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Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50>1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50>1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC>1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.

NACs are plant-specific transcription factors involved in controlling plant development, stress responses, and senescence. As senescence-associated genes (SAGs), NACs integrate age- and stress-dependent pathways that converge to programmed cell death (PCD). In Arabidopsis, NAC-SAGs belong to well-characterized regulatory networks, poorly understood in soybean. Here, we interrogated the soybean genome and provided a comprehensive analysis of senescence-associated Glycine max (Gm) NACs. To functionally examine GmNAC-SAGs, we selected GmNAC065 , a putative ortholog of Arabidopsis ANAC083/VNI2 SAG , and the cell death-promoting GmNAC085, an ANAC072 SAG putative ortholog, for analyses . Expression analysis of GmNAC065 and GmNAC085 in soybean demonstrated (i) these cell death-promoting Gm NACs display contrasting expression changes during age- and stress-induced senescence; (ii) they are co-expressed with functionally different gene sets involved in stress and PCD, and (iii) are differentially induced by PCD inducers. Furthermore, we demonstrated GmNAC065 expression delays senescence in Arabidopsis, a phenotype associated with enhanced oxidative performance under multiple stresses, higher chlorophyll, carotenoid and sugar contents, and lower stress-induced PCD compared to wild-type. In contrast, GmNAC085 accelerated stress-induced senescence, causing enhanced chlorophyll loss, ROS accumulation and cell death, decreased antioxidative system expression and activity. Accordingly, GmNAC065 and GmNAC085 targeted functionally contrasting sets of downstream At SAGs, further indicating that GmNAC85 and GmNAC065 regulators function inversely in developmental and environmental PCD.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe.

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in genetics and biotechnology, in which gene expression analysis is one of the most important fields. Notwithstanding, relatively few works have focused on the analysis of gene expression in coffee. Moreover, most of these works have used less accurate techniques such as northern blot assays instead of more accurate techniques (e.g., qPCR) that have already been extensively used in other plant species. Aiming to boost the use of qPCR in studies of gene expression in coffee, we uncovered reference genes to be used in a number of different experimental conditions. Using two distinct algorithms implemented by geNorm and Norm Finder, we evaluated a total of eight candidate reference genes (psaB, PP2A, AP47, S24, GAPDH, rpl39, UBQ10, and UBI9) in four different experimental sets (control versus drought-stressed leaves, control versus drought-stressed roots, leaves of three different coffee cultivars, and four different coffee organs). The most suitable combination of reference genes was indicated in each experimental set for use as internal control for reliable qPCR data normalization. This study also provides useful guidelines for reference gene selection for researchers working with coffee plant samples under conditions other than those tested here.

Early plants began colonizing earth about 450 million years ago. During the process of coevolution, their metabolic cellular pathways produced a myriad of natural chemicals, many of which remain uncharacterized biologically. Popular preparations containing some of these molecules have been used medicinally for thousands of years. In Brazilian folk medicine, plant extracts from the bamboo plant Guadua paniculata Munro have been used for the treatment of infections and pain. However, the chemical basis of these therapeutic effects has not yet been identified. Here, we performed protein biochemistry and downstream pharmacological assays to determine the mechanisms underlying the anti-inflammatory and antinociceptive effects of an aqueous extract of the G. paniculata rhizome, which we termed AqGP. The anti-inflammatory and antinociceptive effects of AqGP were assessed in mice. We identified and purified a protein (AgGP), with an amino acid sequence similar to that of thaumatins (~20 kDa), capable of repressing inflammation through downregulation of neutrophil recruitment and of decreasing hyperalgesia in mice. In conclusion, we have identified the molecule and the molecular mechanism responsible for the anti-inflammatory and antinociceptive properties of a plant commonly used in Brazilian folk medicine.

Plant dehydrins (DNHs) belong to the LEA (Late Embryogenesis Abundant) protein family and are involved in responses to multiple abiotic stresses. DHNs are classified into five subclasses according to the organization of three conserved motifs (K-; Y-; and S-segments). In the present study, the DHN protein family was characterized by molecular phylogeny, exon/intron organization, protein structure, and tissue-specificity expression in eight Fabaceae species. We identified 20 DHN genes, encompassing three (Y SK , SK , and K ) subclasses sharing similar gene organization and protein structure. Two additional low conserved DHN Φ-segments specific to the legume SK -type of proteins were also found. The expression patterns of DHN genes in four legume species ( , and ) revealed that their tissue-specific regulation is associated with the presence or absence of the Y-segment. Indeed, DHN genes containing a Y-segment are mainly expressed in seeds, whereas those without the Y-segment are ubiquitously expressed. Further qRT-PCR analysis revealed that, amongst stress responsive dehydrins, a SK -type DHN gene from ( ) showed opposite response to biotic and abiotic stress with a positive regulation under water deficit and negative regulation upon nematode infection. Furthermore, transgenic lines overexpressing (OE) displayed improved tolerance to multiple abiotic stresses (freezing and drought) but increased susceptibility to the biotrophic root-knot nematode (RKN) . This contradictory role of in responses to abiotic and biotic stresses was further investigated by qRT-PCR analysis of transgenic plants using a set of stress-responsive genes involved in the abscisic acid (ABA) and jasmonic acid (JA) signaling pathways and suggested an involvement of DHN overexpression in these stress-signaling pathways.

Summary MicroRNAs (miRNAs) modulate the abundance and spatial–temporal accumulation of target mRNAs and indirectly regulate several plant processes. Transcriptional regulation of the genes encoding miRNAs (MIR genes) can be activated by numerous transcription factors, which themselves are regulated by other miRNAs. Fine‐tuning of MIR genes or miRNAs is a powerful biotechnological strategy to improve tolerance to abiotic or biotic stresses in crops of economic importance. Current approaches for miRNA fine‐tuning are based on the down‐ or up‐regulation of MIR gene transcription and the use of genetic engineering tools to manipulate the final concentration of these miRNAs in the cytoplasm. Transgenesis, cisgenesis, intragenesis, artificial MIR genes, endogenous and artificial target mimicry, MIR genes editing using Meganucleases, ZNF proteins, TALENs and CRISPR/Cas9 or CRISPR/Cpf1, CRISPR/dCas9 or dCpf1, CRISPR13a, topical delivery of miRNAs and epigenetic memory have been successfully explored to MIR gene or miRNA modulation and improve agronomic traits in several model or crop plants. However, advantages and drawbacks of each of these new biotechnological tools (NBTs) are still not well understood. In this review, we provide a brief overview of the biogenesis and role of miRNAs in response to abiotic or biotic stresses, we present critically the main NBTs used for the manipulation of MIR genes and miRNAs, we show current efforts and findings with the MIR genes and miRNAs modulation in plants, and we summarize the advantages and drawbacks of these NBTs and provide some alternatives to overcome. Finally, challenges and future perspectives to miRNA modulating in important crops are also discussed.

Nematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis . Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene ( AsECHI ) from Arachis stenosperma , reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance.

Agrobacterium rhizogenes -mediated transformation has long been explored as a versatile and reliable method for gene function validation in many plant species, including soybean ( Glycine max ). Likewise, detached-leaf assays have been widely used for rapid and mass screening of soybean genotypes for disease resistance. The present study combines these two methods to establish an efficient and practical system to generate transgenic soybean hairy roots from detached leaves and their subsequent culture under ex vitro conditions. We demonstrated that hairy roots derived from leaves of two (tropical and temperate) soybean cultivars could be successfully infected by economically important species of root-knot nematodes ( Meloidogyne incognita and M . javanica ). The established detached-leaf method was further explored for functional validation of two candidate genes encoding for cell wall modifying proteins (CWMPs) to promote resistance against M . incognita through distinct biotechnological strategies: the overexpression of a wild Arachis α-expansin transgene ( AdEXPA24 ) and the dsRNA-mediated silencing of an endogenous soybean polygalacturonase gene ( GmPG ). AdEXPA24 overexpression in hairy roots of RKN-susceptible soybean cultivar significantly reduced nematode infection by approximately 47%, whereas GmPG downregulation caused an average decrease of 37%. This novel system of hairy root induction from detached leaves showed to be an efficient, practical, fast, and low-cost method suitable for high throughput in root analysis of candidate genes in soybean.