Background The presence of cyclic nucleotides (CNs) such as cGMP and cAMP in plant cells has unequivocally been demonstrated as have cyclase and cyclic nucleotide phosphodiesterase activity, two key enzymes of cAMP metabolism (1). Accumulating evidence suggests crucial roles for both cAMP and cGMP in plant cellular homeostasis (e.g. 2;3;4).

In the plasma membrane of Arabidopsis thalianacells, we have identified ion channels that are regulated by CNs (see Cell Signalling and Cyclic Nucleotides) and 5). These so-called non-selective ion channels are likely to be involved in transport of ions such as Na+ and Ca2+. The channel function in plant Na+ entry is reflected in growth experiments where the level of salt tolerance exhibited by A. thaliana plants increases in the presence of externally applied CNs.


-------Control---------100 mM NaCl-----NaCl plus cGMP
The effect of NaCl and cyclic GMP on Arabidopsis salt tolerance

However, this response also depends on external Ca2+ suggesting a role for this second messenger in CN plant signalling. Interestingly, plant many CN regulated ion channels contain a calmodulin binding site in the same region as the CN binding domain. In addition the role of Ca2+ and calmodulin in plant salt tolerance has been extensively studied (e.g. 6;7).

Project aims The objective of this project is to establish how CN signalling forms part of the perception of salt stress and mechanisms of salt tolerance in plants. To this end, endogenous CN levels will be determined as a function of external [NaCl] and time. The effects of Ca/CaM on CN levels and salt tolerance will be assessed by imposing different external Ca concentrations and the use of CaM antagonists such as TFP and W-13. Cyclic nucleotides will be determined using immunoassays. In addition, we will use a transgenic approach to express the fluorescent, PKA-based, cAMP reporter (4). This is an innovative strategy that has not been attempted in plants and would allow for the first time the non-destructive real-time measurement of cAMP in living cells. To achieve this,Arabidopsis will be transformed with the catalytic and regulatory PKA subunits fused to respectively fluorescein and rhodamine or other suitable reporters. Measurement of the cAMP induced reduction in FRET between the reporters allows quantification of cellular cAMP.

Methods We will use growth assays, biochemical assays to determine endogenous CN levels and molecular approaches to transform Arabidopsis. If Arabidopsis is successfully expressing endogenous cAMP reporter, we will use confocal microscopy to measure cellular cAMP levels.

References:
(1) Bolwell (1995) TIBS 20:492-495
(2) Ehsan et al (1998) FEBS letters 422:165-169
(3) Penson et al (1996) Plant Cell 8:2325-2333
(4) Moutinho et al (2001) PNAS (USA) 98: 10481-10486
(5) Maathuis & Sanders (2001) Plant Physiol 127:1617-1625
(6) Elphick et al (2001) Plant Cell Environ 24:733-740
(7) Zhu (2001) TIPS 6:66-71.