Unmoving and uninflamed: Characterizing neuroinflammatory dysfunction in the Wistar-Kyoto rat model of depression.

Reductionistic research on depressive disorders has been hampered by the limitations of animal models. Recently, it has been hypothesized that neuroinflammation is a key player in depressive disorders. The Wistar-Kyoto (WKY) rat is an often-used animal model of depression, but no information so far exists on its neuroinflammatory profile. As such, we compared male young adult WKY rats to Wistar (WS) controls, with regard to both behavioral performance and brain levels of key neuroinflammatory markers. We first assessed anxiety- and depression-like behaviors in a battery consisting of the Elevated Plus Maze (EPM), the Novelty Suppressed Feeding (NSFT), Open Field (OFT), Social Interaction (SIT), Forced Swim (FST), Sucrose Preference (SPT), and Splash tests (ST). We found that WKY rats displayed increased NSFT feeding latency, decreased OFT center zone permanence, decreased EPM open arm permanence, decreased SIT interaction time, and increased immobility in the FST. However, WKY rats also evidenced marked hypolocomotion, which is likely to confound performance in such tests. Interestingly, WKY rats performed similarly, or even above, to WS levels in the SPT and ST, in which altered locomotion is not a significant confound. In a separate cohort, we assessed prefrontal cortex (PFC), hippocampus and amygdala levels of markers of astrocytic (GFAP, S100A10) and microglial (Iba1, CD86, Ym1) activation status, as well as of three key proinflammatory cytokines (IL-1ß, IL-6, TNF-α). There were no significant differences between strains in any of these markers, in any of the regions assessed. Overall, results highlight that behavioral data obtained with WKY rats as a model of depression must be carefully interpreted, considering the marked locomotor activity deficits displayed. Furthermore, our data suggest that, despite WKY rats replicating many depression-associated neurobiological alterations, as shown by others, this is not the case for neuroinflammation-related alterations, thus representing a novel limitation of this model.

The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

Cognitive comorbidities of experimental absence seizures are independent of anxiety.

Typical absence seizures (ASs) are brief periods of lack of consciousness, associated with 2.5-4 Hz spike-wave discharges (SWDs) in the EEG, which are highly prevalent in children and teenagers. The majority of probands in these young epileptic cohorts show neuropsychological comorbidities, including cognitive, memory and mood impairments, even after the seizures are pharmacologically controlled. Similar cognition and memory deficits have been reported in different, but not all, genetic animal models of ASs. However, since these impairments are subtle and highly task-specific their presence may be confounded by an anxiety-like phenotype and no study has tested anxiety and memory in the same animals. Moreover, the majority of studies used non-epileptic inbred animals as the only control strain and this may have contributed to a misinterpretation of these behavioural results. To overcome these issues, here we used a battery of behavioural tests to compare anxiety and memory in the same animals from the well-established inbred model of Genetic Absence Epilepsy Rats from Strasbourg (GAERS), their inbred strain of Non-Epileptic Control (NEC) strain (that lack ASs) and normal outbred Wistar rats. We found that GAERS do not exhibit increased anxiety-like behavior and neophobia compared to both NEC and Wistar rats. In contrast, GAERS show decreased spontaneous alternation, spatial working memory and cross-modal object recognition compared to both NEC and Wistar rats. Furthermore, GAERS preferentially used egocentric strategies to perform spatial memory tasks. In summary, these results provide solid evidence of memory deficits in GAERS rats that do not depend on an anxiety or neophobic phenotype. Moreover, the presence of differences between NEC and Wistar rats stresses the need of using both outbred and inbred control rats in behavioural studies involving genetic models of ASs.

TARGETing Transcriptional Regulation in the Medicinal Plant Catharanthus roseus.

Transcriptional regulation is a central piece of the highly valuable monoterpenoid indole alkaloid pathway of C. roseus , and the ultimate tool for its understanding and manipulation. Here, we describe the adaptation of the TARGET methodology to identify specific and genome-wide leaf targets of C. roseus candidate transcription factors (TFs).

Isolation of Specialized Plant Cells by Fluorescence-Activated Cell Sorting.

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells.

Pervasive hybridization with local wild relatives in Western European grapevine varieties.

Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid Pathway Branch Choice in Catharanthus roseus.

Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.

Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells.

The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10 mM CaCl2 did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality.

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

MAIN CONCLUSION: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca2+, and Na+ in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.

Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants.

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.

Protoplast Transformation as a Plant-Transferable Transient Expression System.

The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.

Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don.

BACKGROUND AND AIMS: Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size. METHODOLOGY: The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology. PRINCIPAL RESULTS: The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp. CONCLUSIONS: The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.

The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.

Fusion with fluorescent proteins for subcellular localization of enzymes involved in plant alkaloid biosynthesis.

To establish the role in alkaloid metabolism of candidate genes identified in silico or by Omics approaches, it may be essential to determine the subcellular localization of the encoded proteins. The fusion with fluorescent proteins (FP) may now be used as a quite effective and reliable tool to investigate this question. The methodology involves the choice of the FP, the design and production of the appropriate FP fusions, and the use of a transient or stable transformation protocol applied to a homologous or heterologous plant system. This chapter describes the application of this methodology to an enzyme involved in indole alkaloid biosynthesis, with general considerations on the development of the approach.

Downregulation of the basic peroxidase isoenzyme from Zinnia elegans by gibberellic acid.

Hypocotyl formation during the epigeal germination of seedlings is under strict hormonal regulation. In a 3 d old Zinnia elegans seedling system, gibberellic acid (GA(3)) exerts an opposite effect to that exerted by light on hypocotyl photomorphogenesis because GA(3) promotes an etiolated-like growth with an inhibition of radial (secondary) growth. For this reason, the effect of GA(3) on the basic peroxidase isoenzyme from Z. elegans (ZePrx), an enzyme involved in hypocotyl lignin biosynthesis, was studied. The results showed that GA(3) reduces ZePrx activity, similarly to the way in which it reduces seedling secondary growth. This hormonal response is supported by the analysis of the ZePrx promoter, which contains four types of GA(3)-responsive cis-elements: the W Box/O2S; the Pyr Box; the GARE; and the Amy Box. Taken together, these results suggest that ZePrx is directly regulated by GA(3), with this effect matching the inhibitory effect of GA on the hypocotyl secondary growth.