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1 Moving Signals and Molecules Through Membranes n 0 0 n Plant Molecular Biology Group Colloquium Organized and Edited by P. Gilmartin (Centre for Plant Studies, University of Leeds). 671st Meeting held at the University of Leeds, I April Cyclic-nucleotide- and Ca*+/calmodulin-regulated channels in plants: targets for manipulating heavy-metal tolerance, and possible physiological roles T. Arazi*, B. Kaplan*, R. Sunkar* and H. Fromm*-/.' *The Weizmann Institute of Science, 76 I00 Rehovot, Israel, and +Centre for Plant Sciences, Leeds Institute for Biotechnology and Agricutture, School of Biology, University of Leeds, Leeds LS2 9JT, U.K. Abstract Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with a decreased accumulation of Ni2+ and an enhanced accumulation of Pb2+ respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role. Introduction Toxic metal pollution, which is due mainly to burning fossil fuels, to manufactured waste and to the indiscriminate use of chemicals, has accelerated markedly in recent years owing to world Key words: ion channel, Pb toxicity, signal transduction. Abbreviations used: CNG, cyclic-nucleotide-gated; cnmp, cyclic nucleotide monophosphate; NtCBP4. Nicotiano tabacurn calmodulin-binding protein. 'To whom correspondence should be addressed at the University of Leeds ( h.fromm@leeds.ac.uk). industrialization. In this context, the intrinsic metal uptake systems of plants render them vulnerable to toxic levels of metals in the environment. Because of the large amount of metal-contaminated land and the persistent nondegradable nature of toxic metals, these problems have received high priority from environmental protection agencies [l]. Elucidating the genes that code for proteins involved in the transport of heavy metals should be useful in developing biotechnologies aimed at cleaning the environment (e.g. phytoremediation [2,3]), as well as in engineering plant tolerance to toxic metals. Moreover, because of the limited ion selectivity of biological metal transport systems, heavy-metal transporters are likely to have other activities and various physiological roles yet to be discovered. However, only a few genes of this kind have been cloned from plants [4,5] and very little is known about their mode of action. We have been studying the molecular components of the plant Ca2+ and calmodulin messenger system, which seems to be involved in plant-environment interactions [6,7]. Recently we reported on the isolation and characterization of a calmodulin-binding tobacco plasma membrane protein (designated NtCBP4, for Nicotiuna tabacum calmodulin-binding protein), which is similar in structure to cyclic-nucleotide-gated (CNG) non-selective cation channels [8,9]. We demonstrated that 2-3-fold overexpression of NtCBP4 in transgenic plants had remarkable effects : enhancing Pb2+ uptake and conferring Pb2+ hypersensitivity, as well as attenuating Ni2+ uptake and improving Ni2+ tolerance [8]. Our current studies focus on Pb2+ uptake and tolerance Biochemical Society

2 Biochemical Society Transactions (2000) Volume 28, part 4 associated with NtCBP4, and on the physiological role and regulation of NtCBP4. Pb2+ is a highly toxic metal that can bind with high affinity to Ca2+-binding sites in regulatory proteins such as calmodulin [lo]. The similarity in Pb2+ and Ca2+ protein-binding sites is consistent with reported evidence that Pb2+ entry into animal cells [11,12] and into plant cells [13] occurs, at least in part, via Ca2+-permeable channels. Engineering Pb2+ tolerance and uptake in plants expressing NtCBP4 mutants We have taken two approaches to engineering tolerance and uptake of heavy metals associated with NtCBP4. One approach is mutation of presumed regulatory domains of the protein ; the second is mutation of the predicted pore region, which functions as the ion-selectivity filter. A presentation of these potential manipulations in NtCBP4 is shown in Figure 1. The presumed regulatory domains include a Ca2+-dependent calmodulin-binding site that coincides with a defined structural element of the NtCBP4 putative cyclic-nucleotide-binding domain [9]. In addition, the overall structural similarities of NtCBP4 to mammalian CNG cation channels suggest that this channel protein is regulated by cyclic nucleo I- tides and by Ca2+/calmodulin. Consistent wit1 h this possibility, calmodulin was shown to bin, d NtCBP4 with high affinity [9]; CNGC2 from n Arabidopsis thaliana, which is highly similar tl o NtCBP4 [8], was reported to be modulated b y cyclic nucleotides in heterologous systems [ 141 I. To test the feasibility of further manipulations o If Pb2+ tolerance in plants, a mutant was prepare1 d from which part of the NtCBP4 C-termina 11 domain, including the calmodulin-binding do - main and part of the cyclic nucleotide mono - phosphate (cnmp)-binding domain had beel n removed. On the basis of the known characteristics o f the CNG animal channels, we assume tha t NtCBP4 is a component of a multimeric-channe 4 complex (homomeric or heteromeric), like it s mammalian counterparts [ 151. Therefore the ex - pression of the truncated protein lacking regu - latory domains, assumed to be necessary fo r NtCBP4 function, might have an inhibitory effec t on the activity of endogenous plant NtCBP4 - associated channels. Expression of this mutant ii I transgenic plants did indeed result in attenuate( ;t Pb2+ uptake and increased Pb2+ tolerance (R. Sunkar, B. Kaplan, T. Arazi, D. Dolev and H. Figure I Schematic structural model of NtCBP4 and potential target sites for modulation of channel properties S 1-56 are presumed membrane-spanning domains [8] and the sites of interaction of second messengers [cyclic nucleotides. cnmp and Ca +/calmodulin (CaM)] [9]. Arrowheads denote sites chosen for mutagenesis in regulatory sequences and in the pore region. The amino acid sequences of the CaM-binding domain (CaMBD; bold letters) and part of the cyclic-nucleotidebinding domain predicted helices ab and ac (underlined) are shown [9]. Asterisks denote invariable residues in known cyclic-nucleotide-binding domains. The shadowed residue (T) determines nucleotide specificrty (cgmp or CAMP [ 191). The numbers ofthe terminal residues in this Figure correspond to GenBank accession number AF Biochemical Society 412

3 Moving Signals and Molecules Through Membranes Fromm, unpublished work). Further studies are planned for investigating specific mutations in NtCBP4 regulatory domains and their effects on heavy metal uptake and tolerance. In particular, it should be of interest to express mutants capable of binding either cyclic nucleotides or calmodulin, rather than eliminating both regulatory properties. The molecular basis for the ion selectivity of channels lies mainly within a region referred to as the pore domain. A functional connection between the pores of distantly related channels has been known for several years [16]. Furthermore, mutations in the pore region of channels have been shown to alter their ion selectivity, such as from K+-selective to non-selective univalent cations [16], from Na+-specific to Ca2+-specific channel characteristics [17], and even from cationic to anionic [ 181. Therefore engineering plant channelion selectivity has great potential in improving tolerance to toxic metals, in phytoremediation and in various traits of agronomic importance related to plant nutrition. The localization of NtCBP4 in the tobacco plasma membrane [8] and its identification as a component of channels permeable to Pb2' open the way to assessing the potential of engineering NtCBP4 ion selectivity. Sequence conservation between K+ channels and CNG cation channels is highest in the amino acids corresponding to the pore and the adjacent S6 transmembrane domain [19]. In this region, NtCBP4 shares similarities with K+-selective and CNG non-selective cation channels (Figure 2). However, NtCBP4 lacks the consensus stretch of amino acids GYGD (single-letter codes) that is essential for K+ selectivity [19]. Instead, the motif GQNL is present at the equivalent position in NtCBP4. In addition, the cluster of threonine residues that also determines K' selectivity [20] is missing from NtCBP4. This raises the possibility that NtCBP4 functions as a component of nonselective cation channels. Because the molecular basis for K+-selectivity is fairly well understood, and because Arabidopsis homologues of NtCBP4 were suggested to be permeable to K+ [14,21], we first chose to test the feasibility of obtaining functional engineered NtCBP4 channels that are K+-selective. The resulting engineered NtCBP4 channels are expected to be relatively impermeable to cations such as CaZ+ and Pb2+. Transgenic plants expressing this mutant channel are therefore expected to exhibit improved tolerance to Pb2+. The effects of these modifications on heavy metal uptake and plant tolerance will be investigated in transgenic plants. Physiological role of NtCBP4 : possible function in Caz+ signal transduction Because Pb2+ is a non-essential toxic metal for plants, plants are unlikely to possess Pb2+-specific channels. Rather, the permeability of the plasma membrane to Pb2+ is attributed to the inherent limited selectivity of certain plant ion channels with other functions and physiological roles. Genes homologous with NtCBP4 were isolated from barley [22], Avabidopsis (AtCNGCl- AtCNGC6 [14,21]), alfalfa, tomato and potato (T. Arazi and H. Fromm, unpublished work). However, the physiological role of these proteins remains unknown. They probably have a role in Ca2+ signal transduction either by regulating NtCBP4-channel activity by Ca2+/calmodulin [9], by Ca2+ permeability through the channel [14] or by both mechanisms, as in the mammalian CNG non-selective cation channels. The relationship between Pb2+ and Ca2+ permeability is consistent with previous reports that Pb2+ entry into animal Figure 2 Amino acid sequences of P and S6 regions of NtCBP4 compared with that of K+-selective channels from Srrepromyces lividam (Kcsa), Drosophila (Dmshaker) and Arabidopsis (AtAKT I) and a non-selective cation channel from rat (RCNG2) (GenBank accession numbers , X0713 I, X93022 and X555 I9 respectively) Structural elements ofthe KcsA channel are indicated above the sequence. Residues ofthe ionselectivity filter, and the lining of the cavity and the inner pore [ 191, are shown in bold and shadowed letters respectively. Conserved amino acid residues are on a grey background. BtCBP4 KCIA RCNG2 AtAxT1 r- PORE REGION I S Biochemical Society

4 Biochemical Society Transactions (2000) Volume 28, part 4 cells [l 1,121 and into plant cells [13] occurs via Ca2+-permeable channels. Furthermore, the expression of AtCNGC2 in embryonic kidney cells evoked an influx of Ca2+ [14]. The results presented so far suggest that NtCBP4 functions as a plasma membrane CNG non-selective cation channel. This and related plant channels might be involved in mediating plant responses to biotic and abiotic stimuli that are transduced via Ca2+ and cnmps. We hypothesize that the binding of cyclic nucleotides (preferentially cgmp [9]) to NtCBP4 will probably take place when the cytoplasmic concentration of cgmp rises above a resting level in response to an external stimulus. Certain stimuli are known to induce a rise in cgmp in plants, including gibberellic acid in barley aleurone cells [23] and NO in spruce needles [24] and tobacco plants [25]. cgmp binding to NtCBP4 will result in an influx of cations (including Caa+) into the cytoplasm, causing an increase in the intracellular Ca2+ concentration. An increase in cytoplasmic Ca2+ concentration will activate calmodulin, resulting in binding to NtCBP4. This will impair the binding of cgmp to NtCBP4 and therefore inhibit channel opening (see the working model in Figure 3). Ion channels that are regulated by cyclic nucleotides and Ca2+/calmodulin might be involved in the reciprocally repressive crosstalk between cgmp and Ca2+/calmodulin-mediated pathways in plants (e.g. in phototransduction [26]). Interestingly, Kurosaki et al. [27] reported on the involvement of plasma-membrane-located calmodulin in the response decay of the CNG cation channel of cultured carrot cells, which could be attributed to channels such as NtCBP4. Figure 3 Working model of NtCBP4 function and regulation The presumptive membrane-spanning regions and pore are shown as S 1-56 and P respectively. The putative cyclic-nucleotide-binding domain and the identified calmodulin (CaM)-binding Site are shown. stimulus K+ f z Biochemical Society 474

5 Moving Signals and Molecules Through Membranes However, one should also consider the possibility that the gene family of CNG channels in plants encodes isoforms that might respond to Ca2+/calmodulin and cyclic nucleotides in different ways. These isoforms might differ in their ability to bind different calmodulin isoforms at different concentrations of Ca2+, or even in the absence of Ca2+. In addition, one cannot exclude the possibility that some of these channels, which might function as multimeric complexes (homomeric or heteromeric) are activated by Ca2+/ calmodulin and/or might be inactivated by cyclic nucleotides. A major challenge is to determine whether any of these features can be attributed to NtCBP4 and the related plant proteins. Conclusions and prospects We have isolated and characterized a plasma membrane tobacco protein involved in Pb2+ uptake and tolerance in plants. The transgenic plants that we prepared provide unique research tools for addressing questions with implications for phytoremediation and for the implemention of metal tolerance in plants. Moreover, combining additional approaches such as electrophysiology and analysis of Ca2+ dynamics in plant cells might help to elucidate the physiological roles of NtCBP4 and its family members and might lead to a better understanding of the interactions of Ca2+ and cyclic-nucleotide signalling pathways in plants. We acknowledge the support of the Israel Science Foundation to H. F. and the Sir Charles Clore Foundation for a postdoctoral fellowship to R S. and a graduate fellowship to T. A. References I Eccles, H. (I 998) Biochem. SOC. Trans. 26, I 2 Raskin, I. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, I66 3 Teny, N. and Banuelos, G. ( 1999) Phytoremediation of Contaminated Soil and Water, CRC Press, London 4 Schachtman. D. P., Kumar, R, Schroeder, J. I. and Marsh, E. L. ( 1997) Proc. Natl. Acad. Sci. U.S.A. 94, I I I084 5 Van der Zaal. B. J., Neuteboom, L. W., Pinas, J. E.. Chardonnens, A. N., Schat, H., Verkleij, J. A C. and Hooykaas, P. J. J. (I 999) Plant Physiol. I 19, I055 6 Snedden, W. A. and Fromm, H. (I 998) Trends Plant Sci. 3, Zielinski, R. E. (I 998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, Arazi, T., Sunkar R, Kaplan, B. and Fromm, H. (I 999) Plant J. 20, Arazi, T., Kaplan, B. and Fromm, H. (2000) Plant Mol. Biol. 42, I 10 Ouyang, H. and Vogel, H. J. ( 1998) BioMetals I I, I1 Simons, T. J. B. and Pocock G. (I 987) J. Neurochem. 48, Tomsig, J. L. and Suszkiw, J. B. ( I99 I) Biochim. Biophys. Acta 1069, Huang, J. W. and Cunningham, S. D. (I 996) New Phytol. 134, Leng. Q., Mercier, R W., Yao, W. and Berkowitz. G. A. (I 999) Plant Physiol. I2 I, I I5 Liu. D. T., Tibbs, G. R and Siegelbaum, S. A. (I 996) Neuron 16, Heginbotham, L., Abramson, T. and MacKinnon. R (I 992) Science 258, I 152- I I55 17 Heinemann, S. H., Terlau, H., Stuehmer, W., Imoto, K. and Numa, S. ( 1992) Nature (London) 356,44 I Galzi. J. L., Devillen-Thiery, A,, Hussy, N., Bertrand, S., Changeux, J.-P. and Bertrand, D. (I 992) Nature (London) 359,50& Doyle, D. A., Morais Cabral, J., Pfuetzner, R A., Kuo, A., Gulbis. J. M., Cohen. S. L, Chak B. T. and MacKinnon, R ( 1998) Science 280, Heginbotham, L, Lu. Z., Abramson, T. and MacKinnon, R (I 994) Biophys. J. 66, I06 I - I Kohler, C., Merkle, T. and Neuhaus, G. (1999) Plant J. 18, 97- I Schuurink R C., Shartzer, S. F., Fath, A. and Jones, R. L. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, Penson. S. P., Schuurink R. C., Fath, A., Gubler, F., Jacobsen, J. V. and Jones, R L. (I 996) Plant Cell 8, Pfeiffer, S., Janistyn, B., Jessner, G., Pichomer, H. and Ebermann, R. (I 994) Phytochemistty 36, Dumer, J., Wendehenne, D. and Klessig, D. F., (I 998) Proc. Natl. Acad. Sci. U.S.A. 95, Bowler, C. and Chua, N.-H. ( 1994) Plant Cell 6, I54 I 27 Kurosaki, F.. Kaburakai, H. and Nishi, A. (I 994) FEES Lett. 340, Received 23 February Biochemical Society