As master transcription factors of stomatal development, SPEECHLESS, MUTE, and FAMA, collectively termed SMFs, are primary targets of molecular genetic analyses in the model plant Arabidopsis thaliana. Studies in other model systems identified SMF orthologs as key players in evolutionary developmental biology studies on stomata. However, recent studies on the astomatous liverwort Marchantia polymorpha revealed that the functions of these genes are not limited to the stomatal development, but extend to other types of tissues, namely sporophytic setal and gametophytic epidermal tissues. These studies provide insightful examples of gene-regulatory network co-opting, and highlight SMFs and related transcription factors as general toolkits for novel trait evolution in land plant lineages. Here, we critically review recent literature on the SMF-like gene in M. polymorpha and discuss their implications for plant evolutionary biology.

Land plant bodies develop from stem cells located in meristems. However, we know little about how meristems initiate from non-meristematic cells. The haploid body of bryophytes develops from unicellular spores in isolation from the parental plant, which allows all stages of development to be observed. We discovered that the Marchantia spore undergoes a series of reproducibly oriented cell divisions to generate a flat prothallus on which a meristem later develops de novo. The young sporeling comprises an early cell mass. One cell of the early cell mass elongates and undergoes a formative division that produces the prothalloblast, which initiates prothallus formation. A symmetric division of the prothalloblast followed by two transverse divisions generates a four-celled plate that expands into a flat disc through oblique divisions in three of the four plate-cell-derived quadrants. One quadrant gives rise to a flat flabellum. A notch with a meristem and apical stem cell develops at the margin of the flabellum. The transcription factor Marchantia class III homeodomain-leucine-zipper (MpC3HDZ) is a marker of the first flat prothallus structure and polarizes to the dorsal tissues of flabella and meristems. Mpc3hdz mutants are defective in setting up dorsoventrality and thallus body flatness. We report how a regular set of cell divisions forms the prothallus-the first dorsoventral structure-and how cells on the margin of the prothallus develop a dorsoventralized meristem de novo.

Manganese (Mn) is considered as an essential element for plant growth. Mn starvation has been shown to affect photosystem II, the site of the Mn4CaO5 cluster responsible for water oxidation. Less is known on the effect of Mn starvation on photosystem I. Here we studied the effects of Mn deficiency in vivo on redox changes of P700 and plastocyanin (Pc) in the liverwort Marchantia polymorpha using the KLAS-NIR spectrophotometer. Far-red illumination is used to excite preferentially photosystem I, thus facilitating cyclic electron transport. Under Mn starvation, we observed slower oxidation of P700 and a decrease in the Pc signal relative to P700. The lower Pc content under Mn deficiency was confirmed by western blots. Re-reduction kinetics of P700+ and Pc+ were faster in Mn deficient thalli than in the control. The above findings show that the kinetics studied under Mn deficiency not only depend on the number of available reductants but also on how quickly electrons are transferred from stromal donors via the intersystem chain to Pc+ and P700+. We suggest that under Mn deficiency a structural reorganization of the thylakoid membrane takes place favoring the formation of supercomplexes between ferredoxin, cytochrome b6f complex, Pc and photosystem I, and thus an enhanced cyclic electron transport.

The endomembrane system in plants is composed of interconnected membrane organelles that contribute to intracellular structure and function. These organelles include the endoplasmic reticulum (ER), Golgi apparatus, vacuole, trans-Golgi network, and prevacuolar compartment or multivesicular body. Through vesicle-mediated transport, secreted proteins are synthesized in the ER and subsequently transported along the secretory pathway to the vacuole or outside of cells to fulfill specialized functions. Genetic screening is a crucial method for studying plant protein secretion. It entails identifying phenotypic differences resulting from genetic mutations, such as ethyl methanesulfonate, T-DNA insertion, and RNAi, to investigate gene function and discover mutants with specific traits or gene functions. Significant progress has been achieved in the study of plant protein secretion through genetic screening. In this protocol, we provide a step-by-step guide to studying the protein secretion pathway using a genetic screen approach. We use the example of the free 1 suppressor of Arabidopsis thaliana and oil body mutants of Marchantia polymorpha. Additionally, we offer an overview of genetic screening and briefly summarize the emerging technologies in the field of protein secretion research.

The conquest of land posed severe problems to plants which they had to cope with by adapting biosynthetic capacities. Adaptations to respond to UV irradiation, water loss, pathogen and herbivore defense, and the earth\'s pull were essential. Chemical compounds alleviating these problems can be synthesized by the phenylpropanoid pathway, the core of which are three enzymes: phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase, and 4-coumaric acid coenzyme A-ligase (4CL). The genomes of model organisms, Chara braunii as aquatic alga and the two bryophytes Physcomitrium patens and Marchantia polymorpha, were searched for sequences encoding PAL and 4CL and selected sequences heterologously expressed in Escherichia coli for biochemical characterization. Several possible isoforms were identified for both enzymes in Marchantia polymorpha and Physcomitrium patens, while only one or two isoforms could be retrieved for Chara braunii. Active forms of both enzymes were found in all three organisms, although the catalytic efficiencies varied in a wide range. l-Phenylalanine was accepted as best substrate by all PAL-like enzymes, despite annotations in some cases suggesting different activities. The substrate spectrum of 4CLs was more diverse, but caffeic and/or 4-coumaric acids generally were the best-accepted substrates. Our investigations show that PAL and 4CL, important enzymes for the formation of phenolic compounds, are present and active in extant charophytes and bryophytes as model organisms for the conquest of land.

The auxin signaling molecule controls a variety of growth and developmental processes in land plants. Auxin regulates gene expression through a nuclear auxin signaling pathway (NAP) consisting of the ubiquitin ligase auxin receptor TIR1/AFB, its Aux/IAA degradation substrate, and DNA-binding ARF transcription factors. Although extensive qualitative understanding of the pathway and its interactions has been obtained, mostly by studying the flowering plant Arabidopsis thaliana, it remains unknown how these translate to quantitative system behavior in vivo, a problem that is confounded by the large NAP gene families in most species. Here, we used the minimal NAP of the liverwort Marchantia polymorpha to quantitatively map NAP protein accumulation and dynamics in vivo through the use of knockin fluorescent fusion proteins. Beyond revealing the dynamic native accumulation profile of the entire NAP protein network, we discovered that the two central ARFs, MpARF1 and MpARF2, are proteasomally degraded. This auxin-independent degradation tunes ARF protein stoichiometry to favor gene activation, thereby reprogramming auxin response during the developmental progression. Thus, quantitative analysis of the entire NAP has enabled us to identify ARF degradation and the stoichiometries of activator and repressor ARFs as a potential mechanism for controlling gemma germination.

Meristems are crucial for organ formation, but our knowledge of their molecular evolution is limited. Here, we show that AINTEGUMENTA (MpANT) in the euANT branch of the APETALA2-like transcription factor family is essential for meristem development in the nonvascular plant Marchantia polymorpha. MpANT is expressed in the thallus meristem. Mpant mutants show defects to maintain meristem identity and undergo meristem duplication, while MpANT overexpressers show ectopic thallus growth. MpANT directly upregulates MpGRAS9 in the SHORT-ROOT (SHR) branch of the GRAS family. In the vascular plant Arabidopsis thaliana, the euANT-branch genes PLETHORAs (AtPLTs) and AtANT are involved in the formation and maintenance of root/shoot apical meristems and lateral organ primordia, and AtPLTs directly target SHR-branch genes. In addition, euANTs bind through a similar DNA-binding motif to many conserved homologous genes in M. polymorpha and A. thaliana. Overall, the euANT pathway has an evolutionarily conserved role in meristem development.

Foliar pigmentation patterns vary among plant species and growth conditions. In this study, we utilize hyperspectral imaging to assess foliar pigmentation in the bryophyte Marchantia polymorpha under nutrient stress and identify associated genetic factors. Using singular value decomposition (SVD) for feature selection, we quantitate color variations induced by deficiencies in phosphate, nitrate, magnesium, calcium, and iron. Pseudo-colored thallus images show that disrupting MpWRKY10 causes irregular pigmentation with auronidin accumulation. Transcriptomic profiling shows that MpWRKY10 regulates phenylpropanoid pathway enzymes and R2R3-MYB transcription factors during phosphate deficiency, with MpMYB14 upregulation preceding pigment accumulation. MpWRKY10 is downregulated in older, pigmented thalli under phosphate deficiency but maintained in young thalli, where it suppresses pigmentation genes. This downregulation is absent in pigmented thalli due to aging. Comparative transcriptome analysis suggests similar WRKY and MYB roles in nutrient response and pigmentation in red-leaf lettuce, alluding to conserved genetic factors controlling foliar pigmentation patterns under nutrient deficiency.

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.

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