Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots

Abstract

Signaling through heterotrimeric G proteins is conserved in diverse eukaryotes. Compared to vertebrates, the simpler repertoire of G-protein complex and accessory components in Arabidopsis (Arabidopsis thaliana) offers a unique advantage over all other multicellular, genetic-model systems for dissecting the mechanism of G-protein signal transduction. One of several biological processes that the G-protein complex regulates in Arabidopsis is cell division. We determined cell production rate in the primary root and the formation of lateral roots in Arabidopsis to define individually the types of modulatory roles of the respective G-protein alpha- and beta-subunits, as well as the heterotrimer in cell division. The growth rate of the root is in part a consequence of cell cycle maintenance in the root apical meristem (RAM), while lateral root production requires meristem formation by founder pericycle cells. Thus, a comparison of these two parameters in various genetic backgrounds enabled dissection of the role of the G-protein subunits in modulation of cell division, both in maintenance and initiation. Cell production rates were determined for the RAM and lateral root formation in gpa1 (Arabidopsis G-protein alpha-subunit) and agb1 (Arabidopsis G-protein beta-subunit) single and double mutants, and in transgenic lines overexpressing GPA1 or AGB1 in agb1 or gpa1 mutant backgrounds, respectively. We found in the RAM that the heterotrimeric complex acts as an attenuator of cell proliferation, whereas the GTP-bound form of the Galpha-subunit's role is a positive modulator. In contrast, for the formation of lateral roots, the Gbetagamma-dimer acts largely independently of the Galpha-subunit to attenuate cell division. These results suggest that Arabidopsis heterotrimeric G-protein subunits have differential and opposing roles in the modulation of cell division in roots.

Figure 1.

GPA1 and AGB1 expression. A, Both GPA1 and AGB1 transcripts are expressed at higher levels in roots than in shoots. Transcripts were analyzed in 7-d-old, light-grown seedlings. B, GPA1 protein level (45 kD) is higher in the root than that in the shoot. GPA1 protein level was analyzed in 7-d-old, light-grown seedlings. The lower band is a nonspecific band recognized by the anti-GPA1 peptide antibody, which often shows up in gpa1 mutant background. C, GPA1 and AGB1 cellular localization. Both GPA1-CFP and YFP-AGB1 localize at the plasma membrane. Both GPA1-CFP and YFP-AGB1 accumulate at the nascent cell plates in dividing Arabidopsis cells. Cells were taken from a population of suspension cells transformed with 35S:GPA1-CFP or 35S:YFP-AGB1 binary vector. No CFP or YFP fluorescence was detected in untransformed cells. 35S:GPA1-CFP and 35S:YFP-AGB1 constructs rescued gpa1-4 and agb1-2 mutants, respectively (data not shown), indicating that the fusion proteins are functional.

Figure 2.

Phenotype of gpa1 agb1 double mutant. A, gpa1 mutants are protein null. B, RT-PCR of gpa1 agb1 double mutants. ACTIN2 primers were added together with GPA1 or AGB1 primers in each PCR reaction. C, Phenotype of 2-d-old, dark-grown seedlings. D, Phenotype of 10-d-old, light-grown seedlings. Arrows indicate that both agb1 and gpa1 agb1 double mutant have large and round cotyledons. E, Lengths of hypocotyls and degrees of hook opening. The lengths of hypocotyls were measured from 2-d-old, dark-grown and 10-d-old, light-grown seedlings, respectively. The degrees of hook opening were measured from 2-d-old, dark-grown seedlings. Shown are the average lengths of hypocotyls from at least 20 seedlings ± sd. F, Phenotype of 43-d-old plants. The plants were grown at 8 h (light)/16 h (dark) short-day conditions. Shown below are the tenth rosette leaves. G, Phenotype of rosette leaves of mature plants. The whole rosette leaves were taken from plants upon flowering. The numbers of rosette leaves are indicated, and the flower buds are asterisked. H, The phenotype of flower. I, The phenotype of the stigma of silique.

Figure 3.

Overexpression of GPA1 and AGB1 in gpa1 and agb1 single and double mutant backgrounds. A, Immunoblot analyses of GPA1 protein levels in 35S:GPA1 transgenic lines. Those independent transgenic lines used in subsequent studies are designated by parentheses. B, Quantitative real-time PCR analysis of AGB1 transcript levels in 35S:AGB1 transgenic lines. The transcript levels of AGB1 in independent transgenic lines harboring 35S:AGB1 in wild-type Col-0 (lines W5 and W13), gpa1-4 (lines a4 and a8), agb1-2 (lines b3 and b14), and gpa1-4 agb1-2 (lines ab7 and ab14) backgrounds were examined.

Figure 4.

Analyses of cell production in the primary root and lateral root formation of double and triple mutants among Atrgs1, gpa1, and agb1 mutants. A, Rate of cell production in the primary root. Primary root elongation rate and cortical root cell length were collected from at least 10 seedlings. At least 20 cortical cells at the mature root region were measured in each seedling. The rate of cell production was calculated as the rate of primary root elongation divided by the cortical cell length. Shown are means ± sd of at least 10 seedlings. B, The number of lateral roots. The numbers of lateral roots were counted on 9-d-old roots. Shown are the average numbers of lateral roots from at least 10 seedlings ± sd. Pairwise Student's t test was used to compare values to the wild type (Col). *, Significant (P = 0.05); **, highly significant (P = 0.01).

Figure 5.

Phenotypic analyses of double and triple mutants among Atrgs1, gpa1, and agb1 mutants. A, Phenotypes of 2-d-old, dark-grown seedlings. B, Lengths of hypocotyls and degrees of hook opening of 2-d-old, dark-grown seedlings. Shown are means ± sd of at least 20 seedlings. C, Phenotype of 43-d-old plants. The plants were grown at 8 h (light)/16 h (dark) short-day conditions. Shown on top are the tenth rosette leaves. In the dark, Atrgs1 mutants had longer hypocotyl and closed hook, whereas gpa1 and agb1 mutants had shorter hypocotyl and partially opened hook. Atrgs1 gpa1 double mutant phenocopied the gpa1 single mutant, and Atrgs1 agb1 and Atrgs1 gpa1 agb1 double and triple mutants phenocopied the agb1 single mutant. Of light-grown plants, Atrgs1 gpa1 and Atrgs1 agb1 double mutants phenocopied the gpa1 and agb1 single mutants, respectively, in terms of shape of rosette leaves and size of the rosette. Atrgs1 gpa1 agb1 triple mutant phenocopied the agb1 single mutant.

Figure 6.

Working model for the heterotrimeric G-protein modes of action in root cell division. Shown here is the classical heterotrimeric G-protein activation-deactivation cycle. Ligand binding (gray ovals) to its cognate 7TM cell surface receptor activates receptor-mediated GDP/GTP exchange on the α-subunit (Gα), causing dissociation of Gα from the βγ-dimer (Gβγ). Both activated Gα-subunit and Gβγ-dimer bind to downstream target proteins, which results in the relevant cellular responses. Intrinsic GTPase activity of Gα hydrolyzes GTP to GDP, thereby allowing Gα to reassociate with the Gβγ-dimer. RGS proteins accelerate the intrinsic GTPase activity of the Gα-subunit, thus returning the heterotrimer to its basal GDP-bound state. Gα, GPA1; Gβ, AGB1; Gγ, AGG1/AGG2. RGS1 is a 7TM protein with a functional RGS box shown to accelerate the intrinsic GTPase activity of GPA1. Shown here are the resting (left and right) and activated states (middle) states of the G-protein complex with RGS1. Arrows depict acceleration and bars indicate attenuation of cell division. It is proposed here that RGS1 controls the G-protein state through its GTPase-accelerating function (GAP activity). The effect of RGS1 on the heterotrimer is depicted by a dashed double line. GPA1 is a positive modulator of cell production in RAM, whereas the heterotrimer is a negative modulator. The heterotrimer may not have a modulatory role in lateral root formation. AGB1 is the primary subunit that regulates lateral root formation. GPA1 inhibits AGB1 action presumably by the sequestration of AGB1 to reform the heterotrimer.