Ligeng Ma

A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid.

The plant hormone abscisic acid (ABA) regulates many physiological and developmental processes in plants. The mechanism of ABA perception at the cell surface is not understood. Here, we report that a G protein–coupled receptor genetically and physically interacts with the G protein α subunit GPA1 to mediate all known ABA responses in Arabidopsis. Overexpressing this receptor results in an ABA-hypersensitive phenotype. This receptor binds ABA with high affinity at physiological concentration with expected kinetics and stereospecificity. The binding of ABA to the receptor leads to the dissociation of the receptor-GPA1 complex in yeast. Our results demonstrate that this G protein–coupled receptor is a plasma membrane ABA receptor.

Genome-Wide ORFeome Cloning and Analysis of Arabidopsis Transcription Factor Genes

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

The COP9 Signalosome Interacts Physically with SCF COI1 and Modulates Jasmonate Responses

The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome–mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCFCOI1 E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCFCOI1 and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced–specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCFCOI1 work together to control genome expression and promote JA responses.

Conservation and Divergence of Light-Regulated Genome Expression Patterns during Seedling Development in Rice and Arabidopsis

Genome-wide 70-mer oligonucleotide microarrays of rice (Oryza sativa) and Arabidopsis thaliana were used to profile genome expression changes during light-regulated seedling development. We estimate that the expression of ∼20% of the genome in both rice and Arabidopsis seedlings is regulated by white light. Qualitatively similar expression profiles from seedlings grown under different light qualities were observed in both species; however, a quantitatively weaker effect on genome expression was observed in rice. Most metabolic pathways exhibited qualitatively similar light regulation in both species with a few species-specific differences. Global comparison of expression profiles between rice and Arabidopsis reciprocal best-matched gene pairs revealed a higher correlation of genome expression patterns in constant light than in darkness, suggesting that the genome expression profile of photomorphogenesis is more conserved. Transcription factor gene expression under constant light exposure was poorly conserved between the two species, implying a faster-evolving rate of transcription factor gene expression in light-grown plants. Organ-specific expression profiles during seedling photomorphogenesis provide genome-level evidence for divergent light effects in different higher plant organs. Finally, overrepresentation of specific promoter motifs in root- and leaf-specific light-regulated genes in both species suggests that these cis-elements are important for gene expression responses to light.

Organ-Specific Expression of Arabidopsis Genome during Development

The development of complex eukaryotic organisms can be viewed as the selective expression of distinct fractions of the genome in different organs or tissue types in response to developmental and environmental cues. Here, we generated a genome expression atlas of 18 organ or tissue types representing the life cycle of Arabidopsis (Arabidopsis thaliana). We showed that each organ or tissue type had a defining genome expression pattern and that the degree to which organs share expression profiles is highly correlated with the biological relationship of organ types. Further, distinct fractions of the genome exhibited expression changes in response to environmental light among the three seedling organs, despite the fact that they share the same photoperception and transduction systems. A significant fraction of the genes in the Arabidopsis genome is organized into chromatin domains exhibiting coregulated expression patterns in response to developmental or environmental signals. The knowledge of organ-specific expression patterns and their response to the changing environment provides a foundation for dissecting the molecular processes underlying development.

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis.

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene (PDS3) encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarf phenotypes of the pds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in the pds3 mutant. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype of the pds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Genomic Evidence for COP1 as a Repressor of Light-Regulated Gene Expression and Development in Arabidopsis

Microarray gene expression profiling was used to examine the role of COP1 in the light control of Arabidopsis genome expression. Qualitatively similar gene expression profiles were observed between wild-type seedlings grown in white light and multiple cop1 mutant alleles grown in the dark. Furthermore, overexpression of the dominant-negative-acting N terminus of COP1 (N282) in darkness produced a genome expression profile similar to those produced by white light and the cop1 mutations. Different cop1 mutant alleles, N282, and light treatment also resulted in distinct expression profiles in a small fraction of the genes examined. In the light, the genome expression of cop1 mutations displayed an exaggerated light response. COP1-regulated genes in the dark were estimated to account for >20% of the genome. Analysis of these COP1-regulated genes revealed that >28 cellular pathways are coordinately but antagonistically regulated by light and COP1. Interestingly, the gene expression regulation attributable to HY5 in the light is included largely within those genes regulated by COP1 in the dark. Thus, this genomic study supports the hypothesis that COP1 acts as a repressor of photomorphogenesis, possibly by controlling the degradation of transcription factors and their target gene expression. The majority of light-controlled genome expression could be accounted for by the negative regulation of COP1 activity.

Arabidopsis Has Two Redundant Cullin3 Proteins That Are Essential for Embryo Development and That Interact with RBX1 and BTB Proteins to Form Multisubunit E3 Ubiquitin Ligase Complexes in Vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein–type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.

The Presence of a Heterotrimeric G Protein and Its Role in Signal Transduction of Extracellular Calmodulin in Pollen Germination and Tube Growth

The role of heterotrimeric G proteins in pollen germination, tube growth, and signal transduction of extracellular calmodulin (CaM) was examined in lily pollen. Two kinds of antibodies raised against animal Gzα, one against an internal sequence and the other against its N terminus, cross-reacted with the same 41-kD protein from lily pollen plasma membrane. This 41-kD protein was also specifically ADP ribosylated by pertussis toxin. Microinjection of the membrane-impermeable G protein agonist GTP-γ-S into a pollen tube increased its growth rate, whereas microinjection of the membrane-impermeable G protein antagonist GDP-β-S and the anti-Gα antibody decreased pollen tube growth. The membrane-permeable G protein agonist cholera toxin stimulated pollen germination and tube growth. Anti-CaM antiserum inhibited pollen germination and tube growth, and this inhibitory effect was completely reversed by cholera toxin. The membrane-permeable heterotrimeric G protein antagonist pertussis toxin completely stopped pollen germination and tube growth. Purified CaM, when added directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in plasma membrane vesicles, and this increase in GTPase activity was completely inhibited by pertussis toxin and the nonhydrolyzable GTP analogs GTP-γ-S and guanylyl-5′-imidodiphosphate. The GTPase activity in plasma membrane vesicles was also stimulated by cholera toxin. These data suggest that heterotrimeric G proteins may be present in the pollen system where they may be involved in the signal transduction of extracellular CaM and in pollen germination and tube growth.

A genome-wide analysis of blue-light regulation of Arabidopsis transcription factor gene expression during seedling development

A microarray based on PCR amplicons of 1,864 confirmed and predicted Arabidopsis transcription factor genes was produced and used to profile the global expression pattern in seedlings, specifically their light regulation. We detected expression of 1,371 and 1,241 genes in white-light- and dark-grown 6-d-old seedlings, respectively. Together they account for 84% of the transcription factor genes examined. This array was further used to study the kinetics of transcription factor gene expression change of dark-grown seedlings in response to blue light and the role of specific photoreceptors in this blue-light regulation. The expression of about 20% of those transcription factor genes are responsive to blue-light exposure, with 249 and 115 genes up or down-regulated, respectively. A large portion of blue-light-responsive transcription factor genes exhibited very rapid expression changes in response to blue light, earlier than the bulk of blue-light-regulated genes. This result suggests the involvement of transcription cascades in blue-light control of genome expression. Comparative analysis of the expression profiles of wild type and various photoreceptor mutants demonstrated that during early seedling development cryptochromes are the major photoreceptors for blue-light control of transcription factor gene expression, whereas phytochrome A and phototropins play rather limited roles.