ProtoColVR: Requirements Gathering and Collaborative Rapid Prototyping of VR Training Simulators for Multidisciplinary Teams.

We present ProtoColVR, a methodology and a plugin designed for gathering requirements and collaborative rapid prototyping of virtual reality training simulators. Our methodology outlines the utilization of current technologies, the involvement of stakeholders during design and development, and the implementation of simulator creation through multiple iterations. We incorporate open-source tools and freely available environments like Twine and Unity to establish a reference implementation for requirements gathering and rapid prototyping. ProtoColVR is the outcome of our collaboration with a hospital and our Navy, and it has undergone testing in a development Jam. From these tests, we have gained valuable insights, including the ability to create functional prototypes within multidisciplinary teams, enhance communication among different roles, and streamline requirements gathering while improving our understanding of the virtualized environment.

Multi-organ point-of-care ultrasound for COVID-19 (PoCUS4COVID): international expert consensus.

COVID-19 has caused great devastation in the past year. Multi-organ point-of-care ultrasound (PoCUS) including lung ultrasound (LUS) and focused cardiac ultrasound (FoCUS) as a clinical adjunct has played a significant role in triaging, diagnosis and medical management of COVID-19 patients. The expert panel from 27 countries and 6 continents with considerable experience of direct application of PoCUS on COVID-19 patients presents evidence-based consensus using GRADE methodology for the quality of evidence and an expedited, modified-Delphi process for the strength of expert consensus. The use of ultrasound is suggested in many clinical situations related to respiratory, cardiovascular and thromboembolic aspects of COVID-19, comparing well with other imaging modalities. The limitations due to insufficient data are highlighted as opportunities for future research.

Guía de recomendaciones para el manejo de pacientes pediátricos con enfermedad severa por SARS-CoV-2 / Recommendations guide for the management of pediatric patients with severe SARS-CoV-2

Resumen: La aparición del SARS-CoV-2 a partir de diciembre de 2019 y su rápida expansión en el mundo alcanzando el estatus de Pandemia, se ha convertido en un gran desafío para los equipos de salud. Aunque la evidencia de la infección en niños aún es escasa comparada a la de los adultos, se ha hecho evidente que a nivel de la población pediátrica, la mayor parte de las veces la infección es asintomática o de curso leve, sin embargo no todos los pacientes tienen esta evolución, lo que ha motivado la discusión en los equipos de Cuidados Criticos Pediátricos respecto a cómo enfrentar estos pacientes con enfermedad grave. Este consenso es fruto del trabajo de la Rama de Cuidados Intensivos Pe diátricos de la Sociedad Chilena de Pediatría, recogiendo la evidencia disponible al momento de la revisión más la opinión de expertos nacionales en Cuidados Intensivos Pediátricos. El propósito de estas recomendaciones, es ofrecer a los equipos que atienden a pacientes críticos pediátricos, una guía para el diagnóstico y tratamiento de pacientes que evolucionen con COVID 19 grave, que puedan ser aplicadas en todas las UPC Pediátricas de nuestro país, haciendo especial énfasis en aquellas medidas que han demostrado mayor efectividad a nivel de estudios diagnósticos, tratamiento y cuidados del personal de salud.

Abstract: The appearance of SARS-CoV-2 from December 2019 and its rapid expansion in the world reaching Pandemic status, has become a great challenge for health teams. Although the evidence of infection in children is still scarce compared to that of adults, it has become evident that at the pediatric po pulation level, most of the time the infection is asymptomatic or mild, but not all the patients have this evolution, which has motivated the discussion in the Pediatric Critical Care teams regarding how to face these patients with a more serious disease. This consensus is the result of the work of the Pediatric Intensive Care Branch of the Chilean Society of Pediatrics, collecting the evidence available at the time of the review plus the opinion of national experts in Pediatric Intensive Care. The purpose of these recommendations is to offer teams that care for critically ill pediatric patients a guide for the diagnosis and treatment of patients who evolve with severe COVID 19, which can be applied in all Pediatric UPCs in our country, with special emphasis in those measures that have shown greater effectiveness at the level of diagnostic studies, treatment and care of health personnel.

Heavy Metal Enrichment Factors in Fluvial Sediments of an Amazonian Basin Impacted by Gold Mining.

Artisanal and small-scale gold mining (ASGM) has been performed in the southern Ecuadorian Amazon since the colonial period. However, its effects on fluvial systems have been poorly investigated. Thus, in order to calculate the normalized enrichment factors (NEF) of several heavy metals in fluvial sediments of the Zamora River basin (Ecuadorian Amazon), we analyzed bottom sediments along ASGM-affected and unaffected river sections. The results indicated that sediments of the Congüime River have NEF between 2.2 and 2.3 for Cu (moderate contamination) and higher than 3 for Mn, Zn, Pb, and Hg (severe contamination). Similarly, a severe contamination is also observed in the lower Nangaritza River, due to sediments of this sector have NEF > 3 for Zn, Pb, and Hg. Bottom sediments from the Nambija and Zamora rivers showed a severe contamination with Hg (NEF > 3), suggesting the existence of ASGM activities in the upper Zamora River basin.

Correction: Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening.

[This corrects the article DOI: 10.1371/journal.pone.0197118.].

Structural analysis of the woodland strawberry COI1-JAZ1 co-receptor for the plant hormone jasmonoyl-isoleucine.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes. Fragaria vesca, the woodland strawberry, is a model plant for the Rosaceae family, in which the JA-Ile perception is poorly understood at the molecular level. JA-Ile promotes binding of JAZ repressor to COI1 protein in Arabidopsis to activate jasmonate (JA)-dependent responses. The aim of this work was to understand the molecular basis of the interaction between the F. vesca COI1 (FvCOI1) and JAZ1 (FvJAZ1) promoted by JA-Ile using a computational approach. Multiple sequence alignments and phylogenetic analyses of amino acid sequences were performed for FvCOI1, FvJAZ1 and their ortholog sequences. 3D structures for FvCOI1 and FvJAZ1 proteins were built by methods of homology modeling, using AtCOI1-JA-Ile-AtJAZ1 as template and then they were further refined and validated by molecular dynamics (MD) simulation. A molecular docking approach along with MDS analysis were used to gain insights into the interaction between a putative degron-like sequence present in FvJAZ1 with the FvCOI1-JA-Ile complex. FvCOI1 and FvJAZ1 showed high and moderate sequence identity, respectively, with the corresponding ortholog proteins from other plant species including apple, grape, tomato and Arabidopsis. Moreover, the FvJAZ1 has a variant C-terminal IPMQRK sequence instead of the canonical LPIARR degron sequence located in the Jas domain of AtJAZ1. The MD simulation results showed that the FvCOI1-JA-Ile-FvJAZ1 complex was stable, and the IPMQRK peptide of FvJAZ1 directly interacted with FvCOI1 and JA-Ile. The present research provides novel insight into the molecular interactions among key JA-signaling components in the model plant F. vesca, being few examples of characterized JA-Ile receptors at a structural level in plants.

Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening.

Jasmonates (JAs) are signalling molecules involved in stress responses, development and secondary metabolism biosynthesis, although their roles in fleshy-fruit development and ripening processes are not well known. In strawberry fruit, it has been proposed that JAs could regulate the early development through the activation of the JAs biosynthesis. Moreover, it has been reported that JA treatment increases anthocyanin content in strawberry fruit involving the bioactive jasmonate biosynthesis. Nevertheless, JA signalling pathway, of which main components are the COI1-JAZ co-receptor and the MYC transcription factors (TFs), has not been characterized in strawberry until now. Here we identified and characterized the woodland strawberry (Fragaria vesca) JAZ and MYC genes as well as studied their expression during development and ripening stages in commercial strawberry (Fragaria × ananassa) fruit. We described twelve putative JAZ proteins and two MYC TFs, which showed high conservation with respect to their orthologs in Arabidopsis thaliana and in other fleshy-fruit species such as Malus × domestica, Vitis vinifera and Solanum lycopersicum as revealed by gene synteny and phylogenetic analyses. Noteworthy, their expression levels exhibited a significant decrease from fruit development to ripening stages in F. × ananassa, along with others of the JA signalling-related genes such as FaNINJA and FaJAMs, encoding for negative regulators of JA responses. Moreover, we found that main JA signalling-related genes such as FaMYC2, and FaJAZ1 are promptly induced by JA treatment at early times in F. × ananassa fruit. These results suggest the conservation of the canonical JA signalling pathway in strawberry and a possible role of this pathway in early strawberry fruit development, which also correlates negatively with the beginning of the ripening process.

Independent Preharvest Applications of Methyl Jasmonate and Chitosan Elicit Differential Upregulation of Defense-Related Genes with Reduced Incidence of Gray Mold Decay during Postharvest Storage of Fragaria chiloensis Fruit.

The Chilean strawberry (Fragaria chiloensis) fruit has interesting organoleptic properties, but its postharvest life is affected by gray mold decay caused by Botrytis cinerea. The effect of preharvest applications of methyl jasmonate (MeJA) or chitosan on the molecular defense-related responses and protection against gray mold decay were investigated in Chilean strawberry fruit during postharvest storage. Specifically, we inoculated harvested fruit with B. cinerea spores and studied the expression of genes encoding for the pathogenesis-related (PR) proteins ß-1,3-glucanases (FcBG2-1, FcBG2-2 and FcBG2-3) and chitinases (FcCHI2-2 and FcCHI3-1), and for polygalacturonase inhibiting proteins (FcPGIP1 and FcPGIP2) at 0, 2, 24, 48, and 72 h post inoculation (hpi). Remarkably, MeJA- and chitosan-treated fruit exhibited a lower incidence of B. cinerea infection than the control-treated at 48 and 72 hpi. At the molecular level, both are efficient elicitors for priming in F. chiloensis fruit since we observed an upregulation of the FcBG2-1, FcBG2-3, FcPGIP1, and FcPGIP2 at 0 hpi. Moreover, a chitosan-mediated upregulation of FcPGIPs at early times post inoculation (2-24 hpi) and MeJA upregulated FcBGs (24-72 hpi) and FcPGIP1 at later times could contribute to reduce B. cinerea incidence by differential upregulation of defense genes. We concluded that preharvest applications of MeJA or chitosan had a long-lasting effect on the reduction of B. cinerea incidence during postharvest as well as an enhancer effect on the induction of PR and PGIP gene expression.

Salt stress response triggers activation of the jasmonate signaling pathway leading to inhibition of cell elongation in Arabidopsis primary root.

Salinity is a severe abiotic stress that affects irrigated croplands. Jasmonate (JA) is an essential hormone involved in plant defense against herbivory and in responses to abiotic stress. However, the relationship between the salt stress response and the JA pathway in Arabidopsis thaliana is not well understood at molecular and cellular levels. In this work we investigated the activation of JA signaling by NaCl and its effect on primary root growth. We found that JA-responsive JAZ genes were up-regulated by salt stress in a COI1-dependent manner in the roots. Using a JA-Ile sensor we demonstrated that activation of JA signaling by salt stress occurs in the meristematic zone and stele of the differentiation zone and that this activation was dependent on JAR1 and proteasome functions. Another finding is that the elongation zone (EZ) and its cortical cells were significantly longer in JA-related mutants (AOS, COI1, JAZ3 and MYC2/3/4 genes) compared with wild-type plants under salt stress, revealing the participation of the canonical JA signaling pathway. Noteworthy, osmotic stress - a component of salt stress - inhibited cell elongation in the EZ in a COI1-dependent manner. We propose that salt stress triggers activation of the JA signaling pathway followed by inhibition of cell elongation in the EZ. We have shown that salt-inhibited root growth partially involves the jasmonate signaling pathway in Arabidopsis.

Male sterility in Arabidopsis induced by overexpression of a MYC5-SRDX chimeric repressor.

Jasmonate hormone (JA) plays critical roles in both plant defense and reproductive development. Arabidopsis thaliana plants deficient in JA-biosynthesis or -signaling are male-sterile, with defects in stamen and pollen development. MYC2, MYC3 and MYC4 are JAZ-interacting bHLH transcription factors that play a major role in controlling JA responses in vegetative tissue, but are not likely to play a role in reproductive tissue. We found that a closely related transcription factor, MYC5 (bHLH28), was able to induce JAZ promoters that control some of the early JA-responsive genes in a Daucus carota (carrot) protoplast expression system. A G-box sequence in the JAZ2 promoter was necessary and sufficient for induction by MYC5 (as it is for MYC2, MYC3 and MYC4), and induction of JAZ genes was repressed by co-expression of a stabilized, JAZ1ΔJas repressor. Two allelic myc5 mutants exhibited no overt phenotype; however, transgenic lines expressing MYC5 fused to an SRDX (SUPERMAN repressive domain X) motif phenocopied mutants defective in JA signaling. In particular, MYC5-SRDX plants were male-sterile, with defects in stamen filament elongation, anther dehiscence and pollen viability. Importantly, expression of MYB21 and other transcription factors required for stamen and pollen maturation was strongly reduced in stamens of MYC5-SRDX plants relative to the wild type. Taken together, these results indicate that MYC5, probably together with other, redundant transcription factors, may be activated by JA signaling to induce the expression of MYB21 and components required for male fertility.

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.

The Arabidopsis JAZ2 promoter contains a G-Box and thymidine-rich module that are necessary and sufficient for jasmonate-dependent activation by MYC transcription factors and repression by JAZ proteins.

Jasmonate (JA) is a critical hormone for both plant defense and reproductive development. Until now, early JA-responsive promoters have not been well characterized. To identify the cis-acting DNA element involved in the early JA response at the transcriptional level, we analyzed the promoter of the Arabidopsis gene encoding jasmonate-ZIM domain2 protein (JAZ2). The full-length JAZ2 promoter in JAZ2::GUS plants is active in vegetative and reproductive tissue, with expression in stamen filaments being dependent on JA biosynthesis. We identified a G-box element in the JAZ2 promoter that is required for JA-mediated promoter activation in carrot protoplasts and Arabidopsis seedlings. Three copies of a G-box and flanking sequences has autonomous JA-dependent activity and was transactivated by MYC2, MYC3 and MYC4 transcription factors in carrot protoplasts. Expression of MYC2, MYC3 or MYC4 fused to an EAR (ETHYLENE RESPONSE FACTOR-associated amphiphilic repression) motif, or expression of JAZ1 or JAZ6 all repressed JA- and MYC-dependent activation of the JAZ2 promoter. A thymidine-rich sequence 3' to the G-box was required for full JA activation of the JAZ2 promoter. Because the G-box is also present in genes unresponsive to JA, we propose that this thymidine-rich sequence together with the G-box provides JA specificity to promoters induced early in the JA response. Together, these results indicate that an extended G-box located in the promoter region of an early JA-responsive gene is required for gene induction in vivo, probably through selective binding of MYC2, MYC3 and MYC4 transcription factors.

Characterization of JAZ-interacting bHLH transcription factors that regulate jasmonate responses in Arabidopsis.

The plant hormone jasmonate (JA) plays important roles in the regulation of plant defence and development. JASMONATE ZIM-DOMAIN (JAZ) proteins inhibit transcription factors that regulate early JA-responsive genes, and JA-induced degradation of JAZ proteins thus allows expression of these response genes. To date, MYC2 is the only transcription factor known to interact directly with JAZ proteins and regulate early JA responses, but the phenotype of myc2 mutants suggests that other transcription factors also activate JA responses. To identify JAZ1-interacting proteins, a yeast two-hybrid screen of an Arabidopsis cDNA library was performed. Two basic helix-loop-helix (bHLH) proteins, MYC3 and MYC4, were identified. MYC3 and MYC4 share high sequence similarity with MYC2, suggesting they may have similar biological functions. MYC3 and MYC4 interact not only with JAZ1 but also with other JAZ proteins (JAZ3 and JAZ9) in both yeast two-hybrid and pull-down assays. MYC2, MYC3, and MYC4 were all capable of inducing expression of JAZ::GUS reporter constructs following transfection of carrot protoplasts. Although myc3 and myc4 loss-of-function mutants showed no phenotype, transgenic plants overexpressing MYC3 and MYC4 had higher levels of anthocyanin compared to the wild-type plants. In addition, roots of MYC3 overexpression plants were hypersensitive to JA. Quantitative real-time RT-PCR expression analysis of nine JA-responsive genes revealed that eight of them were induced in MYC3 and MYC4 overexpression plants, except for a pathogen-responsive gene, PDF1.2. Similar to MYC2, MYC4 negatively regulates expression of PDF1.2. Together, these results suggest that MYC3 and MYC4 are JAZ-interacting transcription factors that regulate JA responses.

Desarrollo de un sistema de entrenamiento médico para artroscopia de rodilla / Knee arthroscopy educational system

Se presenta el desarrollo de un prototipo de simulador para entrenamiento en los procedimientos médicos de artroscopia de rodilla. El objetivo es implementar un sistema que permita simular algunos procedimientos artroscópicos empleando un dispositivo háptico de retorno de fuerza. Para esto se construyó un modelo tridimensional de la articulación de la rodilla. Con base en imágenes de resonancia magnética, se simulan algunos de los efectos percibidos durante una artroscopia real y se implementan las escenas gráficas y hápticas que permitan contrastar las sensaciones que se perciben en un proceso artroscópico.

Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development.

The transcription factor LONG HYPOCOTYL5 (HY5) acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here, we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using a high-density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis thaliana genome, we mapped genome-wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed >3000 chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light-responsive genes and transcription factor genes. Our data thus support a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis.

Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis Cullin3-based E3 ligases.

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex that regulates a variety of biological processes. Among its eight subunits, CSN5 and CSN6 contain a characteristic MPN (for Mpr1p and Pad1p N-terminal) domain and, in Arabidopsis thaliana, are each encoded by two genes: CSN5A, CSN5B and CSN6A, CSN6B, respectively. We characterized both MPN subunits using a series of single and double mutants within each gene family. Our results indicate that although CSN6A and CSN6B retain mostly redundant functions, CSN5A and CSN5B play unequal roles in the regulation of plant development. Complete depletion of either of the two MPN members results in CSN instability and the decay of various CSN components, along with the complete loss of CUL1, CUL3, and CUL4 derubylation. Furthermore, we demonstrate that CSN interacts with CUL3, in addition to CUL1 and CUL4, and that the lack of CSN activity differentially affects the stability of those three cullins. Interestingly, we also show that optimal CUL3 activity is required to maintain the cellular pool of CSN5, through a posttranscriptional mechanism. Our data suggest the existence of reciprocal regulation between CUL3 and CSN5 accumulation. This study thus completes the genetic analysis of all CSN subunits and confirms the structural interdependence between PCI and MPN subunits in functional CSN complex formation.

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo.

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

Transfer of RPS14 and RPL5 from the mitochondrion to the nucleus in grasses.

Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an on-going phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. To better understand the strategies developed by higher plants to transfer organellar genes during evolution, we investigated the fate of the mitochondrial RPL5-RPS14 locus in grasses. While maize mitochondrial genome does not contain RPS14 and RPL5 genes, wheat mitochondrial DNA contains an intact RPL5 gene and a nonfunctional RPS14 pseudogene. RPL5 and PsiRPS14 are co-transcribed and their transcripts are edited. In wheat, the functional RPS14 gene is located in the nucleus, within the intron of the respiratory complex II iron-sulfur subunit gene (SDH2). Its organization and expression mechanisms are similar to those previously described in maize and rice, allowing us to conclude that RPS14 transfer and nuclear activation occurred before divergence of these grasses. Unexpectedly, we found evidence for a more recent RPL5 transfer to the nucleus in wheat. This nuclear wheat RPL5 acquired its targeting information by duplication of an existing targeting presequence for another mitochondrial protein, ribosomal protein L4. Thus, mitochondrial and nuclear functional RPL5 genes appear to be maintained in wheat, supporting the hypothesis that in an intermediate stage of the transfer process, both nuclear and mitochondrial functional genes coexist. Finally, we show that RPL5 has been independently transferred to the nucleus in the maize lineage and has acquired regulatory elements for its expression and a mitochondrial targeting peptide from an unknown source.

The four subunits of mitochondrial respiratory complex II are encoded by multiple nuclear genes and targeted to mitochondria in Arabidopsis thaliana.

Mitochondrial respiratory complex II contains four subunits: a flavoprotein (SDHI), an iron-sulphur subunit (SDH2) and two membrane anchor subunits (SDH3 and SDH4). We have found that in Arabidopsis thaliana SDH I and SDH3 are encoded by two, and SDH4 by one nuclear genes, respectively. All these encoded polypeptides are found to be imported into isolated plant mitochondria. While both SDHI proteins are highly conserved when compared to their counterparts in other organisms, SDH3 and SDH4 share little similarity with non-plant homologues. Expression of SDH1-1, SDH3 and SDH4 genes was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the second SDH1 gene (SDH1-2) is expressed at a low level.