Biology subjects

Germain, H.

Publications and source records attributed to Germain, H..

9 recordsLinked to original sources

bioRxiv2025

Branch-Enabling N-Methyltransferase in Golgi Reconciles Divergent Models of Galanthamine Biosynthesis

Galanthamine, a therapeutic Amaryllidaceae alkaloid produced exclusively by species within the Amaryllidoideae subfamily, is a key treatment for early-stage symptoms of Alzheimers disease. Elucidating its biosynthetic pathway is essential for strategies aimed at enhancing production through metabolic engineering. Galantamine derives from the metabolic precursor 4'-O-methylnorbelladine, which undergoes cytochrome P450-mediated para-ortho C-C phenol coupling to yield nornarwedine. Two competing terminal routes have been proposed: (i) reduction of nornarwedine to norgalanthamine, followed by N-methylation, or (ii) N-methylation of nornarwedine to narwedine prior to reduction. Here, we identify three N-methyltransferase (NMT) candidates from Leucojum aestivum: LaNMT, related to coclaurine NMTs, and two {gamma}-tocopherol methyltransferases (TMT) homologs, LaTMT1 and LaTMT2. Subcellular localization studies revealed distinct compartmentalization, with LaNMT targeted to the ER-cytosol, LaTMT1 to plastids, and LaTMT2 to the Golgi apparatus. In vitro enzyme assays demonstrated that LaTMT2 methylates both nornarwedine and norgalanthamine, with a kinetic preference for nornarwedine. Agroinfiltration for transient expression in Nicotiana benthamiana further confirmed LaTMT2 as a catalytically efficient and substrate-promiscuous enzyme that supports both terminal routes. These findings identify LaTMT2 as a key branch-enabling N-methyltransferase that reconcile long-standing models of galanthamine biosynthesis and provides a strategic target for metabolic engineering strategies to enhance galanthamine production. Significant statementThis study identifies LaTMT2, a Golgi-localized {gamma}-tocopherol methyltransferase homolog, as a branch-enabling N-methyltransferase that resolves competing models of galanthamine biosynthesis. By revealing an unanticipated Golgi-associated step in Amaryllidaceae alkaloid metabolism, it redefines the subcellular organization of specialized metabolic pathways and provides a strategic enzymatic target for metabolic engineering of high-value therapeutic alkaloids.

plant biology↗

bioRxiv2025

Insights into cannabinoid biosynthesis in Chlamydomonas reinhardtii : successes with NphB and limitations of CBDAS expression

The growing legalization of Cannabis has increased demand for cannabinoids (CBs). Currently, pharmaceutically relevant CBs are primarily extracted from Cannabis, a process that presents challenges related to yield variability and purity. To overcome these limitations, microbial platforms are being explored for the sustainable production of specific CBs. Compared to conventional microbial hosts, the photosynthetic microalga Chlamydomonas reinhardtii offers plant-like post-translational modifications, low-cost cultivation, CO2 fixation, and low endotoxin contamination, making it a potential chassis for CB biosynthesis. Here, we expressed a codon-optimized, soluble aromatic prenyltransferase (NphBG286S/Y288A) from Streptomyces and Cannabis sativa cannabidiolic acid synthase (CBDAS) in the nuclear genome of C. reinhardtii; transformants were screened for integration, gene expression, protein accumulation, enzymatic activity, and CB production. Our results provide evidence of functional NphB expression in C. reinhardtii, with in vitro CBGA production reaching up to 633 {+/-} 58 {micro}g/L. Although CBDAS transcripts were detected under multiple construct designs, neither protein nor CBDA was accumulated, suggesting limitations in expression, localization, or post-translational processing in C. reinhardtii. Our study provides the first demonstration of in vitro CBGA biosynthesis in the photosynthetic model microalga C. reinhardtii, highlighting its potential as a future platform for CB production. It also highlights key challenges in nuclear expression of plant-derived enzymes, such as CBDAS, emphasizing that improved regulatory control, subcellular targeting, and mRNA processing will be required to achieve full pathway reconstruction in C. reinhardtii. HighlightsO_LIFirst report of active NphB expression in Chlamydomonas reinhardtii C_LIO_LINphB exhibits measurable in vitro activity, producing cannabigerolic acid in algal extracts C_LIO_LIEnzyme localization, construct design, and substrate availability critically influence functionality. C_LI

bioengineering↗

bioRxiv2024

Seed specific overexpression of a modified wheat Or gene leads to enhanced β-carotene in rice and wheat grains

Vitamin A deficiency is a major public health problem affecting up to 50% of the worlds population, as either wheat or rice, which are poor in many essential micronutrients such as vitamin A, are major staple food crops. Biofortification of cereal crops with {beta}-carotene (provitamin A) through genetic engineering is a potential solution to overcome vitamin A deficiency. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and previous studies demonstrated high carotenoid accumulation due to a single-nucleotide polymorphism (SNP) in the CDS leading to substitution of Arg to His in the OR protein results in carotenoid accumulation. In the present study, we showed that this substitution of a single amino acid at position 110 (Arg to His) of wild-type wheat TaOr (referred to as TaOrHis110) increased {beta}-carotene accumulation in transgenic wheat and rice plants overexpressing TaOrHis110 under the control of the seed-specific promoter Glu-1D1. HPLC analysis revealed increase in {beta}-carotene content in rice grain up to 8-fold in case of TP309 (japonica) cultivar, 13-fold in case of IET10364 (indica) cultivar and 7-fold in wheat cv. CPAN1676. Additionally, most of the carotenoid biosynthetic pathway genes were found to be upregulated in TaOrHis110 overexpressing seeds of TP309 and IET10364, which positively correlated with maximum increase in {beta}-carotene content.

plant biology↗

bioRxiv2024

Sanctuary: A Starship transposon facilitating the movement of the virulence factor ToxA in fungal wheat pathogens

There is increasing evidence that mobile genetic elements can drive the emergence of pathogenic fungal species by moving virulence genes horizontally. The 14 kbp ToxhAT transposon has been shown to be moving the necrotrophic effector, ToxA, horizontally between fungal species that infect Triticum aestivum (wheat), namely Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana. All three species utilise the ToxA protein to infect wheat. Previous genomic evidence found ToxhAT in distinct chromosomal positions in two isolates of B. sorokiniana, indicating that the transposon is still active in this species. Here we confirm the movement of ToxhAT using long-read Nanopore MinION sequencing of eight novel and one previously published B. sorokiniana isolates. One event of independent transposition of ToxhAT was observed, and target site duplications of "TA" were identified, confirming this was an autonomous movement facilitated by a yet unidentified transposase. Whole genome analysis revealed that ToxhAT is a passenger embedded in a much larger, conserved 170-196 kbp mobile genetic element. This element, termed Sanctuary, belongs to the newly described Starship transposon superfamily. This classification is based on the presence of short direct repeats, empty insertion sites, a putative tyrosine recombinase gene and other features of Starship transposons. We also show that ToxhAT has been independently captured by two different Starships, Sanctuary and Horizon which share little to no sequenced identity, outside of ToxhAT. This classification makes Horizon and Sanctuary part of a growing number of Starships involved in the horizontal gene transfer of adaptive genetic material between fungal species. ImportanceThe work presented here expands our understanding of a novel group of mobile genetic elements called Starships that facilitate the horizontal exchange of virulence genes in fungal pathogens. Our analysis shows that Sanctuary and ToxhAT are likely active and autonomous transposons in the B. sorokiniana genome. We also show that the smaller ToxhAT transposon has been independently captured by two different Starships, viz. Sanctuary in B. sorokiniana and Horizon in P. tritici-repentis and P. nodorum. Outside of ToxhAT these two Starships share no sequence identity. The capture of ToxhAT by two different mobile elements in three different fungal wheat pathogens demonstrates how horizontal transposon transfer is driving the evolution of virulence in these important wheat pathogens.

genomics↗

bioRxiv2024

Overexpression of barley heat stress transcription factor HvHsfA6a provide thermotolerance by thermopriming

Adverse impacts of climate change, including high temperature on cereal crop production, have been evidenced globally. In plants, heat shock factors (HSFs) are crucial components of heat stress associated rescue mechanisms and are also required for normal biological processes. Here, we functionally characterized a highly heat stress responsive HvHSFA6a in barley by developing constitutively overexpressing transgenic lines. These transgenic lines showed heat tolerant phenotype via improved photosynthesis, antioxidants and upregulation of HSPs and metabolites involved in stress amelioration and keeping thermomemory as compared to wild type plants. Global transcriptomics and ChIP sequencing revealed that HvHSFA6a orchestrates the expression of several genes through direct binding with other HSFs containing consensus HSE in their promoter regions. A GC-MS based metabolomics analysis also revealed the alterations in key metabolic processes such as carbohydrate metabolism, citric acid cycle, amino acids and secondary metabolism. Higher accumulation of key metabolites such as sucrose, galactinol, shikimate and ascorbate has been observed under both control and heat stress in transgenic lines as compared to wild type plants. Taken together, the results suggest that overexpression of HvHsfA6a prime the plants for heat stress conditions by alteration in gene expression and metabolic status. HighlightPriming is a mechanism by which plants respond to various abiotic and biotic stresses. Through multi omics approach we found that barley HsfA6a provide thermotolernce in transgenic plants through priming effect on transcriptome and metabolome.

molecular biology↗

bioRxiv2022

Chloroplast activity provides in vitro regeneration capability in contrasting cultivars

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. We know little about why only some cultivars in crop plants are tissue culture friendly. In this study, tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency. Multiomics studies involving transcriptomics, proteomics, metabolomics, and biochemical analysis were performed using GP and D91 callus to unravel the regulatory mechanisms. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MSMS revealed 3062 protein groups and 16989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis also revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. HighlightsMulti omics analysis revealed chloroplast play crucial role in providing in vitro regeneration capability in contrasting genotypes

plant biology↗

bioRxiv2020

Differential alteration of plant functions by homologous fungal candidate effectors

Rust fungi are plant pathogens that cause epidemics that threaten the production of important plant species, such as wheat, soy, coffee and poplar. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1 184 candidate effectors (CEs), however their functions are poorly known. In this study, we used Arabidopsis plants constitutively expressing CEs of Mlp to discover processes targeted by these fungal proteins. For this purpose, we sequenced the transcriptome and used mass spectrometry to analyse the metabolome of Arabidopsis plants expressing individually one of the 14 selected CEs and of a control line. We found 2 299 deregulated genes across the experiment. Among the down-regulated genes, the KEGG pathways "MAPK signaling pathway" and "Plant-pathogen interaction" were respectively over-represented in six and five of the 14 transgenic lines. Moreover, genes related to hormone response and defense were down-regulated across all transgenic lines are. We further observed that there were 680 metabolites deregulated in at least one CE-expressing transgenic line, with highly unsaturated and phenolic compounds enriched in up-regulated metabolites and peptides enriched among down-regulated metabolites. Interestingly, we found that transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Taken together, our results indicate that the sequence of effectors and their belonging to families may not be a good predictor of their impact on the plant. ImportanceRust fungi are plant pathogens that threaten the production of important crops, including wheat, soy, coffee and poplar. Effectors are used by pathogens to control the host, however in the case of Melampsora larici-populina, the causal agent of the poplar rust, and other rust fungi these proteins are poorly known. We used Arabidopsis plants expressing candidate effectors (CEs) of Mlp to better understand the interaction between this pathogen and its hosts. We found that expression of unrelated CEs led to similar patterns of gene and metabolite deregulation, while transgenic lines expressing CEs belonging to the same family showed different groups of different genes and metabolites deregulated. Thus, our results suggest that functional annotation of effectors based on sequence similarity may be misleading.

plant biology↗

bioRxiv2020

Optimization of SARS-CoV-2 detection by RT-QPCR without RNA extraction

Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study we evaluated the reliability and the efficiency of a direct RT-QPCR method (without RNA extraction) using SeeGene Allplex 2019-nCoV RT-QPCR and the influence of swab storage media composition on further viral detection. We show that SeeGenes assay provides similar efficiency as the RealStar(R) SARS-CoV-2 RT-PCR kit (Altona Diagnostics), and that RNA extraction is not necessary nor advantageous if samples are stored in UTM or molecular water but is recommended if samples are stored in saline solution and in Hanks medium.

molecular biology↗

bioRxiv2017

Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces.\n\nL. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to a lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

plant biology↗