Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis

Abstract

SummaryAbscisic acid (ABA) is perceived by several different types of receptors in plant cells. At the cell surface, the ABA signal is proposed to be perceived by GCR2, which mediates ABA responses in seed germination, early seedling development and stomatal movement. GCR2 was also proposed to be a seven-transmembrane (7TM) G-protein-coupled receptor (GPCR). Here we characterize GCR2 and one of its two homologs, GCR2-LIKE 1 (GCL1), in ABA-mediated seed germination and early seedling development in Arabidopsis. We show that lossof-function mutations in GCL1 did not confer ABA insensitivity. Similarly, we did not observe ABA insensitivity in three independent gcr2 alleles. Furthermore, we generated gcr2 gcl1 double mutants and found that the double mutants still had near wild-type responses to ABA. Consistent with this, we found that the transcription of ABA marker genes was induced by ABA to levels that were comparable in wild type and gcr2 and gcl1 single and double mutants. On the other hand, the loss-of-function alleles of the sole Arabidopsis heterotrimeric G protein a subunit, GPA1, were hypersensitive to ABA in the ABA-inhibition of seed germination and early seedling development, disfavoring a genetic coupling of GCR2 by GPA1. Using multiple robust transmembrane prediction systems, GCR2 was predicted not to be a 7TM protein, a structural hallmark of GPCRs. Taken together, our results do not support the notion that GCR2 is an ABA-signaling GPCR in seed germination and early seedling development.

The multifaceted role of ABA in disease resistance

“…Recently, two putative ABA receptors have been identified on the basis of biochemical binding assays. One of these, a G-protein-coupled receptor, is located in the plasma membrane [61], although subsequent genetic characterization failed to confirm its role in the ABA response [62]. Another putative ABA receptor was identified as a chloroplast-located magnesium-chelatase that is involved in chlorophyll synthesis [63].…”

Control of stomatal aperture

“…58 However, it is important to mention that the characterization of GCR2 as a GPCR and ABA receptor is still a matter of discussion. [59][60][61] More recently, other GPCR-like proteins (GTG1 and GTG2) were identified and its characterization revealed that the GTG proteins represent a novel class of proteins with topology similar to GPCRs but with classic GTP-binding/GTPase activity. 62 Further analysis providing biochemical and phenotypic evidences suggest that GTG1 and GTG2 proteins are redundantly involved in G protein-coupled ABA signaling and are, or are parts of, ABA receptor complexes 62 and their detailed characterization will likely clarify the mechanism underlying the ABA response.…”

Criteria for confirming sequence periodicity identified by Fourier transform analysis: Application to GCR2, a candidate plant GPCR?

“…TMPro did identify 5 of the 7 transmembrane hydrophobic regions, but TMPro only highlights transmembrane regions, it does not determine whether these are sufficiently long to span the membrane. The results of the transmembrane prediction algorithms are given as supporting information and are similar to those given elsewhere [28]. Given the negative results from the BLAST search and the transmembrane prediction algorithms, it is difficult to see why GCR2 has been proposed as a GPCR, particularly given its alignment to the Lanthionine synthetase C-like protein family.…”

“…However, the LANCL1 protein was later re-classified by the original authors as a peripheral membrane protein with enzyme activity [24], an observation now justified by the recent X-ray crystal structure of a lantibiotic cyclase (PDB codes 2g02, 2g0d). Recently the status of GCR2 as a GPCR has also been queried [28] and in an attempt to understand the origin of this confusion, we have analysed the GCR2 -Lanthionine synthetase C-like protein family using Fourier transform analysis, using hydrophobicity as the transformed property.…”

“…Indeed, it is worth noting that Moriyama et al used hidden Markov and related methods to identify novel plant GPCRs but they did not detect GCR2 [70]. Nevertheless, while some aspects of the original report that GCR2 is the GPCR receptor for abscisic acid [21] have been seriously questioned [28], there remains the option that GCR2 may retain an indirect role in signaling in plants since not all of the experiments have been disproved in all plant tissues. essentially equally-spaced hydrophobic helices, leading to a significant spike related to hydrophobicity score at a frequency of 7 in the cross-spectral function.…”