Plant regeneration capacity in seeds of three species of Miconia (Melastomataceae) may be related to endogenous polyamine profiles.

In plant tissue culture, differences in endogenous levels of species-specific plant growth regulators (PGRs) may explain differences in regenerative capacity. In the case of polyamines (PAs), their dynamics and distribution may vary between species, genotypes, tissues, and developmental pathways, such as sexual reproduction and apomixis. In this study, for the first time, we aimed to assess the impact of varying endogenous PAs levels in seeds from distinct reproductive modes in Miconia spp. (Melastomataceae), on their in vitro regenerative capacity. We quantified the free PAs endogenous content in seeds of Miconia australis (obligate apomictic), Miconia hyemalis (facultative apomictic), and Miconia sellowiana (sexual) and evaluated their in vitro regenerative potential in WPM culture medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The morphogenic responses were characterized by light microscopy and scanning electron microscopy and discussed regarding the endogenous PAs profiles found. Seeds of M. sellowiana presented approximately eight times more putrescine than M. australis, which was associated with a higher percentage of regenerated calluses (76.67%) than M. australis (5.56%). On the other hand, spermine levels were significantly higher in M. australis. Spermine is indicated as an inhibitor of auxin-carrying gene expression, which may have contributed to its lower regenerative capacity under the tested conditions. These findings provide important insights into in vitro morphogenesis mechanisms in Miconia and highlight the significance of endogenous PA levels in plant regeneration. These discoveries can potentially optimize future regeneration protocols in Miconia, a plant group still underexplored in this area.

Salacia spp.: recent insights on biotechnological interventions and future perspectives.

The plants of the genus Salacia L. are the storehouse of several bioactive compounds, and are involved in treating human diseases and disorders. Hitherto, a number of reports have been published on in vitro biotechnology as well as microbial involvement in the improvement of Salacia spp. The present review provides comprehensive insights into biotechnological interventions such as tissue culture for plant propagation, in vitro cultures, and endophytic microbes for up-scaling the secondary metabolites and biological potential of Salacia spp. Other biotechnological interventions such as molecular markers and bio-nanomaterials for up-grading the prospective of Salacia spp. are also considered. The in vitro biotechnology of Salacia spp. is largely focused on plant regeneration, callus culture, cell suspension culture, somatic embryogenesis, and subsequent ex vitro establishment of the in vitro-raised plantlets. The compiled information on tissue cultural strategies, involvement of endophytes, molecular markers, and nanomaterials will assist the advanced research related to in vitro manipulation, domestication, and commercial cultivation of elite clones of Salacia spp. Moreover, the genetic diversity and other molecular-marker based assessments will aid in designing conservation policies as well as support upgrading and breeding initiatives for Salacia spp. KEY POINTS: • Salacia spp. plays a multifaceted role in human health and disease management. • Critical and updated assessment of tissue culture, endophytic microbes, metabolites, molecular markers, and bio-nanomaterials of Salacia spp. • Key shortcomings and future research directions for Salacia biotechnology.

Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

Age affects the production of secondary metabolites, but how developmental cues regulate secondary metabolism remains poorly understood. The achiote tree (Bixa orellana L.) is a source of bixin, an apocarotenoid used in diverse industries worldwide. Understanding how age-dependent mechanisms control bixin biosynthesis is of great interest for plant biology and for economic reasons. Here we overexpressed miRNA156 (miR156) in B. orellana to comprehensively study the effects of the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module on age-dependent bixin biosynthesis in leaves. Overexpression of miR156 in annatto plants (miR156ox) reduced BoSPL transcript levels, impacted leaf ontogeny, lessened bixin production, and increased abscisic acid levels. Modulation of expression of BoCCD4-4 and BoCCD1, key genes in carotenoid biosynthesis, was associated with diverting the carbon flux from bixin to abscisic acid in miR156ox leaves. Proteomic analyses revealed an overall low accumulation of most secondary metabolite-related enzymes in miR156ox leaves, suggesting that miR156-targeted BoSPLs may be required to activate several secondary metabolic pathways. Our findings suggest that the conserved BomiR156-BoSPL module is deployed to regulate leaf dynamics of bixin biosynthesis, and may create novel opportunities to fine-tune bixin output in B. orellana breeding programs.

Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 µM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

In vitro micropropagation and tiliroside production in Paratecoma peroba (Record) Kuhlm, an endemic and endangered Brazilian tree.

In vitro tissue culture can be an alternative method for endangered species propagation, biodiversity conservation and secondary metabolite studies. Paratecoma peroba (Record) Kuhlm. (Bignoniaceae) is an endemic and endangered Brazilian species. This work aimed to establish in vitro morphogenesis and callus induction and to perform a phytochemical analysis of P. peroba callus extract. Higher seed germination (43%) was obtained in Wood Plant Medium culture without activated charcoal (AC). Combination of 5 µM benzyladenine + 10 µM gibberellic acid, without AC, resulted in a higher number of shoots (2 shoots/explant). A callus culture was stabilised from zygotic embryos using 2,4-dichlorophenoxyacetic acid. A callus methanolic extract was used for phytochemical analysis. The isolated substance was identified as tiliroside (kaempferol 3-O-ß-D-(6''-O-E-p-coumaroyl)-glucopyranoside) by NMR and quantified in callus and leaf extracts by HPLC. This study adds to the chemical knowledge of this species and it is the first report of a flavonol in Paratecoma.

Exploring genetic diversity in inbred papaya lines for fruit quality in advanced stage of improvement.

Despite the relevance of the global scenario regarding the papaya (Carica papaya L.) trade, there is still a limited number of papaya cultivars with different fruit patterns. Therefore, it is essential to explore the genetic variability at all levels of the germplasm used in the development of new papaya cultivars to meet its marketing goal. Thus, this study measured and explored the potential of genetic variability based on related to fruit quality traits, of a population of papaya lines in the F5 generation through several statistical analyzes. For this, 97 inbred lines obtained using the Single Seed Descent method, resulting from a cross between the JS-12 and Sekati genotypes, both with Formosa fruit pattern, were evaluated. Results indicated there was genetic variability in the fruit quality. The traits that most contributed to the variability were related to the fruit shape. The diverse population of 97 inbred papaya lines in the F5 generation showed promise for producing commercial-sized fruits in Formosa, Intermediate, and Solo patterns. Additionally, the selection of inbred papaya lines based on fruit shape using morpho-anatomical traits does not compromise physical and chemical parameters related to fruit quality.

Omics analyses in citrus reveal a possible role of RNA translation pathways and Unfolded Protein Response regulators in the tolerance to combined drought, high irradiance, and heat stress.

Environmental changes derived from global warming and human activities increase the intensity and frequency of stressful conditions for plants. Multiple abiotic factors acting simultaneously enhance stress pressure and drastically reduce plant growth, yield, and survival. Stress combination causes a specific stress situation that induces a particular plant response different to the sum of responses to the individual stresses. Here, by comparing transcriptomic and proteomic profiles to different abiotic stress combinations in two citrus genotypes, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) and Cleopatra mandarin (Citrus reshni), with contrasting tolerance to different abiotic stresses, we revealed key responses to the triple combination of heat stress, high irradiance and drought. The specific transcriptomic response to this stress combination in Carrizo was directed to regulate RNA metabolic pathways and translation processes, potentially conferring an advantage with respect to Cleopatra. In addition, we found endoplasmic reticulum stress response as common to all individual and combined stress conditions in both genotypes and identified the accumulation of specific groups of heat shock proteins (HSPs), such as small HSPs and HSP70s, and regulators of the unfolded protein response, BiP2 and PDIL2-2, as possible factors involved in citrus tolerance to triple stress combination. Taken together, our findings provide new insights into the acclimation process of citrus plants to multiple stress combination, necessary for increasing crop tolerance to the changing climatic conditions.

Intra-genotypic variability in elite parent lines of papaya.

This study aimed to characterize papaya lines via microsatellite markers, and select genotypes based on the fixation index in order to promote the genetic purification of important commercial hybrids parent lines. Overall, 400 genotypes from three parental lines (JS-12, SS-72/12, and Sekati) were genotyped. Expected (HE), observed (HO) heterozygosity, and fixation index (F), were estimated. Genetic distances were estimated using an unweighted index, which was graphically presented via cluster analysis using the UPGMA and PCoA. Intra-genotypic variability was detected in both JS-12 and Sekati lines, while an absence of it was observed in SS-72/12. Such variability may positively contribute to the fitting of 'UENF/Caliman 01' and 'UC-10' hybrids into the traits of commercial interest how size and weight fruit. Regarding the fixation index, 293 genotypes showed maximum values (F=1) facilitating the genotypes selection process. Concerning population analysis, a close proximity between heterotic group 'Formosa' lines was observed, while a greater distance among 'Solo' group ones, and this enables systematic exploitation of such material. The fixation index maximum enabled the 80 genotypes selection thereby contributing to the parents genetic purification, since, the selected genotypes will be used in future hybridization steps to generate hybrids fitted into the traits of commercial interest.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

Reduction of heat stress pressure and activation of photosystem II repairing system are crucial for citrus tolerance to multiple abiotic stress combination.

Drought, heat and high irradiance are abiotic stresses that negatively affect plant development and reduce crop productivity. The confluence of these three factors is common in nature, causing extreme situations for plants that compromise their viability. Drought and heat stresses increase the saturation of the photosystem reaction centers, increasing sensitivity to high irradiance. In addition, these stress conditions affect photosystem II (PSII) integrity, alter redox balance of the electron transport chain and decrease the photosynthetic rate. Here, we studied the effect of the stress combinations on the photosynthetic apparatus of two citrus genotypes, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) and Cleopatra mandarin (Citrus reshni). Results obtained showed that physiological responses, such as modulation of stomatal aperture and transpiration rate, aimed to reduce leaf temperature, are key to diminishing heat impact on photosynthetic apparatus and increasing tolerance to double and triple combinations of drought, high irradiance and high temperatures. By using transcriptomic and proteomic analyses, we have demonstrated that under these abiotic stress combinations, Carrizo plants were able to increase expression of genes and proteins related to the photosystem repairing machinery (which better maintained the integrity of PSII) and other components of the photosynthetic apparatus. Our findings reveal crucial physiological and genetic responses in citrus to increase tolerance to the combination of multiple abiotic stresses that could be the basis for breeding programs that ensure a sustainable citrus production.

Somatic Embryogenesis in Sugarcane (Saccharum spp.).

Somatic embryogenesis is the process by which embryos are formed from a single or small group of somatic cells in response to specific stimuli. Somatic embryogenesis has been applied to achieve mass clonal propagation on an industrial scale and to increase the agronomic performance of species of economic interest, including sugarcane. The use of somatic embryogenesis in sugarcane stands out as a biotechnological tool with a high potential for application in the clonal propagation of disease-free elite varieties, as an essential part of genetic transformation protocols, and in the production of synthetic seeds. A better understanding of each phase of somatic embryogenesis can help to optimize the process to enhance yields and produce high-quality emblings. In this chapter, we describe a detailed protocol for somatic embryogenesis in sugarcane (Saccharum sp.) to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment of sugarcane emblings.

Integrative proteomics and phosphoproteomics reveals phosphorylation networks involved in the maintenance and expression of embryogenic competence in sugarcane callus.

Plant embryogenic cell culture allows mass propagation and genetic manipulation, but the mechanisms that determine the fate of these totipotent cells in somatic embryos have not yet been elucidated. Here, we performed label-free quantitative proteomics and phosphoproteomics analyses to determine signaling events related to sugarcane somatic embryo differentiation, especially those related to protein phosphorylation. Embryogenic calli were compared at multiplication (EC0, dedifferentiated cells) and after 14 days of maturation (EC14, onset of embryo differentiation). Metabolic pathway analysis showed enriched lysine degradation and starch/sucrose metabolism proteins during multiplication, whereas the differentiation of somatic embryos was found to involve the enrichment of energy metabolism, including the TCA cycle and oxidative phosphorylation. Multiplication-related phosphoproteins were associated with transcriptional regulation, including SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH). The regulation of multiple light harvesting complex photosystem II proteins and phytochrome interacting factor 3-LIKE 5 were predicted to promote bioenergetic metabolism and carbon fixation during the maturation stage. A motif analysis revealed 15 phosphorylation motifs. The [D-pS/T-x-D] motif was overrepresented during somatic embryo differentiation. A protein-protein network analysis predicted interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid-responsive element-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence. The predicted interactions between TOPLESS (TPL) and histone deacetylase 19 (HD19) may be involved in posttranslational protein regulation during somatic embryo differentiation. These results reveal the protein regulation dynamics of somatic embryogenesis and new players in somatic embryo differentiation, including their predicted phosphorylation motifs and phosphosites.

Citrus rootstocks modify scion antioxidant system under drought and heat stress combination.

KEY MESSAGE: The activation of the antioxidant system under stress combination is a transmissible trait from the rootstock to the scion. Therefore, rootstock selection is key to improve crop performance and a sustainable production under changing climate conditions. Climate change is altering weather conditions such as mean temperatures and precipitation patterns. Rising temperatures, especially in certain regions, accelerates soil water depletion and increases drought risk, which affects agriculture yield. Previously, our research demonstrated that the citrus rootstock Carrizo citrange (Citrus sinensis × Poncirus trifoliata) is more tolerant than Cleopatra mandarin (C. reshni) to drought and heat stress combination, in part, due to a higher activation of the antioxidant system that alleviated damage produced by oxidative stress. Here, by using reciprocal grafts of both genotypes, we studied the importance of the rootstock on scion performance and antioxidant response under this stress combination. Carrizo rootstock, under stress combination, positively influenced Cleopatra scion by reducing H2O2 accumulation, increasing superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymatic activities and inducing SOD1, APX2 and catalase (CAT) protein accumulations. On the contrary, Cleopatra rootstock induced decreases in APX2 expression, CAT activity and SOD1, APX2 and CAT contents on Carrizo scion. Taken together, our findings indicate that the activation of the antioxidant system under stress combination is a transmissible trait from the rootstock to the scion and highlight the importance of the rootstock selection to improve crop performance and maintain citrus yield under the current scenario of climate change.

Mitochondrial proteomics reveals new insights into embryogenic competence acquisition in Carica papaya L. callus.

Understanding the mechanisms that endow a somatic cell with the ability to differentiate into a somatic embryo, which could result in numerous biotechnological applications, is still a challenge. The objective of this work was to identify some of the molecular and physiological mechanisms responsible for the acquisition of embryogenic competence during somatic embryogenesis in Carica papaya L. We performed a broad characterization of embryogenic (EC) and nonembryogenic calli (NEC) of using global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide (H2O2) contents. EC and NEC presented remarkable differences in anatomical and histochemical characteristics, with EC showing a higher reactivity for the presence of proteins and neutral polysaccharides. Our results demonstrate that mitochondrial metabolism affects the embryogenic competence of C. papaya callus. The EC presented higher participation of alternative oxidase (AOX) enzymes, higher total cell respiration and presented a stronger accumulation of mitochondrial stress response proteins. Differential accumulation of auxin-responsive Gretchen Hagen 3 (GH3) family proteins in EC was related to a decrease in the content of free 2,4-dichlorophenoxyacetic acid (2,4-D). EC also showed higher endogenous H2O2 contents. H2O2 is a promising molecule for further investigation in differentiation protocols for C. papaya somatic embryos. SIGNIFICANCE: To further advance the understanding of somatic embryogenesis, we performed a broad characterization of embryogenic and nonembryogenic callus, through global and mitochondrial proteomic approaches, histomorphology, histochemistry, respiratory activity, and endogenous hormonal and hydrogen peroxide contents. Based on these results, we propose a working model for the competence of papaya callus. This model suggests that GH3 proteins play an important role in the regulation of auxins. In addition, embryogenic callus showed a greater abundance of stress response proteins and folding proteins. Embryogenic callus respiration occurs predominantly via AOX, and the inhibition of its activity is capable of inhibiting callus differentiation. Although the embryogenic callus presented greater total respiration and a greater abundance of oxidative phosphorylation proteins, they had less COX participation and less coupling efficiency, indicating less ATP production.

Stage-specific protein regulation during somatic embryo development of Carica papaya L. 'Golden'.

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.

Análise da sífilis em gestantes nos anos de 2010 a 2019 em Joinville - SC / Syphilis' analysis in pregnant women from years 2010 to 2019 in joinville - SC

A sífilis é uma infecção sexualmente transmissível comum em todo o mundo. Objetivo: Analisar os casos notificados/confirmados de sífilis gestacional em Joinville - SC. Método: Estudo descritivo, realizado por meio da análise de dados secundários de forma retrospectiva, no período de 2010 a 2019. Foram investigadas 1039 gestantes com sífilis, a partir dos dados extraídos do Sistema de Processamento de Dados do DATASUS. Resultados: A maioria das gestantes possuía idade igual ou inferior a 29 anos, sem ocupação remunerada e de baixa renda. A maior parte teve o diagnóstico da sífilis e foi notificada no 3o trimestre de gestação. A classificação clínica da sífilis predominante foi sífilis latente. 92% das gestantes foram tratadas e 52% dos parceiros. Conclusões: Observou-se que mulheres jovens, sem ocupação remunerada e baixa renda estão mais suscetíveis a Infecções Sexualmente Transmissíveis. Outros achados foram o diagnóstico tardio da sífilis entre as gestantes e a condição de parceiros não tratados.

O processo de desenvolvimento docente no ensino superior da área de saúde: revisão integrativa / Faculty development process in higher education for healthcare professions: an integrative review

O estudo teve como objetivo evidenciar as produções científicas acerca do processo de Desenvolvimento Docente no ensino superior na área da saúde, no período de 2009 a 2019. Trata-se de uma revisão integrativa, realizada nas bases de dados: Medical Literature Analysis and Retrievel System Online (MEDLINE); Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS); e Scientific Electronic Library Online (SciElo), com descritores, desenvolvimento docente, educação e escola médica. A partir dos critérios de inclusão, foram selecionados doze artigos (inglês e português) para discussão e resultados. Surgiram três categorias: percepção docente, papel da Instituição de Ensino Superior e formação, educação continuada e Desenvolvimento Docente. Concluiu-se que, apesar de um aumento expressivo de programas de Desenvolvimento Docente, muito ainda precisa ser realizado, especialmente no que se refere à gestão, planejamento e envolvimento do corpo docente.

Programas de desenvolvimento docente como ativadores da prática reflexiva de processos de aprendizagem: revisão integrativa / Faculty development programs as activators of the reflective practice of learning processes: an integrative review

A reflexão da prática de processos de aprendizagem, por meio da participação em programas de desenvolvimento docente, contribui para a emancipação profissional e para a consolidação de uma profissão autônoma. Objetivou analisar as evidências científicas sobre a utilização de programas de desenvolvimento docente, para ativar a prática reflexiva de processos de aprendizagem. Trata-se de uma revisão integrativa realizada nas bases de dados SciELO, DOAJ e ERIC, no período de 2016 a 2020, resultando em 17 estudos relacionados com a temática. Foram elegíveis 9 (53%) publicações resgatadas na base DOAJ, 6 (35%) na ERIC e 2 (12%) na SciELO. Os estudos apontaram que os programas de formação continuada são opções para auxiliar os docentes no processo de reflexão sobre a prática.