Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

Development of a mite bioassay to evaluate plant resistance and its use in determining regeneration of spider mite resistance

The zonal geranium (Pelargonium xhortorum) possesses tall glandular trichomes that secrete anacardic acids, a viscous, sticky exudate which has been suggested as the primary mechanism in two-spotted spider mite (Tetranychus urticae Koch) resistance. A new bioassay was developed using small Plexiglas^® cylinders as chambers for evaluating the resistance of geranium leaves to the two-spotted spider mite. This bioassay was easy to prepare, required only 24 hours to conduct, exhibited no problems with desiccation, condensation, or mite accountability, and yielded reproducible results. This bioassay was then used to study the regeneration of resistance of attached geranium leaves after they were made mite-susceptible by removing the excreted anacardic acids with water. Washed leaves regained full resistance after 14 days.     

Isolation of an Arabidopsis homologue of the maize homeobox Knotted-1 gene

The shoot apical meristem (SAM) is responsible for forming most of the above-ground portion of the plant. We sought to isolate regulatory genes expressed in the Arabidopsis SMA by screening a Brassica oleracea (cauliflower) meristem cDNA library with the homeobox fragment from the maize Knotted-1 (Kn1) gene. We isolated and characterized the corresponding clone, Merihb1, from Arabidopsis. Analysis shows that the predicted MERIHB1 protein exhibits strong homology to KN1 and RS1 from maize, SBH1 from soybean, and KNAT1 and KNAT2 from Arabidopsis. Merihb1 is highly expressed in mRNA from cauliflower meristems and also accumulates in stem and flower mRNA. Based on the similarity of the Merihb1 and Kn1 sequences, expression patterns, and in situ hybridizations, we suggest that Merihb1 represents an Arabidopsis homologue of the maize Kn1 gene.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

Alleviation of water and osmotic stress-induced changes in nitrogen metabolizing enzymes in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars by potassium

Present communication reports laboratory and pot experiments conducted to study the influence of water and osmotic stress on nitrogen uptake and metabolism in two wheat (Triticum aestivum L) cultivars with and without potassium supplementation. Polyethylene glycol 6000-induced osmotic stress/restricted irrigation caused a considerable decline in the activity of nitrate reductase, glutamate synthase, alanine and aspartate aminotransferases, and glutamate dehydrogenase. Potassium considerably improved nitrogen metabolism under normal water supply conditions and also resulted in amelioration of the negative impact of water and osmotic stresses indicating that potassium supplementation can be used as a potential tool for enhancing the nitrogen use efficiency in wheat for exploiting its genetic potential.     

Cascading costs:An economic nitrogen cycle

The chemical nitrogen cycle is becoming better characterized in terms of fluxes and reservoirs on a variety of scales. Galloway has demonstrated that reactive nitrogen can cascade through multiple ecosystems causing environmental damage at each stage before being denitrified to N2. We propose to construct a parallel economic nitrogen cascade (ENC) in which economic impacts of nitrogen fluxes can be estimated by the costs associated with each stage of the chemical cascade. Using economic data for the benefits of damage avoided and costs of mitigation in the Chesapeake Bay basin, we have constructed an economic nitrogen cascade for the region. Since a single tonne of nitrogen can cascade through the system, the costs also cascade. Therefore evaluating the benefits of mitigating a tonne of reactive nitrogen released needs to consider the damage avoided in all of the ecosystems through which that tonne would cascade. The analysis reveals that it is most cost effective to remove a tonne of nitrogen coming from combustion since it has the greatest impact on human health and creates cascading damage through the atmospheric, terrestrial, aquatic and coastal ecosystems. We will discuss the implications of this analysis for determining the most cost effective policy option for achieving environmental quality goals.     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.