Kinesins have a dual function in organizing microtubules during both tip growth and cytokinesis in Physcomitrella patens

Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components

Molecular and physiological responses to desiccation indicate the abscisic acid pathway is conserved in the peat moss, Sphagnum

Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum

A Lin28 homologue reprograms differentiated cells to stem cells in the moss Physcomitrella patens

Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3′-untranslated region (3′-UTR). Removal of the 3′-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSPgenes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28

Genome of the pitcher plant <i>Cephalotus </i>reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory

Identification of Genes Expressed in Apical Cellsof the Moss Physcomitrella patens Using Gene-trapand Enhancer-trap Systems

Postembryonic growth in land plants occurs from the meristem, a localized region that gives rise to all adult structures, such as a stem and leaves. Meristems control the continuous development of plant organs by balancing the maintenance and proliferation of undifferentiated stem cells, and directing their differentiation. Meristem establishment and maintenance is a fundamental question in plant development research. Mosses have two types of meristems: a protonema apical cell and a gametophore apical cell. The gametophore apical cell is a single meristematic cell that is maintained through self-renewal, and gives rise to such organs as the stem and leaves. In the moss Physcomitrella patens, the developmental process of the apical cell is well defined at the cellular level, and gene targeting based on homologous recombination is feasible. Thus, apical cell differentiation in P. patens is used as a model system for studies of meristem development in land plants. This study investigated apical cell differentiation of gametophores in P. patens by identifying the genes expressed during this differentiation. First, gene-trap and enhancer-trap systems were established in P. patens. These techniques are useful for cloning genes and enhancers that function in specific tissues or cells. In addition, the systems are convenient for obtaining molecular markers specific to certain developmental processes. Elements for the two systems were constructed using a uidA reporter gene with a splice acceptor in the case of the gene-trap system, and a minimal promoter for the enhancer-trap system. The homologous recombination method allowed a high rate of transformation, finding 235 gene-trap and 1073 enhancer-trap lines with variable expression patterns from a total of 5637 gene-trap and 3726 enhancer-trap transgenic lines. To assess the feasibility of isolating a trapped gene, one gene-trap line, YH209 with rhizoid-specific GUS expression, was characterized. UidA-fused fragments were amplified by the 5\u27 RACE method using nil-specific primers. One of the amplified fragments was used to screen the mini-transposon-tagged genomic DNA library that was used to generate the P. patens gene-trap lines. A genomic fragment containing the sequence of 5\u27 RACE fragments was obtained. This fragment was re-integrated into the P. patens genome by homologous recombination, confirming that the fragment-integrated transformants exhibited rhizoid-specific expression patterns observed in the YH2O9 line. In addition, a full-length cDNA was isolated by the 3\u27 RACE method, and the gene was named PpGLU. PpGLU forms a clade with the acidic alpha-glucosidase genes of plants. The gene-trap and enhancer-trap systems should be useful for identifying cell-type and tissue-specific genes in P. patens. From the 235 gene-trap lines and the 1073 enhancer-trap lines, three and four lines, respectively, were isolated. The isolated lines were exhibiting GUS activity preferentially in the apical cells of buds. One gene-trap line, Apicar1, showing GUS activity predominantly in the apical cell of caulonemata, rhizoids, and gametophores, was further characterized. The candidate trapped gene was isolated by both the 5\u27 and 3\u27 RACE methods, using the same approach as used with the YH209 line. A sequence analysis of the isolated cDNA revealed that the trapped gene encoded a kinesin-like protein, API1 (Apicar1). According to a phylogenetic analysis of API1 and kinesin superfamily genes, the API1 gene formed a new family of kinesin-like proteins with one of the Arabidopsis kinesin-like genes. This suggests that API1 may have a novel function that is different from those of kinesins of other subfamilies. API1 will also be useful as a molecular marker in studies of the establishment and maintenance of the apical cell

Kinesins are indispensable for interdigitation of phragmoplast microtubules in the moss Physcomitrella patens

Microtubules form arrays with parallel and antiparallel bundles and function in various cellular processes, including subcellular transport and cell division. The antiparallel bundles in phragmoplasts, plant-unique microtubule arrays, are mostly unexplored and potentially offer new cellular insights. Here, we report that the Physcomitrella patens kinesins KINID1a and KINID1b (for kinesin for interdigitated microtubules 1a and 1b), which are specific to land plants and orthologous to Arabidopsis thaliana PAKRP2, are novel factors indispensable for the generation of interdigitated antiparallel microtubules in the phragmoplasts of the moss P. patens. KINID1a and KINID1b are predominantly localized to the putative interdigitated parts of antiparallel microtubules. This interdigitation disappeared in double-deletion mutants of both genes, indicating that both KINID1a and 1b are indispensable for interdigitation of the antiparallel microtubule array. Furthermore, cell plates formed by these phragmoplasts did not reach the plasma membrane in ∼20% of the mutant cells examined. We observed that in the double-deletion mutant lines, chloroplasts remained between the plasma membrane and the expanding margins of the cell plate, while chloroplasts were absent from the margins of the cell plates in the wild type. This suggests that the kinesins, the antiparallel microtubule bundles with interdigitation, or both are necessary for proper progression of cell wall expansion

A dibasic amino acid pair conserved in the activation loop directs plasma membrane localization and is necessary for activity of plant type I/II phosphatidylinositol phosphate kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific