In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics.

The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 µM vs. 0.05 µM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 µM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones.

Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress.

For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca(2+)]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca(2+)] in the apoplast and in the cytoplasm were studied under salt and "drought" (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells.

Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension.

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal approximately 30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.

Cyclic nucleotides: the current dilemma!

Significant advances in understanding plant cyclic nucleotide signalling have been made in the past two years. The roles of these molecules in the regulation of ionic channels, defence responses and the apical growth of cells are being uncovered.

Reorientation of seedlings in the earth's gravitational field induces cytosolic calcium transients.

The gravitational field controls plant growth, morphology, and development. However, the underlying transduction mechanisms are not well understood. Much indirect evidence has implicated the cytoplasmic free calcium concentration ([Ca(2+)](c)) as an important factor, but direct evidence for changes in [Ca(2+)](c) is currently lacking. We now have made measurements of [Ca(2+)](c) in groups of young seedlings of Arabidopsis expressing aequorin in the cytoplasm and reconstituted in vivo with cp-coelenterazine, a synthetic high-affinity luminophore. Distinct [Ca(2+)](c) signaling occurs in response to gravistimulation with kinetics very different from [Ca(2+)](c) transients evoked by other mechanical stimuli (e.g. movement and wind). [Ca(2+)](c) changes produced in response to gravistimulation are transient but with a duration of many minutes and dependent on stimulus strength (i.e. the angle of displacement). The auxin transport blockers 2,3,5-tri-iodo benzoic acid and N-(1-naphthyl) phthalamic acid interfere with gravi-induced [Ca(2+)](c) responses and addition of methyl indole-3-acetic acid to whole seedlings induces long-lived [Ca(2+)](c) transients, suggesting that changes in auxin transport may interact with [Ca(2+)](c). Permanent nonaxial rotation of seedlings on a two-dimensional clinostat, however, produced a sustained elevation of the [Ca(2+)](c) level. This probably reflects permanent displacement of gravity-sensing cellular components and/or disturbance of cytoskeletal tension. It is concluded that [Ca(2+)](c) is part of the gravity transduction mechanism in young Arabidopsis seedlings.