Improved super-resolution ribosome profiling reveals prevalent translation of upstream ORFs and small ORFs in Arabidopsis.

A crucial step in functional genomics is identifying actively translated ORFs and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed noncoding RNAs. Proteomic data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of trans-acting small interfering RNAs (TAS1-4) and microRNAs (pri-MIR163 and pri-MIR169) and periodic ribosome stalling supporting cotranslational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2 to 10 amino acids) and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.

The Association between Gestational Diabetes and the Microbiome: A Systematic Review and Meta-Analysis.

Gestational diabetes, affecting about 10% of pregnancies, is characterized by impaired glucose regulation and can lead to complications for health of pregnant women and their offspring. The microbiota, the resident microbes within the body, have been linked to the development of several metabolic conditions. This systematic review with meta-analysis aims to summarize the evidence on the differences in microbiota composition in pregnant women with gestational diabetes and their offspring compared to healthy pregnancies. A thorough search was conducted in the PubMed, Scopus, and Web of Science databases, and data from 21 studies were analyzed utilizing 41 meta-analyses. In the gut microbiota, Bifidobacterium and Alistipes were found to be more abundant in healthy pregnancies, while Roseburia appears to be more abundant in gestational diabetes. The heterogeneity among study findings regarding the microbiota in the meconium is considerable. The placental microbiota exhibited almost no heterogeneity, with an increased abundance of Firmicutes in the gestational diabetes group and a higher abundance of Proteobacteria in the control. The role of the microbiota in gestational diabetes is reinforced by these findings, which additionally point to the potential of microbiome-targeted therapies. To completely comprehend the interactions between gestational diabetes and the microbiome, standardizing methodologies and further research is necessary.

Pneumococcal Serotype Evolution and Burden in European Adults in the Last Decade: A Systematic Review.

Pneumococcal disease is a major cause of morbidity/mortality worldwide, and vaccination is an important measure in its prevention. Despite European children being vaccinated with pneumococcal conjugate vaccines (PCVs), pneumococcal infections are still a major cause of morbidity/mortality in adults with risk conditions and their vaccination might be an important prevention strategy. New PCVs have been approved, but information is lacking on their potential impact in European adults. In our review, we searched PubMed, MEDLINE, and Embase for studies on the additional PCV20 serotypes (concerning incidence, prevalence, disease severity, lethality, and antimicrobial resistance) in European adults, between January 2010 and April 2022, having included 118 articles and data from 33 countries. We found that these serotypes have become more prevalent in both invasive and non-invasive pneumococcal disease (IPD and NIPD), representing a significant proportion of cases (serotypes 8, 12F, 22F) and more serious disease and/or lethality (10A, 11A, 15B, 22F), showing antimicrobial resistance (11A, 15B, 33F), and/or affecting more vulnerable individuals such as the elderly, immunocompromised patients, and those with comorbidities (8, 10A, 11A, 15B, 22F). The relevance of pneumococcal adult carriers (11A, 15B, 22F, and 8) was also identified. Altogether, our data showed an increase in the additional PCV20 serotypes' prevalence, accounting for a proportion of approximately 60% of all pneumococcal isolates in IPD in European adults since 2018/2019. Data suggest that adults, as older and/or more vulnerable patients, would benefit from vaccination with higher-coverage PCVs, and that PCV20 may address an unmet medical need.

Cork Development: What Lies Within.

The cork layer present in all dicotyledonous plant species with radial growth is the result of the phellogen activity, a secondary meristem that produces phellem (cork) to the outside and phelloderm inwards. These three different tissues form the periderm, an efficient protective tissue working as a barrier against external factors such as environmental aggressions and pathogen attacks. The protective function offered by cork cells is mainly due to the abundance of suberin in their cell walls. Chemically, suberin is a complex aliphatic network of long chain fatty acids and alcohols with glycerol together with aromatic units. In most woody species growing in temperate climates, the first periderm is replaced by a new functional periderm upon a few years after being formed. One exception to this bark development can be found in cork oak (Quercus suber) which display a single periderm that grows continuously. Quercus suber stands by its thick cork layer development with continuous seasonal growth. Cork raw material has been exploited by man for centuries, especially in Portugal and Spain. Nowadays, its applications have widened vastly, from the most known product, stoppers, to purses or insulating materials used in so many industries, such as construction and car production. Research on how cork develops, and the effect environmental factors on cork oak trees is extremely important to maintain production of good-quality cork, and, by maintaining cork oak stands wealthy, we are preserving a very important ecosystem both by its biodiversity and its vital social and economic role in areas already showing a population declination.

Nitrogen Acquisition and Transport in the Ectomycorrhizal Symbiosis-Insights from the Interaction between an Oak Tree and Pisolithus tinctorius.

In temperate forests, the roots of various tree species are colonized by ectomycorrhizal fungi, which have a key role in the nitrogen nutrition of their hosts. However, not much is known about the molecular mechanisms related to nitrogen metabolism in ectomycorrhizal plants. This study aimed to evaluate the nitrogen metabolic response of oak plants when inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. The expression of candidate genes encoding proteins involved in nitrogen uptake and assimilation was investigated in ectomycorrhizal roots. We found that three oak ammonium transporters were over-expressed in root tissues after inoculation, while the expression of amino acid transporters was not modified, suggesting that inorganic nitrogen is the main form of nitrogen transferred by the symbiotic fungus into the roots of the host plant. Analysis by heterologous complementation of a yeast mutant defective in ammonium uptake and GFP subcellular protein localization clearly confirmed that two of these genes encode functional ammonium transporters. Structural similarities between the proteins encoded by these ectomycorrhizal upregulated ammonium transporters, and a well-characterized ammonium transporter from E. coli, suggest a similar transport mechanism, involving deprotonation of NH4+, followed by diffusion of uncharged NH3 into the cytosol. This view is supported by the lack of induction of NH4+ detoxifying mechanisms, such as the GS/GOGAT pathway, in the oak mycorrhizal roots.

Erratum: Velada et al. Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA. Biology 2021, 10, 209.

The author wishes to make an erratum to the published version of the paper [...].

Neonatal Atypical Hemolytic Uremic Syndrome in the Eculizumab Era.

The atypical hemolytic uremic syndrome (aHUS) in the newborn is a rare disease, with high morbidity. Eculizumab, considered a first-line drug in older children, is not approved in neonates and in children weighing less than 5 kg. We present a 5-day-old female newborn, born at 36 weeks' twin gestation, by emergency cesarean section due to cord prolapse, with birth weight of 2,035 g and Apgar score of 7/7/7, who develops microangiopathic hemolytic anemia, thrombocytopenia, and progressive acute renal failure. In day 5, after diagnosis of aHUS, a daily infusion of fresh frozen plasma begins, with improvement of thrombocytopenia and very slight improvement in renal function. The etiologic study (congenital infection, Shiga toxin, ADAMTS13 activity, directed metabolic study) was normal. C3c was slightly decreased. On day 16 for maintenance of anemia and severe renal failure, she started 300 mg/dose eculizumab. Anemia resolves in 10 weeks and creatinine has normal values after 13 weeks of treatment. The genetic study was normal. In this case, eculizumab is effective in controlling microangiopathy and in the recovery of renal function. Diagnosis of neonatal aHUS can be challenging because of phenotypic heterogeneity and potential overlap with other manifestations that may confound it, such as perinatal asphyxia or sepsis/disseminated intravascular coagulation.

Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA.

Higher plants are composed of different tissue and cell types. Distinct cells host different biochemical and physiological processes which is reflected in differences in gene expression profiles, protein and metabolite levels. When omics are to be carried out, the information provided by a specific cell type can be diluted and/or masked when using a mixture of distinct cells. Thus, studies performed at the cell- and tissue-type level are gaining increasing interest. Laser microdissection (LM) technology has been used to isolate specific tissue and cell types. However, this technology faces some challenges depending on the plant species and tissue type under analysis. Here, we show for the first time a LM protocol that proved to be efficient for harvesting specific tissue types (phloem, cortex and epidermis) from olive stem nodal segments and obtaining RNA of high quality. This is important for future transcriptomic studies to identify rooting-competent cells. Here, nodal segments were flash-frozen in liquid nitrogen-cooled isopentane and cryosectioned. Albeit the lack of any fixatives used to preserve samples' anatomy, cryosectioned sections showed tissues with high morphological integrity which was comparable with that obtained with the paraffin-embedding method. Cells from the phloem, cortex and epidermis could be easily distinguished and efficiently harvested by LM. Total RNA isolated from these tissues exhibited high quality with RNA Quality Numbers (determined by a Fragment Analyzer System) ranging between 8.1 and 9.9. This work presents a simple, rapid and efficient LM procedure for harvesting specific tissue types of olive stems and obtaining high-quality RNA.

Distinct Responses to Light in Plants.

The development of almost every living organism is, to some extent, regulated by light. When discussing light regulation on biological systems, one is referring to the sun that has long been positioned in the center of the solar system. Through light regulation, all life forms have evolved around the presence of the sun. As soon our planet started to develop an atmospheric shield against most of the detrimental solar UV rays, life invaded land, and in the presence of water, it thrived. Especially for plants, light (solar radiation) is the source of energy that controls a high number of developmental aspects of growth, a process called photomorphogenesis. Once hypocotyls reach soil's surface, its elongation deaccelerates, and the photosynthetic apparatus is established for an autotrophic growth due to the presence of light. Plants can sense light intensities, light quality, light direction, and light duration through photoreceptors that accurately detect alterations in the spectral composition (UV-B to far-red) and are located throughout the plant. The most well-known mechanism promoted by light occurring on plants is photosynthesis, which converts light energy into carbohydrates. Plants also use light to signal the beginning/end of key developmental processes such as the transition to flowering and dormancy. These two processes are particularly important for plant´s yield, since transition to flowering reduces the duration of the vegetative stage, and for plants growing under temperate or boreal climates, dormancy leads to a complete growth arrest. Understanding how light affects these processes enables plant breeders to produce crops which are able to retard the transition to flowering and avoid dormancy, increasing the yield of the plant.

Phellem Cell-Wall Components Are Discriminants of Cork Quality in Quercus suber.

Cork is a renewable, non-wood high valued forest product, with relevant ecological and economic impact in the Mediterranean-type ecosystems. Currently, cork is ranked according to its commercial quality. The most valuable planks are chosen for cork stoppers production. Cork planks with adequate thickness and porosity are classified as stoppable quality cork (SQC). The chemical composition of cork is known, but the regulation of metabolic pathways responsible of cork production and composition, hence of cork quality, is largely unknown. Here, we tested the hypothesis that post-genomic events may be responsible for the development of SQC and N-SQC (non-stoppable quality cork). Here, we show that combined proteomics and targeted metabolomics (namely soluble and cell wall bound phenolics) analyzed on recently formed phellem allows discriminate cork planks of different quality. Phellem cells of SQC and N-SQC displayed different reducing capacity, with consequential impact on both enzymatic pathways (e.g., glycolysis) and other cellular functions, including cell wall assembly and suberization. Glycolysis and respiration related proteins were abundant in both cork quality groups, whereas the level of several proteins associated to mitochondrial metabolism was higher in N-SQC. The soluble and cell wall-bound phenolics in recently formed phellem clearly discriminated SQC from N-SCQ. In our study, SQC was characterized by a high incorporation of aromatic components of the phenylpropanoid pathway in the cell wall, together with a lower content of hydrolysable tannins. Here, we propose that the level of hydrolysable tannins may represent a valuable diagnostic tool for screening recently formed phellem, and used as a proxy for the quality grade of cork plank produced by each tree.

Characterization of the cork formation and production transcriptome in Quercus cerris × suber hybrids.

Cork oak is the main cork-producing species worldwide, and plays a significant economic, ecological and social role in the Mediterranean countries, in particular in Portugal and Spain. The ability to produce cork is limited to a few species, hence it must involve specific regulation mechanisms that are unique to these species. However, to date, these mechanisms remain largely understudied, especially with approaches involving the use of high-throughput sequencing technology. In this study, the transcriptome of cork-producing and non-cork-producing Quercus cerris × suber hybrids was analyzed in order to elucidate the differences between the two groups of trees displaying contrasting phenotypes for cork production. The results revealed the presence of a significant number of genes exclusively associated with cork production, in the trees that developed cork. Moreover, several gene ontology subcategories, such as cell wall biogenesis, lipid metabolic processes, metal ion binding and apoplast/cell wall, were only detected in the trees with cork production. These results indicate the existence, at the transcriptome level, of mechanisms that seem to be unique and necessary for cork production, which is an advancement in our knowledge regarding the genetic regulation behind cork formation and production.

Transcriptional profiling of cork oak phellogenic cells isolated by laser microdissection.

MAIN CONCLUSION: The phenylpropanoid pathway impacts the cork quality development. In cork of bad quality, the flavonoid route is favored, whereas in good quality, cork lignin and suberin production prevails. Cork oaks develop a thick cork tissue as a protective shield that results of the continuous activity of a secondary meristem, the cork cambium, or phellogen. Most studies applied to developmental processes do not consider the cell types from which the samples were extracted. Here, laser microdissection (LM) coupled with transcript profiling using RNA sequencing (454 pyrosequencing) was applied to phellogen cells of trees producing low- and good quality cork. Functional annotation and functional enrichment analyses showed that stress-related genes are enriched in samples extracted from trees producing good quality cork (GQC). This process is under tight transcriptional (transcription factors, kinases) regulation and also hormonal control involving ABA, ethylene, and auxins. The phellogen cells collected from trees producing bad quality cork (BQC) show a consistent up-regulation of genes belonging to the flavonoid pathway as a response to stress. They also display a different modulation of cell wall genes resulting into a thinner cork layer, i.e., less meristematic activity. Based on the analysis of the phenylpropanoid pathway regulating genes, in GQC, the synthesis of lignin and suberin is promoted, whereas in BQC, the same pathway favors the biosynthesis of free phenolic compounds. This study provided new insights of how cell-specific gene expression can determine tissue and organ morphology and physiology and identified robust candidate genes that can be used in breeding programs aiming at improving cork quality.

Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses.

GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (Vitis vinifera L.), an economically important woody crop. We performed a model curation of GRAS genes identified in the latest genome annotation leading to the identification of 52 genes. Gene models were improved and three new genes were identified that could be grapevine- or woody-plant specific. Phylogenetic analysis showed that GRAS genes could be classified into 13 groups that mapped on the 19 V. vinifera chromosomes. Five new subfamilies, previously not characterized in other species, were identified. Multiple sequence alignment showed typical GRAS domain in the proteins and new motifs were also described. As observed in other species, both segmental and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family in grapevine. Expression patterns across a variety of tissues and upon abiotic and biotic conditions revealed possible divergent functions of GRAS genes in grapevine development and stress responses. By comparing the information available for tomato and grapevine GRAS genes, we identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming at improving the quality of fleshy fruits.

Complex Interplay of Hormonal Signals during Grape Berry Ripening.

Grape and wine production and quality is extremely dependent on the fruit ripening process. Sensory and nutritional characteristics are important aspects for consumers and their development during fruit ripening involves complex hormonal control. In this review, we explored data already published on grape ripening and compared it with the hormonal regulation of ripening of other climacteric and non-climacteric fruits. The roles of abscisic acid, ethylene, and brassinosteroids as promoters of ripening are discussed, as well as the role of auxins, cytokinins, gibberellins, jasmonates, and polyamines as inhibitors of ripening. In particular, the recently described role of polyamine catabolism in grape ripening is discussed, together with its putative interaction with other hormones. Furthermore, other recent examples of cross-talk among the different hormones are presented, revealing a complex interplay of signals during grape development and ripening.

Comparison of good- and bad-quality cork: application of high-throughput sequencing of phellogenic tissue.

Cork is one of the most valuable non-wood forest products and plays an important role in Mediterranean economies. The production of high-quality cork is dependent on both genome and environment, posing constraints on the industry because an ever-growing amount of bad-quality cork (BQC) development has been observed. In order to identify genes responsible for production of cork of superior quality we performed a comparative analysis using the 454 pyrosequencing approach on phellogenic tissue of good- and bad-quality samples. The transcriptional profiling showed a high number of genes differentially expressed (8.48%) from which 78.8% displayed annotation. Genes more highly represented in BQC are involved in DNA synthesis, RNA processing, proteolysis, and transcription factors related to the abiotic stress response. Putative stomatal/lenticular-associated genes which may be responsible for the disadvantageous higher number of lenticular channels in BQC are also more highly represented. BQC also showed an elevated content of free phenolics. On the other hand, good-quality cork (GQC) can be distinguished by highly expressed genes encoding heat-shock proteins. Together the results provide valuable new information about the molecular events leading to cork formation and provide putative biomarkers associated with cork quality that can be useful in breeding programmes.

Anatomy and development of the endodermis and phellem of Quercus suber L. roots.

Quercus suber L. has been investigated with special attention to the stem bark and its cork formation layer, but excluding the roots. Roots are the location of infection by pathogens such as Phytophthora cinnamomi responsible for the tree's sudden death. It is widely accepted that suberin establishes boundaries within tissues, serves as a barrier against free water and ion passage, and works as a shield against pathogen attacks. We followed the suberization of young secondary roots of cork oak. The first suberin deposition detectable by transmission electron microscopy (TEM) and neutral red (NR) was in the endoderm Casparian strips. Casparian strips are not detected by Sudan red 7B and Fluorol yellow (FY) that specifically stain lamellae suberin. Reaction to Sudan was verified in the endodermis and later on in phellem cells that resulted from the phellogen. Under TEM, the Sudan and FY-stained cells showed clear suberin lamellae while the newer formed phellem cells displayed a distinct NR signal compared to the outermost phellem cells. We concluded that suberin chemical components are arranged differently in the cell wall according to the physiological role or maturation stage of a given tissue.

Suberized cell walls of cork from cork oak differ from other species.

Plants have suberized cells that act as protective interfaces with the environment or between different plant tissues. A lamellar structure of alternating dark and light bands has been found upon transmission electron microscopy (TEM) observation of cork cells and considered a typical feature of the suberized secondary wall. We observed cork cells from periderms of Quercus suber, Quercus cerris, Solanum tuberosum, and Calotropis procera by TEM after uranyl acetate and lead citrate staining. A lamellated structure was observed in S. tuberosum and C. procera but not in Q. suber and Q. cerris where the suberized cell wall showed a predominantly hyaline aspect with only a dark dotted staining. Removal of suberin from Q. suber cells left a thinner secondary wall that lost the translucent aspect. We hypothesize that the species' specific chemical composition of suberin will result in different three-dimensional macromolecular development and in a different spatial location of lignin and other aromatics. A lamellated ultrastructure is therefore not a general feature of suberized cells.

Ultrastructural observations reveal the presence of channels between cork cells.

The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

Adaptabilidade e estabilidade de genótipos de soja avaliados para resistência ao oídio / Adaptability and stability of soybean genotypes evaluated for resistance to powdery mildew

O objetivo desta pesquisa foi identificar genótipos de soja com adaptabilidade e estabilidade de comportamento para resistência ao oídio em condições de campo. O trabalho foi realizado no Campo Experimental Professor Diogo Alves de Mello, do Departamento de Fitotecnia, da Universidade Federal de Viçosa (UFV), em Viçosa, Minas Gerais. O delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições, disposto em parcelas subdivididas, com 15 genótipos (parcelas) e sete épocas de avaliação (subparcela). A avaliação da incidência e severidade do oídio nos genótipos foi realizada por meio da quantificação visual do nível de infecção (NI) provocado pelo oídio, sendo avaliados o nível de infecção da área foliar infectada pelo oídio (NIAFI) e o nível de infecção do folíolo mais infectado pelo oídio (NIFI). Foram realizadas a análise de variância e análise de adaptabilidade e estabilidade conforme EBERHART & RUSSELL (1966). Os resultados permitiram tirar as seguintes conclusões: os genótipos que apresentaram os melhores níveis de resistência, em geral, também foram os de melhor adaptabilidade e estabilidade, tanto para NIAFI quanto para NIFI, com destaque para: UFV 89-361826 T2, UFV 94-334268, UFV-16 e UFV-19, FT-10 RC5 (F3), FT-Abyara RC6 (F2), Doko RC, FT-Abyara RC5 (F4), enquanto que os mais suscetíveis foram os de pior previsibilidade (estabilidade) de comportamento, com destaque para UFV 94-5126, FT-104, UFV 94-3500, FT-Cristalina, FT-Estrela e BR-16.

The objective of this research was to identify soybean genotypes with good adaptability and stability of behavior for resistance to powdery mildew in field conditions. The work was carried out at the Professor Diogo Alves de Mello Experimental Station, Agronomy Department of the Federal University of Viçosa (UFV), Viçosa, Minas Gerais. The experiment was arranged in a randomized block design, in split-plots, with 15 genotypes (plots) and seven evaluation times (subplots), with four repetitions. Evaluation of incidence and severity of powdery mildew in the genotypes were accomplished by visual quantification of fungus infection level (IL), assessing the level of infection of the leaf area infected (NIAFI) by the powdery mildew and the level of infection of the most infected leaflet (NIFI). Data were examined by analysis of variance and analysis of adaptability and stability according to EBERHART & RUSSELL (1966). The results allowed to get to the following conclusions: the genotypes that showed the highest resistance levels, were, in general, also the ones of higher adaptability and stability, both for NIAFI and NIFI, standing out UFV 89-361826 T2, UFV 94-334268, UFV-16 and UFV-19, FT-10 RC5 (F3), FT-Abyara RC6 (F2), Doko RC, FT-Abyara RC5 (F4), whereas the most susceptible genotypes were the ones of worse predictability, standing out UFV 94-5126, FT-104, UFV 94-3500, FT-Cristalina, FT-Estrela and BR-16.