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The virulence of encapsulated fungal conidia against Aedes aegypti larvae was investigated. Molecular studies confirmed that the fungal isolate used here was Beauveria bassiana. Different conidial concentrations were tested. A concentration of 1 × 10 8 conidia mL - 1 was the most effective, resulting in 7% larval survival after 7 days. Next, alginate capsules (0.65%) containing conidia were prepared with different densities of calcium chloride (0.01 M, 0.009 M, and 0.008 M CaCl₂) and tested against larvae. Furthermore, groups of capsules were prepared with bird diet to act as an attractant. All capsule densities tested reduced larval survival (ranging from 22 to 67%). However, capsules with 0.008 M CaCl₂ were the most effective. Furthermore, fungus-only capsules were more efficient when compared to those containing bird diet. Laboratory and semi-field bioassays were conducted using mixtures of capsules with different densities. In the laboratory, survival ranged from 26 to 53%, whereas in semi-field conditions, 35%, and 80% survival was observed for groups exposed to fungus-only capsules or capsules containing diet, respectively. Histopathological studies of larvae exposed to capsules showed the presence of the fungus in the digestive tract and visible damage to enterocytes. These findings offer new insights into the biological control of Ae. aegypti larvae., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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-β-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 .

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Genomic DNA methylation patterns play a crucial role in the developmental processes of plants and mammals. In this study, we aimed to investigate the significant effects of epigenetic mechanisms on the development of soybean seedlings and metabolic pathways. Our analyses show that 5-azaC-treatment affects radicle development from two Days After Imbibition (DAI), as well as both shoot and root development. We examined the expression levels of key genes related to DNA methylation and demethylation pathways, such as DRM2, which encodes RNA-directed DNA Methylation (RdDM) pathway, SAM synthase, responsible for methyl group donation, and ROS1, a DNA demethylase. In treated seedling roots, we observed an increase in DRM2 expression and a decrease in ROS1 expression. Additionally, 5-azaC treatment altered protein accumulation, indicating epigenetic control over stress response while inhibiting nitrogen assimilation, urea cycle, and glycolysis-related proteins. Furthermore, it influenced the levels of various phytohormones and metabolites crucial for seedling growth, such as ABA, IAA, ethylene, polyamines (PUT and Cad), and free amino acids, suggesting that epigenetic changes may shape soybean responses to pathogens, abiotic stress, and nutrient absorption. Our results assist in understanding how hypomethylation shapes soybean responses to pathogens, abiotic stress, and nutrient absorption crucial for seedling growth, suggesting that the plant's assimilation of carbon and nitrogen, along with hormone pathways, may be influenced by epigenetic changes., (© 2024 Scandinavian Plant Physiology Society.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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.

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., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., (© 2022 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

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 H 2 O 2 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., (© 2021. The Author(s).)

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 (H 2 O 2 ) 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 H 2 O 2 contents. H 2 O 2 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)