[Show abstract][Hide abstract] ABSTRACT: We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.
[Show abstract][Hide abstract] ABSTRACT: The homeodomain transcription factor KNAT7 has been reported to be involved in the regulation of secondary cell wall biosynthesis. Previous work suggested that KNAT7 can interact with members of the Ovate Family Protein (OFP) transcription co-regulators. However, it remains unknown whether such an OFP-KNAT7 complex could be involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. We re-tested OFP1 and OFP4 for their abilities to intact with KNAT7 using yeast two-hybrid assays, and verified KNAT7-OFP4 interaction but found only weak interaction between KNAT7 and OFP1. Further, the interaction of KNAT7 with OFP4 appears to be mediated by the KNAT7 homeodomain. We used bimolecular fluorescence complementation to confirm interactions and found that OFP1 and OFP4 both interact with KNAT7 in planta. Using a protoplast transient expression system we showed that KNAT7 as well as OFP1 and OFP4 act as transcriptional repressors. Furthermore, in planta interactions between KNAT7 and both OFP1 and OFP4 enhance KNAT7's transcriptional repression activity. An ofp4 mutant exhibited similar irx and fiber cell wall phenotypes as knat7, and the phenotype of a double ofp4 knat7mutant was similar to those of the single mutants, consistent with the view that KNAT7 and OFP function in a common pathway or complex. Furthermore, the pleiotropic OFP1 and OFP4 overexpression phenotype was suppressed in a knat7 mutant background, suggesting that OFP1 and OFP4 functions depend at least partially on KNAT7 function. We propose that KNAT7 forms a functional complex with OFP proteins to regulate aspects of secondary cell wall formation.
The Plant Journal 04/2011; 67(2):328-41. · 6.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs.
We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes.
Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed their possible roles in plant growth and development.
PLoS ONE 01/2011; 6(8):e23896. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Earlier studies have shown that RACK1 functions as a negative regulator of abscisic acid (ABA) responses in Arabidopsis (Arabidopsis thaliana), but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, coexpression analysis revealed that more than 80% of the genes coexpressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1's function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and Saccharomyces cerevisiae RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B, and RACK1C, complemented the growth defects of the S. cerevisiae cross pathway control2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor6 (eIF6), whose mammalian homolog is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The heterotrimeric GTP-binding protein (G-protein) complex is a conserved signaling module found in all eukaryotes. G-proteins
function as molecular switches to regulate diverse signal transduction pathways. Although in contrast to its counterpart in
mammals, the repertoire of G-protein complex in plants is much simpler, G-proteins play important roles in plant development,
hormonal signaling, and biotic and abiotic stress responses. Gene expression and protein localization studies, pharmacological
analysis, and genetic characterization demonstrated that G-proteins are critical modulators of plant cell division. Many of
these studies have been concentrated on the model plant Arabidopsis thaliana, and the role of G-proteins in cell division has been best characterized in hypocotyls, rosette leaves, and roots. However,
little is known about the upstream and downstream components coupled to G-proteins in the regulation of cell division. Future
studies are expected to reveal the molecular mechanism through which G-proteins exert their modulatory roles in plant cell
[Show abstract][Hide abstract] ABSTRACT: *The patterning of epidermal cell types in Arabidopsis is an excellent model for studying the molecular basis of cell specification. Trichome and root hair formation is controlled by a transcriptional activator complex that induces the homeobox gene GLABRA2 (GL2) and some single-repeat R3 MYB genes (single MYB). However, it remains unclear how the actions of GL2 and single MYBs are coordinated to regulate epidermal patterning. *GL2 is thought to act downstream of single MYBs to regulate trichome and root hair development. In order to test this hypothesis genetically, double and higher order mutants between gl2 and single myb were generated. *In these mutants, the glabrous phenotypes observed in the gl2 single mutants were partially recovered, suggesting that single MYBs may not act solely through GL2 to regulate trichome development. On the other hand, double and higher order mutants between gl2 and single myb phenocopied the root hair phenotype of gl2 single mutants, suggesting that GL2 and single MYBs act in a common pathway to regulate root hair patterning. *These findings reveal distinct relationships between GL2 and single MYBs in the regulation of trichome vs root hair development, and provide new insights into the molecular mechanism of epidermal patterning.
New Phytologist 10/2009; 185(2):387-400. · 6.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian receptor for activated C kinase 1 (RACK1) is a versatile scaffold protein, playing regulatory roles in multiple signal transduction pathways. Moreover, RACK1 interacts with the heterotrimeric G-proteins (G-proteins) and regulates some specific functions of Gbetagamma. Although the protein sequences of both RACK1 and G-proteins are highly conserved in Arabidopsis, their relationship remains elusive. Here we provide genetic and biochemical evidence that Arabidopsis RACK1 and G-proteins may act through a mechanism that is distinct from their counterparts in mammals. Loss-of-function alleles of RACK1A (the most abundantly expressed RACK1 gene in Arabidopsis) do not appear to share morphological and developmental phenotypes with loss-of-function alleles of GPA1 (encoding the sole Galpha in Arabidopsis) or AGB1 (encoding the sole Gbeta in Arabidopsis). The analysis of gpa1 rack1a and agb1 rack1a double mutants suggested that the effect of RACK1A on morphological and developmental traits may occur independently of the presence or absence of the G-protein subunits. Although both RACK1A and G-protein subunits are negative regulators of ABA responses in the ABA inhibition of early seedling development, an additive ABA hypersensitivity was observed in gpa1 rack1a and agb1 rack1a double mutants. Biochemical analysis suggested that unlike their counterparts in mammals, RACK1 may not physically interact with AGB1. Taken together, these findings revealed some fundamental differences in the relationship of RACK1 and G-proteins between Arabidopsis and mammals.
Plant and Cell Physiology 09/2009; 50(9):1681-94. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Receptor for Activated C Kinase 1 (RACK1) is viewed as a versatile scaffold protein in mammals. The protein sequence of RACK1 is highly conserved in eukaryotes. However, the function of RACK1 in plants remains poorly understood. Accumulating evidence suggested that RACK1 may be involved in hormone responses, but the precise role of RACK1 in any hormone signalling pathway remains elusive. Molecular and genetic evidence that Arabidopsis RACK1 is a negative regulator of ABA responses is provided here. It is shown that three RACK1 genes act redundantly to regulate ABA responses in seed germination, cotyledon greening and root growth, because rack1a single and double mutants are hypersensitive to ABA in each of these processes. On the other hand, plants overexpressing RACK1A displayed ABA insensitivity. Consistent with their proposed roles in seed germination and early seedling development, all three RACK1 genes were expressed in imbibed, germinating and germinated seeds. It was found that the ABA-responsive marker genes, RD29B and RAB18, were up-regulated in rack1a mutants. Furthermore, the expression of all three RACK1 genes themselves was down-regulated by ABA. Consistent with the view that RACK1 negatively regulates ABA responses, rack1a mutants lose water significantly more slowly from the rosettes and are hypersensitive to high concentrations of NaCl during seed germination. In addition, the expression of some putative RACK1-interacting, ABA-, or abiotic stress-regulated genes was mis-regulated in rack1a rack1b double mutants in response to ABA. Taken together, these findings provide compelling evidence that RACK1 is a critical, negative regulator of ABA responses.
Journal of Experimental Botany 08/2009; 60(13):3819-33. · 5.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seven transmembrane G-protein-coupled receptors (GPCRs) are commonly used by eukaryotes to sense extracellular signals to switch on cellular responses through the activation of cognate heterotrimeric G-proteins. In Arabidopsis thaliana, GCR2 has been proposed as a GPCR for the plant hormone abscisic acid. On the other hand, biochemical analysis demonstrates that the sole Arabidopsis heterotrimeric G-protein alpha subunit, GPA1, is in the activated state (GTP-bound) by default, suggesting that the heterotrimeric G-proteins may act without any GPCRs.
[Show abstract][Hide abstract] ABSTRACT: Mitogen-activated protein kinase (MAPK) phosphatases are important negative regulators in the MAPK signaling pathways responsible for many essential processes in plants, including development, stress management and hormonal responses. A mutation in INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5), which is predicted to encode a dual-specificity MAPK phosphatase, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis roots. To further characterize IBR5, and to understand how it might help integrate MAPK cascades with hormone signaling, we searched for IBR5-interacting MAPKs. Yeast two-hybrid assays, in vitro binding assays and in vivo protein co-immunoprecipitation studies demonstrated that MPK12 and IBR5 are physically coupled. The C-terminus of MPK12 appears to be essential for its interaction with IBR5, and in vitro dephosphorylation and immunocomplex kinase assays indicated that activated MPK12 is efficiently dephosphorylated and inactivated by IBR5. MPK12 and IBR5 mRNAs are both widely expressed across Arabidopsis tissues, and at the subcellular level each protein is predominantly localized in the nucleus. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, but shows normal ABA sensitivity. MPK12 suppression in an ibr5 background partially complements the ibr5 auxin-insensitivity phenotype. Our results demonstrate that IBR5 is a bona fide MAPK phosphatase, and suggest that MPK12 is both a physiological substrate of IBR5 and a novel negative regulator of auxin signaling in Arabidopsis.
The Plant Journal 12/2008; 57(6):975-85. · 6.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transcription factors regulate gene expression by directly binding the cis-acting regulatory elements of target genes via their DNA-binding domains or by interacting with other transcription factors. Trichome cell fate determination in Arabidopsis utilizes a lateral inhibition mechanism that relies on the interplay of transcription factors. GLABRA1 (GL1), an R2R3 MYB transcription factor, GLABRA3 (GL3), a basic helix-loop-helix (bHLH) transcription factor, and TRANSPARENT TESTA GLABRA1 (TTG1), a WD40 protein, are believed to form a transcriptional activator complex to control the transcription of GLABRA2 (GL2), which in turn induces trichome formation in shoots. However, the molecular mechanism of the regulation of GL2 expression by this activator complex is still poorly understood. Here we report that GL1 and GL3 control GL2 expression by a previously unrecognized mechanism in which in addition to the protein-protein interaction between GL1 and GL3, concurrent binding of GL1 and GL3 to the promoter of GL2 via their own DNA-binding domains is probably required to activate GL2. We demonstrate that disruption or deletion of the DNA-binding domains in either GL1 or GL3 completely abolishes the transcriptional activity of the GL1-GL3 complex in activating GL2. These results provide new insight into the interplay of GL1 and GL3 transcription factors in the activation of GL2.
Plant and Cell Physiology 11/2008; 49(12):1792-804. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RACK1 is a versatile scaffold protein in mammals, regulating diverse developmental processes. Unlike in non-plant organisms where RACK1 is encoded by a single gene, Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively. Previous studies indicated that the loss-of-function alleles of RACK1A displayed multiple defects in plant development. However, the functions of RACK1B and RACK1C remain elusive. Further, the relationships between three RACK1 homologous genes are unknown.
We isolated mutant alleles with loss-of-function mutations in RACK1B and RACK1C, and examined the impact of these mutations on plant development. We found that unlike in RACK1A, loss-of-function mutations in RACK1B or RACK1C do not confer apparent defects in plant development, including rosette leaf production and root development. Analyses of rack1a, rack1b and rack1c double and triple mutants, however, revealed that rack1b and rack1c can enhance the rack1a mutant's developmental defects, and an extreme developmental defect and lethality were observed in rack1a rack1b rack1c triple mutant. Complementation studies indicated that RACK1B and RACK1C are in principle functionally equivalent to RACK1A. Gene expression studies indicated that three RACK1 genes display similar expression patterns but are expressed at different levels. Further, RACK1 genes positively regulate each other's expression.
These results suggested that RACK1 genes are critical regulators of plant development and that RACK1 genes function in an unequally redundant manner. Both the difference in RACK1 gene expression level and the cross-regulation are likely the molecular determinants of their unequal genetic redundancy.
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function alleles of the sole heterotrimeric G-protein alpha subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development. Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response. Here we provide genetic evidence that loss-of-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis. gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively. By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants. Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants. Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots. Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner.
Plant and Cell Physiology 08/2008; 49(7):1013-24. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In mammals, basal currents through G protein-coupled inwardly rectifying K(+) (GIRK) channels are repressed by Galpha(i/o)GDP, and the channels are activated by direct binding of free Gbetagamma subunits released upon stimulation of Galpha(i/o)-coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana, is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis Galpha (AtGPA1), Gbeta (AGB1), and Regulator of G protein Signaling (AtRGS1) genes for impacts on ion channel regulation. In guard cells, where K(+) fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K(+) (K(in)) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in Atgpa1 and agb1 single mutants and the Atgpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The Atrgs1 mutation did not affect K(in) current magnitude or ABA sensitivity, but K(in) voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the Galpha subunit or regulation of the heterotrimer to mediate K(in) channel modulation after ligand perception. In contrast, outwardly rectifying (K(out)) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.
Proceedings of the National Academy of Sciences 07/2008; 105(24):8476-81. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GCR2 was recently proposed to represent a G-protein-coupled receptor (GPCR) for the plant hormone, abscisic acid (ABA). We and others provided evidence that GCR2 is unlikely to be a bona fide GPCR because it is not clearly predicted to contain seven transmembrane domains, a structural hallmark for classical GPCRs. Instead, GCR2 shows significant sequence similarity to homologs of bacterial lanthionine synthetase component C (LanC). Here, we provide additional analysis of GCR2 and LanC-like (LANCL) proteins in plants, and propose that GCR2 is a new member of the eukaryotic LANCL protein family.
[Show abstract][Hide abstract] ABSTRACT: The plant hormone abscisic acid (ABA) regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22 degrees C, continuous white light with 150 micromol m(-2) s(-1)) but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23 degrees C, 14/10 hr photoperiod with 120 micromol m(-2) s(-1)). It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2.
In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes.
These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development.
PLoS ONE 02/2008; 3(8):e2982. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signaling through heterotrimeric G-proteins (G-proteins) is a conserved mechanism found in all eukaryotes. In plants, the repertoire of G-protein signaling complex is much simpler than in metazoans. Specifically, the genome of the model plant, Arabidopsis, encodes only one canonical Galpha, one Gbeta, and two Ggamma subunits. Similarly, only one Regulator of G-protein Signaling (RGS) protein is encoded by the Arabidopsis genome, and no bona fide G-protein-coupled receptor (GPCR) together with its ligand has been unequivocally identified. Nonetheless, several proteins, including AtPIRIN1, PLDa 1, PD1, and THF1, have been shown to physically interact with the Arabidopsis heterotrimeric G-protein alpha subunit (GPA1), and are potential downstream effectors for GPA1. The smaller repertoire of the heterotrimeric G-protein complex in plants offers a unique advantage over its counterpart in mammals for dissecting their roles in development. The analyses of loss-of-function alleles and gain-of-function transgenic lines of G-protein subunits and signaling components suggest that the G-proteins play regulatory roles in multiple developmental processes ranging from seed germination and early seedling development to root development and organ shape determination. Future studies are expected to reveal more components of the heterotrimeric G-protein signal transduction pathways, and to identify the mechanisms by which G-proteins regulate phenotypic and developmental plasticity.
Frontiers in Bioscience 02/2008; 13:3321-33. · 3.29 Impact Factor