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Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca²⁺ sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca²⁺ levels. When global Ca²⁺ responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

While exogenous toxic compounds such as herbicides are thought to be sequestered into vacuoles in the form of glutathione (GSH) conjugates, little is understood about natural plant products conjugated with GSH. To identify natural products conjugated with GSH in plants, metabolites in the Arabidopsis γ-glutamyl transpeptidase (ggt) 4 knockout mutants that are blocked in the degradation of GSH conjugates in the vacuole were compared with those in wild-type plants. Among the metabolites identified, one was confirmed to be the 12-oxo-phytodienoic acid (OPDA)-GSH conjugate, indicating that OPDA, a precursor of jasmonic acid (JA), is transported into the vacuole as a GSH conjugate.

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a 'sulphur-free' solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance.

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

Reactive oxygen species (ROS), such as O2- and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2- and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H2O2, nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels.

The effects of ageing on autonomic nervous responses have been investigated in 29 young adults, 64 healthy elderly in the age range 66 to 86 years, and 20 elderly in-patients with hypothermia, instability of bladder function or marked orthostatic hypotension. In the healthy elderly group, the beat-to-beat variation in response to postural change was significantly diminished, the vasoconstrictor response to cooling reduced and baroreflex sensitivity during lower-body negative pressure was decreased compared with young adults. Patients with an atronic bladder, determined by urodynamic tests, showed an even more marked decrease in baroreflex sensitivity. The multisystem nature of these autonomic disturbances suggests that physiological impairment may occur in autonomic neural pathways with ageing.