Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

Background: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. Results: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 59-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. Conclusion: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner

A Conserved Multi-Gene Family Induces Cross-Reactive Antibodies Effective in Defense against Plasmodium falciparum

BACKGROUND: Two related merozoite surface proteins, MSP3 and MSP6, have previously been identified as targets of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. Both MSP3 and MSP6 share a common characteristic small N-terminal signature amino-acid stretch (NLRNA/G), a feature similar to MSP3-like orthologs identified in other human and primate malaria parasites. METHODS/RESULTS: This signature amino-acid sequence led to the identification of eight ORFs contiguously located on P. falciparum chromosome 10. Our subsequent investigations on their expression, localization, sequence conservation, epitope sharing, immunogenicity and the functional role of antibodies in defense are reported here. Six members of P. falciparum MSP3-multigene family share similar sequence organization within their C-terminal regions, are simultaneously expressed as merozoite surface proteins and are highly conserved among parasite isolates. Each of these proteins is a target of naturally occurring antibodies effective at parasite killing in ADCI assays. Moreover, both naturally occurring antibodies and those generated by immunization display cross-reactivity with other members of the family and exhibit varied binding avidities. CONCLUSIONS/SIGNIFICANCE: The unusual characteristics of the MSP3 multi-gene family lead us to hypothesize that the simultaneous expression of targets eliciting cross-reactive antibody responses capable of controlling parasite densities could represent an immune process selected through evolution to maintain homeostasis between P. falciparum and human hosts; a process that allows the continuous transmission of the parasite without killing the host. Our observations also have practical consequences for vaccine development by suggesting MSP3 vaccine efficacy might be improved when combined with the various C-terminus regions of the MSP3 family members to generate a wider range of antibodies acting and to increase vaccine immunogenicity in varied human genetic backgrounds

Rapid Identification of Malaria Vaccine Candidates Based on α-Helical Coiled Coil Protein Motif

To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens

Diurnal and Circadian Rhythms in the Tomato Transcriptome and Their Modulation by Cryptochrome Photoreceptors

BACKGROUND: Circadian clocks are internal molecular time-keeping mechanisms that provide living organisms with the ability to adjust their growth and physiology and to anticipate diurnal environmental changes. Circadian clocks, without exception, respond to light and, in plants, light is the most potent and best characterized entraining stimulus. The capacity of plants to respond to light is achieved through a number of photo-perceptive proteins including cryptochromes and phytochromes. There is considerable experimental evidence demonstrating the roles of photoreceptors in providing light input to the clock. METHODOLOGY: In order to identify genes regulated by diurnal and circadian rhythms, and to establish possible functional relations between photoreceptors and the circadian clock in tomato, we monitored the temporal transcription pattern in plants entrained to long-day conditions, either by large scale comparative profiling, or using a focused approach over a number of photosensory and clock-related genes by QRT-PCR. In parallel, focused transcription analyses were performed in cry1a- and in CRY2-OX tomato genotypes. CONCLUSIONS: We report a large series of transcript oscillations that shed light on the complex network of interactions among tomato photoreceptors and clock-related genes. Alteration of cryptochrome gene expression induced major changes in the rhythmic oscillations of several other gene transcripts. In particular, over-expression of CRY2 had an impact not only on day/night fluctuations but also on rhythmicity under constant light conditions. Evidence was found for widespread diurnal oscillations of transcripts encoding specific enzyme classes (e.g. carotenoid biosynthesis enzymes) as well as for post-transcriptional diurnal and circadian regulation of the CRY2 transcript

Réponse de la vigne (Vitis vinifera L) aux températures inférieures à 0°C. III. Effets d'un refroidissement contrôlé sur des bourgeons au cours du débourrement

L'évolution du profil de cristallisation des bourgeons latents d'un cépage champenois a été suivie en analyse thermique différentielle de novembre 1987 à mai 1988. De novembre à début mars, le profil exothermique des bourgeons en repos végétatif (stade 1) est stable et apparaît toujours composé de 3 pics. À partir de la mi-mars, alors qu'aucune modification morphologique du bourgeon n'est décelable, les températures d'initiation des 3 pics s'élèvent. Au stade 2, montrant les premiers signes visibles d'une reprise de croissance du bourgeon, le troisième exotherme disparaît. Du stade 3 au stade 9, marqués par la croissance du bourgeon primaire, la remontée progressive de la température d'apparition des 2 exothermes suit l'évolution du stade phénologique. Cette élévation des températures de cristallisation se poursuit jusqu'au stade 9, lorsque le deuxième exotherme fusionne presque avec le premier. Au stade 2, seul le bourgeon primaire est affecté par le gel. La nature et l'intensité des dégâts varient ensuite en fonction du stade phénologique. Les déchirures tissulaires qui apparaissent perturbent profondément l'anatomie des bourgeons, dont la vascularisation est désorganisée. L'apex et les primordia inflorescentiels sont nécrosés.Analysis of the response of grapevine buds to controlled freezing. The response of latent grapevine buds (Vitis vinifera L cv Chardonnay) from a Champagne vineyard was studied by means of differential thermal analysis from November 1987 to May 1988. During the rest period between November 1987 and March 15th, freezing thermograms (1°C/min) displayed 3 crystallization peaks (E1, E2 and E3) as previously described (fig 1). On and after the 15th of March, before any apparent morphological change in the bud, the freezing point (first exotherm) and starting temperatures of the 2 last exotherms (E2 and E3) increased progressively. At stage 2, corresponding to the swelling of the bud according to Eichhorn and Lorenz terminology, the starting temperature of the third exotherm increased, leading to the merging of the 2 peaks E1 and E2: the thermograms displayed only 2 crystallization peaks. From stage 3 to stage 9 which corresponded to rapid elongation of the shoot and to growth of the leaf and inflorescence, the starting temperatures of the 2 remaining peaks continued to increase. This evolution, which was related to phenological stages, affected the second crystallization peak and led to the fusion of the 2 peaks: freezing thermograms at the end of the growth recovery period only displayed 1 exotherm. At stage 2, the first exotherm induced irreversible histological alterations in the main bud, whereas histological organization of the axillary bud remained apparently unaltered. At stage 5, which was particularly studied in this work, destruction affected every tissue: bark (fig 2), xylem and liber (fig 3), meristematic tissues of the apical dome (fig 4), and the leaf and inflorescence (fig 5)

Réponse de la vigne ( Vitis vinifera L) aux températures inférieures à 0°C. II. Effets d'un refroidissement contrôlé sur des bourgeons latents avant le débourrement

Des bourgeons latents de vigne (Vitis vinifera L cv Chardonnay) soumis à un refroidissement progressif (1°C•min -1) entre +5°C et -50°C et étudiés en analyse thermique présentent un profil de cristallisation constitué de 3 exothermes successifs. Le premier se situe vers -12,9°C, le second, plus important, vers -17,0°C et le troisième, plus étendu, vers -21,4°C. À l'échelle histologique, les observations réalisées en microscopie photonique montrent que les cristallisations affectent successivement les tissus de la base du bourgeon (1er exotherme), ceux du bourgeon primaire (2e exotherme) et enfin ceux du bourgeon secondaire (3 e exotherme).Analysis of the response of grapevine latent buds to controlled freezing. The effects of controlled freezing on latent grapevine buds (Vitis vinifera L Chardonnay) were studied by means of differential scanning micro-calorimetry and histological techniques. Buds were excised from shoots taken from plants in a Champagne vineyard and frozen progressively from + 5°C to -50°C (1 °C/min). Figure 1 presents a simplified scheme of the organization of a latent bud. Each bud includes a primary bud with a secondary and eventually a tertiary bud. Thermograms of an entire bud (fig 2A) generally display 3 exotherms at ≈ -12.9, -17.0°C and -21.4°C. Exothermic peaks obtained during programmed freezing of different parts of the buds differed markedly. Bud scales (fig 2B) showed no exotherm. Two exotherms at -18.2°C and -26°C respectively were obtained when the bud was devoid of its basal part (fig 2C), whereas the 2 peaks of the basal part of the bud occurred at -13.3 and -20°C (fig 2D). When the principal and the secondary buds were frozen separately without their basal parts, they presented only 1 crystallization peak which occurred 3.2 degrees higher in the main bud than in the axillary bud (fig 2E, F). The first exotherm induced histological alterations in the pith of the basal part of the bud (figs 3-5). The second exotherm induced some ruptures of the cell wall (fig 6) and an extension of the cytoplasm contraction to the cells through the basal third of the pith; the other parts of the bud remained apparently intact (fig 7). The third exotherm produced irreversible alterations throughout the main bud (figs 8-11), whereas the meristematic cells seemed to remain intact (fig 12)

Réponse de la vigne (Vitis vinifera L) aux températures inférieures à 0°C. I. Effets d'un refroidissement contrôlé sur des sarments aoûtés

Afin de simuler les effets d'un froid hivernal, des fragments de sarments aoûtés de vigne ont été soumis artificiellement à un refroidissement programmé de 1°C.mm-1, entre +5° et -50°C. Les thermogrammes obtenus en microcalorimétrie différentielle à balayage présentent 2 exothermes successifs : le premier, nettement marqué vers -9°C, le second, plus étalé, débute vers -17°C et persiste jusqu'à -30°C. Corrélativement à ces pics de cristallisation les analyses histo-cytologiques ont montré des seuils thermiques de dégradation sélective des différents tissus caulinaires : parenchyme cortical et vaisseaux conducteurs du bois (-10°C), liber (-17°C) et cellules médullaires (-30°C).Analysis of the response of grapevine mature shoots to controlled freezing. The effects of controlled freezing on mature shoots of grapevine (Vitis vinifera L cv Chardonnay) were studied by differential scanning microcalorimetry and histological techniques. One-yr-old shoots were taken from plants in a Champagne vineyard and frozen progressively from +5°C to -50°C (1°C/min). Figure 1 presents thermograms obtained from different parts of the shoot. Thermograms generally displayed 2 exotherms at ≈ -10°C and -17°C respectively, whatever the nature of the explant: a piece of shoot, bark or pith. However, secondary xylem thermograms presented only 1 crystallization peak. Histological alterations due to the formation of ice appeared after the first exotherm and involved the bark (figs 2, 3) and the secondary xylem vessels (figs 9, 10 and 11), whereas other tissues (the cambium and liber) remained unaltered (fig 6). After the second exotherm (between -17 and -30°C) every tissue was affected: the bark (fig 4), the liber and cambium (figs 5, 7 and 8), and the secondary xylem (fig 12). In conclusion, although some destruction inside the shoot seemed to have been induced by freezing at -9°C, it only concerned the superficial tissues and did not affect further development of the plant. Further freezing < -17°C appeared necessary to destroy liber and secondary xylem vessels