ABSTRACT
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.
Subject(s)
Bryopsida , Bryopsida/genetics , Calpain/genetics , Cell Membrane , Plant Proteins/geneticsABSTRACT
High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.
Subject(s)
Bryopsida/genetics , Datasets as Topic , Genes, Plant/genetics , Chromosome Mapping , Genome, Plant/genetics , High-Throughput Nucleotide Sequencing , Transcriptome/geneticsABSTRACT
The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.
Subject(s)
Biological Evolution , Bryopsida/genetics , Chromosomes, Plant , Genome, Plant , Centromere , Chromatin/genetics , DNA Methylation , DNA Transposable Elements , Genetic Variation , Polymorphism, Single Nucleotide , Recombination, Genetic , SyntenyABSTRACT
In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gß and Gγ genes, but no canonical Gα Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gß proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGß2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGß2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.
Subject(s)
Bryopsida/genetics , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein beta Subunits/genetics , Plant Proteins/genetics , Amino Acid Sequence , Bryopsida/growth & development , Bryopsida/metabolism , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/metabolism , Gametogenesis, Plant/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Germ Cells, Plant/growth & development , Heterotrimeric GTP-Binding Proteins/classification , Heterotrimeric GTP-Binding Proteins/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , Life Cycle Stages/genetics , Microscopy, Fluorescence , Mutation , Phylogeny , Plant Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction/geneticsABSTRACT
Early land plants like moss Physcomitrella patens have developed remarkable drought tolerance. Phytohormone abscisic acid (ABA) protects seeds during water stress by activating genes through transcription factors such as ABSCISIC ACID INSENSITIVE (ABI3). Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are key gene regulators in eukaryotes, playing critical roles in stress tolerance in plants. Combining next-generation sequencing and computational analysis, we profiled and characterized sncRNA species from two ABI3 deletion mutants and the wild type P. patens that were subject to ABA treatment in dehydration and rehydration stages. Small RNA profiling using deep sequencing helped identify 22 novel miRNAs and 6 genomic loci producing trans-acting siRNAs (ta-siRNAs) including TAS3a to TAS3e and TAS6. Data from degradome profiling showed that ABI3 genes (ABI3a/b/c) are potentially regulated by the plant-specific miR536 and that other ABA-relevant genes are regulated by miRNAs and ta-siRNAs. We also observed broad variations of miRNAs and ta-siRNAs expression across different stages, suggesting that they could potentially influence desiccation tolerance. This study provided evidence on the potential roles of sncRNA in mediating desiccation-responsive pathways in early land plants.
Subject(s)
Bryopsida/genetics , RNA, Small Untranslated/genetics , Abscisic Acid , Arabidopsis/genetics , Desiccation/methods , Gene Expression Regulation, Plant/genetics , Genes, Plant/genetics , MicroRNAs/genetics , Plant Growth Regulators/genetics , Plant Proteins/genetics , RNA, Small Interfering/genetics , Seeds/genetics , Transcription Factors/genetics , Transcriptional Activation/geneticsABSTRACT
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.
Subject(s)
Bryopsida/genetics , Gene Deletion , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Plant Proteins/genetics , Bryopsida/growth & development , Bryopsida/ultrastructure , Germ Cells, Plant/growth & development , Germ Cells, Plant/metabolism , Germ Cells, Plant/ultrastructure , Microscopy, Confocal , Microscopy, Electron , Morphogenesis/genetics , Multigene Family , Phenotype , Plant Epidermis/genetics , Plant Epidermis/growth & development , Plant Epidermis/ultrastructure , Protein Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Seeds/genetics , Seeds/growth & development , Seeds/ultrastructureABSTRACT
Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation tolerance might have evolved in ancestral land plants before the separation of bryophytes and vascular plants.
Subject(s)
Abscisic Acid/metabolism , Adaptation, Physiological , Bryopsida/physiology , Droughts , Plant Proteins/metabolism , Transcription Factors/metabolism , Abscisic Acid/pharmacology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/physiology , Bryopsida/genetics , Bryopsida/metabolism , Desiccation , Gene Deletion , Mutation , Plant Growth Regulators/metabolism , Plant Proteins/genetics , Proteome/genetics , Proteome/metabolism , Proteomics , Seeds/metabolism , Transcription Factors/genetics , TranscriptomeABSTRACT
Novel developmental programs often evolve via cooption of existing genetic networks. To understand this process, we explored cooption of the TAS3 tasiRNA pathway in the moss Physcomitrella patens. We find an ancestral function for this repeatedly redeployed pathway in the spatial regulation of a conserved set of Auxin Response Factors. In moss, this results in stochastic patterning of the filamentous protonemal tissue. Through modeling and experimentation, we demonstrate that tasiRNA regulation confers sensitivity and robustness onto the auxin response. Increased auxin sensitivity parallels increased developmental sensitivity to nitrogen, a key environmental signal. We propose that the properties lent to the auxin response network, along with the ability to stochastically modulate development in response to environmental cues, have contributed to repeated cooption of the tasiRNA-ARF module during evolution. The signaling properties of a genetic network, and not just its developmental output, are thus critical to understanding evolution of multicellular forms.
Subject(s)
Gene Expression Regulation, Plant/genetics , Gene Regulatory Networks/genetics , Indoleacetic Acids/metabolism , RNA, Plant/genetics , RNA, Small Interfering/genetics , MicroRNAs/genetics , Plant Proteins/genetics , Signal Transduction/physiologyABSTRACT
The bryophytes are a morphologically and ecologically diverse group of plants that have recently emerged as major model systems for a variety of biological processes. In particular, the genome sequence of the moss, Physcomitrella patens, has significantly enhanced our understanding of the evolution of developmental processes in land plants. However, to fully explore the diversity within bryophytes, we need additional genomic resources. Here, we describe analyses of the transcriptomes of a male and a female isolate of the moss, Ceratodon purpureus, generated using the 454 FLX technology. Comparative analyses between C. purpureus and P. patens indicated that this strategy generated nearly complete coverage of the protonemal transcriptome. An analysis of the overlap in gene sets between C. purpureus and P. patens provides new insights into the evolution of gene family composition across the land plants. In spite of the overall transcriptomic similarity between the two species, Ka /Ks analysis of P. patens and C. purpureus suggests considerable physiological and developmental divergence. Additionally, while the codon usage was very similar between these two mosses, C. purpureus genes showed a slightly greater codon usage bias than P. patens genes potentially because of the contrasting mating system of the two species. Finally, we found evidence of a genome doubling ~65-76 MYA that likely coincided with the contemporaneous polyploidy event inferred for P. patens but postdates the divergence of P. patens and C. purpureus. The powerful laboratory tools now available for C. purpureus will enable the research community to fully exploit these genomic resources.
Subject(s)
Bryopsida/genetics , Gene Expression Profiling , Evolution, Molecular , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
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.
Subject(s)
Body Patterning , Bryopsida/genetics , Genes, Plant , Plant Proteins/genetics , Amino Acid Sequence , Molecular Sequence Data , Plant Proteins/chemistry , Plant Proteins/physiology , Sequence Homology, Amino AcidABSTRACT
One of the most common post-translational modifications is protein phosphorylation, which controls many activities of plant life. However, its role in the reprogramming of developmental pathways of plant cells remains elusive. Here, using Physcomitrella patens, we characterize the phospho-proteome for protonemata, protoplasts made therefrom, and protoplasts regenerated for 2d. Through a titanium dioxide (TiO2)-based phospho-peptide enrichment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS), more than 2000 phospho-proteins were identified. Among the 519 proteins with functional annotation in fresh protoplasts and protoplasts regenerated for 2d, proteins involved in epigenetic modification, post-transcriptional gene regulation, hormone signal transduction, and meristem maintenance have been previously reported to be important for developmental reprogramming. Several novel transcription factors including SWI/SNF complex protein, SNF2 family protein and MADS-domain transcription factor appear to be important in developmental reprogramming plant cells. Phosphorylation of marker proteins such as somatic embryogenesis receptor kinase and NAC transcription factor, suggests that this post-translational modification is vital for the cell's ability to adjust its developmental program. Together, our study presents a more complete understanding of the plant cell's developmental reprogramming. BIOLOGICAL SIGNIFICANCE: Protoplast regeneration is an ideal model system for investigating developmental reprogramming in plants. Here, for Physcomitrella patens, we characterize the phospho-proteome for protonemata, protoplasts made therefrom, and for protonemata regenerated from the protoplasts for 2d. Among the 519 proteins with functional annotation in fresh protoplasts and protoplasts regenerated for 2d, proteins involved in epigenetic modification, post-transcriptional gene regulation, hormone signal transduction, and meristem maintenance have been reported to be important for expression of developmental reprogramming. Together, our study presents a more complete understanding of the plant cell's developmental reprogramming.
Subject(s)
Bryopsida/metabolism , Peptides/metabolism , Phosphoproteins/metabolism , Plant Proteins/metabolism , Proteomics , Transcription Factors/metabolism , Bryopsida/genetics , Chromatography, Liquid , Mass Spectrometry , Peptides/genetics , Phosphoproteins/genetics , Plant Proteins/genetics , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Bryopsida/growth & development , Bryopsida/metabolism , Plant Proteins/metabolism , DNA, Complementary/genetics , Gene Deletion , Genetic Complementation Test , Germ Cells, Plant/growth & development , Germ Cells, Plant/metabolism , Molecular Sequence Data , Open Reading Frames/genetics , Phenotype , Plant Proteins/chemistry , Protein Structure, TertiaryABSTRACT
Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P. patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P. patens development and differentiation processes.
Subject(s)
Bryopsida/cytology , Bryopsida/metabolism , Plant Proteins/metabolism , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Animals , Arabidopsis/genetics , Bryopsida/genetics , Cell Adhesion , Cell Differentiation , Cell Polarity , Cell Wall/metabolism , Gene Knockdown Techniques , Genetic Complementation Test , Light , Mutation , Plant Proteins/genetics , Protein PrenylationABSTRACT
Sex chromosomes evolve from ordinary autosomes through the expansion and subsequent degeneration of a region of suppressed recombination that is inherited through one sex. Here we investigate the relative timing of these processes in the UV sex chromosomes of the moss Ceratodon purpureus using molecular population genetic analyses of eight newly discovered sex-linked loci. In this system, recombination is suppressed on both the female-transmitted (U) sex chromosome and the male-transmitted (V) chromosome. Genes on both chromosomes therefore should show the deleterious effects of suppressed recombination and sex-limited transmission, while purifying selection should maintain homologs of genes essential for both sexes on both sex chromosomes. Based on analyses of eight sex-linked loci, we show that the nonrecombining portions of the U and V chromosomes expanded in at least two events (~0.6-1.3 MYA and ~2.8-3.5 MYA), after the divergence of C. purpureus from its dioecious sister species, Trichodon cylindricus and Cheilothela chloropus. Both U- and V-linked copies showed reduced nucleotide diversity and limited population structure, compared to autosomal loci, suggesting that the sex chromosomes experienced more recent selective sweeps that the autosomes. Collectively these results highlight the dynamic nature of gene composition and molecular evolution on nonrecombining portions of the U and V sex chromosomes.
Subject(s)
Bryopsida/genetics , Evolution, Molecular , Genetic Variation , Selection, Genetic , Sex Chromosomes/genetics , Chromosome Mapping , Genetics, Population , Genotype , Mid-Atlantic Region , North Carolina , Recombination, Genetic/genetics , Time Factors , VirginiaABSTRACT
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 Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.
Subject(s)
Calpain/chemistry , Plant Proteins/chemistry , Plants/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/physiology , Calcium/metabolism , Calpain/genetics , Calpain/physiology , Charophyceae/genetics , Charophyceae/metabolism , Conserved Sequence , Evolution, Molecular , Genetic Complementation Test , Models, Molecular , Molecular Sequence Data , Mutagenesis , Plant Proteins/genetics , Plant Proteins/physiology , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, ProteinABSTRACT
Two Δ(12)-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from Physcomitrella patens and their functions identified. The open reading frames (ORFs) of PpFAD2-1 and PpFAD2-2 consisted of 1,128 bp and code for 375 amino acids. Their deduced polypeptides showed 62-64 % identity to microsomal Δ(12)-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. Yeast cells transformed with plasmid constructs containing PpFAD2-1 or PpFAD2-2 produced an appreciable amount of hexadecadienoic (16:2 Δ(9,12)) and linoleic acids (18:2 Δ(9,12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ(12)-desaturase enzymes. In addition, reduction of the growth temperature from 30 to 15 °C resulted in increased accumulation of unsaturated fatty acid products.
Subject(s)
Bryopsida/enzymology , Fatty Acid Desaturases/metabolism , Linoleic Acid/biosynthesis , Amino Acid Sequence , Bryopsida/genetics , Cloning, Molecular , Fatty Acid Desaturases/chemistry , Fatty Acid Desaturases/genetics , Fatty Acids, Unsaturated/biosynthesis , Molecular Sequence Data , Open Reading Frames , Sequence Homology, Amino AcidABSTRACT
The phytohormone ABA and the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1) function in protecting embryos during the desiccation stage of seed development. In a similar signaling pathway, vegetative tissue of the moss Physcomitrella patens survives desiccation by activating downstream genes (e.g. LEA1) in response to ABA and ABI3. We show that the PpLEA1 promoter responds to PpABI3 primarily through the ACTT-core element (5'-TCCACTTGTC-3'), while the ACGT-core ABA-responsive element (ABRE) appears to respond to ABA alone. We also found by yeast-two-hybrid screening that PpABI3A interacts with PpNF-YC1, a subunit of CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y). PpNF-YC1 increased the activation of the PpLEA1 promoter when incubated with PpABI3A, as did NF-YB, NF-YC, and ABI3 from Arabidopsis. This new response element (ACTT) is responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. These results further define the regulatory interactions at the transcriptional level for the expression of this network of genes required for drought/desiccation tolerance. This gene regulatory set is in large part conserved between vegetative tissue of bryophytes and seeds of angiosperms and will shed light on the evolution of this pathway in the green plant lineage.
Subject(s)
Abscisic Acid/metabolism , Bryopsida/genetics , CCAAT-Binding Factor/metabolism , Gene Expression Regulation, Plant , Plant Proteins/metabolism , Response Elements , Transcription Factors/metabolism , Bryopsida/metabolism , CCAAT-Binding Factor/genetics , Gene Regulatory Networks , Genes, Plant , Plant Proteins/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
PREMISE OF THE STUDY: We developed and tested primers for 218 nuclear loci for studying population genetics, phylogeography, and genome evolution in bryophytes. ⢠METHODS AND RESULTS: We aligned expressed sequence tags (ESTs) from Ceratodon purpureus to the Physcomitrella patens genome sequence, and designed primers that are homologous to conserved exons but span introns in the P. patens genome. We tested these primers on four isolates from New York, USA; Otavalo, Ecuador; and two laboratory isolates from Austria (WT4 and GG1). The median genome-wide nucleotide diversity was 0.008 substitutions/site, but the range was large (0-0.14), illustrating the among-locus heterogeneity in the species. ⢠CONCLUSIONS: These loci provide a valuable resource for finely resolved, genome-wide population genetic and species-level phylogenetic analyses of C. purpureus and its relatives.
ABSTRACT
Plants have two classes of myosins. While recent work has focused on class XI myosins showing that myosin XI is responsible for organelle motility and cytoplasmic streaming, much less is known about the role of myosin VIII in plant growth and development. We have used a combination of RNAi and insertional knockouts to probe myosin VIII function in the moss Physcomitrella patens. We isolated Δmyo8ABCDE plants demonstrating that myosin VIII is not required for plant viability. However, myosin VIII mutants are smaller than wild-type plants in part due to a defect in cell size. Additionally, Δmyo8ABCDE plants produce more side branches and form gametophores much earlier than wild-type plants. In the absence of nutrient media, Δmyo8ABCDE plants exhibit significant protonemal patterning defects, including highly curved protonemal filaments, morphologically defective side branches, as well as an increase in the number of branches. Exogenous auxin partially rescues protonemal defects in Δmyo8ABCDE plants grown in the absence of nutrients. This result, together with defects in protonemal branching, smaller caulonemal cells, and accelerated development in the Δmyo8ABCDE plants, suggests that myosin VIII is involved in hormone homeostasis in P. patens.