CHUP1 mediates actin-based light-induced chloroplast avoidance movement in the moss Physcomitrella patens

Chloroplasts change their intracellular distribution in response to light intensity. CHUP1 (CHLOROPLAST UNUSUAL POSITIONING1) is indispensable for this response in Arabidopsis thaliana. However, involvement of CHUP1 in light-induced chloroplast movement is unknown in other plants. In this study, CHUP1 orthologues were isolated from a moss, Physcomitrella patens, and a fern, Adiantum capillus-veneris, by cDNA library screening and PCR cloning based on the P. patens genome sequence. Functional motifs found in CHUP1 of A. thaliana were conserved among the CHUP1 orthologues. In addition to the putative functional regions, the C-terminal regions (approximately 250 amino acids), which are unique in CHUP1s, were highly conserved. Green fluorescent protein (GFP) fusions of P. patens CHUP1s (PpCHUP1A, PpCHUP1B and PpCHUP1C) were transiently expressed in protoplast cells. All GFP fusions were localized on the chloroplasts. Light-induced chloroplast avoidance movement of chup1 disruptants of P. patens was examined in the presence of cytoskeletal inhibitors because of the utilization of both microtubules and actin filaments for the movement in P. patens. When actin filaments were disrupted by cytochalasin B, the wild type (WT) and all chup1 disruptants showed chloroplast avoidance movement. However, when microtubules were disrupted by Oryzalin, chloroplasts in ?chup1A and ?chup1A/B rarely moved and stayed in the strong light-irradiated area. On the other hand, WT, ?chup1B and ?chup1C showed chloroplast avoidance movement. These results suggest that PpCHUP1A predominantly mediates the actin-based light-induced chloroplast avoidance movement. This study reveals that CHUP1 functions on the chloroplasts and is involved in the actin-based light-induced chloroplast avoidance movement in P. patens.     

Phytochrome modulation of blue light-induced chloroplast movements in Arabidopsis

Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 micromol m-2 s-1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5-15 micromol m-2 s-1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.     

Phototropins and chloroplast activity in plant blue light signaling

In plants, phototropins 1 (phot1) and 2 (phot2) mediate chloroplast movement to blue light (BL). A recent report showed that phototropins (phot) are required for the expression of chloroplast genes in rice. The light-induced responses of phot1a rice mutants result in H₂O₂-mediated damage to chloroplast photosystems, indicating that phot-regulated responses might be associated with the other photoreceptor, such as cryptochrome (cry) BL receptor. This suggests diversification and specialization of photoreceptor signaling in plants.Key words: blue light, blue light receptor, chloroplast, cryptochrome, H₂O₂, phototropin, signalingIn order to counteract the adverse effects of environmental ight, plants have evolved sensory mechanisms that monitor their surroundings and adapt their growth and development through the use of a complex signaling network.¹ Plants sense their environmental light conditions by using three principal families of signal-transducing photoreceptors; the red/far-red (R/FR) light-absorbing phytochromes (phy) and the UV-A/blue light (BL)-absorbing cryptochromes (cry) and phototropins (phot).² The phys are reversibly photochromic biliproteins that absorb maximally in the R and FR light regions of the spectrum. Cry and phot possess a pair of flavin derivates. Two cry and two phot family members have been identified and well characterized in Arabidopsis. Photoreceptors regulate development throughout the plant lifecycle, from seed germination through to plant maturation and the onset of reproduction. BL regulates a wide variety of photoresponses in higher plants, including chloroplast movement, inhibition of hypocotyls elongation, circadian timing, regulation of gene expression and stomatal opening.^3–5 The roles of individual photoreceptors in mediating plant development have, however, often been confounded by redundant, synergistic and in some cases mutually antagonistic mechanisms of action. The mechanisms of photoreceptor signal transduction are far from being completely elucidated, but are believed to involve both cytosolic and nuclear components. The presence of putative kinase domains within photoreceptor proteins has suggested a role for phosphorylation in light signaling. The action of cry1 and cry2 has been demonstrated to involve BL-mediated autophosphorylation.^6,7 Phot1 was originally identified as a 120 kDa-membrane associated protein displaying BL-mediated autophosphorylation.⁸ It is now well accepted that phot mediates chloroplast movement, phototropism and stomatal opening in plants in response to BL.     

Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis

One of the most important functions of blue light (BL) is to induce chloroplast movements in order to reduce the damage to the photosynthetic machinery under excess light. Hydrogen peroxide (H(2)O(2)), which is commonly generated under various environmental stimuli, can act as a signalling molecule that regulates a number of developmental processes and stress responses. To investigate whether H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements, a laser scanning confocal microscope and a luminescence spectrometer were used to observe H(2)O(2) generation in situ with the assistance of the fluorescence probe dichlorofluorescein diacetate (H(2)DCF-DA). After treatment with high-fluence BL, an enhanced accumulation of H(2)O(2), indicated by the fluorescence intensity of DCF, can be observed in leaf cells of Arabidopsis thaliana. Exogenously applied H(2)O(2) promotes the high-fluence BL-induced chloroplast movements in a concentration-dependent manner within the range of 0-10(-4) M, not only increasing the degree of movements but also accelerating the start of migrations. Moreover, the high-fluence BL-induced H(2)O(2) generation and the subsequent chloroplast movements can be largely abolished by the administration of the H(2)O(2)-specific scavenger catalase and other antioxidants. In addition, in-depth subcellular experiments indicated that high-fluence BL-induced H(2)O(2) generation can be partly abolished by the addition of diphenyleneiodonium (DPI), which is an NADPH oxidase inhibitor, and the blocker of electron transport chain dichlorophenyl dimethylurea (DCMU), respectively. The results presented here suggest that high-fluence BL can induce H(2)O(2) generation at both the plasma membrane and the chloroplast, and that the production of H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements.     

HAK transporters from Physcomitrella patens and Yarrowia lipolytica mediate sodium uptake

The widespread presence of Na(+)-specific uptake systems across plants and fungi is a controversial topic. In this study, we identify two HAK genes, one in the moss Physcomitrella patens and the other in the yeast Yarrowia lipolytica, that encode Na(+)-specific transporters. Because HAK genes are numerous in plants and are duplicated in many fungi, our findings suggest that some HAK genes encode Na(+) transporters and that Na(+) might play physiological roles in plants and fungi more extensively than is currently thought.     

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.     

Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement

Red light-induced chloroplast movement in Physcomitrella patens (Pp) is mediated by dichroic phytochrome in the cytoplasm. To analyze the molecular function of the photoreceptor in the cytoplasm, we developed a protoplast system in which chloroplast photomovement was exclusively dependent on the expression of phytochrome cDNA constructs introduced by polyethylene glycol (PEG) transformation. YFP was fused to the phytochrome constructs and their expression was detected by fluorescence. The chloroplast avoidance response was induced in the protoplasts expressing a YFP fusion of PHY1-PHY3, but not of PHY4 or YFP alone. Phy::yfp fluorescence was detected in the cytoplasm. No change in the location of phy1::yfp or phy2::yfp was revealed before and after photomovement. When phy1::yfp and phy2::yfp were targeted to the nucleus by fusing a nuclear localization signal to the constructs, red light avoidance was not induced. To determine the domains of PHY2 essential for avoidance response, various partially-deleted PHY2::YFP constructs were tested. The N-terminal extension domain (NTE) was found to be necessary but the C-terminal histidine kinase-related domain (HKRD) was dispensable. An avoidance response was not induced under expression of phytochrome N-terminal half domain [deleting both the PAS (Per, Arnt, Sim)-related domain (PRD) and HKRD]. GUS fusion of this N-terminal half domain, reported to be fully functional in Arabidopsis for several phyA- and phyB-regulated responses was not effective in chloroplast avoidance movement. Domain requirement and GUS fusion effect were also confirmed in PHY1. These results indicate that Pp phy1-Pp phy3 in the cytoplasm mediate chloroplast avoidance movement, and that NTE and PRD, but not HKRD, are required for their function.     

Gene targeting in Physcomitrella patens

Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Sequencing programs and microbiological molecular genetic approaches are now being developed to unravel the precise function of plant genes. Physcomitrella patens, as the new 'green yeast', might well become a major tool for functional genomic studies of multicellular eukaryotes.     

A cytokinin-sensitive mutant of the moss,Physcomitrella patens,defective in chloroplast division

Summary An X-ray induced mutant (PC22) of the moss,Physcomitrella patens was analysed with respect to its morphology, physiology and suitability for microculture techniques. The mutant protonemata are defective in bud formation and in chloroplast division. As a consequence of the latter, giant chloroplasts are formed which disturb the development of the phragmoplast, the formation of regular cross walls, and cell division. Abnormal cross walls are rich in callose. The actin cytoskeleton was found to be less regularly developed in the mutant than in the wild type. Three-dimensional analysis of the microtubular arrangement with confocal laser scan microscopy demonstrates a close association between spindle- or phragmoplast- and interphase-microtubules. The deficiencies in chloroplast division and in bud formation can partly be compensated for by exogeneously applied cytokinin. The suitability of this particular developmental mutant for further studies was shown by regeneration of protoplasts in microculture and microinjection of the fluorochrome Lucifer yellow into the chloroplast.Abbreviations CLSM confocal laser scan microscope - DAPI diamidinophenyl indole - DiOC 3,3-dihexyloxacarbocyanine iodide - EGTA ethylene glycol-bis-(-amino-ethylether-N,N,N,N-tetraacetic acid - i⁶Ade N⁶-(²-isopentenyladenine) - PIPES piperazine-N, N-bis-2-ethanesulfonic acid - ptDNA chloroplast DNA Devoted to the memory of Prof. Dr. O. Kiermayer, our colleague and friend.     

RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens

Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear‐encoded chloroplast‐localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13–63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.     

MicroRNAs in the moss Physcomitrella patens

Having diverged from the lineage that lead to flowering plants shortly after plants have established on land, mosses, which share fundamental processes with flowering plants but underwent little morphological changes by comparison with the fossil records, can be considered as an evolutionary informative place. Hence, they are especially useful for the study of developmental evolution and adaption to life on land. The transition to land exposed early plants to harsh physical conditions that resulted in key physiological and developmental changes. MicroRNAs (miRNAs) are an important class of small RNAs (sRNAs) that act as master regulators of development and stress in flowering plants. In recent years several groups have been engaged in the cloning of sRNAs from the model moss Physcomitrella patens. These studies have revealed a wealth of miRNAs, including novel and conserved ones, creating a unique opportunity to broaden our understanding of miRNA functions in land plants and their contribution to the latter??s evolution. Here we review the current knowledge of moss miRNAs and suggest approaches for their functional analysis in P. patens.     

Expansins in the bryophyte Physcomitrella patens

Expansins are cell wall proteins which play a key function in basic processes of plant growth and differentiation. It has been proposed that expansins are likely to be present in all land plants and, to date, they have been reported in angiosperms, gymnosperms and pteridophytes. In this paper, we provide the first report and analysis of genes encoding expansin-like proteins in the bryophyte, Physcomitrella patens. Our analysis indicates that both - and -expansins are present as gene families in this plant and expression analysis indicates that these genes are subject to a complex regulation by both hormonal and environmental factors. In particular, the expression of many expansin genes in P. patens is upregulated by stress conditions, suggesting that they play a role in the specific cellular differentiation displayed by P. patens in response to such stress. Finally, we provide the first report on the generation and analysis of a series of knockout mutants for individual expansin genes. Abbreviations: IAA, indole-acetic acid; BAP, 6-benzylaminopurine; ABA, abscisic acid; npt, neomycin phospotransferase; KO, knockout     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication     

基因打靶技术在模式植物小立碗藓的应用

介绍了基因打靶技术在新型模式植物小立碗藓(Physcomitrella patens)中的应用.     

A hypergravity environment increases chloroplast size,photosynthesis, and plant growth in the moss Physcomitrella patens

The physiological and anatomical responses of bryophytes to altered gravity conditions will provide crucial information for estimating how plant physiological traits have evolved to adapt to significant increases in the effects of gravity in land plant history. We quantified changes in plant growth and photosynthesis in the model plant of mosses, Physcomitrella patens, grown under a hypergravity environment for 25 days or 8 weeks using a custom-built centrifuge equipped with a lighting system. This is the first study to examine the response of bryophytes to hypergravity conditions. Canopy-based plant growth was significantly increased at 10×g, and was strongly affected by increases in plant numbers. Rhizoid lengths for individual gametophores were significantly increased at 10×g. Chloroplast diameters (major axis) and thicknesses (minor axis) in the leaves of P. patens were also increased at 10×g. The area-based photosynthesis rate of P. patens was also enhanced at 10×g. Increases in shoot numbers and chloroplast sizes may elevate the area-based photosynthesis rate under hypergravity conditions. We observed a decrease in leaf cell wall thickness under hypergravity conditions, which is in contrast to previous findings obtained using angiosperms. Since mosses including P. patens live in dense populations, an increase in canopy-based plant numbers may be effective to enhance the toughness of the population, and, thus, represents an effective adaptation strategy to a hypergravity environment for P. patens.

     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

RNA interference in the moss Physcomitrella patens

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.     

小立碗藓愈伤组织诱导和培养

小立碗藓已经成为植物功能基因组学的模式系统,其材料的大量培养是所有工作的基础.文章探讨了小立碗藓愈伤组织诱导和组织培养的基本条件,并观察了小立碗藓愈伤组织的亚显微结构.     

A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.     

小立碗藓（Physcomitrella patens）植物microRNA结合靶位预测

利用857条植物miRNA序列对27546条小立碗藓mRNA序列进行搜索,预测出162个植物miRNA家族在小立碗藓中存在结合靶位。miRNA结合靶位数目和miRNA协同作用网络分析结果同时显示,miR482和miR1168在小立碗藓中结合靶位多、协同作用广,提示它们对于小立碗藓可能具有重要生物学功能。52个菜茵衣藻特有的miRNA被预测在小立碗藓中存在结合靶位,显示小立碗藓在从藻类向种子植物进化过程中处在独特演化位置。