Integrative analysis of green ash phloem transcripts and proteins during an emerald ash borer infestation.

BACKGROUND: Emerald ash borer (Agrilus planipennis; EAB) is an Asian insect species that has been invasive to North America for 20 years. During this time, the emerald ash borer has killed tens of millions of American ash (Fraxinus spp) trees. Understanding the inherent defenses of susceptible American ash trees will provide information to breed new resistant varieties of ash trees. RESULTS: We have performed RNA-seq on naturally infested green ash (F. pennsylvanica) trees at low, medium and high levels of increasing EAB infestation and proteomics on low and high levels of EAB infestation. Most significant transcript changes we detected occurred between the comparison of medium and high levels of EAB infestation, indicating that the tree is not responding to EAB until it is highly infested. Our integrative analysis of the RNA-Seq and proteomics data identified 14 proteins and 4 transcripts that contribute most to the difference between highly infested and low infested trees. CONCLUSIONS: The putative functions of these transcripts and proteins suggests roles of phenylpropanoid biosynthesis and oxidation, chitinase activity, pectinesterase activity, strigolactone signaling, and protein turnover.

Salas de Conversa: atividade integrativa de mentoria no contexto da Covid-19 / Chat Rooms: integrative mentoring activity in the context of Covid-19

Resumo: Introdução: O Programa de Mentoria da Universidade Federal do Rio Grande do Norte (UFRN) busca promover o desenvolvimento pessoal e profissional do aluno durante seu período acadêmico. Por meio do método da escuta e do diálogo entre mentores e mentorados, a atividade de mentoria propicia o desenvolvimento da empatia e de outras habilidades emocionais importantes para uma prática médica humanizada e o bom convívio profissional. Busca também trazer para reflexão temas relativos à ética médica, à inserção do médico na comunidade, entre outros. Dentro dessa proposta, no contexto da pandemia do novo coronavírus (Covid-19), o programa passou a desenvolver, além dos encontros on-line regulares, uma atividade integrativa intitulada "Salas de Conversa". Relato de experiência: Entre junho e dezembro de 2020, realizaram-se, mensalmente, seis salas de conversa com temas diversos. Os encontros ocorreram em salas virtuais e contaram com a participação de todos os grupos de mentoria e mentores cadastrados no programa, além de convidados que estiveram à frente das discussões. Discussão: O retorno positivo de discentes e docentes mostrou o potencial dessa modalidade de interação de complementar os encontros já desenvolvidos no Programa de Mentoria. A interação virtual apresentou entraves por limitações tecnológicas, todos contornados em tempo hábil, sem prejuízo dessa nova forma de experiência. Conclusão: As Salas de Conversa mostraram-se eficazes na superação das dificuldades de interação impostas pelo distanciamento físico da pandemia de Covid-19. Essa atividade abordou temáticas atuais e pertinentes não apenas à vida do estudante de Medicina, mas também à vida em sociedade, de modo a contribuir tanto para a formação médica quanto para a formação pessoal dos alunos e professores.

Abstract: Introduction: The Mentoring Program of the Federal University of Rio Grande do Norte seeks to promote the student's personal and professional development during his academic training. Through the method of listening and dialogue between mentors and mentees, the mentoring activity promotes the development of empathy and other emotional skills important for a humanized medical practice and good professional life. It also seeks to bring up themes related to medical ethics, insertion of the doctor in the community, among others. Within this proposal, and against the backdrop of the new coronavirus pandemic, an integrative activity entitled "Chat Rooms" was implemented in addition to the regular online meetings. Experience report: From June to December 2020, six chat rooms with different themes were held monthly. The meetings were held in virtual rooms for all mentoring groups and mentors registered on the program, in addition to guests who were at the forefront of the discussions. Discussion: The positive feedback from students and mentors showed the potential of this type of interaction to complement the meetings already developed in the mentoring program. The virtual interaction presented obstacles due to technological limitations, all of which were overcome in a timely manner, without detriment to this new form of experience. Conclusion: The Chat Rooms proved to be effective in resolving the interaction difficulties imposed by the social distancing measures enforced by the Covid-19 pandemic. Bringing current and pertinent themes not only to the medical student's life but to life in the wider society, they contributed both to the medical training and personal training of students and mentors.

The large repertoire of conifer NLR resistance genes includes drought responsive and highly diversified RNLs.

The NLRs or NBS-LRRs (nucleotide-binding, leucine-rich-repeat) form the largest resistance gene family in plants, with lineage-specific contingents of TNL, CNL and RNL subfamilies and a central role in resilience to stress. The origin, evolution and distribution of NLR sequences has been unclear owing in part to the variable size and diversity of the RNL subfamily and a lack of data in Gymnosperms. We developed, searched and annotated transcriptomes assemblies of seven conifers and identified a resource of 3816 expressed NLR sequences. Our analyses encompassed sequences data spanning the major groups of land plants and determinations of NLR transcripts levels in response to drought in white spruce. We showed that conifers have among the most diverse and numerous RNLs in tested land plants. We report an evolutionary swap in the formation of RNLs, which emerged from the fusion of an RPW8 domain to a NB-ARC domain of CNL. We uncovered a quantitative relationship between RNLs and TNLs across all land plants investigated, with an average ratio of 1:10. The conifer RNL repertoire harbours four distinct groups, with two that differ from Angiosperms, one of which contained several upregulated sequences in response to drought while the majority of responsive NLRs are downregulated.

Functional Analysis of the PgCesA3 White Spruce Cellulose Synthase Gene Promoter in Secondary Xylem.

Cellulose is an essential structural component of the plant cell wall. Its biosynthesis involves genes encoding cellulose synthase enzymes and a complex transcriptional regulatory network. Three cellulose synthases have been identified in conifers as being potentially involved in secondary cell wall biosynthesis because of their preferential expression in xylem tissues; however, no direct functional association has been made to date. In the present work, we characterized the white spruce [Picea glauca (Moench) Voss] cellulose synthase PgCesA3 gene and 5' regulatory elements. Phylogenetic analysis showed that PgCesA1-3 genes grouped with secondary cell wall-associated Arabidopsis cellulose synthase genes, such as AtCesA8, AtCesA4, and AtCesA7. We produced transgenic spruce expressing the GUS reporter gene driven by the PgCesA3 promoter. We observed blue staining in differentiating xylem cells from stem and roots, and in foliar guard cells indicating that PgCesA3 is clearly involved in secondary cell wall biosynthesis. The promoter region sequence of PgCesA3 contained several putative MYB cis-regulatory elements including AC-I like motifs and secondary wall MYB-responsive element (SMRE); however, it lacked SMRE4, 7 and 8 that correspond to the sequences of AC-I, II, and III. Based on these findings and results of previous transient trans-activation assays that identified interactions between the PgCesA3 promoter and different MYB transcription factors, we performed electrophoretic mobility shift assays with MYB recombinant proteins and cis-regulatory elements present in the PgCesA3 promoter. We found that PgMYB12 bound to a canonical AC-I element identified in the Pinus taeda PAL promoter and two AC-I like elements. We hypothesized that the PgMYB12 could regulate PgCesA3 in roots based on previous expression results. This functional study of PgCesA3 sequences and promoter opens the door for future studies on the interaction between PgMYBs and the PgCesA3 regulatory elements.

Expansion of the dehydrin gene family in the Pinaceae is associated with considerable structural diversity and drought-responsive expression.

Temperatures are expected to increase over the next century in all terrestrial biomes and particularly in boreal forests, where drought-induced mortality has been predicted to rise. Genomics research is helping to develop hypotheses regarding the molecular basis of drought tolerance and recent work proposed that the osmo-protecting dehydrin proteins have undergone a clade-specific expansion in the Pinaceae, a major group of conifer trees. The objectives of this study were to identify all of the putative members of the gene family, trace their evolutionary origin, examine their structural diversity and test for drought-responsive expression. We identified 41 complete dehydrin coding sequences in Picea glauca, which is four times more than most angiosperms studied to date, and more than in pines. Phylogenetic reconstructions indicated that the family has undergone an expansion in conifers, with parallel evolution implicating the sporadic resurgence of certain amino acid sequence motifs, and a major duplication giving rise to a clade specific to the Pinaceae. A variety of plant dehydrin structures were identified with variable numbers of the A-, E-, S- and K-segments and an N-terminal (N1) amino acid motif including assemblages specific to conifers. The expression of several of the spruce dehydrins was tissue preferential under non-stressful conditions or responded to water stress after 7-18 days without watering, reflecting changes in osmotic potential. We found that dehydrins with N1 K2 and N1 AESK2 sequences were the most responsive to the lack of water. Together, the family expansion, drought-responsive expression and structural diversification involving loss and gain of amino acid motifs suggests that subfunctionalization has driven the diversification seen among dehydrin gene duplicates. Our findings clearly indicate that dehydrins represent a large family of candidate genes for drought tolerance in spruces and in other Pinaceae that may underpin adaptability in spatially and temporally variable environments.

Evolution of gene structure in the conifer Picea glauca: a comparative analysis of the impact of intron size.

BACKGROUND: A positive relationship between genome size and intron length is observed across eukaryotes including Angiosperms plants, indicating a co-evolution of genome size and gene structure. Conifers have very large genomes and longer introns on average than most plants, but impacts of their large genome and longer introns on gene structure has not be described. RESULTS: Gene structure was analyzed for 35 genes of Picea glauca obtained from BAC sequencing and genome assembly, including comparisons with A. thaliana, P. trichocarpa and Z. mays. We aimed to develop an understanding of impact of long introns on the structure of individual genes. The number and length of exons was well conserved among the species compared but on average, P. glauca introns were longer and genes had four times more intronic sequence than Arabidopsis, and 2 times more than poplar and maize. However, pairwise comparisons of individual genes gave variable results and not all contrasts were statistically significant. Genes generally accumulated one or a few longer introns in species with larger genomes but the position of long introns was variable between plant lineages. In P. glauca, highly expressed genes generally had more intronic sequence than tissue preferential genes. Comparisons with the Pinus taeda BACs and genome scaffolds showed a high conservation for position of long introns and for sequence of short introns. A survey of 1836 P. glauca genes obtained by sequence capture mostly containing introns <1 Kbp showed that repeated sequences were 10× more abundant in introns than in exons. CONCLUSION: Conifers have large amounts of intronic sequence per gene for seed plants due to the presence of few long introns and repetitive element sequences are ubiquitous in their introns. Results indicate a complex landscape of intron sizes and distribution across taxa and between genes with different expression profiles.

Variações espaço-temporais no estoque de sementes do solo na floresta amazônica / Space-time variations in soil seed stock in an amazon rainforest

The dispersion efficiency, longevity and the ability of seeds to remain latent waiting for suitable conditions for germination in the forest soil seed bank ensures the presence of pioneer tree species in disturbed areas. The seasonal and spatial variations in the density and floristic composition of the seed bank in tropical rainforests is a subject still little understood. This work verified the existence of spatio-temporal changes of the seed bank present in areas of humid tropical rain forest located near Manaus, Amazonas. In each of the six study areas, 40 circular samples of topsoil (10 cm in diameter and 2 cm deep) were randomly collected every two months, from August 2004 to June/2005. Inside a greenhouse, the collected soil samples were distributed in trays and the emergence of seeds present in the soil was accompanied during four months. There was a significant reduction (H: 14.09112, p <0.05) in the mean density of seeds presents in the soil in June (early dry season) compared to February (middle of the rainy season). There was also a significant difference (H: 188.7245, p <0.05) in the mean density of soil seeds present in the different samples areas. Similar to other tropical forest areas, the permanent seed bank in the forest was dominated by pioneer species, mainly from the family Melastomataceae, while typical tropical forest species were rare in the forest soil.

A dispersão eficiente, a longevidade e a capacidade das sementes de permanecer em estado latente a espera de condições adequadas de germinação no banco de sementes do solo da floresta garantem a presença de espécies arbóreas pioneiras nas áreas perturbadas. As variações estacionais e espaciais na densidade e na composição florística do banco de sementes em Florestas Tropicais Úmidas são assuntos ainda pouco compreendidos. Este trabalho verificou a existência de modificações espaço-temporais do banco de sementes presente em áreas de Floresta Tropical úmida localizadas próximas a Manaus, AM. Em cada uma das seis áreas estudadas, foram coletadas 40 amostras circulares de solo superficial (10 cm de diâmetro e 2 cm de profundidade) ao acaso. Essas amostras foram coletadas a cada dois meses, entre agosto/2004 e junho/2005,. As amostras de solo foram distribuídas em bandejas em casa de vegetação e a emergência das sementes presentes no solo foi acompanhada por 4 meses. Houve uma redução significativa (H: 14,09, p < 0,05) na densidade média de sementes no solo em junho (início da estação seca) em relação a fevereiro (meio da estação chuvosa). Houve também diferença significativa (H: 188,72, p < 0,05) na densidade média de sementes do solo presente nas diferentes áreas amostradas. Assim como para outras áreas de florestas tropicais, o banco de sementes permanente da floresta foi dominado por espécies pioneiras, principalmente da família Melastomataceae, enquanto as espécies típicas da Floresta Tropical madura foram raras no solo florestal.

The Norway spruce genome sequence and conifer genome evolution.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Efeito da calagem e da correção dos teores de Ca e Mg do solo sobre o crescimento de mudas de angelim-pedra (Dinizia excelsa Ducke) / Liming effect and correction of the Ca and Mg contents of the soil on angelim-pedra (Dinizia excelsa Ducke) seedling growth

O conhecimento dos requerimentos nutricionais das espécies e de suas respostas à correção do substrato é fundamental para a produção de mudas de qualidade. O objetivo deste trabalho foi avaliar o efeito de diferentes tipos de calcário e da correção da deficiência de Ca e Mg sobre o desenvolvimento de mudas de angelim-pedra (D. excelsa) em casa de vegetação, utilizando-se como substrato Latossolo Amarelo. Foram testados três tipos de calcário e fornecimento de Ca e Mg por meio de fontes não-corretivas da acidez em três relações Ca:Mg. Os tratamentos consistiram de T0 & testemunha; T1 & calcário dolomítico; T2 & calcário magnesiano; T3 & calcário calcítico; T4 & Ca e Mg na relação 3:1; T5 & Ca e Mg na relação 9:1; e T6 & Ca e Mg na relação 15:1. O delineamento experimental foi de blocos ao acaso, com cinco repetições, totalizando 35 parcelas, cada uma delas com 3 mudas. O substrato foi adubado com doses equivalentes a 100-250-150 e 15 kg ha-1 de N, P2O5, K2O e S, respectivamente e com solução de micronutrientes (3 mL de Chelamix L-1 de água destilada). Foram avaliadas: a altura da planta, o diâmetro do colo, matéria seca da parte aérea, matéria seca da raiz, matéria seca total, relação raiz/parte aérea e conteúdos de nutrientes da parte aérea das plantas. De modo geral, a aplicação de calcário favoreceu o crescimento de D. excelsa, sendo os melhores resultados obtidos com a aplicação de calcário magnesiano na relação 9:1.

Knowledge of nutritional requirements of the species and their answers to correcting the substratum is fundamental for the production of quality seedlings. In this work, we aimed to evaluate the effect of different limestone types and the correction of Ca and Mg deficiency on the development of angelim-pedra (Dinizia excelsa) seedlings, under glasshouse conditions. We tested three types of limestone and three types of Ca:Mg provisioning on a Yellow Oxisol (with non-corrective acidity sources): T0 & control; T1 & soil limed with dolomitic lime (3:1); T2 & soil limed with magnesium lime (9:1); T3 & soil limed with calcitic lime (15:1); T4 & a mixture of calcium and magnesium, in the 3:1 proportion; T5 & calcium and magnesium, in the 9:1 proportion; and T6 & calcium and magnesium, in the 15:1 proportion. The experimental design was of random blocks, with 5 repetitions, totaling 35 parcels with 3 seedlings each. The substratum was fertilized with doses equivalent to 100-250-150 and 15 k ha-1 of N, P2O5, K2O and S respectively, and micronutrient solution (3 mL of Chelamix L-1 of distilled water). We evaluated the height, diameter of the stem, dry biomass of the aerial part, dry biomass of the root, dry biomass of the drought, aerial part/root relation and nutrient contents of the aerial part of the plants. The limestone application affected positively the height, the diameter, the matter total biomass and the absorption of Ca of the D. excelsa seedlings (p<0,05). The application of magnesium lime with Ca:Mg 9:1 proportion showed the best results.

Automatic identification of mycobacterium tuberculosis with conventional light microscopy.

This article presents an automatic identification method of mycobacterium tuberculosis with conventional microscopy images based on Red and Green color channels using global adaptive threshold segmentation. Differing from fluorescence microscopy, in the conventional microscopy the bacilli are not easily distinguished from the background. The key to the bacilli segmentation method employed in this work is the use of Red minus Green (R-G) images from RGB color format. In this image, the bacilli appear as white regions on a dark background. Some artifacts are present in the (R-G) segmented image. To remove them we used morphological, color and size filters. The best sensitivity achieved was about 76.65%. The main contribution of this work was the proposal of the first automatic identification method of tuberculosis bacilli for conventional light microscopy.