Injection-based hairy root induction and plant regeneration techniques in Brassicaceae.

BACKGROUND: Hairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media. RESULTS: We initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies. CONCLUSION: We developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Transcriptome analysis of thermomorphogenesis in ovules and during early seed development in Brassica napus.

BACKGROUND: Plant sexual reproduction is highly sensitive to elevated ambient temperatures, impacting seed development and production. We previously phenotyped this effect on three rapeseed cultivars (DH12075, Topas DH4079, and Westar). This work describes the transcriptional response associated with the phenotypic changes induced by heat stress during early seed development in Brassica napus. RESULTS: We compared the differential transcriptional response in unfertilized ovules and seeds bearing embryos at 8-cell and globular developmental stages of the three cultivars exposed to high temperatures. We identified that all tissues and cultivars shared a common transcriptional response with the upregulation of genes linked to heat stress, protein folding and binding to heat shock proteins, and the downregulation of cell metabolism. The comparative analysis identified an enrichment for a response to reactive oxygen species (ROS) in the heat-tolerant cultivar Topas, correlating with the phenotypic changes. The highest heat-induced transcriptional response in Topas seeds was detected for genes encoding various peroxidases, temperature-induced lipocalin (TIL1), or protein SAG21/LEA5. On the contrary, the transcriptional response in the two heat-sensitive cultivars, DH12075 and Westar, was characterized by heat-induced cellular damages with the upregulation of genes involved in the photosynthesis and plant hormone signaling pathways. Particularly, the TIFY/JAZ genes involved in jasmonate signaling were induced by stress, specifically in ovules of heat-sensitive cultivars. Using a weighted gene co-expression network analysis (WGCNA), we identified key modules and hub genes involved in the heat stress response in studied tissues of either heat-tolerant or sensitive cultivars. CONCLUSIONS: Our transcriptional analysis complements a previous phenotyping analysis by characterizing the growth response to elevated temperatures during early seed development and reveals the molecular mechanisms underlying the phenotypic response. The results demonstrated that response to ROS, seed photosynthesis, and hormonal regulation might be the critical factors for stress tolerance in oilseed rape.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus).

Our study examined the mutation efficiency of the CRISPR/Cas9 method for tryptophan aminotransferase BnaTAA1 genes involved in the auxin biosynthesis pathway. We made nine CRISPR/Cas9 constructs with various promoters driving the expression of a Cas9 from Staphylococcus aureus (SaCas9) or a plant-codon-optimized Streptococcus pyogenes Cas9 (pcoCas9). We developed a fast and efficient system for evaluating the variety and frequency of mutations caused by each construct using Brassica napus hairy roots. We showed that pcoCas9 is more efficient in mutating the targeted loci than SaCas9 and the presence of the NLS signal enhanced the chance of mutagenesis by 25%. The mutations were studied further in regenerated lines, and we determined the BnaTAA1 gene expression and heritability of the gene modifications in transgenic plants. Hairy root transformation combined with CRISPR/Cas9-mediated gene editing represents a fast and straightforward system for studying target gene function in the important oilseed crop B. napus.

On the trail of auxin: Reporters and sensors.

The phytohormone auxin is a master regulator of plant growth and development in response to many endogenous and environmental signals. The underlying coordination of growth is mediated by the formation of auxin maxima and concentration gradients. The visualization of auxin dynamics and distribution can therefore provide essential information to increase our understanding of the mechanisms by which auxin orchestrates these growth and developmental processes. Several auxin reporters have been developed to better perceive the auxin distribution and signaling machinery in vivo. This review focuses on different types of auxin reporters and biosensors used to monitor auxin distribution and its dynamics, as well as auxin signaling, at the cellular and tissue levels in different plant species. We provide a brief history of each reporter and biosensor group and explain their principles and utilities.

Characterization and expression analysis of adipokinetic hormone and its receptor in eusocial aphid Pseudoregma bambucicola.

Aphids display an extraordinary phenotypic plasticity ranging from widespread reproductive and wing polyphenisms to the occurrence of sterile or subfertile soldier morphs restricted to eusocial species of the subfamilies Eriosomatinae and Hormaphidinae. Individual morphs are specialized by their behavior, anatomy, and physiology to perform different roles in aphid societies at different stages of the life cycle. The capacity of the insects to cope with environmental stressors is under the control of a group of neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH) that bind to a specific receptor (AKHR). Here, we describe the molecular characteristics of AKH and AKHR in the eusocial aphid Pseudoregma bambucicola. The sequence of the bioactive AKH decapeptide and the intron position in P. bambucicola AKH preprohormone were found to be identical to those in a phylogenetically distant aphid Dreyfusia spp. (Adelgidae). We detected four transcript variants of AKHR that are translated into three protein isoforms. Further, we analyzed AKH/AKHR expression in different tissues and insects of different castes. In wingless females, a remarkable amount of AKH mRNA was only expressed in the heads. In contrast, AKHR transcript levels increased in the order gut

Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species.

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution.

Expression of stress-related genes in the parthenogenetic forms of the pea aphid, Acyrthosiphon pisum.

Aphids are an economically important group of insects that have an intricate life cycle with seasonal polyphenism. This study aimed to explore the physiological background of aphid migration from unfavorable nutritional conditions to a new, intact host plant. Specifically, the relative expression of stress/metabolism-related genes and changes in metabolic reserves were determined for the winged and wingless forms of female pea aphids, Acyrthosiphon pisum, under two different nutritional conditions. The expression level was determined for the following sets of genes: the adipokinetic hormone (AKH) and its receptor, enzymes involved in carbohydrate and lipid metabolism, detoxifying enzymes, and genes encoding exoskeleton/cuticular proteins and cytoskeleton proteins. In both forms, the transcription of the adipokinetic hormone was upregulated during nutritional stress, whereas its receptor mRNA levels remained unchanged. Similarly, the expression of genes engaged in glycogen and triglyceride degradation was elevated. Glycogen reserves and phospholipids appeared to be used during stress. In comparison, nutrient rich reproductively active females of both forms appeared to use triglycerides. Moreover, we revealed changes in the mRNA level of the detoxifying genes delta-class glutathione S-transferase (GST-δ) and cytochrome P450 monooxygenase (CYP450), as well as the CP gene (which encodes exoskeleton/cuticular proteins) and the cofilin gene (the products of which influence cytoskeleton organization). These results indicate the possible correlation between nutritional stress, energy content, AKH, and the stress-related enzymes of different metabolic pathways in winged and wingless forms of A. pisum.

Molecular characterization, tissue distribution, and ultrastructural localization of adipokinetic hormones in the CNS of the firebug Pyrrhocoris apterus (Heteroptera, Insecta).

Adipokinetic hormones (AKHs) are a group of insect metabolic neurohormones, synthesized and released from an endocrine retrocerebral gland, the corpus cardiacum (CC). Small amounts of AKH have also been identified in the brain, although their role in this organ is not clear. To address this gap in the knowledge about insect brain biology, we studied the nucleotide sequence, tissue distribution, and subcellular localization of AKHs in the brain and CC of the firebug Pyrrhocoris apterus. This insect expresses two AKHs; the octapeptides Pyrap-AKH and Peram-CAH-II, the presence of which was documented in the both studied organs. In situ hybridization and quantitative reverse-transcription (q-RT)-PCR revealed the expression of the genes encoding for both AKHs not only in the CC, but also in brain. Electron microscopy analysis of the brain revealed the presence of these hormones in specialized secretory granules localized predominantly in the cellular bodies of neurons. The hormones might be transported from the granules into the axons, where they could play a role in neuronal signaling. Under acute stress induced by the injection of 3µmol KCl, the level of AKHs in the brain increased to a greater extent than that in the CC. These results might indicate an enhanced role of brain-derived AKHs in defence reaction under acute stress situations.

The metabolic fate of ivermectin in host (Ovis aries) and parasite (Haemonchus contortus).

Ivermectin (IVE), one of the most important anthelmintics, is often used in the treatment of haemonchosis in ruminants. The objective of our work was (1) to find and identify phase I and II metabolites of IVE formed by the Barber's pole worm (Haemonchus contortus), and (2) to compare IVE metabolites in helminths with IVE biotransformation in sheep (Ovis aries) as host species. Ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, microsomes (from adult worms or from ovine liver) and a primary culture of ovine hepatocytes were incubated with IVE. In the ex vivo study, living H. contortus adults were incubated in the presence of 1 µM IVE for 24 h. The results showed that the H. contortus enzymatic system is not able to metabolize IVE. On the other hand, 7 different phase I as well as 9 phase II IVE metabolites were detected in ovine samples using UHPLC/MS/MS analyses. Most of these metabolites have not been described before. Haemonchus contortus is not able to deactivate IVE through biotransformation; therefore, biotransformation does not contribute to the development of IVE-resistance in the Barber's pole worm.

The inability of tapeworm Hymenolepis diminuta and fluke Dicrocoelium dendriticum to metabolize praziquantel.

Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 µM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance.