Stress-related levels of abscisic acid in guard cell protoplasts of Vicia faba L.

Levels of abscisis acid (ABA) were determined in isolated guard cell (GCP) and mesophyll cell (MCP) protoplasts of Vicia faba L. in relation to water stress. Incubation of GCP and MCP in 0.4 M or 0.8 M mannitol resulted in an average increase in the level of free abscisic acid (ABA) in the cells of 34% (GCP) and 38% (MCP) within 15–60 min. It is concluded that guard cell protoplasts form ABA in response to osmotic stress.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - GCP guard cell protoplasts - MCP mesophyll cell protoplasts - MES [2-(N-morpholino)-ethanesulfonic acid] - TLC thin layer chromatography Part 20 in the series, Use of Immunoassay in Plant Science     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

Determination of phytohormones in phloem exudate from tree species by radioimmunoassay

A sensitive and specific radioimmunoassay was used to determine quantitatively four of the most important phytohormones in the phloem exudate from 14 different tree species of 8 genera. For cytokinins and indole-3-acetic acid (IAA) we found higher concentrations than those reported previously for other species. The gibberellin values were of the same order of magnitude as in earlier analyses (with different methods) of tree phloem exudates, but lower than the ones reported for Ricinus. Free abscisic acid (ABA) was found in tree phloem exudates in similar concentrations as before in Yucca or palm phloem exudate, but at considerably lower ones than reported for Ricinus and in Lupinus phloem exudate.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA gibberellic acid     

Radioimmunoassay for the determination of limonin in Citrus

An ¹²⁵I-radioimmunoassay technique has been developed for the triterpenoid bitter principle, limonin. Synthesis of the iodinated tracer and the limonin—bovine serum albumin conjugate are described. The antibody has a high affinity (K_a 1.1 x 10⁹l/mol) and specificity for limonin and the detection limit of the assay is 0.07 ng or 0.7 ppb. Standard curves are linear over a range of 0.5–100 ng limonin, assays can be performed in crude extracts, and several hundred samples can be processed per day. The distribution of limonin in fruits and vegetative parts of Citrus paradisi has been determined, highest values (0.92%) being found in the seeds, lowest (0.0007%) in the juice vesicles of ripe fruits. The potential of this assay method in citrus research is discussed.     

Reducing Biotic and Abiotic Land‐Use Legacies to Restore Invaded,Abandoned Citrus Groves

Land‐use legacies associated with agriculture, such as increased soil fertility and elevated soil pH, promote invasions by non‐native plant species on former agricultural lands. Restoring natural soil conditions (i.e. low fertility and low pH) may be an effective, long‐term method to control and reduce the abundance of non‐native and ruderal species that invade abandoned agricultural lands. In this study, we examined how soil manipulation treatments of lowering soil fertility with carbon additions and lowering soil pH by applying sulfur affect non‐native and ruderal native plant species abundance in two former citrus groves in central Florida. Non‐native plant biomass was removed by one of two methods (tilling or topsoil removal), and was combined with a soil amendment of sulfur, carbon, sulfur + carbon, or none. The biomass removal treatments significantly decreased non‐native abundance, with topsoil removal as the most effective. Carbon additions did not affect soil fertility or vegetation. Sulfur and sulfur + carbon additions significantly decreased soil pH in both groves for at least 1 year post‐treatment; however, we did not see a significant vegetation response. Overall, our results suggest that removing vegetation by tilling and topsoil removal is an effective method for reducing non‐target species cover. Although we did not see a response of vegetation to our treatments, we were able to restore the initial soil characteristics, which can be a first step toward complete restoration.     

Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Testing for a Gap Junction-Mediated Bystander Effect in Retinitis Pigmentosa: Secondary Cone Death Is Not Altered by Deletion of Connexin36 from Cones

Retinitis pigmentosa (RP) relates to a group of hereditary neurodegenerative diseases of the retina. On the cellular level, RP results in the primary death of rod photoreceptors, caused by rod-specific mutations, followed by a secondary degeneration of genetically normal cones. Different mechanisms may influence the spread of cell death from one photoreceptor type to the other. As one of these mechanisms a gap junction-mediated bystander effect was proposed, i.e., toxic molecules generated in dying rods and propagating through gap junctions induce the death of healthy cone photoreceptors. We investigated whether disruption of rod-cone coupling can prevent secondary cone death and reduce the spread of degeneration. We tested this hypothesis in two different mouse models for retinal degeneration (rhodopsin knockout and rd1) by crossbreeding them with connexin36-deficient mice as connexin36 represents the gap junction protein on the cone side and lack thereof most likely disrupts rod-cone coupling. Using immunohistochemistry, we compared the progress of cone degeneration between connexin36-deficient mouse mutants and their connexin36-expressing littermates at different ages and assessed the accompanied morphological changes during the onset (rhodopsin knockout) and later stages of secondary cone death (rd1 mutants). Connexin36-deficient mouse mutants showed the same time course of cone degeneration and the same morphological changes in second order neurons as their connexin36-expressing littermates. Thus, our results indicate that disruption of connexin36-mediated rod-cone coupling does not stop, delay or spatially restrict secondary cone degeneration and suggest that the gap junction-mediated bystander effect does not contribute to the progression of RP.     

A method for in situ monitoring of the isotope composition of tree xylem water using laser spectroscopy

Field studies analyzing the stable isotope composition of xylem water are providing important information on ecosystem water relations. However, the capacity of stable isotopes to characterize the functioning of plants in their environment has not been fully explored because of methodological constraints on the extent and resolution at which samples could be collected and analysed. Here, we introduce an in situ method offering the potential to continuously monitor the stable isotope composition of tree xylem water via its vapour phase using a commercial laser‐based isotope analyser and compact microporous probes installed into the xylem. Our technique enables efficient high‐frequency measurement with intervals of only a few minutes per sample while eliminating the need for costly and cumbersome destructive collection of plant material and laboratory‐based processing. We present field observations of xylem water hydrogen and oxygen isotope compositions obtained over several days including a labelled irrigation event and compare them against results from concurrent destructive sampling with cryogenic distillation and mass spectrometric analysis. The data demonstrate that temporal changes as well as spatial patterns of integration in xylem water isotope composition can be resolved through direct measurement. The new technique can therefore present a valuable tool to study the hydraulic architecture and water utilization of trees.