Glutamate-Receptor Genes in Plants

scientific correspondence Glutamate-receptor genes in plants n animal brains, ionotropic glutamate transmembrane domains (M1 to M4) and GlnH1 (including the ligand-binding Ireceptors (GluRs) function as glutamate- the putative ligand-binding domains (GlnH1 residue R), GlnH2, and M1 to M4, especially activated ion channels in rapid synaptic and GlnH2), previously shown to be con- within M3 (Fig. 1a). The degree of identity transmission. We have now discovered that served between Escherichia coli glutamine between Arabidopsis GLRs and animal genes encoding putative ionotropic GluRs permease (GlnH) and animal ionotropic ionotropic GluRs within these domains exist in plants, and we present preliminary GluRs1 (Fig. 1). Hydropathy plots, trans- (63% to 16%) is similar to that between ani-8 evidence for their involvement in light- membrane prediction2 and protein-sorting3 mal ionotropic GluR subtypes that have signal transduction. It may be that sig- programs predict that the Arabidopsis GLR kainate/AMPA rather than NMDA as ago- nalling between cells by excitatory amino cDNAs encode a plasma-membrane signal nists (60% to 32%). Arabidopsis, like ani- acids in animal brains evolved from a prim- peptide and four transmembrane domains mals, seems to have a family of expressed itive signalling mechanism that existed (M1 to M4), of which M2 is predicted not genes encoding ionotropic GluRs, as judged before the divergence of plants and animals. to span the membrane (Fig. 1b). This mem- by the existence of other GLR genes (Fig. 1a) Our findings also help to explain why neu- brane topology is analogous to animal and northern blot analysis (data not roactive compounds made by plants work ionotropic GluRs in which the putative shown). GLR-related genes also seem to on receptors in human brains. ligand-binding GlnH domains are exposed exist in a variety of higher plants, inclu- We isolated from Arabidopsis two full- to the external side of the membrane4. ding both dicotyledons (tobacco and pea) length complementary DNAs, GLR1 and In addition to their similar secondary and monocotyledons (rice and maize), as GLR2. Each of these cDNAs encodes all of structures, the Arabidopsis GLRs also share judged by heterologous Southern blot the signature domains of animal ionotro- extensive sequence identity with animal analysis (not shown). pic GluRs, including the ‘three-plus-one’ ionotropic GluRs in the signature domains To investigate the possible in vivo a

Figure 1 Arabidopsis GLR cDNAs encode proteins with b sequence identity to animal ionotropic glutamate receptors. a, Sequence alignment of signature + Arabidopsis GLR1 ionotropic GluR domains conserved between Ara- S M1 M3 M4 M2 bidopsis GLR genes (AtGLR1-4) and animal ionotropic GlnH2 GluR subtypes using kainate/AMPA or NMDA as ago- GlnH1 (S2) 0 (S1) nists. Numbers above the sequences correspond to Out residues of Arabidopsis GLR1; numbers underneath correspond to residues in rat GluRK1. Residues M1 M2 M3 M4 between the conserved domains are shown as dots. - Arabidopsis GLR1 (Genbank accession no. AF079998) In and GLR2 (AF079999) sequences are from full-length 100 200 300 400 500 600 700 800 900 cDNAs obtained by screening cDNA libraries with expressed sequence tag clones (accession nos T22862 and R29880, respective- b, Transmembrane organization of Arabidopsis GLR1 based on hydropathy plot, ly). The GLR2 cDNA corresponds to Arabidopsis genomic sequence (accession TMPred2 and PSORT3 analysis. Hydrophobic domains include a putative plasma- no. AC002329). GLR3 and GLR4 sequences were derived from Arabidopsis membrane signal sequence (hatched box) and four transmembrane domains genomic sequences of a bacterial and yeast artificial chromosome clones M1–M4 (filled boxes), of which M2 is predicted not to span the membrane. Open (AF007271 and AC000098, respectively). Animal ionotropic GluR genes used in boxes denote the two putative extracellular glutamate-binding domains GlnH1 the alignment are: AMPA/kainate subtypes, accession numbers M36418, X17184, and GlnH2 (also called S1 and S2), identified in animal ionotropic GluR genes as X57498, M83552, X59996, S40369, U34661, M73271; or NMDA subtypes, accession sharing sequence identity with Escherichia coli glutamine permease1. GlnH1C numbers X63255, L05666, M91561. Sequences were aligned using Clustal X8. denotes identity within the carboxy-terminal half of the GlnH1 domain.

NATURE | VOL 396 | 12 NOVEMBER 1998 | www.nature.com Nature © Macmillan Publishers Ltd 1998 125 scientific correspondence function(s) of putative ionotropic GluRs in deficient in the synthesis of the phyto- light-signal transduction in plants. Because plants, Arabidopsis seedlings were grown in chrome photoreceptor chromophore (hy1 Arabidopsis, like animals, seems to possess a the presence of DNQX, an antagonist of and hy2; 1.5- to 2.5-fold)5,6. family of GLR genes, the in planta effects of animal kainate/AMPA ionotropic GluRs. Because light and hormones interact to DNQX-treated plants may be due to inhibi- Plants grown on media containing DNQX control hypocotyl elongation, one or both tion of one or more ionotropic GluR recep- phenocopy Arabidopsis long-hypocotyl (hy) may be responsible for the DNQX effect. tors in vivo. mutants impaired in light-signal transduc- We therefore measured the effects of DNQX The existence of putative ionotropic tion5, as judged by two independent crite- on a second light-dependent process, GluRs in plants is surprising, and provides ria: DNQX at least partly blocks the ability chlorophyll synthesis. DNQX treatment an insight into why plants make chemicals of light to inhibit hypocotyl elongation (Fig. impairs the ability of light to induce the that act on human brains. Several classical8 2a–c) and to induce chlorophyll synthesis synthesis of chlorophyll in plants grown in ionotropic GluR agonists that activate and (Fig. 2d–f). Plants grown in the light in the the dark and exposed to light for 5 hours define specific ionotropic GluR subtypes presence of DNQX display a significant (Fig. 2d–f). DNQX-treated plants exhibited in brains are natural plant products. light- and dose-dependent increase in a 60% reduction in light-induced chloro- Examples include kainate, synthesized in hypocotyl elongation compared with con- phyll accumulation (Fig. 2e) compared with the seaweed Digenea simplex, and trol untreated plants (Fig. 2b,c). This effect controls (Fig. 2d). This effect is light spe- quisqualic acid, found in Quisqualis indica is specific for light, as the same DNQX con- cific as growth and chlorophyll levels are seeds7. Other less well known plant- centration has no such effect in the dark unaffected in DNQX-treated plants grown derived ionotropic GluR agonists include (Fig. 2a). The increase in hypocotyl length in the dark (Fig. 2a,f). ȋ-N-methyl-amino-L-alanine, produced in DNQX-treated plants (1.5- to 2-fold) is Taken together, these findings indicate in cycads, and ȋ-N-oxalylamino-L-alanine, similar to that of Arabidopsis hy mutants that ionotropic GluRs may be involved in from chick-peas, both of which are associ- a Dark d ated with neurodegenerative diseases in ÐDNQX +DNQX ÐDNQX (5h light) animals7. It is traditionally believed that plants produce such neurotoxins for defence against herbivory. However, the discovery of putative ionotropic GluRs in plants and their possible role in light-signal transduction suggest an alternative theory: that plants may synthesize ionotropic GluR agonists in order to regulate their endogenous Light b ÐDNQX +DNQX e +DNQX (5h light) ionotropic GluR receptors, and that selec- tive pressure in certain species then led to high-level production for defence against herbivores. The existence of putative glutamate receptors in plants raises the possibility that other neurologically active compounds made by plants (such as nicotine, cocaine and caffeine), which act on receptors in animal brains, may also act as ligands for c n = 51 f Dark Light 0.6 ) endogenous receptors in plants to regulate Light g control

n 80 i n = 53 l a variety of as yet unknown cell-to-cell sig- d ) e e m nalling systems in higher plants. s c

( r

h 60

t Hon-Ming Lam*, Joanna Chiu†, p

0.4 g g n n = 44 n = 53 n

e +DNQX Ming-Hsiun Hsieh†, Lee Meisel†, ( l

l t y n t Igor C. Oliveira†, Michael Shin†, e 40 o t c n o o Gloria Coruzzi† p

0.2 c

y l l H y *The Chinese University of Hong Kong, h 20 p o

r Shatin, New Territories, Hong Kong o l

h control 0.0 +DNQX †Department of Biology, 0 0.1 0.2 0.4 C DNQX concentration (mM) 1 2 3 4 New York University, 1009 Main Building, 100 Washington Square East, Figure 2 Effects of ionotropic GluR antagonist DNQX on Arabidopsis seedlings. Seedlings were sown on MS New York, New York 10003, USA medium (Gibco BRL Murashige and Skoog basal medium containing 3% sucrose and 0.9% Difco bactoagar) e-mail: [email protected] in the absence or presence of the ionotropic GluR antagonist DNQX. All plates contained the solvent 1. Nakanishi, N., Schneider, N. A. & Axel, R. Neuron 5, 569–581 dimethyl sulphoxide at 1.6 Ȗl per ml medium. a–c, Effect of DNQX on hypocotyl length. Seedlings were ger- (1990). minated for 7 days either in the dark (a) or in a normal day–night cycle (16 h light: 8 h dark) (b) at 22 °C in the 2. Hofmann, K. & Stoffel, W. Biol. Chem. Hoppe-Seyler 374, 166 presence (400 ȖM) or absence of DNQX. Hypocotyl length was determined for light-grown seedlings (n, (1993). 3. Nakai, K. & Kanehisa, M. Genomics 14, 897–911 (1992). 44–51) germinated on 0, 100, 200 and 400 ȖM DNQX (c). An unpaired t-test comparing values from control 4. Barinaga, M. Science 267, 177–178 (1995). and DNQX treatments: 100 ȖM DNQX (no significant difference); 200 ȖM DNQX (P Ͻ 0.0001); 400 ȖM DNQX 5. Koornneef, M., Rolff, E. & Spruit, C. J. P. Z. Pflanzenphysiol. (P Ͻ 0.0001). d–f, Effect of DNQX on light-induced chlorophyll levels. Seedlings were germinated in total dark- 100, 147–160 (1980). ness in the absence (d) or presence (e) of 400 ȖM DNQX for 7 days. Plants were transferred to light for 5 h 6. Goto, N., Yamamoto, K. T. & Watanabe, M. Photochem. and chlorophyll levels quantified, in three separate pools of 30–40 seedlings grown in the dark (columns 1, 2) Photobiol. 57, 867–871 (1993). 7. Neurotoxins (eds Adams, M. & Swanson, G.) Trends Neurosci. and subsequently exposed to light for 5 h (columns 3, 4) (f). An unpaired t-test indicates a significant differ- (suppl.) 20–22 (1996). ence (P ϭ 0.0031) in chlorophyll levels between DNQX-treated and untreated controls in light-treated plants, 8. Thompson, J., Higgins, D. G. & Gibson, T. J. Nucleic Acids Res. and no difference in dark-grown plants. 22, 4673–4680 (1994).