Limits in the use of cPTIO as nitric oxide scavenger and EPR probe in plant cells and seedlings.

Over the last decade the importance of nitric oxide (NO) in plant signaling has emerged. Despite its recognized biological role, the sensitivity and effectiveness of the methods used for measuring NO concentration in plants are still under discussion. Among these, electron paramagnetic resonance (EPR) is a well-accepted technique to detect NO. In the present work we report the constraints of using 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in biological samples as spin trap for quantitative measurement of NO. EPR analyses on Arabidopsis cell cultures and seedlings show that cPTIO(NNO) is degraded in a matter of few minutes while the (INO) compound, produced by cPTIO and NO reaction, has not been detected. Limitations of using this spin trap in plant systems for quantitative measurements of NO are discussed. As NO scavenger, cPTIO is widely used in combination with 4-amino-5-methylamino-2('),7(')-difluorofluorescein (DAF-FM) fluorescent dye in plant research. However, the dependence of DAF-FM fluorescence on cPTIO and NO concentrations is not clearly defined so that the range of concentrations should be tightly selected. In this context, a systematic study on cPTIO NO scavenging properties has been performed, as it was still lacking for plant system applications. The results of this systematic analysis are discussed in terms of reliability of the use of cPTIO in the quantitative determination and scavenging of NO in plants and plant cultured cells.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride­proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²âº handling relationship in plant cells.

Here we describe use of a mitochondrial targeted Cameleon to produce stably transformed Arabidopsis plants that enable analyses of mitochondrial Ca²âº dynamics in planta and allow monitoring of the intra-mitochondrial Ca²âº concentration in response to physiological or environmental stimuli. Transgenic plants co-expressing nuclear and mitochondrial targeted Cameleons were also generated and analyzed. Here we show that mitochondrial Ca²âº accumulation is strictly related to the intensity of the cytoplasmic Ca²âº increase, demonstrating a tight association between mitochondrial and cytoplasmic Ca²âº dynamics. However, under all experimental conditions, mitochondrial Ca²âº dynamics were substantially different from those monitored in the cytoplasm, demonstrating that mitochondria do not passively sense cytosolic Ca²âº, but actively modulate the intra-mitochondrial level of the cation. In particular, our analyses show that the kinetics of Ca²âº release from mitochondria are much slower than in the cytoplasm and nucleus. The mechanisms and functional implications of these differences are discussed.

Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L.

Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 microg arsenic g(-1), and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5'-end, where the similarity is as high as 85-95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation.

The metabolic basis for 2,4-D resistance in two variant cell lines of carrot.

In the present work, the characterization of two variant cell lines of carrot capable of growing in high (92 µmol L-1) concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. Both these cell lines (4w77 and 4w13) show a significantly lower uptake of 2,4-D with respect to wild-type (wt) cells. In contrast to wt cells, influx of IAA is not reduced by the addition of 100 µM 2,4-D and the presence of this compound appears to stimulate IAA uptake. When grown in the presence of high concentrations of 2,4-D, both 4w77 and 4w13 cells show behavioural differences: instead of lowering the endogenous level of free IAA, the two resistant lines react to the high exogenous concentrations of auxin by raising the level of the free hormone. In 4w77 cells, this is accomplished by reduction of auxin released in the external medium or converted to amide-linked conjugates. In 4w13 cells, the final level of endogenous IAA is an equilibrium between increased synthesis of IAA and a massive release into the medium of the ester- and free-forms of IAA. Both cell lines show disturbances in embryogenesis: line 4w77 forms globular embryos that only mature into aberrant forms having multiple axes, whereas line 4w13 has completely lost its morphogenic capacity.