Characterization of the complete chloroplast genome sequence of Daphne retusa Hemsl. (Thymelaeaceae), a rare alpine plant species in northwestern China.

Daphne retusa Hemsl. (Thymelaeaceae) is an evergreen shrub plant. First, we characterized the complete nucleotide sequence of chloroplast (cp) genome of D. retusa. The total length of cp genome was found to be 170,553 bp, including a large single copy (LSC) region of 84,886 bp, a small single copy (SSC) region of 2,437 bp, and a pair of inverted repeats (IRs) of 41,617 bp. The cp genome of Daphne retusa Hemsl. contains 134 genes, including 90 protein-coding genes (75 PCG species), 37 transfer RNA genes (29 tRNA species), and 6 rRNA genes (3 RNA species). A total of 13 genes (trnK-UUU, trnS-CGA, atpF, rpoC1, trnL-UAA, trnC-ACA, petD, rpl16, rpl2, ndhB, trnE-UUC, ndhA, and trnA-UGC) contain a single intron, and one gene (ycf3) contains two introns. The GC content in whole cp genome, LSC region, SSC region, and IR region was 36.75%, 34.83%, 28.19%, and 38.96% respectively, like other Thymelaeaceae plants. Phylogenetic analysis suggested that D. retusa has a close relationship with congeneric Daphne tangutica.

The complete chloroplast genome sequence of the medicinal shrub Daphne Giraldii Nitsche. (Thymelaeaceae).

Daphne giraldii Nitsche. (Thymelaeaceae) is a slow-growing shrub which has been used in Chinese folk medicine and commonly called 'Zu Shima'. In this study, we assembled the complete chloroplast (cp) genome of D. giraldii using data from high-throughput Illumina sequencing. The D. giraldii cp genome is 171,643 bp in size and includes two inverted repeat regions of 41,798 bp each, which is separated by a large single copy region of 85,171 bp and a small single copy region of 2876 bp. A total of 137 genes were predicted, including 38 tRNA, 8 rRNA, and 90 protein-coding genes. In addition, 10 PCG genes possess a single intron, 92 PCG genes no intron, 1 gene harbor two introns. Six tRNA genes harbor a single intron. Phylogenetic analysis indicated that D. giraldii is closer to Daphne kiusiana and Daphne tangutica than other taxa.

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization.

Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.

A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.

Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ∼100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

Disruption of actin filaments induces mitochondrial Ca2+ release to the cytoplasm and [Ca2+]c changes in Arabidopsis root hairs.

BACKGROUND: Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca2+ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca2+ storage, cytoplasmic Ca2+ concentration ([Ca2+]c), and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in Arabidopsis root hairs. RESULTS: In this study, we found that treatments with latrunculin B (Lat-B) and jasplakinolide (Jas), which depolymerize and polymerize actin filaments respectively, decreased membrane potential and Ca2+ stores in the mitochondria of Arabidopsis root hairs. Simultaneously, these treatments induced an instantaneous increase of cytoplasmic Ca2+, followed by a continuous decrease. All of these effects were inhibited by pretreatment with cyclosporin A (Cs A), a representative blocker of the mitochondrial permeability transition pore (mPTP). Moreover, we found there was a Ca2+ concentration gradient in mitochondria from the tip to the base of the root hair, and this gradient could be disrupted by actin-acting drugs. CONCLUSIONS: Based on these results, we concluded that the disruption of actin filaments caused by Lat-B or Jas promoted irreversible opening of the mPTP, resulting in mitochondrial Ca2+ release into the cytoplasm, and consequent changes in [Ca2+]c. We suggest that normal polymerization and depolymerization of actin filaments are essential for mitochondrial Ca2+ storage in root hairs.

Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis.

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co-immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2-CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two-parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.

The speed of mitochondrial movement is regulated by the cytoskeleton and myosin in Picea wilsonii pollen tubes.

Strategic control of mitochondrial movements and cellular distribution is essential for correct cell function and survival. However, despite being a vital process, mitochondrial movement in plant cells is a poorly documented phenomenon. To investigate the roles of actin filaments and microtubules on mitochondrial movements, Picea wilsonii pollen tubes were treated with two microtubule-disrupting drugs, two actin-disrupting drugs and a myosin inhibitor. Following these treatments, mitochondrial movements were characterized by multiangle evanescent wave microscopy and laser-scanning confocal microscopy. The results showed that individual mitochondria underwent three classes of linear movement: high-speed movement (instantaneous velocities >5.0 microm/s), low-speed movement (instantaneous velocities <5.0 microm/s) and variable-speed movement (instantaneous velocities ranging from 0.16 to 10.35 microm/s). 10 nM latrunculin B induced fragmentation of actin filaments and completely inhibited mitochondrial vectorial movement. Jasplakinolide treatment induced a 28% reduction in chondriome motility, and dramatically inhibition of high-speed and variable-speed movements. Treatment with 2,3-butanedione 2-monoxime caused a 61% reduction of chondriome motility, and the complete inhibition of high-speed and low-speed movements. In contrast to actin-disrupting drugs, microtubule-disrupting drugs caused mild effects on mitochondrial movement. Taxol increased the speed of mitochondrial movement in cortical cytoplasm. Oryzalin induced curved mitochondrial trajectories with similar velocities as in the control pollen tubes. These results suggest that mitochondrial movement at low speeds in pollen tubes is driven by myosin, while high-speed and variable-speed movements are powered both by actin filament dynamics and myosin. In addition, microtubule dynamics has profound effects on mitochondrial velocity, trajectory and positioning via its role in directing the arrangement of actin filaments.

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth.

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca(2+) gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.

No detectable maternal effects of elevated CO(2) on Arabidopsis thaliana over 15 generations.

Maternal environment has been demonstrated to produce considerable impact on offspring growth. However, few studies have been carried out to investigate multi-generational maternal effects of elevated CO(2) on plant growth and development. Here we present the first report on the responses of plant reproductive, photosynthetic, and cellular characteristics to elevated CO(2) over 15 generations using Arabidopsis thaliana as a model system. We found that within an individual generation, elevated CO(2) significantly advanced plant flowering, increased photosynthetic rate, increased the size and number of starch grains per chloroplast, reduced stomatal density, stomatal conductance, and transpiration rate, and resulted in a higher reproductive mass. Elevated CO(2) did not significantly influence silique length and number of seeds per silique. Across 15 generations grown at elevated CO(2) concentrations, however, there were no significant differences in these traits. In addition, a reciprocal sowing experiment demonstrated that elevated CO(2) did not produce detectable maternal effects on the offspring after fifteen generations. Taken together, these results suggested that the maternal effects of elevated CO(2) failed to extend to the offspring due to the potential lack of genetic variation for CO(2) responsiveness, and future plants may not evolve specific adaptations to elevated CO(2) concentrations.

Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs.

BACKGROUND: Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. CONCLUSIONS/SIGNIFICANCE: Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.

Calmodulin binds to extracellular sites on the plasma membrane of plant cells and elicits a rise in intracellular calcium concentration.

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca(2+) fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca(2+) levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.

Carbon nanotubes as molecular transporters for walled plant cells.

We have investigated the capability of single-walled carbon nanotubes (SWNTs) to penetrate the cell wall and cell membrane of intact plant cells. Confocal fluorescence images revealed the cellular uptake of both SWNT/fluorescein isothiocyanate and SWNT/DNA conjugates, demonstrating that SWNTs also hold great promise as nanotransporters for walled plant cells. Moreover, the result suggested that SWNTs could deliver different cargoes into different plant cell organelles.

Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

Protein phosphatases 1 and 2A and the regulation of calcium uptake and pollen tube development in Picea wilsonii.

To investigate the roles of protein phosphatases 1 and 2A in the development of pollen tubes of Picea wilsonii Mast., pollen grains were cultured in standard medium in the presence and absence of the protein phosphatase inhibitors okadaic acid and calyculin A. At nanomolar concentrations, these compounds blocked pollen tube growth, causing abnormal morphologies of the pollen tubes. Studies with Fluo-3 revealed that the inhibitors reduced the pollen-tube tip-to-base cytoplasmic calcium (Ca(2+)) gradient and arrested extracellular Ca(2+) uptake. The transmission electron microscope observations indicated that the fusion of paramural bodies with plasma membranes occurred frequently in the tip and sub-tip regions of control pollen tubes, but fusion rarely occurred in inhibitor-treated pollen tubes. Staining with aniline blue showed that callose accumulated in the tip regions of inhibitor-treated pollen tubes. Immunolabeling of pollen tubes revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present in the tip region and on the flanks of the sub-tip in normal pollen tubes. In inhibitor-treated pollen tubes, these epitopes existed only in the extreme tip region and at higher concentrations than in control pollen tubes. The esterified pectin recognized by JIM7 was located preferentially at the extreme tip region in normal pollen tubes, but at basal sites in inhibitor-treated tubes. Fourier transform infrared (FTIR) analysis further confirmed the changes in acidic and esterified pectin distributions and their relative contents. These results suggest that protein phosphatase 1 or 2A, or both, are involved in the regulation of Ca(2+) uptake across the plasma membrane, in exocytotic activity and in the biosynthesis of cell wall components, all processes that occur in the tip region of pollen tubes and that control pollen tube development.

Effects of brefeldin A on pollen germination and tube growth. Antagonistic effects on endocytosis and secretion.

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 microg mL(-1)) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.

Boron influences pollen germination and pollen tube growth in Picea meyeri.

To study the role of boron in pollen germination and pollen tube growth of Picea meyeri Rehd. et Wils., pollen grains were cultured in standard medium or boron-deficient medium. Effects of boron on the localization of pectins and callose in the walls of pollen tubes were observed by laser scanning confocal microscopy after staining with aniline blue or immunolabeling with antibodies JIM5 and JIM7. Changes in the structures of pectins and phenolics were investigated by fourier transform infrared (FTIR) microspectroscopy. Pollen germination in boron-deficient medium ranged from 18 to 24%, whereas pollen germination in standard medium reached 61%. Callose accumulated in the tip-regions of pollen tubes cultured in boron-deficient medium, but not in standard medium. Immunolabeling with antibody JIM5 revealed that acidic pectin preferentially accumulated in the tip regions of pollen tubes cultured in boron-deficient medium, whereas acidic pectin was weakly distributed along the entire lengths of pollen tubes cultured in standard medium. Esterified pectin, detected by immunolabeling with antibody JIM7, showed a similar distribution pattern in pollen tubes in both the boron-deficient and standard treatments. The FTIR spectra indicated slight increases in contents of phenolics and carboxylic acids and a substantial decrease in the content of saturated esters in boron-deficient pollen tubes compared with normal pollen tubes. The FTIR spectra confirmed that boron deficiency enhanced acidic pectin accumulation in pollen tubes, which may be associated with the increased content of carboxylic acid. We conclude that boron has a regulatory role in pollen germination and pollen tube growth.

Relationship between tumor necrosis factor-alphaand liver fibrosis.

INTRODUCTION:To intrestigate the relationship between tumor necrosis factor-alphaand liver fibrosis in patients with chronic liver disease.METHODS:Radioimmunoassay was made in 20 patients with mild chronic hepatitis(CMH),20 patients with severe chronic hepatitis(CSH),51 patients with liver cirrhosis(LC)and 32-normal persons to determine the contents of tumor necrosis factor-alpha(TNF-alpha),laminin-(LN) and hyaluronate (HA) in serum.The changes in and relationship between TNF-alpha,LN and HA were analyzed.The TNF-alphaand collagen III were determined using mmunohistochemical studies in liver tissues from 32 persons including 7 normal persons,3 patients with MCH,5 patients with SCH and 17 with LC.RESULTS:TNF-alpha,LN and HA levels in serum of CSH and LC patients were significantly higher than those in healthy controls (SCH:1.11plus minus0.59 130.7plus minus17.2,219.1plus minus121.3;LC:0.92plus minus0.66,156.8plus minus31.7,400.5plus minus183.7,P<0.05-0.01),which increased gradually,and correlated positively with each other in all patients with liver diseases (n=91,gamma=0.3149 P <0.01).TNF-alpha contents-showed a remarkably positive correlation with HA and LN levels in CMH and CSH (LN:n=40,gamma=0.3404,P <0.05 HA n=40,gamma=0.3847 P <0.05).The total collagen content of MCH,SCH and LC increased gradually in liver biopsy specimens.The number of TNF-alphapositive cells increased significantly in liver tissues from patients with SCH and LC (62%;45%;P <0.01).TNF-alphapositive cells were mainly located in the periportal areas.CONCLUSION:TNF-alphamay be related to liver fibrosis,and might promote liver fibrosis.