Emerging technologies for non-invasive quantification of physiological oxygen transport in plants.

Oxygen plays a critical role in plant metabolism, stress response/signaling, and adaptation to environmental changes (Lambers and Colmer, Plant Soil 274:7-15, 2005; Pitzschke et al., Antioxid Redox Signal 8:1757-1764, 2006; Van Breusegem et al., Plant Sci 161:405-414, 2001). Reactive oxygen species (ROS), by-products of various metabolic pathways in which oxygen is a key molecule, are produced during adaptation responses to environmental stress. While much is known about plant adaptation to stress (e.g., detoxifying enzymes, antioxidant production), the link between ROS metabolism, O2 transport, and stress response mechanisms is unknown. Thus, non-invasive technologies for measuring O2 are critical for understanding the link between physiological O2 transport and ROS signaling. New non-invasive technologies allow real-time measurement of O2 at the single cell and even organelle levels. This review briefly summarizes currently available (i.e., mainstream) technologies for measuring O2 and then introduces emerging technologies for measuring O2. Advanced techniques that provide the ability to non-invasively (i.e., non-destructively) measure O2 are highlighted. In the near future, these non-invasive sensors will facilitate novel experimentation that will allow plant physiologists to ask new hypothesis-driven research questions aimed at improving our understanding of physiological O2 transport.

TSO1 is a novel protein that modulates cytokinesis and cell expansion in Arabidopsis.

Previous analyses of tso1 mutants revealed a loss of control of directional cellular expansion and coordination of growth of adjacent cells, and defects in karyokinesis and cytokinesis. We isolated TSO1 using a map-based approach, and show that it is a member of a family of at least three genes in Arabidopsis. Consistent with the mutant phenotype, TSO1 transcript was most abundant in flowers, where it accumulated to the highest levels in developing ovules and microspores. The putative TSO1 protein has two cysteine-rich regions that are similar to the CXC domains of a variety of proteins from plants and animals, including a class of kinesins involved in chromosome segregation, and enhancer of zeste-type proteins. Visualization of TSO1-fusion proteins indicated that TSO1 is a nuclear protein. The tso1 mutant phenotypes and the novelty of the TSO1 sequence suggest the existence of previously unknown participants in regulation of directional processes in eukaryotic cells.

INNER NO OUTER regulates abaxial- adaxial patterning in Arabidopsis ovules.

The Arabidopsis INNER NO OUTER (INO) gene is essential for formation and asymmetric growth of the ovule outer integument. INO encodes a member of the newly described YABBY family of putative transcription factors that contain apparent Cys(2)-Cys(2) zinc-finger domains and regions of similarity to the high mobility group (HMG) transcription factors. In wild-type plants, INO is expressed specifically on one side of the central region of each ovule primordium in the cells that give rise to the outer integument. Alterations in the INO expression pattern in mutant backgrounds implicate INO as a positive regulator of its own expression, and ANT, HLL, BEL1, and SUP as direct or indirect negative regulators that help to establish the spatial pattern of INO expression. We hypothesize that INO is necessary for polarity determination in the central part of the ovule. Maintenance of polarity in other parts of ino ovules indicates the existence of additional regulators and provides further evidence that the ovule is a compound structure.

Arabidopsis TSO1 regulates directional processes in cells during floral organogenesis.

Flowers of the previously described Arabidopsis tso1-1 mutant had aberrant, highly reduced organs in place of petals, stamens, and carpels. Cells of tso1-1 flowers had division defects, including failure in cytokinesis, partial cell wall formation, and elevated nuclear DNA content. We describe here two new tso1 alleles (tso1-3 and tso1-4), which caused defects in ovule development, but had little effect on gross floral morphology. Early ovule development occurred normally in tso1-3 and tso1-4, but the shapes and alignments of integument cells became increasingly more disordered as development progressed. tso1-3 ovules usually lacked embryo sacs due to a failure to form megaspore mother cells. The cell division defects described for the strong tso1-1 mutant were rarely observed in tso1-3 ovules. The aberrations in tso1-3 mutants primarily resulted from a failure in directional expansion of cells and/or coordination of this process among adjacent cells. Effects of tso1-3 appeared to be independent of effects of other ovule development mutations, with the exception of leunig, which exhibited a synergistic interaction. The data are consistent with TSO1 acting in processes governing directional movement of cellular components, indicating a likely role for TSO1 in cytoskeletal function.

Temporal and photoregulated expression of five tomato phytochrome genes.

Quantitative measurements of the absolute amounts of mRNAs transcribed from each of five phytochrome genes (PHYA, PHYB1, PHYB2, PHYE, PHYF) throughout the life cycle of a tomato plant and in response to changes in ambient light conditions are reported here. From their lowest level in unimbibed seed, all five transcripts increase by from 10- to 1000-fold during the first 24 h following the onset of imbibition, both in continuous darkness and in a greenhouse. In a greenhouse and on a whole-plant basis, all continue to increase throughout day 6, after which all but PHYE decline over the next week to a plateau at about one-half of the maximal value. PHYE mRNA differs in that in continues to increase in abundance during the first 2-3 weeks and thereafter remains at that maximal level. In adult plants, on a whole-plant basis and in decreasing order of abundance, PHYA, PHYB1, PHYE, PHYB2, and PHYF transcripts were present at approximately 120, 40, 40, 15, and 8 mumol microgram-1 of poly(A)(+)-enriched RNA, respectively. The data are consistent with the demonstrated roles of phytochromes A and B1 during seedling development and lead to the suggestion that phytochrome E might have a more important role in mature plants. Somewhat unexpectedly, PHYA and PHYB2 expression patterns are very similar. In seedlings, PHYA and PHYB2 exhibit the greatest increase in expression following a light-to-dark transition, as well as the greatest decrease following a dark-to-light transition. PHYA and PHYB2 are also similar in that both exhibit comparable variation on a natural diurnal cycle, while PHYB1 also exhibits variation but with a markedly different phase. The diurnal variation in expression of PHYA, PHYB1 and PHYB2 is consistent with the possibility that one or more of the phytochromes they encode is important with respect not only to photoperiodic behavior but also to the regulation of other events whose photosensitivity varies during a diurnal cycle.

GENETIC ANALYSIS OF OVULE DEVELOPMENT.

Ovules are the direct precursors of seeds and thus play central roles in sexual plant reproduction and human nutrition. Extensive classical studies have elucidated the evolutionary trends and developmental processes responsible for the current wide variety of ovule morphologies. Recently, ovules have been perceived as an attractive system for the study of genetic regulation of plant development. More than a dozen regulatory genes have now been identified through isolation of ovule mutants. Characterization of these mutants shows that some aspects of ovule development follow independent pathways, while other processes are interdependent. Some of these mutants have ovules resembling those of putative ancestors of angiosperms and may help in understanding plant evolution. Clones of several of the regulatory genes have been used to determine expression patterns and putative biochemical functions of the gene products. Newly constructed models of genetic regulation of ovule development provide a framework for interpretation of future discoveries.

Absolute quantification of five phytochrome transcripts in seedlings and mature plants of tomato (Solanum lycopersicum L.).

Described here are the first quantitative measurements of absolute amounts of mRNAs transcribed from individual members of a phytochrome gene (PHY) family. The abundances of PHY mRNAs were determined for dry seed and for selected organs of green-house-grown tomato (Solanum lycopersicum L.) seedlings and mature plants. With a Phosphoimager, absolute amounts of PHYA, PHYB1, PHYB2, PHYE and PHYF transcripts were measured with reference to standard curves prepared from mRNA fragments synthesized in vivo. Methodology was developed permitting the use of polymerase chain reaction (PCR)-generated probes derived from a highly conserved region of PHY, obviating the necessity to clone cDNAs and to isolate probes derived from their 3' non-coding regions. In dry seeds, PHYB1 mRNA appeared to be most abundant (4-5 mumol/mol mRNA) while in all other instances PHYA mRNA predominated. In seedlings, PHYB1, PHYB2, PHYE, and PHYF mRNAs were most abundant in the shoot (25-87 mumol/mol mRNA) while PHYA mRNA was most abundant in the root (325 mumol/mol mRNA). In adult plants, the levels of PHYA. PHYB1 and PHYE mRNAs were relatively uniform among different organs (approx. 100, 75, and 10 mumol/mol mRNA, respectively). In contrast, PHYB2 and PHYF were expressed preferentially in ripening fruits (35 and 47 mumol/mol mRNA, respectively), indicative of a possible role in fruit ripening for the phytochromes they encode. In general, the order of decreasing abundance of the five mRNAs for both seedlings and mature plants was PHYA, PHYB1, PHYE, PHYB2 and PHYF. Based upon observations that relatively modest changes in the extent of PHY expression result in changes in phenotype, the differential expression of each of the five tomato PHY described here is predicted to impact upon the spatial expression of biological activity of each phytochrome.

The phytochrome gene family in tomato includes a novel subfamily.

Data presented here define five tomato phytochrome genes (PHY) and indicate the existence of additional PHY in the tomato genome. Portions of each gene, encoding amino acids 203 through 315 in a consensus amino acid sequence, were amplified by polymerase chain reaction. Four of these genes, PHYA, PHYB1, PHYB2 and PHYE, are members of previously identified PHY subfamilies, while the fifth, PHYF, is identified as a member of a new PHY subfamily. PHYA, PHYB1, PHYB2 and PHYE fragments encode amino acid sequences that share 88% to 98% sequence identity with their Arabidopsis counterparts. The PHYF fragment, however, encodes a polypeptide that shares only 65% to 74% sequence identity with previously identified Arabidopsis phytochromes. A phylogenetic analysis suggests that PHYF arose soon after, or perhaps prior to, the origin of angiosperms. This analysis leads to the prediction that PHYF might be widespread among angiosperms, including both monocotyledons and dicotyledons. Each of the five tomato PHY is expressed as a transcript of sufficient size to encode a full-length phytochrome apoprotein. Two PHYF transcripts, 4.4 and 4.7 kb in length, have been detected in 9-day-old light-grown seedlings, consistent with either multiple transcription start sites or differential processing. Analyses of genomic Southern blots hybridized with radiolabelled RNA probes derived from the five tomato PHY, as well as Arabidopsis PHYC, indicate that the tomato genome contains as many as 9 to 13 PHY. The tomato PHY family is apparently not only different from, but also larger than, the PHY family presently described for Arabidopsis.

Rational and irrational use of antibiotics in a Canadian teaching hospital.

Parenteral therapy with gentamicin, cloxacillin, ampicillin and cephalothin was surveyed on a surgical, a gynecologic and medical ward of a teaching hospital. During a 3-month period 219 patients (12.9% of the total number admitted to the three wards) received at least one of the four antibiotics parenterally. Ampicillin and gentamicin were used most frequently on the three wards when the indication for therapy was either infection or empirical use. Cephalothin was used most frequently for prophylaxis in the gynecologic and surgical patients; no medical patient received this drug. Overall, therapy was assessed to be irrational in 42.0, 50.0 and 12.0% of the surgical, gynecologic and medical patients, respectively. Prophylaxis was the indication for therapy in 76.9 and 86.8% of the surgical and gynecologic patients, respectively, for whom the therapy was assessed to be irrational.