RNA In Situ Hybridization of Paraffin Sections to Characterize the Multicellular Compartmentation of Plant Secondary Metabolisms.

As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus. RNA in situ hybridization has proven a key technological approach to unravel this complex spatial organization. Pioneer work in 1999 discovered the involvement of epidermis and laticifer/idioblasts in the intermediate and late steps of the pathway, respectively. The localization of the early steps of the pathway to the internal phloem-associated parenchyma later came to complete the three-tissular block organization of the pathway. Since then, RNA in situ hybridization was routinely used to map the gene expression profile of most of the nearly 30 genes involved in this pathway. We introduce here a comparison of advantages and drawbacks of in situ hybridization and more popular promoter: GUS strategies. Two main advantages of in situ hybridization are the suitability to any plant species and the direct localization of transcripts rather than the localization of a promoter activity. We provide a step-by-step protocol describing every details allowing to reach a medium throughput including riboprobe synthesis, paraffin-embedded plant tissue array preparation, prehybridization, in situ hybridization, stringent washing and immunodetection of hybridized probes, and imaging steps. This should be helpful for new comers willing to domesticate the technique. This protocol has no species limitation and is particularly adapted to the increasingly studied model, nonmodel species, nonamenable to promoter::GUS transformation, such as C. roseus.

Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories.

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

Dietary inclusion of Peptiva, a peptide-based feed additive, can accelerate the maturation of the fecal bacterial microbiome in weaned pigs.

BACKGROUND: Weaning is one of the most critical transition stages of the swine production cycle, as the piglet gut physiology and microbiome need to rapidly adapt to changes in diet and environmental conditions. Based on their potential for producing a vast array of bioactive molecules, peptide formulations represent a largely untapped source of compounds that could be developed into feed additives to benefit animal health and nutrition. In this context, a commercial-scale nursery trial was performed to evaluate the impact of low inclusion of a peptide-based feed additive (Peptiva, Vitech Bio-Chem Corporation) on the performance and fecal microbiome of weaned pigs. RESULTS: While no significant differences in body weight, daily gain, daily feed intake nor gain:feed were observed between control and treatment animals (P > 0.05), an effect of Peptiva on the fecal bacterial composition of weaned pigs was observed. The first main observation was that the fecal bacterial profiles from pigs fed Control-Phase II and Control Phase III diets were found to be very distinct, suggesting that a transition or succession stage had occurred between the two phases. Lactobacilli, represented by four main OTUs (Ssd-00002, Ssd-00019, Ssd-00025, and Ssd-00053), were more abundant at the end of Phase II (P < 0.05), while Streptococci, mostly represented by OTUs Ssd-00039 and Ssd-00048, were in higher abundance at the end of Phase III (P < 0.05). Secondly, the fecal bacterial composition from pigs fed Peptiva Phase II diets showed similarities to both Control-Phase II and Control Phase III samples, while there was no difference in fecal bacterial composition between Control-Phase III and Peptiva Phase III samples. For instance, OTUs Ssd-00019,and Ssd-00053 were in lower abundance in Peptiva Phase II samples compared to Control Phase II (P < 0.05), but no significant difference was observed in the abundance of these two OTUs when comparing Peptiva Phase II to Control Phase III (P > 0.05). CONCLUSIONS: Together, these results suggest that Peptiva can modulate the composition of the swine microbiome during a specific window of the nursery stage, potentially by accelerating its maturation.

A BAHD acyltransferase catalyzing 19-O-acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids.

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

Folivory elicits a strong defense reaction in Catharanthus roseus: metabolomic and transcriptomic analyses reveal distinct local and systemic responses.

Plants deploy distinct secondary metabolisms to cope with environment pressure and to face bio-aggressors notably through the production of biologically active alkaloids. This metabolism-type is particularly elaborated in Catharanthus roseus that synthesizes more than a hundred different monoterpene indole alkaloids (MIAs). While the characterization of their biosynthetic pathway now reaches completion, still little is known about the role of MIAs during biotic attacks. As a consequence, we developed a new plant/herbivore interaction system by challenging C. roseus leaves with Manduca sexta larvae. Transcriptomic and metabolic analyses demonstrated that C. roseus respond to folivory by both local and systemic processes relying on the activation of specific gene sets and biosynthesis of distinct MIAs following jasmonate production. While a huge local accumulation of strictosidine was monitored in attacked leaves that could repel caterpillars through its protein reticulation properties, newly developed leaves displayed an increased biosynthesis of the toxic strictosidine-derived MIAs, vindoline and catharanthine, produced by up-regulation of MIA biosynthetic genes. In this context, leaf consumption resulted in a rapid death of caterpillars that could be linked to the MIA dimerization observed in intestinal tracts. Furthermore, this study also highlights the overall transcriptomic control of the plant defense processes occurring during herbivory.

Virus-induced gene silencing in Rauwolfia species.

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

In a Neonatal Piglet Model of Intestinal Failure, Administration of Antibiotics and Lack of Enteral Nutrition Have a Greater Impact on Intestinal Microflora Than Surgical Resection Alone.

BACKGROUND: Data are limited on how short bowel syndrome (SBS) affects the healthy developing intestinal microbiome, with even less assessing different SBS anatomical surgical models. This study was conducted to describe the "ileal" and "colonic" microflora in 2 surgical models of SBS. MATERIALS AND METHODS: Neonatal piglets (2-5 days old) underwent intestinal resection, leaving the ileum (JI anatomy, n = 6) or removing the ileum and ileocecal valve (JC anatomy, n = 5), or sham surgery (sham; n = 4). JI, JC, and sham piglets commenced parenteral nutrition on day 0 and received ampicillin and trimethoprim-sulfadoxine on days 0-4 for prevention of line sepsis. At day 7, ileal and colonic digesta were collected, and they were also collected from age-matched sow-fed piglets (n = 6). DNA extraction, sequencing, and annotation followed standard procedures. RESULTS: Colonic and ileal bacterial genus diversity and relative bacterial abundance were greater ( P < .05) in sow-fed compared with JI, JC, and sham piglets; however, minor differences were observed in either location between sham, JI, and JC piglets and within the surgical model. In the colon, sow-fed piglets had higher ( P < .05) abundance of Lactobacillus (26%) and tended to have lower ( P = .06) abundance of Enterococcus (<.1%) than JI, JC, or sham piglets, in which Lactobacillus and Enterococcus abundance averaged <.1% and 9%, respectively. CONCLUSIONS: Intestinal resection reduces bacterial diversity in the large bowel, and the difference is associated with the presence/absence of the ileum and ileocecal valve. The lack of enteral nutrition and antibiotic administration (ie, sow-fed vs surgery) had a greater influence on the observed shift in diversity and relative abundance than intestinal resection.

Phytochemical genomics of the Madagascar periwinkle: Unravelling the last twists of the alkaloid engine.

The Madagascar periwinkle produces a large palette of Monoterpenoid Indole Alkaloids (MIAs), a class of complex alkaloids including some of the most valuable plant natural products with precious therapeutical values. Evolutionary pressure on one of the hotspots of biodiversity has obviously turned this endemic Malagasy plant into an innovative alkaloid engine. Catharanthus is a unique taxon producing vinblastine and vincristine, heterodimeric MIAs with complex stereochemistry, and also manufactures more than 100 different MIAs, some shared with the Apocynaceae, Loganiaceae and Rubiaceae members. For over 60 years, the quest for these powerful anticancer drugs has inspired biologists, chemists, and pharmacists to unravel the chemistry, biochemistry, therapeutic activity, cell and molecular biology of Catharanthus roseus. Recently, the "omics" technologies have fuelled rapid progress in deciphering the last secret of strictosidine biosynthesis, the central precursor opening biosynthetic routes to several thousand MIA compounds. Dedicated C. roseus transcriptome, proteome and metabolome databases, comprising organ-, tissue- and cell-specific libraries, and other phytogenomic resources, were developed for instance by PhytoMetaSyn, Medicinal Plant Genomic Resources and SmartCell consortium. Tissue specific library screening, orthology comparison in species with or without MIA-biochemical engines, clustering of gene expression profiles together with various functional validation strategies, largely contributed to enrich the toolbox for plant synthetic biology and metabolic engineering of MIA biosynthesis.

Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

Functional analysis of the DAT gene promoter using transient Catharanthus roseus and stable Nicotiana tabacum transformation systems.

The Catharanthus roseus DAT gene encodes the enzyme acetyl-CoA:deacetylvindoline-4-O-acetyltransferase involved in the last step of the indole alkaloid pathway leading to vindoline. This gene is characterized by specific cell type expression in idioblasts and laticifers. To understand the specific transcriptional regulation mechanism(s) of DAT, several DAT promoter GUS constructs were cloned into pCAMBIA1305.1. Agroinfiltration of different explant types of C. roseus resulted in organ-specific accumulation of GUS, albeit at various levels. Heterologous accumulation of GUS in transgenic tobacco revealed both general and non-specific expression with the exception of a stomata-specific expression when 2.3 kb of the DAT promoter was coupled with a portion of the DAT ORF. These results suggest that in addition to the 2.3 kb upstream of the DAT transcriptional start site, additional cis-acting elements may be responsible for the specific spatial expression of DAT in vivo. Furthermore, hairy roots transformed with DAT promoter GUS constructs demonstrated GUS expression in root tissues (visualized through GUS enzyme activity), even though DAT is repressed in non-transformed roots.

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs.

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid-monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid-monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.

CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells.

CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common alpha subunit and a specific beta subunit. We have established RNA interference cell lines targeting the beta subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.

Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus.

Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the alpha/beta hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed.

Conserved and specific functions of mammalian ssu72.

We describe the cloning and characterization of a human homolog of the yeast transcription/RNA-processing factor Ssu72, following a yeast two-hybrid screen for pRb-binding factors in the prostate gland. Interaction between hSsu72 and pRb was observed in transfected mammalian cells and involved multiple domains in pRb; however, so far, mutual effects of these two factors could not be demonstrated. Like the yeast counterpart, mammalian Ssu72 associates with TFIIB and the yeast cleavage/polyadenylation factor Pta1, and exhibits intrinsic phosphatase activity. Mammals contain a single ssu72 gene and a few pseudogenes. During mouse embryogenesis, ssu72 was highly expressed in the nervous system and intestine; high expression in the nervous system persisted in adult mice and was also readily observed in multiple human tumor cell lines. Both endogenous and ectopically expressed mammalian Ssu72 proteins resided primarily in the cytoplasm and only partly in the nucleus. Interestingly, fusion to a strong nuclear localization signal conferred nuclear localization only in a fraction of transfected cells, suggesting active tethering in the cytoplasm. Suppression of ssu72 expression in mammalian cells by siRNA did not reduce proliferation/survival, and its over-expression did not affect transcription of candidate genes in transient reporter assays. Despite high conservation, hssu72 was unable to rescue an ssu72 lethal mutation in yeast. Together, our results highlight conserved and mammalian specific characteristics of mammalian ssu72.

High expression during neurogenesis but not mammogenesis of a murine homologue of the Deleted in Breast Cancer2/Rhobtb2 tumor suppressor.

Deleted in Breast Cancer-2, Dbc2, identified as a candidate tumor suppressor gene for breast cancer and other human malignancies, is a member of a novel gene family encoding putative small GTPases designated as Rhobtb. In this report, we describe the spatio-temporal expression of mDbc2/Rhobtb2 mRNA during mammary gland development and embryogenesis in the mouse. We have first determined that mDbc2/Rhobtb2 transcripts are expressed at low, but seemingly constant levels during mammopoiesis. Secondly, we have found that mDbc2/Rhobtb2 is expressed highly and specifically in the central and peripheral nervous systems during mouse embryogenesis. While our results do not reveal any connection between elevated expression of mDbc2/Rhobtb2 and a specific stage of mammary gland development, they strongly support a role for this gene during development of the nervous system.

The Hand1, Stra13 and Gcm1 transcription factors override FGF signaling to promote terminal differentiation of trophoblast stem cells.

The trophoblast cell lineage is an interesting model system because it is composed of a limited number of cell types that are spatially patterned. Trophoblast stem (TS) cells reside within a layer called the chorion and either remain as stem cells or differentiate into spongiotrophoblast (SpT), trophoblast giant (TG) cells or syncytiotrophoblast cells (SynT) of the labyrinth. Maintenance of the TS phenotype is dependent on stimulation by FGF4, whereas differentiation and/or maintenance of the differentiated derivatives are dependent on key transcription factors: Mash2 for SpT, Hand1 for TG cells and Gcm1 for SynT cells. TS cells proliferate and retain their stem cell phenotype in culture in response to FGF4 and an additional factor(s) that can be provided by conditioned medium from embryonic fibroblast feeder cells (CM). To understand the functions of Hand1, Mash2 and Gcm1 at a cellular level, we tested the effects of their ectopic and over-expression on the ability of TS cells to either continue to proliferate or differentiate into their alternative fates. Expression of Mash2 alone had no effects on TS cell differentiation. However, Mash2-transfected cells continued to divide longer after withdrawal of FGF/CM. Hand1 promoted TGC differentiation, even in the continued presence of FGF4/CM. Stra13, another bHLH factor gene that is expressed in TG cells, also induced TG differentiation. Gcm1 induced a rapid arrest of TS proliferation but, in contrast to Hand1 and Stra13, blocked TG cell differentiation. Although Gcm1 was not sufficient to promote SynT formation, expression of an antisense Gcm1 transcript blocked SynT differentiation. These data suggest that Mash2 functions to promote transient FGF4-independent amplification of trophoblast cells that are progressing towards the SpT and TG cell phenotype. By contrast, Hand1 and Stra13 promote cell cycle exit and restrict cells towards the TG fate, whereas Gcm1 promotes cell cycle exit and restriction towards the SynT fate.