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STUDY OF ACTIVITY OF ANION CHANNELS IN TOMATO MUTANTS AFFECTED IN RESPONSIVENESS
TO BLUE LIGHT
1. Introduction
The rapid initial changes of developmental processes are induced by a distinctly different light response in
which plants use the light as an environmental signal (photomorphogenesis) Kendrick and Kronenberg 1994).
The importance of blue light (B) in plant photomorphogenesis was recognized earlier (Darwin 1881; Vines
1886). On the basis of molecular genetic studies, it is now clear that there are at least two basic types of B
receptors in plants: cryptochromes and phototropins. Cryptochromes (CRY1 and CRY2) work together with
phytochromes to regulate cell elongation and photoperiodic flowering (Lin 2002). Electrophysiological studies
have demonstrated that rapid decrease in B-induced growth rate is always preceded by a depolarization of the
hypocotyl cell plasma membrane in irradiated tissues (Spalding and Cosgrove 1989), and that anion channels
are involved in B-controlled hypocotyl growth (Cho and Spalding 1996). A genetic and molecular approach
allowed identification of cryptochromes in tomato (Weller et al. 2001; Perrotta et al. 2001). It was shown that
CRY1 in tomato encodes a protein, which shows high similarity to Arabidopsis CRY1, and that it controls
hypocotyl elongation in response to B (Ninu et al. 1999).
In field conditions, plants are often exposed to various abiotic stresses such as low temperature, high
salinity, or osmotic stress. Abiotic stresses substantially reduce crop plant productivity (Epstein et al. 1980).
Therefore, an understanding of the underlying mechanisms involved in the plant responses to abiotic stresses is
essential to solve this important agronomic problem. Blue light has a role in the biosynthesis/metabolism of ABA
(Volmaro et al. 1998; Baraldi et al. 1995), which plays important role in ability of plants to resist abiotic stress.
Also, water channels could represent a prerequisite for strategies against osmotic stress. It was shown in
Arabidopsis, that gene PIP1b coding for aquaporin is highly expressed in B and by ABA (Kaldenhoff and Ecker
1999). These facts raise the question of the role of B in plant responses to osmotic stress.
2. Preliminary results and hypotheses
In tomato, mannitol (up to 140 mM) strongly reduces seed germination in light, but not in dark. We previously
reported that spontaneous mutant 7B-1 in tomato is resistant to osmotic stress in seed germination specifically
under blue light (Fellner and Sawhney 2001, 2002). Relative to WT, 7B-1 seedlings develop longer hypocotyl in
W and B, and contain elevated levels of endogenous ABA (Fellner et al. 2001). We found that in tomato, B
strongly inhibits seed germination in WT, but essentially less in the 7B-1 mutant. Interestingly, B-induced
inhibition of seed germination in 7B-1 can be completely restored by an anion channel blocker, 9-AC (Fellner,
unpublished results). Analysis of cry1-1 and cry1-2 mutants in tomato suggests that CRY1 is not most likely
involved in B-induced inhibition of seed germination. It is also supported by the fact that expression of CRY1
gene is not affected in 7B-1 mutant in comparison with WT (Fellner, unpublihed results).
We hypothesize that similarly to B-induced inhibition of hypocotyl growth mediated by CRY1 receptor in
Arabidopsis (Cho and Spalding 1996), B can inhibit seed germination in tomato by similar mechanism, i.e. via
activation of anion channels and consequent plasma membrane depolarization. But we also hypothesize that B
inhibits seed germination through an unknown receptor, possibly coding by 7B-1 gene. Putative resistance of
7B-1 to B-stimulated activation of anion channels can help the mutant to tolerate osmotic stress in blue light.
3. Project objectives
The long-term objective of our work is to contribute to understanding of the role of light signaling pathways in
plant growth and development, and to determine whether the mutation in 7B-1 gene affect a step involved in
blue light perception or signal transduction? In comparison with Arabidopsis, the only B-insensitive mutant, cry1,
and transgenic plants, tcry1 and tcry2 have been identified or constructed in tomato so far. Therefore, the
availability of B-resistant mutant 7B-1 offers a great opportunity to identify a new gene involved in B signaling.
Although in some systems B was shown to be involved in cell osmoregulation, to the best of our knowledge,
direct involvement of light including B in plant tolerance to osmotic stress was not reported.
The basic project question is:
- Do 7B-1, cry1-1, and cry1-2 differ from corresponding WTs in activity of anion channels and their
responsiveness to anion channel blockers?
Conditional project question is:
- Does blue light regulate the activity of anion channels in tomato protoplasts?
4. Experimental
Plant material: Experiments will be conducted with tomato Lycopersicon esculentum (Mill.). The following
tomato mutants and their corresponding WT will be used in the experiments: 7B-1 mutant (genetic background
cv. Rutgers; Sawhney 1997; Fellner and Sawhney, 2002); cry1-1 and cry1-2 mutants (γ-ray-generated mutant;
cv. Moneymaker; Weller et al. 2001; kindly provided by R. E. Kendrick, Wageningen University, Netherlands).
Experiments: Determination of activity of anion channels in protoplasts derived from tomato mutants
impaired in blue light sensing.
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Patch clamping will be used for study of activity of anion channels in protoplasts isolated from just
germinating tomato seeds. Ion currents across the plasma membrane will be measured on protoplasts using
established patch clamp electrophysiological methods (Hamill et al. 1981; Thomine et al. 1995). The effect of
anion channel blockers (9-AC, NPPB, etc) on activity of anion channels will be tested in tomato mutants 7B-1,
cry1-1, cry1-2, and corresponding WTs.
If possible, we will also study activation of anion channels in tomato protoplasts by blue light. Effect of
blue light on the activity of anion channels will be measured as described by Assmann et al. (1985) and Cho
and Spalding (1996).
Protoplast isolation will be carried out by a modified method described by Dorin et al. (1999) and Cho
and Spalding (1996). Protoplasts will be prepared from just germinating tomato seeds. Protoplasts prepared in
this way will be checked for high viability as determined by fluorescein diacetate fluorescence, and a high
capacity to form gigseals with patch pipettes.
Expression of genes coding for Arabidopsis anion channels AtCLC and AtVDAC will be studied in young
tomato seedlings grown in dark or under B. We will first look for AtCLC and AtVDAC homologues in tomato
using BLAST homology search. Afterwards, possible effect of B on expression of the genes in germinating
seeds will be studied by RT-PCR. RT-PCR will be carried out using a one step RT-PCR kit (InVitrogen)
according the manufacture’s instructions. Total RNA will be extracted from plant tissues using an RNA
extraction kit (RNeasy Plant Minikit Qiagen). cDNA will be synthesized using oligo(dT) 20 primer and
ThermoScript RT-PCR System (InVitrogen). WT and mutant cDNAs will be amplified with forward and reverse
primers specific for tomato genes found to code the anion channel proteins.
5. Literature cited
Assmann SM et al. (1995) Nature 318: 285-287; Baraldi R et al. (1995) Photochem Photobiol 62: 800-803; Cho MH,
Spalding EP (1996) Proc Natl Acad Sci USA 93: 8134-8138; Darwin C (1881) The power of movements in plants (New
York: D. Appleton and Company); Dorin N et al. (1999) Plant Cell Tissue Organ Culture 56: 9-16; Epstein E et al. (1980)
Science 210: 399-404; Fellner M, Sawhney VK (2001) Theor Appl Genet 102: 215-221; Fellner M, Sawhney VK (2002)
Planta 214: 675-682; Fellner M et al. (2001) J Exp Botany 52: 725-738; Hamill OP et al. (1981) Pflugers Arch 391: 85-100;
Kaldenhoff R, Ecker M (1999) J Photochem Photobiol B. Biol 52: 1-6; Kendrick RE, Kronenberg GHM (1994)
Photomorphogenesis in Plant, 2nd ed., Kluwer Academic Publishers, Dordrecht, Netherlands; Lin C (2002) Plant Cell Suppl:
S207-S225; Ninu L et al. (1999) Plant J 18: 551-556; Perrotta et al. (2001) Plant Cell Environ 24: 991-997; Sawhney VK
(1997) In: Shivanna KR, Sawhney VK, ed, Pollen Biotechnology for Crop Production and Improvement, Cambridge
University Press, Cambridge, UK, pp. 183-198; Spalding EP, Cosgrove DJ (1989) Planta 178: 407-410; Thomine et al.
(1995) Plant Cell 7: 2091-2100; Vines SH (1886) Lectures on the physiology of plants Cambridge University Press,
Cambridge. UK; Volmaro C et al. (1998) Plant Growth Regul 26: 165-173; Weller JM et al. (2001) Plant J 25: 427-440;
6. Timeline, project significance and supporting circumstances
The project will take place over 2 years and expects 8-week-stay in total in French laboratory. The
electrophysiological experiments described above will be performed by Martin Fellner or his diploma student,
Zdenek Svoboda in laboratory of Dr. Hélène Barbier-Brygoo at Institute of Plant Sciences (Institut des Sciences
du Végétales, CNRS) in Gif-sur-Yvette (http://www.isv.cnrs-gif.fr/groupes/groupegb.html). In reverse, study of
gene expression will be performed by a young French fellow in Martin Fellner’s laboratory at Institute of
Experimental Botany and Palacky University in Olomouc.
To the best of my knowledge, direct involvement of B in plant tolerance to osmotic stress was not
reported. Scientists dealing with B found this subject of study interesting. M. Fellner was invited as a symposium
speaker to present his results and new ideas at the International meeting The Blue Light Syndrome IV, held in
Okazaki, Japan, in December 16-18, 2002. The fact that in tomato two genes, Arabidopsis homologues, CRY1
and CRY2 were identified so far, makes the 7B-1 mutant very interesting, with respect to the possibility to
identify a new gene involved in B signaling. We will first investigate whether 7B-1 and the other B-resistant
mutants differ in basic activity of anion channels. If this result is not what we expect, we still have a chance to
study their activity under blue light and, in a possible future project(s), to contribute to revealing the function of
the 7B-1 gene in plant growth. If we obtain the results confirming our hypothesis, in the future project we plan to
map and clone the 7B-1 gene.
In the laboratory of Dr. Hélène Barbier-Brygoo I spent almost 5 years during my Ph.D. study at
Université Paris-Sud in period from 1992 to 1997. Dr. Hélène Barbier-Brygoo is now director of the Institute of
Plant Sciences. Her research group “Membrane transporters” (http://www.isv.cnrs-gif.fr/hbb/indexgb.html)
extended and deals now with four research areas, such as Molecular and functional characterization of anion
channels, and osmotic stress signalling. Research focus of Hélène’s lab, top quality of the team, our similar
research interest, and finally, our friendly connections are very suitable for performing experiments mentioned in
the project above.
Abbreviations: ABA = abscisic acid; A-9C = anthracene-9-carboxylic acid; B = blue light; NPPB = 9-nitro-2-(3phenylpropylamino)benzoic acid; W = white light; WT = wild-type.
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