Biology subjects

Flores-Sandoval, E.

Publications and source records attributed to Flores-Sandoval, E..

6 recordsLinked to original sources

bioRxiv2026

Neryl diphosphate-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha

Monoterpenes (C10) are a large group of specialized metabolites important for plant interactions with the environment. Their biosynthesis is well understood in seed plants, where geranyl diphosphate serves as the canonical substrate, but knowledge of monoterpene biosynthesis outside seed plants remains very limited. Here, we report neryl diphosphate (NPP)-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha. MpMTPSL2, a microbial-type terpene synthase, converts NPP into -phellandrene and D-limonene in vitro. CRISPR knockout lines showed reduced production of both monoterpenes, providing direct genetic evidence for its in planta function. MpCPT5, a cis-prenyltransferase (CPT) family member identified through co-expression with MpMTPSL2, was confirmed to encode NPP synthase, as its knockout plants abolished -phellandrene and D-limonene production. Subcellular localization analyses in protoplasts and stable transgenic plants demonstrated that both MpCPT5 and MpMTPSL2 localize to plastids, co-localizing across all cell types with markedly stronger signals in non-green plastids of oil-body cells. Consistent with this, expression of both genes under their respective promoters was nearly abolished in oil-body-deficient mutants and strongly upregulated in a gain-of-function line for oil-body formation. MpMTPSL2 and MpCPT5 are physically linked through a shared bidirectional promoter that drives their coexpression specific to oil body cells, forming a unique biosynthetic gene cluster whose coordinated expression is maintained by PRC2-mediated H3K27me3 repression. Phylogenetic analysis implies that NPP synthases in M. polymorpha and in flowering plants evolved independently from their respective long-chain CPT ancestors. These findings provide new insights into the mechanisms and evolution of monoterpene biosynthesis in non-seed plants. Significance statementMonoterpenes are a diverse group of specialized metabolites produced widely among land plants, yet our understanding of their biosynthesis outside seed plants remains limited. Here we report that in the liverwort Marchantia polymorpha, the non-canonical substrate neryl diphosphate is used for monoterpene biosynthesis. The functions of the monoterpene synthase gene MpMTPSL2 and the neryl diphosphate synthase gene MpCPT5 were demonstrated through CRISPR knockouts. These two genes are physically linked and share a bidirectional promoter. Promoter assays show both genes function in plastids within oil-body cells, revealing cell-type specificity. These findings shew new light on the mechanisms and evolution of monoterpene biosynthesis in non-seed plants.

plant biology↗

bioRxiv2025

Carbon availability acts via cytokinins to promote gemma cup formation in Marchantia polymorpha

Liverworts can clonally propagate by producing compact shoot structures called gemmae, which develop within basket-like structures known as gemma cups. It was previously reported in Marchantia nepalensis that carbon availability promotes gemma cup formation. However, the mechanisms by which carbon availability controls this process remains largely unexplored. To address this knowledge gap, we investigated how carbon promotes gemma cup formation using Marchantia polymorpha as a model species. Through a series of pharmacological and genetic experiments, we found that carbon availability promotes gemma cup formation by inducing the cytokinin pathway, thereby increasing the expression of MpGCAM1 and MpSTG, which encode two transcription factors involved in forming the basal floor of gemma cups. Indeed, our data show that cytokinins accumulate in marchantia thallus in response to sucrose and to high light treatments. In addition, constitutive induction of cytokinin signalling could overcome the repressive effect of low sucrose on gemma cup formation, whereas suppression of this hormonal pathway led to inhibition of sucrose-induced gemma cup formation. Furthermore, our results indicate that sucrose can induce gemma cup formation independently of KAI2A and MAX2, two molecular components of karrikin signalling known to control this developmental process by inducing cytokinin synthesis. Interestingly, in flowering plants, carbon availability also promotes cytokinin accumulation to induce axillary bud outgrowth, a process involving the transcription factors AtRAX and AtLOF1, the Arabidopsis thaliana orthologues of MpGCAM1 and MpSTG, respectively. Collectively, these observations indicate that the interactions between carbon and cytokinins are critical for the developmental plasticity of land plants in response to their environment.

plant biology↗

bioRxiv2025

Modulation of the GT Family 47 clade B gene affects arabinan deposition in elaters of Marchantia polymorpha

The plant cell wall polymer -1,5-linked arabinan is associated with important functions in plant physiology, such as cell wall flexibility and desiccation tolerance in angiosperms. However, its biosynthetic mechanism remains obscure. A putative arabinan arabinosyltransferase (AraT) is the Arabidopsis ARABINAN-DEFICIENT1 in GT family 47 clade B (GT47B), but its role is inferred by its mutant cell wall phenotypes, and not through in vitro assays. With seven other genes in the clade, investigating the function of the members in GT47B is hampered by the high genetic redundancy in Arabidopsis. Instead, we probe the function of the only two genes in GT47B in the liverwort model organism Marchantia polymorpha, named MpARAD-Like 1 (MpARADL1) and MpARADL2. Mparadl1 and Mparadl2 loss-of-function mutants and overexpression lines were generated, and their cell wall composition was probed using Comprehensive Microarray Polymer Profiling (CoMPP), glycosyl linkage analysis and immunolabelling. Interestingly, Mparadl2 mutants have much less of the 1,5--L-arabinan epitope in elaters since LM6 antibody recognition showed a notable reduction. However, 5-linked Araf levels in the Marchantia thallus tested with glycosyl linkage analysis are comparable between mutants and wild type, suggesting that there is another enzyme forming 5-Araf linkages in Marchantia. Our attempts to obtain the biochemical activity of these enzymes through the expression and purification of AtARAD1, MpARADL1 and MpARADL2 proteins in the HEK293 cell heterologous expression system were unsuccessful. Therefore, we suspect that an evolutionarily conserved, and clade-specific mechanism is required to confer solubility for purification and subsequent activity characterisation of GT47B proteins. Previous studies have shown that co-expression of protein interaction partners can enhance protein solubility in the HEK293 cell system, but co-expression of MpARADL2 with AtARAD1 and MpARADL1 did not yield soluble proteins, prompting further investigation into the protein-protein interactors of GT47B proteins.

plant biology↗

bioRxiv2025

The Building Blocks of Early Land Plants: Glycosyltransferases and Cell Wall Architecture in the model liverwort Marchantia polymorpha

The liverwort Marchantia polymorpha has emerged as an important plant model for developmental studies and may become central to elucidate the complex process of cell wall polysaccharide biosynthesis. This study comprehensively analyses the composition and structure of cell wall glycans across eight different M. polymorpha tissue types. We show that while the cell walls largely mirror known land plant cell wall composition, they also exhibit some unique characteristics. For example, -(1,5)-arabinan was prominently present in the sporophyte tissue, which may indicate a specialised role in this life stage. Furthermore, M. polymorpha cell walls displayed a remarkably low overall pectin content, yet the abundance of pectic arabinan in sporophytes hint at its putative role in the evolution and complexity of spermatophyte cell walls. Through comparative analyses of glycosyltransferase (GT) families across plant species, we found that M. polymorpha has a diversified GT repertoire compared for example, to the model plant Arabidopsis thaliana, indicating uniqueness in its cell wall biosynthesis pathways. To support research underpinning cell wall biosynthesis, we developed a Gateway compatible compendium of 93 M. polymorpha GTs, providing a valuable resource for genetic and functional studies. Our study thus works as a foundation to drive new insights into cell wall evolution, structure and function across the plant kingdom.

plant biology↗

bioRxiv2024

The MpANT-auxin loop modulates Marchantia polymorpha development

AINTEGUMENTA-LIKE/PLETHORA (APB) genes are considered part of the ancestral developmental toolkit in land plants. In Arabidopsis thaliana, these transcription factors are induced by auxin and are primarily expressed in tissues with actively dividing cells, where they play essential roles in organ development. Marchantia polymorpha, a liverwort that diverged from A. thaliana early in embryophyte evolution, possesses a single APB ortholog, MpAINTEGUMENTA (MpANT), encoded in its genome. In this study, we aimed to characterize the function of MpANT. Analysis of a transcriptional fusion line indicates that MpANT is predominantly expressed in the meristematic region. We report that the MpANT promoter region contains several cis-acting Auxin Responsive Elements (AREs) and demonstrate that its expression, which occurs predominantly in meristematic regions, is significantly altered by addition of exogenous auxin and inhibition of auxin transport. These findings indicate that MpANT acts downstream of Auxin Response Factors (ARFs) and auxin signaling. Analyses of loss- and gain-of-function MpANT alleles highlight the importance of this transcription factor in meristem maintenance and cell proliferation. Additionally, we found that MpANT acts upstream of the auxin transporter MpPIN1 by influencing auxin distribution. Taken together, our findings reveal a feedforward regulatory loop involving auxin, MpANT, and MpPIN1 that is important for Marchantia development.

plant biology↗

bioRxiv2023

Control of cell fate specification and patterning by an ancestral microRNA

The formation of an organized body requires the establishment and maintenance of cells with structural and functional distinctive characteristics. A central question in developmental biology is how changes in the regulation of genes drive cell specification and patterning1. microRNAs (miRNAs) are small non-coding RNAs that regulate development through mRNA cleavage and/or translational repression2. In plants, miRNAs regulate key aspects including growth, development, stem cell maintenance, vegetative phase change, leaf morphogenesis, floral organ formation and flowering time3. Biogenesis of plant miRNAs depends on the activity of DICER-LIKE 1 (DCL1), an RNase type III endonuclease that processes double stranded RNA to give rise to mature miRNAs 4. The genomes of todays flora contain at least one bona fide copy of DCL1 5,6. Using Marchantia polymorpha -a model bryophyte that allows comparative approaches to infer characteristics of the ancestral land plant-, we demonstrate that MpDCL1a is required for the biogenesis of miRNAs and uncovered a central role for miR166/Homeodomain Zipper Class III-regulated auxin synthesis in the specification of cell identity, patterning, meristem function, laminar expansion and the development of the body in the last common ancestor of extant land plants.

plant biology↗