Arabidopsis mutants resistant to S(+)-beta-methyl-alpha, beta-diaminopropionic acid, a cycad-derived glutamate receptor agonist

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are the predominant neuroreceptors in the mammalian brain. Genes with high sequence similarity to animal iGluRs have been identified in Arabidopsis. To understand the role of Arabidopsis glutamate receptor-like (AtGLR) genes in plants, we have taken a pharmacological approach by examining the effects of BMAA [S(+)-beta-methyl-alpha, beta-diaminopropionic acid], a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When applied to Arabidopsis seedlings, BMAA caused a 2- to 3-fold increase in hypocotyl elongation and inhibited cotyledon opening during early seedling development. The effect of BMAA on hypocotyl elongation is light specific. Furthermore, BMAA effects on early morphogenesis of Arabidopsis can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. To determine the targets of BMAA action in Arabidopsis, a genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA, bim mutants exhibited short hypocotyls compared with wild type. bim mutants were grouped into three classes based on their morphology when grown in the dark in the absence of BMAA. Class-I bim mutants have a normal, etiolated morphology, similar to wild-type plants. Class-II bim mutants have shorter hypocotyls and closed cotyledons when grown in the dark. Class-III bim mutants have short hypocotyls and open cotyledons when grown in the dark, resembling the previously characterized constitutively photomorphogenic mutants (cop, det, fus, and shy). Further analysis of the bim mutants should help define whether plant-derived iGluR agonists target glutamate receptor signaling pathways in plants.

Figure 1

BMAA causes hypocotyl elongation and inhibits cotyledon unfolding in light-grown Arabidopsis. Arabidopsis plants were germinated and cultivated on Murashige and Skoog media and 0.7% (w/v) agar containing 50 μm BMAA (A) or no BMAA (B) for 8 d in the light. The average hypocotyl length for each treatment, as well as the arc-angle of opening for the cotyledons, is shown (n = 30).

Figure 2

Quantification of BMAA-induced hypocotyl elongation in light-grown and etiolated plants. Arabidopsis plants were grown on increasing concentrations of BMAA (0–100 μm), in the light (A) or in continuous dark (B) for 5 d. The average hypocotyl length for each treatment is shown. Error bars show the se of the mean (n = 30).

Figure 3

BMAA-induced hypocotyl elongation, as well as inhibition of cotyledon opening is reversed in a dose-dependent manner by Glu. Arabidospsis seedlings were cultivated in the light on Murashige and Skoog media containing 1, 3, or 10 mm Glu in the absence (left, A and B) or presence (right, A and B) of 25 μm BMAA. A, The average hypocotyl length for each treatment. B, The arc of cotyledon opening. Error bars show the se of the mean (n = 30)

Figure 4

A genetic screen to isolate BMAA insensitive morphology (bim) mutants. A, Strategy to isolate Arabidopsis mutants resistant to the effects of BMAA is shown. Individual EMS mutagenized M2 seedlings are grown for up to 2 weeks on Murashige and Skoog media containing 50 μm BMAA in the light. M2 individuals with short hypocotyls, compared with neighboring plants, are recovered from the BMAA-containing media and allowed to produce seed for analysis in the M3 generation. B, Representative M2 bim plant (bim26) is shown as detected in the primary screen.

Figure 5

bim mutants are insensitive to BMAA-induced hypocotyl elongation. Two representative M3 bim mutants (bim 131 and bim 26) were cultivated for 5 d in the light on Murashige and Skoog media in the presence of 50 μm BMAA (A) or in the absence of BMAA (B). A, Both bim 131 and bim 26 have a short hypocotyl phenotype (right) when compared with wild type (left), which has an elongated hypocotyl in the presence of BMAA. B, Plants grown in the absence of BMAA where bim 131 and bim 26 appear similar to wild type at the early seedling stage.

Figure 6

Quantification of hypocotyl lengths of bim mutants grown in the light in the presence or absence of BMAA. bim 18, 40, 77, 50, 26, 136, 59, 131, 167, and 175 were grown for 5 d in the light in the presence of 50 μm BMAA (A) or with no BMAA (B). Hypocotyl lengths of bim seedlings and wild type were measured and quantified. Wild type is indicated with a black bar on the far right of each graph. cop1-6 mutant is indicated with a white bar on the far left of each graph. The average hypocotyl length for each treatment is shown (n = 30). Error bars show the se of the mean.

Figure 7

bim mutants are subgrouped into three separate classes based on their dark-grown morphology. bim mutants were cultivated on Murashige and Skoog media in the absence of BMAA and grown in the dark for 5 d. Class-I bim mutants exhibit a normal etiolated phenotype (Fig. 7A, right) when grown in the dark compared with wild type (Fig. 7A, left). Class-II bim mutants have a short hypocotyl and closed cotyledons (Fig. 7B, right) when compared with wild type (Fig. 7B, left). Class-III bim mutants have a short hypocotyl and open cotyledons (Fig. 7C, left) when compared with wild type (Fig. 7C, right).

Figure 8

Quantification of hypocotyl lengths of bim mutants grown in the dark in the absence or presence of BMAA. bim 18, 40, 77, 50, 26, 136, 59, 131, 167, and 175 and wild type were grown for 5 d in the dark in the absence (Fig. 8A) or presence of 50 μm BMAA (Fig. 8B). Hypocotyl lengths were quantified. Wild type is indicated with black bars. Class-I bim mutants (bim 131 and bim 175) have wild-type length hypocotyls and are indicated with hatched bars. Class-II and -III bim mutants, which have short hypocotyls in the dark, are marked with gray shaded bars. cop1-6 is shown in the far left side of the graph (white bar). The average hypocotyl length for each treatment is shown (n = 30). Error bars show the se of the mean.