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Project
I. Changing calcium homeostasis in transgenic plants: A reverse genetics
approach (Thompson,
Robertson
and Boss).
This research effort is one of five integrated projects that in their
totality comprise the NSCORT in Gravitational Biology. As part of the
reverse genetic approach to study the role of phosphoinositide signaling
in plant gravitropic responses, we are producing Arabidopsis plants transformed
with genes encoding either the Arabidopsis phosphatidylinositol phosphate
5-kinase or the human type I inositol polyphosphate 5'phosphatase. The
gravitropic responses of T4 generation plants are currently under investigation.
The inositol polyphosphate 5'phosphatase was also expressed in tobacco
cell cultures, where changes in phosphoinositide metabolism were characterized
biochemically (Perera et al., 2002).
To investigate the effect of Ca++ storage and localization by over-expressed calreticulin calcium-binding peptides (CBP), we have now produced transgenic Arabidopsis lines expressing these peptides as ER-targeted GFP fusions (ER-CBP). Interestingly, we found that constitutive expression of a GFP fusion with the ER-CBP can increase calcium stores resulting in higher tissue levels of calcium (Wyatt, et al., 2001, Tsou, 2001). Similar constructs tested in primary maize transformants demonstrated that increased levels of calcium accumulated in transgenic ER-CBP plants compared to GFP control transgenics. In this trial, plants were grown under standard conditions in soil. We are now analyzing the gravity response in the Arabidopsis transformants that constitutively overproduce the C-terminal domain of calreticulin, the calcium binding peptide (CBP). As these plants carry the CBP as a fusion protein with green fluorescent protein (GFP), all analyses of the transgenics have used this reporter to verify expression of the transgene. These plants have a two-fold reduction in root gravitropism when grown on media with no added calcium and this inhibition is not observed when 10 mM calcium is added back to the media, suggesting that the free levels of Ca++ are important in the intensity of the gravitropic response. Additional experiments are examining auxin transport in these transgenics under low, high, or low returned to high calcium conditions. A reproducible delay in the gravitropic response of about 30 mins was seen in roots of Arabidopsis carrying the CBP-GFP fusion construct compared to GFP controls. As might be predicted for plants with high calcium stores, these same plants show better growth than GFP controls after transfer to high sodium chloride (0.1 - 0.2 M salt) (Tsou, 2002, Patent Application No. PCT/US01/13563).
A third project was stimulated by our need for high-resolution analysis of the free Ca++ distribution in living cells. In this project, we have produced greatly improved 'cameleon' genes that function as sensitive in vivo reporters of free Ca++ concentration (Love et al. in preparation). In these chimeric genes, different variants of the GFP fluorophore are linked to opposite ends of a calmodulin molecule. Free Ca++ influences the conformation of the calmodulin, leading to changes in fluorescence resonance energy transfer (FRET) between the two fluorophores. FRET patterns can then be used to obtain a map of Ca concentration in living cells. The new cameleon construct permits high resolution, real time imaging of Ca++ concentrations at the subcellular, cellular, or tissue level, using confocal microscopy or high resolution multiphoton imaging techniques.