The Cell Wall Proteome of Marchantia polymorpha Reveals Specificities Compared to Those of Flowering Plants

Primary plant cell walls are composite extracellular structures composed of three major classes of polysaccharides (pectins, hemicelluloses, and cellulose) and of proteins. The cell wall proteins (CWPs) play multiple roles during plant development and in response to environmental stresses by remodeling the polysaccharide and protein networks and acting in signaling processes. To date, the cell wall proteome has been mostly described in flowering plants and has revealed the diversity of the CWP families. In this article, we describe the cell wall proteome of an early divergent plant, Marchantia polymorpha, a Bryophyte which belong to one of the first plant species colonizing lands. It has been possible to identify 410 different CWPs from three development stages of the haploid gametophyte and they could be classified in the same functional classes as the CWPs of flowering plants. This result underlied the ability of M. polymorpha to sustain cell wall dynamics. However, some specificities of the M. polymorpha cell wall proteome could be highlighted, in particular the importance of oxido-reductases such as class III peroxidases and polyphenol oxidases, D-mannose binding lectins, and dirigent-like proteins. These proteins families could be related to the presence of specific compounds in the M. polymorpha cell walls, like mannans or phenolics. This work paves the way for functional studies to unravel the role of CWPs during M. polymorpha development and in response to environmental cues.

Circadian and diel regulation of photosynthesis in the bryophyte Marchantia polymorpha

Circadian rhythms are 24-hour biological cycles that align metabolism, physiology and development with daily environmental fluctuations. Photosynthetic processes are governed by the circadian clock in both flowering plants and cyanobacteria, but it is unclear how extensively this is conserved throughout the green lineage. We investigated the contribution of circadian regulation to photochemistry in Marchantia polymorpha, a liverwort that diverged from flowering plants early in the evolution of land plants. First, we identified in M. polymorpha the circadian regulation of several measures of photosynthetic biochemistry (delayed fluorescence, the rate of photosynthetic electron transport, and non-photochemical quenching of chlorophyll fluorescence). Second, we identified that light-dark cycles increase the robustness of the 24 h cycles of photosynthesis in M. polymorpha, which might be due to the masking of underlying circadian rhythms of photosynthesis by light-dark cycles. Finally, we used a pharmacological approach to identify that chloroplast translation might be necessary for clock control of light harvesting in M. polymorpha. We infer that the circadian regulation of photosynthesis might be well-conserved amongst terrestrial plants.

Improved clearing method contributes to deep imaging of plant organs

Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain.

Phytochemical Profile and Anticancer Potential of Endophytic Microorganisms from Liverwort Species, Marchantia polymorpha L.

Liverwort endophytes could be a source of new biologically active substances, especially when these spore-forming plants are known to produce compounds that are not found in other living organisms. Despite the significant development of plant endophytes research, there are only a few studies describing liverwort endophytic microorganisms and their metabolites. In the presented study, the analysis of the volatile compounds obtained from thallose liverwort species, Marchantia polymorpha L., and its endophytes was carried out. For this purpose, non-polar extracts of plant material and symbiotic microorganisms were obtained. The extracts were analyzed using gas chromatography coupled to mass spectrometry. Compounds with the structure of diketopiperazine in the endophyte extract were identified. Liverwort volatile extract was a rich source of cuparane-, chamigrane-, acorane-, and thujopsane-type sesquiterpenoids. The cytotoxicity of ethyl acetate extracts from endophytic microorganisms was evaluated on a panel of cancer (FaDu, HeLa, and SCC-25) cell lines and normal (VERO), and revealed significant anticancer potential towards hypopharyngeal squamous cell carcinoma and cervical adenocarcinoma.

Functions of COP1/SPA E3 Ubiquitin Ligase Mediated by MpCRY in the Liverwort Marchantia Polymorpha under Blue Light

COP1/SPA1 complex in Arabidopsis inhibits photomorphogenesis through the ubiquitination of multiple photo-responsive transcription factors in darkness, but such inhibiting function of COP1/SPA1 complex would be suppressed by cryptochromes in blue light. Extensive studies have been conducted on these mechanisms in Arabidopsis whereas little attention has been focused on whether another branch of land plants bryophyte utilizes this blue-light regulatory pathway. To study this problem, we conducted a study in the liverwort Marchantia polymorpha and obtained a MpSPA knock-out mutant, in which Mpspa exhibits the phenotype of an increased percentage of individuals with asymmetrical thallus growth, similar to MpCRY knock-out mutant. We also verified interactions of MpSPA with MpCRY (in a blue light-independent way) and with MpCOP1. Concomitantly, both MpSPA and MpCOP1 could interact with MpHY5, and MpSPA can promote MpCOP1 to ubiquitinate MpHY5 but MpCRY does not regulate the ubiquitination of MpHY5 by MpCOP1/MpSPA complex. These data suggest that COP1/SPA ubiquitinating HY5 is conserved in Marchantia polymorpha, but dissimilar to CRY in Arabidopsis, MpCRY is not an inhibitor of this process under blue light.

Functional Analyses of the Two Distinctive Types of Two-Pore Channels (TPCs) and the Slow Vacuolar Channel in Marchantia polymorpha

The two-pore channel (TPC) family is widely conserved in eukaryotes. Many vascular plants, including Arabidopsis and rice, possess a single TPC gene which functions as a slow vacuolar (SV) channel—voltage-dependent cation-permeable channel located in the vacuolar membrane (tonoplast). On the other hand, a liverwort Marchantia polymorpha genome encodes three TPC homologs: MpTPC1 is similar to TPCs in vascular plants (type 1 TPC), while MpTPC2 and MpTPC3 are classified into a distinctive group (type 2 TPC). Phylogenetic analysis suggested that the type 2 TPC emerged before the land colonization in plant evolution and was lost in vascular plants and hornworts. All of the three MpTPCs were shown to be localized at the tonoplast. We generated knockout mutants of tpc1, tpc2, tpc3, and tpc2 tpc3 double mutant by CRISPR/Cas9 genome editing and performed patch-clamp analyses of isolated vacuoles. The SV channel activity was abolished in the Mptpc1 loss-of-function mutant (Mptpc1-1KO), while Mptpc2-1KO, Mptpc3-1KO, and Mptpc2-2/tpc3-2KO double mutant exhibited similar activity to the wild type, indicating that MpTPC1 (type 1) is solely responsible for the SV channel activity. Activators of mammalian TPC channels, PI(3,5)P2, and, NAADP, did not affect the ion channel activity of any MpTPCs. These results indicate that the type 1 TPCs, which are well conserved in all land plant species, encode the SV channel, while the type 2 TPCs likely encode other tonoplast cation channel(s) distinct from the SV channel and animal TPC channels.

Post-fire peatland vegetation recovery: A case study in open rich fens of the Canadian boreal forest

Fire plays a major role in structuring and the functioning of boreal ecosystems. As peatlands are important components of boreal forests, the impact of fire upon these wetter ecosystems is increasingly studied, but with the main focus on treed peatlands and Sphagnum-dominated bogs so far. Important fires occurring more frequently in the past decade in southern Northwest Territories (Canada) provide the opportunity to assess early post-fire vegetation regeneration in open rich fens (1, 2 and 5-years post-fire) and to better understand early recovery succession. We aimed to 1) evaluate whether and how open rich fens are affected by fire 2) describe short-term vegetation regeneration, for both bryophytes and vascular species. A shift was observed between pioneer bryophytes and brown mosses between the second and fifth-year post-fire. Vascular plants, especially slow-growing species and the ones reproducing mainly by seeds, recovered partially. The first bryophyte species recovering were pioneer species adapted to colonize burned environment such as Marchantia polymorpha or Ceratodon purpureus. For vascular plant species, the ones previously present and capable to regrowth rapidly from unburned plant structures (base of tussocks, rhizomes, roots) were represented by species like Betula glandulosa or Carex aquatilis. The wetter conditions and lower fuel availability of fen depressional biotopes were important factors controlling the resistance and regeneration of species associated with them.

Roles of a Cryptochrome in Carbon Fixation and Sucrose Metabolism in the Liverwort Marchantia polymorpha

In vascular plants, blue-light photoreceptors and cryptochromes contain a myriad of roles and functions to adapt to different ambient light conditions, while the roles of cryptochromes in bryophytes have been rarely reported. In this study, we investigated functions of a single-copy ortholog of cryptochrome (MpCRY) in the liverwort Marchantia polymorpha. Knock-out of MpCRY showed that a large number of the mutant plants exhibited asymmetric growth of thalli under blue light. Transcriptome analyses indicated that MpCRY is mainly involved in photosynthesis and sugar metabolism. Further physiological analysis showed that Mpcry mutant exhibited a reduction in CO2 uptake and sucrose metabolism. In addition, exogenous application of sucrose or glucose partially restored the symmetrical growth of the Mpcry mutant thalli. Together, these results suggest that MpCRY is involved in the symmetrical growth of thallus and the regulation of carbon fixation and sucrose metabolism in M. polymorpha.