DRMY1 promotes robust morphogenesis in Arabidopsis by sustaining the translation of cytokinin-signaling inhibitor proteins.

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals robustly initiate and grow to a constant size to enclose and protect the inner floral organs. We previously characterized the mutant development-related myb-like 1 (drmy1), where 3-5 sepals initiate variably and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), two cytokinin-signaling inhibitors that are normally rapidly produced before sepal initiation. The resultant upregulation of cytokinin signaling disrupts robust auxin patterning and sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

DRMY1 promotes robust morphogenesis by sustaining translation of a hormone signaling protein.

Robustness is the invariant development of phenotype despite environmental changes and genetic perturbations. In the Arabidopsis flower bud, four sepals initiate at robust positions and times and grow to equal size to enclose and protect the inner floral organs. We previously characterized the mutant development related myb-like1 (drmy1), where 3-5 sepals initiate at irregular positions and variable times and grow to different sizes, compromising their protective function. The molecular mechanism underlying this loss of robustness was unclear. Here, we show that drmy1 has reduced TARGET OF RAPAMYCIN (TOR) activity, ribosomal content, and translation. Translation reduction decreases the protein level of ARABIDOPSIS RESPONSE REGULATOR7 (ARR7), a rapidly synthesized and degraded cytokinin signaling inhibitor. The resultant upregulation of cytokinin signaling disrupts the robust positioning of auxin signaling, causing variable sepal initiation. Our work shows that the homeostasis of translation, a ubiquitous cellular process, is crucial for the robust spatiotemporal patterning of organogenesis.

Translating across kingdoms: target of rapamycin promotes protein synthesis through conserved and divergent pathways in plants.

mRNA translation is the growth rate-limiting step in genome expression. Target of rapamycin (TOR) evolved a central regulatory role in eukaryotes as a signaling hub that monitors nutrient availability to maintain homeostasis and promote growth, largely by increasing the rate of translation initiation and protein synthesis. The dynamic pathways engaged by TOR to regulate translation remain debated even in well-studied yeast and mammalian models, however, despite decades of intense investigation. Recent studies have firmly established that TOR also regulates mRNA translation in plants through conserved mechanisms, such as the TOR-LARP1-5'TOP signaling axis, and through pathways specific to plants. Here, we review recent advances in our understanding of the regulation of mRNA translation in plants by TOR.

Light regulates alternative splicing outcomes via the TOR kinase pathway.

For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Light-triggered chloroplast retrograde signals control alternative splicing in Arabidopsis thaliana. Here, we provide evidence that light regulates the expression of a core set of splicing-related factors in roots. Alternative splicing responses in roots are not directly caused by light but are instead most likely triggered by photosynthesized sugars. The target of rapamycin (TOR) kinase plays a key role in this shoot-to-root signaling pathway. Knocking down TOR expression or pharmacologically inhibiting TOR activity disrupts the alternative splicing responses to light and exogenous sugars in roots. Consistently, splicing decisions are modulated by mitochondrial activity in roots. In conclusion, by activating the TOR pathway, sugars act as mobile signals to coordinate alternative splicing responses to light throughout the whole plant.

TOR coordinates nucleotide availability with ribosome biogenesis in plants.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic Ser/Thr protein kinase that coordinates growth and metabolism with nutrient availability. We conducted a medium-throughput functional genetic screen to discover essential genes that promote TOR activity in plants, and identified a critical regulatory enzyme, cytosolic phosphoribosyl pyrophosphate (PRPP) synthetase (PRS4). PRS4 synthesizes cytosolic PRPP, a key upstream metabolite in nucleotide synthesis and salvage pathways. We found that prs4 knockouts are embryo-lethal in Arabidopsis thaliana, and that silencing PRS4 expression in Nicotiana benthamiana causes pleiotropic developmental phenotypes, including dwarfism, aberrant leaf shape, and delayed flowering. Transcriptomic analysis revealed that ribosome biogenesis is among the most strongly repressed processes in prs4 knockdowns. Building on these results, we discovered that TOR activity is inhibited by chemical or genetic disruption of nucleotide biosynthesis, but that this effect can be reversed by supplying plants with nucleobases. Finally, we show that TOR transcriptionally promotes nucleotide biosynthesis to support the demands of ribosomal RNA synthesis. We propose that TOR coordinates ribosome biogenesis with nucleotide availability in plants to maintain metabolic homeostasis and support growth.

Parallel global profiling of plant TOR dynamics reveals a conserved role for LARP1 in translation.

Target of rapamycin (TOR) is a protein kinase that coordinates eukaryotic metabolism. In mammals, TOR specifically promotes translation of ribosomal protein (RP) mRNAs when amino acids are available to support protein synthesis. The mechanisms controlling translation downstream from TOR remain contested, however, and are largely unexplored in plants. To define these mechanisms in plants, we globally profiled the plant TOR-regulated transcriptome, translatome, proteome, and phosphoproteome. We found that TOR regulates ribosome biogenesis in plants at multiple levels, but through mechanisms that do not directly depend on 5' oligopyrimidine tract motifs (5'TOPs) found in mammalian RP mRNAs. We then show that the TOR-LARP1-5'TOP signaling axis is conserved in plants and regulates expression of a core set of eukaryotic 5'TOP mRNAs, as well as new, plant-specific 5'TOP mRNAs. Our study illuminates ancestral roles of the TOR-LARP1-5'TOP metabolic regulatory network and provides evolutionary context for ongoing debates about the molecular function of LARP1.

TOR dynamically regulates plant cell-cell transport.

The coordinated redistribution of sugars from mature "source" leaves to developing "sink" leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) metabolic signaling network restricts PD transport in leaves. Genetic approaches and chemical or physiological treatments to either promote or disrupt TOR activity demonstrate that glucose-activated TOR decreases PD transport in leaves. We further found that TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and that this increase in TOR activity correlates with decreased rates of PD transport. We conclude that leaf cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.

[Development and application of a method for isolating Escherichia coli O157:H7 in the city of Gualeguaychú]. / Desarrollo y aplicación de una metodología de aislamiento de Escherichia coli O157:H7 en el Municipio de Gualeguaychú.

Culture media, reagents, and commercial kits were compared on artificially contaminated food samples. The objective was to find an isolation method for Escherichia coli O157:H7 sensitive, specific and accessible in terms of cost, requirements of equipments and qualification of the analyst. The adopted scheme consisted in a selective enrichment at 42 degrees C during 18 to 24 h, using an appropriate medium, in accordance with the nature of the sample, followed by a step of immunomagnetic separation and simultaneous isolation on a chromogenic agar and MacConkey sorbitol agar with potassium tellurite and cefixime, during 18-24 h at 37 degrees C. The presumptive colonies were confirmed as E. coli O157 by serological and biochemical tests. Secondly, this methodology was applied to food samples, water, bovine gastric content and manure. A total of 410 samples were studied: 279 from meat, 54 milk and dairy products, 6 from vegetables, 27 water samples and 44 bovine gastric content and manure. The frequency of isolation of E. coli O157:H7 was of 3.9%. The phenotypic and genotypic characterization of the isolates was performed. A simple isolation methodology for E. coli O157 was developed, which proved accessible to food laboratories of lower complexity. This methodology allowed the detection of this pathogen in food and environmental samples in Gualeguaychú City. The role of water as vehicle of infection was also established. The strains harbored the same virulence factors as those recovered from human disease.

Desarrollo y aplicación de una metodología de aislamiento de Escherichia coli O157:H7 en el Municipio de Gualeguaychú / [Development and application of a method for isolating Escherichia coli O157:H7 in the city of Gualeguaychú]

Culture media, reagents, and commercial kits were compared on artificially contaminated food samples. The objective was to find an isolation method for Escherichia coli O157:H7 sensitive, specific and accessible in terms of cost, requirements of equipments and qualification of the analyst. The adopted scheme consisted in a selective enrichment at 42 degrees C during 18 to 24 h, using an appropriate medium, in accordance with the nature of the sample, followed by a step of immunomagnetic separation and simultaneous isolation on a chromogenic agar and MacConkey sorbitol agar with potassium tellurite and cefixime, during 18-24 h at 37 degrees C. The presumptive colonies were confirmed as E. coli O157 by serological and biochemical tests. Secondly, this methodology was applied to food samples, water, bovine gastric content and manure. A total of 410 samples were studied: 279 from meat, 54 milk and dairy products, 6 from vegetables, 27 water samples and 44 bovine gastric content and manure. The frequency of isolation of E. coli O157:H7 was of 3.9

. The phenotypic and genotypic characterization of the isolates was performed. A simple isolation methodology for E. coli O157 was developed, which proved accessible to food laboratories of lower complexity. This methodology allowed the detection of this pathogen in food and environmental samples in Gualeguaychú City. The role of water as vehicle of infection was also established. The strains harbored the same virulence factors as those recovered from human disease.

Isolation and characterization of Escherichia coli O157:H7 from retail meats in Argentina.

Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychú City, Argentina. Samples were assayed for Escherichia coli O157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium. Eleven E. coli O157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages. E. coli O157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed. The isolates were tested for virulence-related genes. Ten additional Shiga toxin-producing E. coli (STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes. All 21 isolates harbored both eae and EHEC-hlyA genes, and 12 (57.1%) encoded stx2/stx2vh-a. The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain). Six strains were nontypable by phage typing. Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles. Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments. The enrichment culture method with IMS was a sensitive procedure to detect E. coli O157:H7 strains in retail meats. Some of the isolates from different stores presented a high clonal relatedness, as determined by XhaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.

[Chondroma and chondrosarcoma of the larynx: apropos of 4 cases]. / Les chondromes et chondrosarcome du larynx: à propos de 4 observations.

Chondroma and laryngeal chondrosarcomas, tumours of variable malignancy, are rare but a source of concern given their location and the surgery involved. Their anatomical limits and classification are still difficult, to the extent that the diagnosis of chondroma is often questioned. Of the four cases reported, two presented a high degree of malignancy, including one with metastasis. Two required a total laryngectomy, and two a partial laryngectomy with a costal cartilage graft in one case.