Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex.

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.

Solute flux into parasitic plants.

Parasitic plants form intimate contacts with host tissue in order to gain access to host solutes. There are a variety of cell types within the host which parasitic plants could access to extract solutes. Depending on the degree to which the parasite has embraced the parasitic lifestyle, the extent of solute flux and the pathways used to transfer solutes from host to parasite will vary. To date, a variety of experimental approaches argue for diversity in the mechanisms and the routes by which parasites accumulate host solutes. Contact between host and parasite ranges from direct lumen-to-lumen links between host and parasite xylem and continuity between the sieve elements of host and parasite, to the involvement of transfer cells between host and parasite. Progress has been slow since Solms-Laubach distinguished types of parasitic plants that fed from host phloem or xylem in 1867, but advances in clearly delineating the pathways that link host and parasite should now be possible using fluorescent proteins expressed and restricted to particular cell types of the host. This will initially necessitate using Arabidopsis, but should allow the types of connection, i.e. symplasmic or apoplasmic, to be determined and then the identification of parasite transporters responsible for solute flux.

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

Unusual carotenoid composition and a new type of xanthophyll cycle in plants.

The capture of photons by the photosynthetic apparatus is the first step in photosynthesis in all autotrophic higher plants. This light capture is dominated by pigment-containing proteins known as light-harvesting complexes (LHCs). The xanthophyll-carotenoid complement of these LHCs (neoxanthin, violaxanthin, and lutein) is highly conserved, with no deletions and few, uncommon additions. We report that neoxanthin, considered an integral component of LHCs, is stoichiometrically replaced by lutein-5,6-epoxide in the parasitic angiosperm Cuscuta reflexa, without compromising the structural integrity of the LHCs. Lutein-5,6-epoxide differs from neoxanthin in that it is involved in a light-driven deepoxidation cycle similar to the deepoxidation of violaxanthin in the xanthophyll cycle, which is implicated in protection against photodamage. The absence of neoxanthin and its replacement by lutein-5,6-epoxide changes our understanding of the structure-function relationship in LHCs, has implications for biosynthetic pathways involving neoxanthin (such as the plant hormone abscisic acid), and identifies one of the early steps associated with the evolution of heterotrophy from autotrophy in plants.

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase.

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

Professional goals and industrial strategies: an international survey.

Although nurses are often uneasy about following industrial strategies, particularly strikes, such strategies can enhance nursing practice and protect the nurses' role in employment relationships. But to what extent can nurses actually include professional goals in their collective agreements? The following report of an exploratory survey of the professional outcomes of collective bargaining by nurses in Australia, Canada, New Zealand and the United Kingdom provides baseline data for progress in this area.

Striving for safety: experiences of nurses in a hospital under siege.

The hospital in which this study took place served as a centre for emergencies, critically ill patients and maternity cases during a nurses' strike in 98 hospitals in Alberta, Canada. Nurses at this hospital were not members of the striking union; they belonged to their own independent union and they worked throughout the 19-day siege. Thirty-two nurses at the hospital responded to posters seeking volunteers for telephone interviews. The objective of the research was to discover the perceptions and experiences of nurses coping with extraordinarily heavy workloads arising out of a labour dispute. Interview data were gathered and analysed according to the conventions of grounded theory. Nurses voiced many concerns about the care patients received and they coped as best they could in a process described as 'striving for safety'. Fearing they would make mistakes that would harm their patients, they engaged in two subordinate processes of 'assessing competence' and 'preserving integrity'. Several organizational factors were identified that, if present, contributed to nurses' ability to continue or 'hang in' but if absent, contributed to despair or 'feeling demoralized'. The study was exploratory in nature and thus limited, but several implications for supporting nurses and managing such crises were discussed.

Implementing shared governance: a false start.

A case study of the implementation of shared governance in a large teaching hospital in Western Canada has been presented. The project was placed in jeopardy due to two major contingencies: turnover in the chief nursing executive position, and a sudden reduction in the operating budget of the nursing division necessitating significant layoffs. Other factors that threatened the survival of shared governance included lack of systematic, long-range planning; the number and diversity of major changes introduced concurrently in the nursing division; and insufficient support systems to sustain organizational change. In particular, some senior and first-line managers could not adapt to or accept the radical philosophical change and so they were unable to empower their staff and to provide the necessary reinforcement needed to ensure the success of shared governance. This combination of these factors contributed to the loss of momentum in the implementation of shared governance. Lowered morale in the wake of layoffs, together with union grievances, and lack of clarity of the role to be played by union representatives in shared governance produced conflict and confrontation within the nursing division and between union and management. Despite the difficulties encountered, there remains optimism and commitment to the challenge of making shared governance succeed. As this article goes to press, remarkable strides have been made in addressing the described issues. A task force composed primarily of staff nurses has developed a "customized" model of governance that meets the needs of the hospital and deals with the identified flaws of the first implementation attempt. The organization is optimistic that by taking time to develop a solid foundation for the proposed change and tending carefully to the details of decision-making processes, an effective structure to support the professional role of the nursing staff will be a reality.

Structure as a causal factor in nurses' strikes.

Nurses employed in Alberta's hospitals have earned a reputation of militancy as a result of four province-wide strikes in the 11-year period 1977-1988. Based on a case study of the first three strikes, the principal causes of the labour disputes are discussed: namely, union ideology, economic factors and inherent constraints in the structure of bargaining in the public sector. Interorganizational relationships of the three main players, the United Nurses of Alberta, the Alberta Hospital Association and the provincial government have been contributing factors in all three strikes. Although issues in the disputes were primarily economic, demands associated with the needs of working women, and demands that threatened the traditional prerogatives of management were at the heart of every impasse.