skoog[7]medium - NAA 1-naphthaleneacetic acid     

Radiation induced formation of giant cells in Saccharomyces uvarum

Summary Thin sections of budding yeast cells and giant gells grown after X-irradiation have been examined by electron microscopy. The different steps of cross-wall formation during budding were documented with unirradiated cells. With X-ray induced giant cells cytokinesis was shown to be absent. Neither primary nor secondary septae appeared thus cell separation did not occur. Despite this fact both macromolecular synthesis and bud growth continued, giving rise to the formation of giant cells.     

Fluorescence recovery spectroscopy as a probe of slow rotational motions.

Pump-and-probe techniques can be used to follow the slow rotational motions of fluorescent labels bound to macromolecules in solution. A strong pulse of polarized light initially anisotropically depletes the ground-state population. A continuous low-intensity beam of variable polarization then probes the anisotropic ground-state distribution. Using an additional emission polarizer, the generated fluorescence can be recorded as it rises towards its prepump value. A general theory of fluorescence recovery spectroscopy (FRS) is presented that allows for irreversible depletion processes like photobleaching as well as slowly reversible processes like triplet formation. In either case, rotational motions modulate recovery through cosine-squared laws for dipolar absorption and emission processes. Certain pump, probe, and emission polarization directions eliminate the directional dependence of either dipole and simplify the resulting expressions. Two anisotropy functions can then be constructed to independently monitor the rotations of either dipole. These functions are identical in form to the anisotropy used in fluorescence depolarization measurements and all rotational models developed there apply here with minor modifications. Several setups are discussed that achieve the necessary polarization alignments. These include right-angle detection equipment that is commonly available in laboratories using fluorescence methods.     

DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.