The involvement of ER calcium in abiotic stress tolerance

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Title: The involvement of ER calcium in abiotic stress tolerance
Author: Lee, Sang Yoon
Advisors: George Allen, Committee Member
heike winter-sederoff, Committee Member
dominique robertson, Committee Chair
nina stromgren allen, Committee Member
Abstract: Calcium application is known to reduce the deleterious effects of NaCl in many plant species. Ca2+ supplementation is thought to act by inhibiting ion channels that allow Na+ influx and by blocking Na+-induced K+ efflux. I generated transgenic Arabidopsis lines that constitutively express a low affinity, high capacity Calcium Binding Peptide (CBP) localized to the endoplasmic reticulum. Four independent transformed lines, two that also contained GFP and two that lacked it, were analyzed and contained up to 10% more total calcium than GFP control and wild-type plants. Each of these lines also showed increased K+ that was balanced by a decrease in Na+ accumulation. There were no significant changes in the relative amounts of other ions. ER-CBP transgenic plants exhibited better salt and osmotic tolerance, increased survival in soil under intermittent drought conditions, longer root growth, higher chlorophyll content, and higher total seed production compared to GFP transgenic plants and control plants. One member of the CIPK (CBL-Interacting Protein Kinase) family, CIPK6, showed higher transcript levels in ER-CBP lines along with other drought-associated genes such as DREB1a and rd29a, even under non-stress conditions. However, DREB2a transcript level was not affected by ER-CBP. CIPK6 interacts with a Calcineurin B-Like Protein(s) (CBL) and was recently shown to interact with the C-terminus of the Arabidopsis Potassium Transporter1 (AKT1) protein. Using cipk6 null mutants, I showed that expression of both ER-CBP and CIPK6 was needed to achieve salt tolerance. I used two methods, confocal ratio imaging of Indo-1 and cytoplasmically-expressed aequorin luminescence, to detect transient changes in [Ca2+]cyt in response to a salt stimulus. There were no significant differences in [Ca2+]cyt measured by confocal ratio imaging between ER-CBP transgenic plants and control Arabidopsis plants in response to a short term salt treatment ( ~ 20 min). However, after three days incubation on 100 mM NaCl, ER-CBP lines had a higher steady state level of [Ca2+]cyt than wild type plants. Similarly, ER-CBP transgenic plants expressing aequorin in the cytoplasm did not show significant differences in Ca2+ spikes in response to 150 mM or 300 mM NaCl. After seedlings were grown on Ca2+ depleted media for 5 days, ER-CBP transgenic plants maintained Ca2+ peak heights similar to that seen before the low-calcium treatment, but control plants showed a decrease in Ca2+ spikes. This suggests that ER-CBP transgenic lines utilized extra ER Ca2+ stores under continual stress to maintain optimal Ca2+ levels in the cytoplasm. Trehalose has been shown to play an important role in drought tolerance. It is one of the most studied osmoprotectants. ER-CBP transgenic lines exhibited increased Trehalose 6-phosphate synthase (TPS) and Trehalose-6-phosphate phosphatase (TPP) expression. Accordingly, the trehalose content of ER-CBP lines was significantly higher compared to control plants. These results strongly implicate ER calcium as being critical for sustained tolerance to drought and salt in Arabidopsis.
Date: 2010-04-15
Degree: PhD
Discipline: Plant Biology

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