Gilles Clément

Mutations in the Arabidopsis Homolog of LST8/GβL, a Partner of the Target of Rapamycin Kinase, Impair Plant Growth, Flowering, and Metabolic Adaptation to Long Days

LST8 is a member of the widely conserved TOR kinase complex. This work shows that LST8 is important for plant growth as well as metabolic and developmental processes linked to changes in light conditions, probably by influencing the activity of the TOR complex. The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein β subunit-like (LST8/GβL) protein is a member of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the synthesis of myo-inositol and raffinose during plant adaptation to long days.

Two-site enzyme immunometric assays for determination of native and denatured β-lactoglobulin

Two enzyme immunometric assays suitable for measuring native and denatured beta-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cows milk and cows milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cows milk in goats milk.

Motor behavior underlying the control of an upside-down vertical posture

SummaryThe present study examines the control of equilibrium by trained acrobats standing balanced upside-down on their hands. The focus of the experiment was to analyze the postural attitudes of various body segments and the underlying motor activity, and to evaluate the influence of vision. We found a strong correlation between the motor activity of two antagonist muscles of the forearm and the anteriorposterior position of the center of gravity. The upside-down standing posture was most stable when the neck was dorsiflexed, indicating that the tonic neck reflex and the tonic labyrinthine reflex play an important role in the stability of this posture. Comparison between known data about the normal standing posture, on the feet, and the upside-down standing posture, on the hands, shows that motor behavior of the two postures is similarly organized.

Characterization of a NADH-Dependent Glutamate Dehydrogenase Mutant of Arabidopsis Demonstrates the Key Role of this Enzyme in Root Carbon and Nitrogen Metabolism

A third isoenzyme of Glu dehydrogenase (GDH) is expressed in mitochondria of Arabidopsis root companion cells. A GDH triple mutant differed greatly from the wild type in continuous darkness, suggesting that the main function of the enzyme is to provide 2-oxoglutarate for the tricarboxylic acid cycle, leading to an accumulation of Ala, γ-aminobutyrate, and Asp in both roots and leaves. The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

Adaptive modifications of postural attitude in conditions of weightlessness

SummaryAdaptation of static posture was studied before, during, and after a 7-day space flight. Body segment orientations, body stability, and muscle activity underlying the reproduction of several postural attitudes were examined in various visual situations either with the shoes attached to the floor or during free floating. In standing or relaxed subjects whose shoes were attached to the floor, the tonic activity of the ankle flexor was enhanced relative to that in the same posture on earth, whereas the extensor activity disappeared. Errors in attempting to reproduce the normal terrestrial upright posture and a forward-leaning posture were accompanied by major changes in the synergies between neck, hip, knee, and ankle joints. These changes are mainly attributed to cumulative adjustments in response to nonvestibular signals such as tactile, articular, and proprioceptive cues.

Ocular counterrolling induced by centrifugation during orbital space flight

Abstract. During the 1998 Neurolab mission (STS-90), four astronauts were exposed to interaural centripetal accelerations (Gy centrifugation) of 0.5g and 1g during rotation on a centrifuge, both on Earth and during orbital space flight. Subjects were oriented either left-ear out or right-ear out, facing or back to motion. Binocular eye movements were measured in three dimensions using a video technique. On Earth, tangential centrifugation that produces 1g of interaural linear acceleration combines with gravity to tilt the gravitoinertial acceleration (GIA) vector 45° in the roll plane relative to the head vertical, generating a summed vector of 1.4g. Before flight, this elicited mean ocular counterrolling (OCR) of 5.7°. Due to the relative absence of gravity during flight, there was no linear acceleration along the dorsoventral axis of the head. As a result, during in-flight centrifugation, gravitoinertial acceleration was strictly aligned with the centripetal acceleration along the interaural axis. There was a small but significant decrease (mean 10%) in the magnitude of OCR in space (5.1°). The magnitude of OCR during postflight 1g centrifugation was not significantly different from preflight OCR (5.9°). Findings were similar for 0.5g centrifugation, but the OCR magnitude was approximately 60% of that induced by centrifugation at 1g. OCR during pre- and postflight static tilt was not significantly different and was always less than OCR elicited by centrifugation on Earth for an equivalent interaural linear acceleration. In contrast, there was no difference between the OCR generated by in-flight centrifugation and by static tilt on Earth at equivalent interaural linear accelerations. These data support the following conclusions: (1) OCR is generated predominantly in response to interaural linear acceleration; (2) the increased OCR during centrifugation on Earth is a response to the head dorsoventral 1g linear acceleration component, which was absent in microgravity. The dorsoventral linear acceleration could have activated either the otoliths or body-tilt receptors that responded to the larger GIA magnitude (1.4g), to generate the increased OCR during centrifugation on Earth. A striking finding was that magnitude of OCR was maintained throughout and after flight. This is in contrast to most previous postflight OCR studies, which have generally registered decreases in OCR. We postulate that intermittent exposure to artificial gravity, in the form of the centripetal acceleration experienced during centrifugation, acted as a countermeasure to deconditioning of this otolith-ocular orienting reflex during the 16-day mission.

Nitrogen metabolism meets phytopathology

Nitrogen (N) is essential for life and is a major limiting factor of plant growth. Because soils frequently lack sufficient N, large quantities of inorganic N fertilizers are added to soils for crop production. However, nitrate, urea, and ammonium are a major source of global pollution, because much of the N that is not taken up by plants enters streams, groundwater, and lakes, where it affects algal production and causes an imbalance in aquatic food webs. Many agronomical data indicate that the higher use of N fertilizers during the green revolution had an impact on the incidence of crop diseases. In contrast, examples in which a decrease in N fertilization increases disease severity are also reported, indicating that there is a complex relationship linking N uptake and metabolism and the disease infection processes. Thus, although it is clear that N availability affects disease, the underlying mechanisms remain unclear. The aim of this review is to describe current knowledge of the mechanisms that link plant N status to the plants response to pathogen infection and to the virulence and nutritional status of phytopathogens.

Stitching together the Multiple Dimensions of Autophagy Using Metabolomics and Transcriptomics Reveals Impacts on Metabolism, Development, and Plant Responses to the Environment in Arabidopsis

We performed transcriptomic and metabolomic analysis of rosette leaves of Arabidopsis autophagy mutants to elucidate the housekeeping process of autophagy, which plays a key role in immunity, senescence, nutrient recycling, and environmental adaptation. We deciphered the wide range of effects generated by autophagy defects in plants and uncovered links between autophagy, metabolism, and signaling. Autophagy is a fundamental process in the plant life story, playing a key role in immunity, senescence, nutrient recycling, and adaptation to the environment. Transcriptomics and metabolomics of the rosette leaves of Arabidopsis thaliana autophagy mutants (atg) show that autophagy is essential for cell homeostasis and stress responses and that several metabolic pathways are affected. Depletion of hexoses, quercetins, and anthocyanins parallel the overaccumulation of several amino acids and related compounds, such as glutamate, methionine, glutathione, pipecolate, and 2-aminoadipate. Transcriptomic data show that the pathways for glutathione, methionine, raffinose, galacturonate, and anthocyanin are perturbed. Anthocyanin depletion in atg mutants, which was previously reported as a possible defect in flavonoid trafficking to the vacuole, appears due to the downregulation of the master genes encoding the enzymes and regulatory proteins involved in flavonoid biosynthesis. Overexpression of the PRODUCTION OF ANTHOCYANIN PIGMENT1 transcription factor restores anthocyanin accumulation in vacuoles of atg mutants. Transcriptome analyses reveal connections between autophagy and (1) salicylic acid biosynthesis and response, (2) cytokinin perception, (3) oxidative stress and plant defense, and possible interactions between autophagy and the COP9 signalosome machinery. The metabolic and transcriptomic signatures identified for the autophagy mutants are discussed and show consistencies with the observed phenotypes.

Effects of static tilt about the roll axis on horizontal and vertical optokinetic nystagmus and optokinetic after-nystagmus in humans

SummaryHorizontal and vertical OKN and OKAN were recorded in four conditions using the EOG technique. Instructions to subjects were aimed at obtaining a “look” type OKN. Two optokinetic stimulators, a stationary sphere and a binocular portable model, were compared with the subject in the upright condition. Three posture orientations, upright, 90° roll (horizontal), and upside-down, were then compared using the portable stimulator to determine the effect of roll-axis tilt on OKN at three velocities and on OKAN. Vertical OKN asymmetry was found to increase in the 90° roll position and to tend toward a reversal in the upside-down position. The time constant of vertical OKAN with slow phase up increased in both the 90° roll and upside-down positions. And finally, cross-coupled vertical eye movements during and after horizontal OKN were clearly observed. These results confirm the data obtained in monkeys, and are in accordance with the hypothesis of a three-dimensional organization of the velocity storage mechanism.

Contribution of eye positioning to control of the upside-down standing posture

SummaryThe present study attempts to clarify the relationship between eye and body positioning during an upside-down standing posture on the hands. In this posture the head was observed to be stable and the eyes were anchored to an earth-fixed target. We measured the variations of body sway when subjects displaced their gaze upward or downward from their anchoring position. They did this voluntarily under instruction, and involuntarily by means of base-down or base-up wedge prisms. Results show that the anchoring point chosen by the subjects corresponds to a perceived limit of their body sway. They suggest that vision is also used to convey the desired optimal zone for the center of gravity in cases where fine balance is required.