Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis

Abstract

Arabidopsis (Arabidopsis thaliana) mutants lacking a functional ERA1 gene, which encodes the beta-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects that establish roles for protein prenylation in abscisic acid (ABA) signaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsis GGB gene, which encodes the beta-subunit of protein geranylgeranyltransferase type I (PGGT I). Stomatal apertures of ggb plants were smaller than those of wild-type plants at all concentrations of ABA tested, suggesting that PGGT I negatively regulates ABA signaling in guard cells. However, germination of ggb seeds in response to ABA was similar to the wild type. Lateral root formation in response to exogenous auxin was increased in ggb seedlings compared to the wild type, but no change in auxin inhibition of primary root growth was observed, suggesting that PGGT I is specifically involved in negative regulation of auxin-induced lateral root initiation. Unlike era1 mutants, ggb mutants exhibited no obvious developmental phenotypes. However, era1 ggb double mutants exhibited more severe developmental phenotypes than era1 mutants and were indistinguishable from plp mutants lacking the shared alpha-subunit of PFT and PGGT I. Furthermore, overexpression of GGB in transgenic era1 plants partially suppressed the era1 phenotype, suggesting that the relatively weak phenotype of era1 plants is due to partial redundancy between PFT and PGGT I. These results are discussed in the context of Arabidopsis proteins that are putative substrates of PGGT I.

Figure 1.

Structure of the GGB (At2g39550) gene and molecular characterization of ggb mutants. A, The genomic sequence of the GGB gene is shown. Exons are in bold type. The ATG initiation and TGA termination codons are in shadow type. The locations of the T-DNA inserts in ggb-1 and ggb-2 are indicated. B, The intron-exon structure of the GGB gene, the locations of T-DNA inserts in ggb-1 and ggb-2, and the primer binding sites used for genomic PCR analysis are shown. C, RT-PCR analysis of GGB transcripts is shown for Ws, ggb-1, Col, and ggb-2, along with 100 bp M_r markers. The 18S rRNA competimer/primer pairs were included in each RT-PCR reaction as an internal control. The 18S RT-PCR product is approximately 300 bp in length, whereas the GGB RT-PCR product is 657 bp in length. D, Genomic PCR of Ws, ggb-1, Col, and ggb-2 is shown along with HindIII-cut λ DNA markers. The top two panels show the results of PCR analysis using genomic DNA from the indicated lines and two GGB gene-specific primers that bind to sequences flanking the T-DNA insertion sites. The bottom two panels show the results of genomic PCR analysis using one T-DNA-specific primer and one GGB gene-specific reverse primer.

Figure 2.

PFT and PGGT I activities in floral extracts of wild-type and ggb mutants of Arabidopsis. Protein prenyltransferase reactions were performed in the presence of floral extracts from the indicated plants, recombinant Ras protein substrates expressed in Escherichia coli, and either [³H]FPP or [³H]GGPP. Ras-CAIM is a well-characterized substrate for PFT, Ras-CAIL is a well-characterized substrate for PGGT I, and Ras-SVLS is a control protein that cannot be prenylated by any known protein prenyltransferase (Randall and Crowell, 1999).

Figure 3.

Growth and morphology of wild-type and ggb mutants of Arabidopsis. Rosettes, inflorescences, and flowers of Ws, ggb-1, Col, and ggb-2 are shown.

Figure 4.

Effects of ggb-2 or constitutive GGB overexpression on the era1-4 phenotype. The floral phenotypes of ggb-2 and era1-4 ggb-2 plants and inflorescence and floral phenotypes of era1-4 and era1-4 35S::GGB plants are shown.

Figure 5.

Analysis of Ws, ggb-1, Col, ggb-2, and era1-2 stomata in response to exogenous ABA. Epidermal peels were prepared from excised leaves treated for 2 h with various concentrations of ABA under high-light conditions. Stomatal apertures were measured from photographic images and recorded as the ratio of width per length. Standard error of the mean is shown for all data points. Asterisks indicate significant differences determined by Student's t test (P < 0.05).

Figure 6.

Leaf and whole-plant dehydration of wild-type and ggb mutants of Arabidopsis. Top two panels: water loss from excised leaves was monitored as a function of time for the indicated plants. Standard error of the mean is shown for all data points. Asterisks indicate significant differences determined by Student's t test (P < 0.05). Bottom panel: individually potted Ws and ggb-1 plants were denied water for approximately 10 d, beginning 1 to 2 d after bolting, and photographed. The data shown are representative of three independent experiments.

Figure 7.

Analysis of Ws, ggb-1, Col, ggb-2, and era1-2 seed germination in response to exogenous ABA. Seeds were imbibed in the presence of the indicated concentrations of cisABA and scored for germination as a function of time. The data indicate the percentage of seeds from which the radical penetrated the seed coat. The ggb-1, ggb-2, and era1-2 data shown are representative of four, two, and four independent experiments, respectively.

Figure 8.

Analysis of Col, ggb-2, plp, and ggb-2 era1-4 seed germination in response to exogenous ABA. Seeds were imbibed in the presence of the indicated concentrations of cisABA and scored for germination as a function of time. The data indicate the percentage of seeds from which the radical penetrated the seed coat.

Figure 9.

Lateral root formation in response to auxin treatment of wild-type and ggb seedlings. Ws, ggb-1, Col, and ggb-2 seedlings were grown on vertically oriented plates in the presence of various concentrations of α-naphthalene acetic acid (α-NAA) for 4 d and scored for number of lateral roots. Standard error of the mean is shown for all data points. Asterisks indicate significant differences determined by Student's t test (P < 0.01, right panel; P < 0.05, left panel).

Figure 10.

Inhibition of primary root growth in response to auxin treatment of wild-type and ggb seedlings. Ws, ggb-1, Col, and ggb-2 seedlings were grown on vertically oriented plates in the presence of various concentrations of α-NAA for 2 d, rotated 90°, grown for two more days, and the length of the root from the bend to the tip was measured. This procedure was followed to rule out differences in germination time. Standard error of the mean is shown for all data points. No significant differences were observed between wild-type and ggb seedlings.