Capacity for microtubule reorganization and cell wall synthesis in cytoplasts of the green alga Mougeotia

A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoplasts (cytoplasts) are formed during the preparation of protoplasts from the filamentous green alga Mougeotia . Regeneration of Mougeotia protoplasts is an orderly process known to involve reorganisation of cortical microtubules into polar arrays centered upon two opposing foci, synthesis of new cell walls and elongation to reform cylindrical cells. The ability of cytoplasts to carry out microtubule reorganisation and cell wall synthesis was investigated by combining Hoechst staining, to distinguish cytoplasts from karyoplasts and protoplasts, with immunofluorescent staining of microtubules and Calcofluor or Tinopal staining of cell walls. Cytoplasts survived at least 20 h in culture, but did not elongate. However, cytoplasts did participate in the first steps of protoplast regeneration. The majority of cytoplasts synthesized some cell wall material, while a small proportion was able to form ordered arrays of cortical microtubules indistinguishable from those in regenerating nucleate protoplasts. These results demonstrate the ability of plant microtubules to form new, orderly arrays in the absence of a nucleus, and suggest that the reestablishment of axiality in the protoplasts does not require a nucleus or nuclear DNA transcription.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41730/1/709_2005_Article_BF01404116.pd

NtGNL1 Plays an Essential Role in Pollen Tube Tip Growth and Orientation Likely via Regulation of Post-Golgi Trafficking

Background: Tobacco GNOM LIKE 1 (NtGNL1), a new member of the Big/GBF family, is characterized by a sec 7 domain. Thus, we proposed that NtGNL1 may function in regulating pollen tube growth for vesicle trafficking. Methodology/Principal Findings: To test this hypothesis, we used an RNAi technique to down-regulate NtGNL1 expression and found that pollen tube growth and orientation were clearly inhibited. Cytological observations revealed that both timing and behavior of endocytosis was disrupted, and endosome trafficking to prevacuolar compartments (PVC) or multivesicular bodies (MVB) was altered in pollen tube tips. Moreover, NtGNL1 seemed to partially overlap with Golgi bodies, but clearly colocalized with putative late endosome compartments. We also observed that in such pollen tubes, the Golgi apparatus disassembled and fused with the endoplasmic reticulum, indicating abnormal post-Golgi trafficking. During this process, actin organization was also remodeled. Conclusions/Significance: Thus, we revealed that NtGNL1 is essential for pollen tube growth and orientation and it likel

A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network

Differential Expression of miRNAs in Response to Topping in Flue-Cured Tobacco (Nicotiana tabacum) Roots

Topping is an important cultivating measure for flue-cured tobacco, and many genes had been found to be differentially expressed in response to topping. But it is still unclear how these genes are regulated. MiRNAs play a critical role in post-transcriptional gene regulation, so we sequenced two sRNA libraries from tobacco roots before and after topping, with a view to exploring transcriptional differences in miRNAs.Two sRNA libraries were generated from tobacco roots before and after topping. Solexa high-throughput sequencing of tobacco small RNAs revealed a total of 12,104,207 and 11,292,018 reads representing 3,633,398 and 3,084,102 distinct sequences before and after topping. The expressions of 136 conserved miRNAs (belonging to 32 families) and 126 new miRNAs (belonging to 77 families) were determined. There were three major conserved miRNAs families (nta-miR156, nta-miR172 and nta-miR171) and two major new miRNAs families (nta-miRn2 and nta-miRn26). All of these identified miRNAs can be folded into characteristic miRNA stem-loop secondary hairpin structures, and qRT-PCR was adopted to validate and measure the expression of miRNAs. Putative targets were identified for 133 out of 136 conserved miRNAs and 126 new miRNAs. Of these miRNAs whose targets had been identified, the miRNAs which change markedly (>2 folds) belong to 53 families and their targets have different biological functions including development, response to stress, response to hormone, N metabolism, C metabolism, signal transduction, nucleic acid metabolism and other metabolism. Some interesting targets for miRNAs had been determined.The differential expression profiles of miRNAs were shown in flue-cured tobacco roots before and after topping, which can be expected to regulate transcripts distinctly involved in response to topping. Further identification of these differentially expressed miRNAs and their targets would allow better understanding of the regulatory mechanisms for flue-cured tobacco response to topping

Central venous access and handwashing: variability in policies and practices.

This study examined variability in handwashing policy between hospitals, variability in handwashing practices in nurses and how practice differed from policy in tertiary paediatric hospitals in Australia and New Zealand. Eight of the possible nine major paediatric hospitals provided a copy of their handwashing and/or central venous access device (CVAD) policies, and 67 nurses completed a survey on their handwashing practices associated with CVAD management. A high degree of variability was found in relation to all the questions posed in the study. There was little consistency between policies and little agreement between policies and clinical practice, with many nurses washing for longer than required by policy. Rigour of handwashing also varied according to the procedure undertaken and the type of CVAD with activities undertaken farther from the insertion site of the device more likely to be performed using a clean rather than an aseptic handwashing technique. As both patients and nursing staff move within and between hospitals, a uniform and evidence-based approach to handwashing is highly desirable

Handwashing practice and policy variability when caring for central venous catheters in paediatric intensive care.

It has been estimated that there may be as many as 150,000 healthcare associated infections (HCAI) in Australia each year, contributing to 7,000 deaths, many of which could be prevented through the implementation of appropriate infection control practices. Contact with contaminated hands is a primary source of HCAI. Intensive care staff have been identified as one of the least adherent groups of health care professionals with handwashing; they are less likely to practise hand antisepsis before invasive procedures than staff working in other patient care specialties. The study examined the self-reported clean and aseptic handwashing practices of nurses working in paediatric intensive care units (PICUs) across Australia and New Zealand, the patterns in variation between nurses' reported handwashing practices and the local policies, and patterns in the duration of procedural handwashing for specific procedures. A survey was undertaken in 2001 in which participating tertiary paediatric hospitals provided copies of their infection control policies pertaining to central venous catheter (CVC) management; five nurses on each unit were asked to provide information in relation to their handwashing practices. Seven hospitals agreed to participate and 30 nurses completed the survey. The study found an enormous level of variation among and between nurses' reported practices and local policies. This variation extended across all aspects of handwashing practices - duration and extent of handwash, type of solution and drying method used. The rigour of handwashing varied according to the procedure undertaken, with some evidence that nurses made their own risk assessments based on the proximity of the procedure to the patient. In conclusion, this study's findings substantiate the need for standardisation of practice in line with the current Centers for Disease Control and Prevention Guidelines, including the introduction of alcohol handrub