Jin-Gui Chen

Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.

Auxin is a multifunctional hormone essential for plant development and pattern formation. A nuclear auxin-signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling, as in its coordination of cell polarization, is unexplored. We found a cytoplasmic auxin-signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 s and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based auxin-signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.

Different Signaling and Cell Death Roles of Heterotrimeric G Protein α and β Subunits in the Arabidopsis Oxidative Stress Response to Ozone

Arabidopsis thaliana plants with null mutations in the genes encoding the α and β subunits of the single heterotrimeric G protein are less and more sensitive, respectively, to O3 damage than wild-type Columbia-0 plants. The first peak of the bimodal oxidative burst elicited by O3 in wild-type plants is almost entirely missing in both mutants. The late peak is normal in plants lacking the Gβ protein but missing in plants lacking the Gα protein. Endogenous reactive oxygen species (ROS) are first detectable in chloroplasts of leaf epidermal guard cells. ROS production in adjacent cells is triggered by extracellular ROS signals produced by guard cell membrane-associated NADPH oxidases encoded by the AtrbohD and AtrbohF genes. The late, tissue damage–associated component of the oxidative burst requires only the Gα protein and arises from multiple cellular sources. The early component of the oxidative burst, arising primarily from chloroplasts, requires signaling through the heterotrimer (or the Gβγ complex) and is separable from Gα-mediated activation of membrane-bound NADPH oxidases necessary for both intercellular signaling and cell death.

The β-Subunit of the Arabidopsis G Protein Negatively Regulates Auxin-Induced Cell Division and Affects Multiple Developmental Processes

Plant cells respond to low concentrations of auxin by cell expansion, and at a slightly higher concentration, these cells divide. Previous work revealed that null mutants of the α-subunit of a putative heterotrimeric G protein (GPA1) have reduced cell division. Here, we show that this prototypical G protein complex acts mechanistically by controlling auxin sensitivity toward cell division. Loss-of-function G protein mutants have altered auxin-mediated cell division throughout development, especially during the auxin-induced formation of lateral and adventitious root primordia. Ectopic expression of the wild-type Gα-subunit phenocopies the Gβ mutants (auxin hypersensitivity), probably by sequestering the Gβγ-subunits, whereas overexpression of Gβ reduces auxin sensitivity and a constitutively active (Q222L) mutant Gα behaves like the wild type. These data are consistent with a model in which Gβγ acts as a negative regulator of auxin-induced cell division. Accordingly, basal repression of approximately one-third of the identified auxin-regulated genes (47 of 150 upregulated genes among 8300 quantitated) is lost in the Gβ transcript-null mutant. Included among these are genes that encode proteins proposed to control cell division in root primordia formation as well as several novel genes. These results suggest that although auxin-regulated cell division is not coupled directly by a G protein, the Gβ-subunit attenuates this auxin pathway upstream of the control of mRNA steady state levels.

Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination

Seed germination is regulated by many signals. We investigated the possible involvement of a heterotrimeric G protein complex in this signal regulation. Seeds that carry a protein null mutation in the gene encoding the alpha subunit of the G protein in Arabidopsis (GPA1) are 100-fold less responsive to gibberellic acid (GA), have increased sensitivity to high levels of Glc, and have a near-wild-type germination response to abscisic acid and ethylene, indicating that GPA1 does not directly couple these signals in germination control. Seeds ectopically expressingGPA1 are at least a million-fold more responsive to GA, yet still require GA for germination. We conclude that the GPA1 indirectly operates on the GA pathway to control germination by potentiation. We propose that this potentiation is directly mediated by brassinosteroids (BR) because the BR response and synthesis mutants,bri1-5 and det2-1, respectively, share the same GA sensitivity as gpa1 seeds. Furthermore,gpa1 seeds are completely insensitive to brassinolide rescue of germination when the level of GA in seeds is reduced. A lack of BR responsiveness is also apparent in gpa1 roots and hypocotyls suggesting that BR signal transduction is likely coupled by a heterotrimeric G protein at various points in plant development.

G-Protein Complex Mutants Are Hypersensitive to Abscisic Acid Regulation of Germination and Postgermination Development

Abscisic acid (ABA) plays regulatory roles in a host of physiological processes throughout plant growth and development. Seed germination, early seedling development, stomatal guard cell functions, and acclimation to adverse environmental conditions are key processes regulated by ABA. Recent evidence suggests that signaling processes in both seeds and guard cells involve heterotrimeric G proteins. To assess new roles for the Arabidopsis (Arabidopsis thaliana) Gα subunit (GPA1), the Gβ subunit (AGB1), and the candidate G-protein-coupled receptor (GCR1) in ABA signaling during germination and early seedling development, we utilized knockout mutants lacking one or more of these components. Our data show that GPA1, AGB1, and GCR1 each negatively regulates ABA signaling in seed germination and early seedling development. Plants lacking AGB1 have greater ABA hypersensitivity than plants lacking GPA1, suggesting that AGB1 is the predominant regulator of ABA signaling and that GPA1 affects the efficacy of AGB1 execution. GCR1 acts upstream of GPA1 and AGB1 for ABA signaling pathways during germination and early seedling development: gcr1 gpa1 double mutants exhibit a gpa1 phenotype and agb1 gcr1 and agb1 gcr1 gpa1 mutants exhibit an agb1 phenotype. Contrary to the scenario in guard cells, where GCR1 and GPA1 have opposite effects on ABA signaling during stomatal opening, GCR1 acts in concert with GPA1 and AGB1 in ABA signaling during germination and early seedling development. Thus, cell- and tissue-specific functional interaction in response to a given signal such as ABA may determine the distinct pathways regulated by the individual members of the G-protein complex.

The Plastid Protein THYLAKOID FORMATION1 and the Plasma Membrane G-Protein GPA1 Interact in a Novel Sugar-Signaling Mechanism in Arabidopsis

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of d-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Gα interaction partner that functions downstream of the plasma membrane–delimited heterotrimeric G-protein (GPA1) in a d-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Förster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous d-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high d-glucose. Additionally, THF1 levels are rapidly degraded by d-glucose but not l-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling

Heterotrimeric G protein signaling is important for cell-proliferative and glucose-sensing signal transduction pathways in the model plant organism Arabidopsis thaliana. AtRGS1 is a seven-transmembrane, RGS domain-containing protein that is a putative membrane receptor for d-glucose. Here we show, by using FRET, that d-glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo. AtGPA1 is a unique heterotrimeric G protein α subunit that is constitutively GTP-bound given its high spontaneous nucleotide exchange coupled with slow GTP hydrolysis. Analysis of a point mutation in AtRGS1 that abrogates GTPase-accelerating activity demonstrates that the regulation of AtGPA1 GTP hydrolysis mediates sugar signal transduction during Arabidopsis development, in contrast to animals where nucleotide exchange is the limiting step in the heterotrimeric G protein nucleotide cycle.

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

Signal recognition by seven-transmembrane (7TM) cell-surface receptors is typically coupled by heterotrimeric G-proteins to downstream effectors in metazoan, fungal, and amoeboid cells. Some responses perceived by 7TM receptors in amoeboid cells and possibly in human cells can initiate downstream action independently of heterotrimeric G-proteins. Plants use heterotrimeric G-protein signaling in the regulation of growth and development, particularly in hormonal control of seed germination, but it is not yet clear which of these responses utilize a 7TM receptor. Arabidopsis GCR1 has a predicted 7TM-spanning domain and other features characteristic of 7TM receptors. Loss-of-function gcr1 mutants indicate that GCR1 plays a positive role in gibberellin- (GA) and brassinosteroid- (BR) regulated seed germination. The null mutants of GCR1 are less sensitive to GA and BR in seed germination. This phenotype is similar to that previously observed for transcript null mutants in the Gα-subunit, gpa1. However, the reduced sensitivities toward GA and BR in the single gcr1, gpa1, and agb1 (heterotrimeric G-protein β-subunit) mutants are additive or synergistic in the double and triple mutants. Thus, GCR1, unlike a typical 7TM receptor, apparently acts independently of the heterotrimeric G-protein in at least some aspects of seed germination, suggesting that this alternative mode of 7TM receptor action also functions in the plant kingdom.

Population genomics of Populus trichocarpa identifies signatures of selection and adaptive trait associations

Forest trees are dominant components of terrestrial ecosystems that have global ecological and economic importance. Despite distributions that span wide environmental gradients, many tree populations are locally adapted, and mechanisms underlying this adaptation are poorly understood. Here we use a combination of whole-genome selection scans and association analyses of 544 Populus trichocarpa trees to reveal genomic bases of adaptive variation across a wide latitudinal range. Three hundred ninety-seven genomic regions showed evidence of recent positive and/or divergent selection and enrichment for associations with adaptive traits that also displayed patterns consistent with natural selection. These regions also provide unexpected insights into the evolutionary dynamics of duplicated genes and their roles in adaptive trait variation.

Differential Roles of Arabidopsis Heterotrimeric G-Protein Subunits in Modulating Cell Division in Roots

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 α- and β-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 α-subunit) and agb1 (Arabidopsis G-protein β-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 Gα-subunits role is a positive modulator. In contrast, for the formation of lateral roots, the Gβγ-dimer acts largely independently of the Gα-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.