Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (P_f) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the P_f of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution

Stomatal pores evolved more than 410 million years ago [1,?2]?and allowed vascular plants to regulate transpirational water loss during the uptake of CO(2) for photosynthesis [3]. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens [2] respond to environmental signals in a similar way to those of flowering plants [4] and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response [5] is?involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) [6], is rescued by substitution with the moss P.?patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P.?patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago [7], prior to the evolution of ferns [8, 9].     

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.     

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate- N -acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE : AtMurE . Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds -tagged mutant alleles of AtMurE in A. thaliana . All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens ( Pp ) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens , transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.     

Exploration and identification of Physcomitrella patens moss peptides

In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins.     

Coulter counter cell size determination of protoplasts fromArabidopsis thaliana PIP1b aquaporin antisense lines under iso- and hypo-osmotic conditions

Summary Water uptake ofArabidopsis thaliana protoplasts was measured after transfer into hypo-osmotic conditions. The time-dependent swelling of protoplast populations was monitored by a Coulter counter device. In order to ascertain the contribution of the plasma membrane intrinsic protein 1b (PIP1b) to the membrane's water permeability, protoplasts of five different plant lines that were transformed with a PIP1b antisense construct were compared to controls. The size distribution of 5 independent protoplast preparations provided similar results for control and antisense lines under iso-osmolar conditions. After transfer into hypo-osmotic conditions, a time difference for the swelling of protoplasts from the different sources was observed. The sizes of control protoplasts changed in less than 20 s, which indicates high water influx rates. In contrast, the protoplast populations obtained from 5 different antisense plants took about 75 s to reach a steady-state cell size distribution. The difference in time by a factor of about 3 confirms the significance of the aquaporin PIP1b for the water permeability of plant plasma membranes and the cellular water transport.     

Modified suppression subtractive hybridization identifies an AP2-containing protein involved in metal responses in Physcomitrella patens

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.     

Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle

The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions. The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1). We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Loss of GH3 function does not affect phytochrome-mediated development in a moss, Physcomitrella patens

Auxin-induced gene expression is described for a variety of different genes including the SAUR-, Aux/IAA- and GH3-families, members of which have been found in seed plants. The precise function of GH3-like proteins in plant development is not well characterised yet. Mutant analysis in Arabidopsis thaliana indicates a possible role for GH3-like proteins in connecting auxin and light signal transduction. Here, we report the isolation of three different GH3-like homologues from a lower land plant, the moss Physcomitrella patens. Two of the GH3-like homologues were chosen for further characterisation. Both genes are expressed in gametophytic tissues, with expression starting very early in moss development. Knockout plants were generated and analysed. In comparison to white-light growth, cultivation of the wild type and knockout plants under red-light conditions resulted in a delay in gametophytic tissue development. The leafy moss plants displayed an elongated phenotype. Growth delay and elongation were even stronger under far-red light conditions. No obvious differences between wild type and knockout plants could be detected under the examined conditions, indicating functional redundancy of the two genes.     

The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens

The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.     

BLADE-ON-PETIOLE genes are not involved in the transition from protonema to gametophore in the moss Physcomitrella patens

Journal of Plant Research - The timing of the transition between developmental phases is a critical determinant of plant form. In the moss Physcomitrella patens, the transition from protonema to...     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana

The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.     

Observation of polarity induction by cytochemical localization of phenylalkylamine-binding sites in regenerating protoplasts of the moss Physcomitrella patens

Different external (e.g., light) and internal (e.g., auxin and calcium gradients) factors control differentiation of the moss protonema. The present investigations demonstrate that exogenously applied auxin, the pharmacological blockade of auxin efflux by naphthylphthalamic acid, and treatment with (-)bepridil, a calcium channel antagonist, inhibit protoplast division without affecting protoplast viability in the moss Physcomitrella patens. A fluorescently labelled phenylalkylamine (DM-Bodipy PAA), another calcium channel antagonist, was used as a probe for in vivo labelling of phenylalkylamine(PAA)-binding sites. The specificity of this binding was demonstrated by competition with (-)bepridil. Confocal laser scanning microscopy visualized PAA-binding sites on the plasma membrane and along the nuclear membrane as uniformly distributed clusters. During asymmetric division of P. patens protoplasts, however, fluorescence labelling particularly increases at the membrane invagination and later along the plate separating the new cells. Intracellular localization of PAA-binding sites, probably at the membranes of vesicles and vacuoles, significantly increases in the smaller daughter cell, destined to later form a polar outgrowth, the first chloronema cell. Thus, a system was established to visualize early events in P. patens protoplast polarization at the subcellular level.