Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

Membrane-anchored calpains - hidden regulators of growth and development beyond plants?

Calpains are modulatory proteases that modify diverse cellular substrates and play essential roles in eukaryots. The best studied are animal cytosolic calpains. Here, we focus on enigmatic membrane-anchored calpains, their structural and functional features as well as phylogenetic distribution. Based on domain composition, we identified four types of membrane-anchored calpains. Type 1 and 2 show broad phylogenetic distribution among unicellular protists and streptophytes suggesting their ancient evolutionary origin. Type 3 and 4 diversified early and are present in brown algae and oomycetes. The plant DEK1 protein is the only representative of membrane-anchored calpains that has been functionally studied. Here, we present up to date knowledge about its structural features, putative regulation, posttranslational modifications, and biological role. Finally, we discuss potential model organisms and available tools for functional studies of membrane-anchored calpains with yet unknown biological role. Mechanistic understanding of membrane-anchored calpains may provide important insights into fundamental principles of cell polarization, cell fate control, and morphogenesis beyond plants.

Challenges of in vivo protein localization in plants seen through the DEK1 protein lens.

During the last 25 y, fluorescent protein tagging has become a tool of choice to investigate protein function in a cellular context. The information gathered with this approach is not only providing insights into protein subcellular localization but also allows contextualizing protein function in multicellular settings. Here we illustrate the power of this method by commenting on the recent successful localization of the large membrane DEK1 protein during three-dimensional body formation in the moss Physcomitrella patens. But as many approaches, protein tagging is not exempt of caveats. The multiple infructuous (failed) attempts to detect DEK1 using a fluorescent protein tag present a good overview of such potential problems. Here we discuss the insertion of different fluorescent proteins at different positions in the PpDEK1 protein and the resulting unintended range of mutant phenotypes. Albeit none of these mutants generated a detectable fluorescent signal they can still provide interesting biological information about DEK1 function.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients.

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

Mesostigma viride Genome and Transcriptome Provide Insights into the Origin and Evolution of Streptophyta.

The Streptophyta include unicellular and multicellular charophyte green algae and land plants. Colonization of the terrestrial habitat by land plants is a major evolutionary event that has transformed the planet. So far, lack of genome information on unicellular charophyte algae hinders the understanding of the origin and the evolution from unicellular to multicellular life in Streptophyta. This work reports the high-quality reference genome and transcriptome of Mesostigma viride, a single-celled charophyte alga with a position at the base of Streptophyta. There are abundant segmental duplications and transposable elements in M. viride, which contribute to a relatively large genome with high gene content compared to other algae and early diverging land plants. This work identifies the origin of genetic tools that multicellular Streptophyta have inherited and key genetic innovations required for the evolution of land plants from unicellular aquatic ancestors. The findings shed light on the age-old questions of the evolution of multicellularity and the origin of land plants.

DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth.

Defective Kernel 1 (DEK1) is genetically at the nexus of the 3D morphogenesis of land plants. We aimed to localize DEK1 in the moss Physcomitrella patens to decipher its function during this process. To detect DEK1 in vivo, we inserted the tdTomato fluorophore into PpDEK1 gene locus. Confocal microscopy coupled with the use of time-gating allowed the precise DEK1 subcellular localization during 3D morphogenesis. DEK1 localization displays a strong polarized signal, as it is restricted to the plasma membrane domain between recently divided cells during the early steps of 3D growth development as well as during the subsequent vegetative growth. The signal furthermore displays a clear developmental pattern because it is only detectable in recently divided and elongating cells. Additionally, DEK1 localization appears to be independent of its calpain domain proteolytic activity. The DEK1 polar subcellular distribution in 3D tissue developing cells defines a functional cellular framework to explain its role in this developmental phase. Also, the observation of DEK1 during spermatogenesis suggests another biological function for this protein in plants. Finally the DEK1-tagged strain generated here provides a biological platform upon which further investigations into 3D developmental processes can be performed.

An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing.

Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5' end of the intron and preserve the nucleotide sequence of the targeted intron.

The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens.

The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain active-site amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site.

The phenotype of the CRINKLY4 deletion mutant of Physcomitrella patens suggests a broad role in developmental regulation in early land plants.

MAIN CONCLUSIONS: Deletion of the ancestral gene of the land plant multigene family of receptor like kinase CR4 in Physcomitrella patens demonstrates involvement in developmental control of gametophytic and sporophytic organs. The CRINKLY4 (CR4) family of receptor kinases in angiosperms consists of three clades, one including CR4, the CR4-related CCR1 and CCR2, a second including CCR3 and CCR4 family members, and a third and more distant clade. In addition to crinkly leaves in maize, which gave rise to the mutant gene name, CR4 is implicated in ovule, embryo, flower and root development in Arabidopsis thaliana. In root tips of the same species the module including a CLAVATA3/ESR-related protein, an Arabidopsis CR4, a CLAVATA1 and a WUSCHEL-related homeobox 5 (CLE40-ACR4-CLV1-WOX5) is implicated in meristem cell regulation. In embryos and shoots, CR4 acts together with A. thaliana MERISTEM LAYER 1 and PROTODERMAL FACTOR 2 to promote A. thaliana epidermis differentiation. Phylogenetic analysis has demonstrated that early land plants, e.g. mosses carry a single ancestral CR4 gene, together with genes encoding the other members of the CLE40-ACR4-CLV1-WOX5 signaling module. Here we show that CR4 serves as a broad regulator of morphogenesis both in gametophyte phyllids, archegonia and in sporophyte epidermis of the moss Physcomitrella patens. The phenotype of the CR4 deletion mutant in moss provides insight into the role of the ancestral CR4 gene as a regulator of development in early land plants.

DEK1; missing piece in puzzle of plant development.

Patterning of land plant bodies is determined by positioning of cell walls. A crucial event in land plant evolution was the ability to utilize spatial information to direct cell wall deposition. Recent studies of DEK1 in Physcomitrella patens support a role for DEK1 in position dependent cell wall orientation.

Genetic analysis of DEFECTIVE KERNEL1 loop function in three-dimensional body patterning in Physcomitrella patens.

DEFECTIVE KERNEL1 (DEK1) of higher plants plays an essential role in position-dependent signaling and consists of a large transmembrane domain (MEM) linked to a protease catalytic domain and a regulatory domain. Here, we show that the postulated sensory Loop of the MEM domain plays an important role in the developmental regulation of DEK1 activity in the moss Physcomitrella patens. Compared with P. patens lacking DEK1 (∆dek1), the dek1∆loop mutant correctly positions the division plane in the bud apical cell. In contrast with an early developmental arrest of ∆dek1 buds, dek1∆loop develops aberrant gametophores lacking expanded phyllids resulting from misregulation of mitotic activity. In contrast with the highly conserved sequence of the protease catalytic domain, the Loop is highly variable in land plants. Functionally, the sequence from Marchantia polymorpha fully complements the dek1∆loop phenotype, whereas sequences from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) give phenotypes with retarded growth and affected phyllid development. Bioinformatic analysis identifies MEM as a member of the Major Facilitator Superfamily, membrane transporters reacting to stimuli from the external environment. Transcriptome analysis comparing wild-type and ∆dek1 tissues identifies an effect on two groups of transcripts connected to dek1 mutant phenotypes: transcripts related to cell wall remodeling and regulation of the AINTEGUMENTA, PLETHORA, and BABY BOOM2 (APB2) and APB3 transcription factors known to regulate bud initiation. Finally, sequence data support the hypothesis that the advanced charophyte algae that evolved into ancestral land plants lost cytosolic calpains, retaining DEK1 as the sole calpain in the evolving land plant lineage.

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (∆dek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

Massive expansion of the calpain gene family in unicellular eukaryotes.

BACKGROUND: Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. RESULTS: Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. CONCLUSIONS: The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

Gabaculine alters plastid development and differentially affects abundance of plastid-encoded DPOR and nuclear-encoded GluTR and FLU-like proteins in spruce cotyledons.

Synthesis of 5-aminolevulinic acid (ALA) represents a rate limiting step in the tetrapyrrole biosynthetic pathway, and is regulated by metabolic feedback control of glutamyl-tRNA reductase (GluTR) activity. The FLU protein has been attributed to this regulation. Later in the biosynthetic pathway, reduction of protochlorophyllide (Pchlide), catalyzed by protochlorophyllide oxidoreductase (POR), ensures another important regulatory step in the chlorophyll biosynthesis. In the present work, we investigated the expression and cellular abundance of nuclear-encoded and plastid-encoded proteins involved in ALA synthesis and Pchlide reduction in Norway spruce (Picea abies L. Karst.) as a representative of plant species with high ability to synthesize chlorophyll in the dark. Using dark-grown, light/dark-grown and gabaculine-treated seedlings, we demonstrated that gabaculine-impaired etiochloroplast and chloroplast development has no negative effect on GluTR accumulation in the cotyledons. However, in contrast to control plants, the relative amount of GluTR was similar both in the dark-grown and light/dark-grown gabaculine-treated seedlings. We identified a partial sequence of the FLU-like gene in Norway spruce, and using antibodies against the FLU-like protein (FLP), we showed that FLP accumulated mostly in the dark-grown control seedlings and gabaculine-treated seedlings. In contrast to nuclear-encoded GluTR and FLP, accumulation of plastid-encoded light-independent POR (DPOR) was sensitive to gabaculine treatment. The levels of DPOR subunits were substantially lower in the light/dark-grown control seedlings and gabaculine-treated seedlings, although the corresponding genes chlL, chlN and chlB were expressed. Since we analyzed the samples with different plastid types, plastid ultrastructure and physiological parameters like Pchlide and chlorophyll contents, in vivo chlorophyll fluorescence and photosynthetic efficiency of the seedlings were characterized. Apart from etiochloroplast-specific accumulation of the DPOR subunits, we described, in some detail, additional specific features of chlorophyll biosynthesis in the spruce seedlings that differ from those known in angiosperms.

Trap closure and prey retention in Venus flytrap (Dionaea muscipula) temporarily reduces photosynthesis and stimulates respiration.

BACKGROUND AND AIMS: The carnivorous plant Venus flytrap (Dionaea muscipula) produces a rosette of leaves: each leaf is divided into a lower part called the lamina and an upper part, the trap, with sensory trigger hairs on the adaxial surface. The trap catches prey by very rapid closure, within a fraction of a second of the trigger hairs being touched twice. Generation of action potentials plays an important role in closure. Because electrical signals are involved in reduction of the photosynthetic rate in different plant species, we hypothesized that trap closure and subsequent movement of prey in the trap will result in transient downregulation of photosynthesis, thus representing the energetic costs of carnivory associated with an active trapping mechanism, which has not been previously described. METHODS: Traps were enclosed in a gas exchange cuvette and the trigger hairs irritated with thin wire, thus simulating insect capture and retention. Respiration rate was measured in darkness (RD). In the light, net photosynthetic rate (AN), stomatal conductance (gs) and intercellular CO2 concentration (ci) were measured, combined with chlorophyll fluorescence imaging. Responses were monitored in the lamina and trap separately. KEY RESULTS: Irritation of trigger hairs resulted in decreased AN and increased RD, not only immediately after trap closure but also during the subsequent period when prey retention was simulated in the closed trap. Stomatal conductance remained stable, indicating no stomatal limitation of AN, so ci increased. At the same time, the effective quantum yield of photosystem II (PSII) decreased transiently. The response was confined mainly to the digestive zone of the trap and was not observed in the lamina. Stopping mechanical irritation resulted in recovery of AN, RD and PSII. CONCLUSIONS: We put forward the first experimental evidence for energetic demands and carbon costs during insect trapping and retention in carnivorous plants, providing a new insight into the cost/benefit model of carnivory.

A novel insight into the regulation of light-independent chlorophyll biosynthesis in Larix decidua and Picea abies seedlings.

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment-protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.

Feeding enhances photosynthetic efficiency in the carnivorous pitcher plant Nepenthes talangensis.

BACKGROUND AND AIMS: Cost-benefit models predict that carnivory can increase the rate of photosynthesis (A(N)) by leaves of carnivorous plants as a result of increased nitrogen absorption from prey. However, the cost of carnivory includes decreased A(N) and increased respiration rates (R(D)) of trapping organs. The principal aim of the present study was to assess the costs and benefits of carnivory in the pitcher plant Nepenthes talangensis, leaves of which are composed of a lamina and a pitcher trap, in response to feeding with beetle larvae. METHODS: Pitchers of Nepenthes grown at 200 micromol m(-2) s(-1) photosynthetically active radiation (PAR) were fed with insect larvae for 2 months, and the effects on the photosynthetic processes were then assessed by simultaneous measurements of gas exchange and chlorophyll fluorescence of laminae and pitchers, which were correlated with nitrogen, carbon and total chlorophyll concentrations. KEY RESULTS: A(N) and maximum (F(v)/F(m)) and effective quantum yield of photosystem II (Phi(PSII)) were greater in the fed than unfed laminae but not in the fed compared with unfed pitchers. Respiration rate was not significantly affected in fed compared with unfed plants. The unfed plants had greater non-photochemical quenching (NPQ) of chlorophyll fluorescence. Higher NPQ in unfed lamina did not compensate for their lower Phi(PSII), resulting in lower photochemical quenching (QP) and thus higher excitation pressure on PSII. Biomass and nitrogen and chlorophyll concentration also increased as a result of feeding. The cost of carnivory was shown by lower A(N) and Phi(PSII) in pitchers than in laminae, but R(D) depended on whether it was expressed on a dry weight or a surface area basis. Correlation between nitrogen and A(N) in the pitchers was not found. Cost-benefit analysis showed a large beneficial effect on photosynthesis from feeding as light intensity increased from 200 to 1000 micromol m(-2) s(-1) PAR after which it did not increase further. All fed plants began to flower. CONCLUSION: Feeding pitchers with insect larvae increases A(N) of leaf laminae, due to higher nutrient acquisition, with strong correlation with nitrogen concentration, but A(N) of pitchers does not increase, despite increased nitrogen concentration in their tissue. Increased A(N) improves growth and reproduction and is likely to increase the competitive advantage of carnivorous over non-carnivorous plants in nutrient-poor habitats.