Heather Walker

Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling

Summary An integral part of global environment change is an increase in the atmospheric concentration of CO2 ([CO2]) [1]. Increased [CO2] reduces leaf stomatal apertures and density of stomata that plays out as reductions in evapotranspiration [2–4]. Surprisingly, given the importance of transpiration to the control of terrestrial water fluxes [5] and plant nutrient acquisition [6], we know comparatively little about the molecular components involved in the intracellular signaling pathways by which [CO2] controls stomatal development and function [7]. Here, we report that elevated [CO2]-induced closure and reductions in stomatal density require the generation of reactive oxygen species (ROS), thereby adding a new common element to these signaling pathways. We also show that the PYR/RCAR family of ABA receptors [8, 9] and ABA itself are required in both responses. Using genetic approaches, we show that ABA in guard cells or their precursors is sufficient to mediate the [CO2]-induced stomatal density response. Taken together, our results suggest that stomatal responses to increased [CO2] operate through the intermediacy of ABA. In the case of [CO2]-induced reductions in stomatal aperture, this occurs by accessing the guard cell ABA signaling pathway. In both [CO2]-mediated responses, our data are consistent with a mechanism in which ABA increases the sensitivity of the system to [CO2] but could also be explained by requirement for a CO2-induced increase in ABA biosynthesis specifically in the guard cell lineage. Furthermore, the dependency of stomatal [CO2] signaling on ABA suggests that the ABA pathway is, in evolutionary terms, likely to be ancestral.

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50u2009mgu2009kg−1 using a combination of HPLC-MS, HPLC-MS/MS, 19F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with 35Cl and 34S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5u2009mgu2009kg−1 with [14C]-labelled compound enabled the comparison of standard radioassay analysis methods with 19F-NMR spectroscopy. 19F-NMR resonances were only readily detectable in the 0–12u2009h post-dose samples. Dosing at 50u2009mgu2009kg−1 allowed the facile and specific detection and quantification of metabolites by 19F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with 35Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using 19F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS (127I)

A combination of 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by 19F-NMR spectroscopy and HPLC-127I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.

A novel root‐to‐shoot stomatal response to very high CO2 levels in the soil: Electrical, hydraulic and biochemical signalling

Investigations were undertaken in the context of the potential environmental impact of carbon capture and storage (CCS) transportation in the form of a hypothetical leak of extreme levels of CO2 into the soil environment and subsequent effects on plant physiology. Laboratory studies using purpose built soil chambers, separating and isolating the soil and aerial environments, were used to introduce high levels of CO2 gas exclusively into the rhizosphere. CO2 concentrations greater than 32% in the isolated soil environment revealed a previously unknown whole plant stomatal response. Time course measurements of stomatal conductance (gs ), leaf temperature and leaf abscisic acid (ABA) show strong coupling between all three variables over a specific period (3 h following CO2 gassing) occurring as a result of CO2 -specific detection by roots. The coupling of gs and ABA subsequently breaks down resulting in a rapid and complete loss of turgor in the shoot. Root access to water is severely restricted as evidenced by the inability to counter turgor loss, however, the plant regains some turgor over time. Recovery of full turgor is not achieved over the longer term. Results suggest an immediate perception and whole plant response as changes in measured parameters (leaf temperature, gs and ABA) occur in the shoot, but the response is solely due to detection of very high CO2 concentration at the root/soil interface which results in loss of stomatal regulation and disruption to control over water uptake.

Understanding metabolism of arginine in biological systems via MALDI imaging.

Arginine is an important amino acid but has been barely studied in plants. The little research that has been done indicates that the pathways of synthesis are similar to those found in animals and procaryotes. However little is known about the cellular and tissue localization of the amino acid in plants. The research reported in this paper was designed to examine whether MALDI‐MSI was sufficiently sensitive to examine the distribution of this amino acid in plant material, and whether the synthetic pathways were co‐located. In wheat and orchid roots, the amount of arginine in tissues varies greatly and the pathways for its synthesis were not always detected with the amino acid.

A metabolite profiling method for diagnosis of precancerous cervical lesions and HPV persistence

AIMnWith the advent of rapid metabolic profiling techniques and of portable mass spectrometers we examined whether cells distinguished by their cytology and persistence of human papillomavirus infection, could be easily differentiated by their metabolite profile.nnnMATERIALS & METHODSnDirect injection electrospray mass spectrometry was used in a nontargeted double-blind experiment. Samples were collected from women diagnosed with one of two grades of cervical cytology and exhibiting either human papilloma virus persistence or clearance. Cell extracts were prepared using a DNA extraction procedure and the resulting supernatant, normally discarded, was analyzed. Data were interpreted using principal component analysis.nnnRESULTSnThe results indicate strongly that a simple metabolite profiling method could be used to rapidly identify women at increased risk of cervical cancer.