Eunkyoo Oh

Integration of Brassinosteroid Signal Transduction with the Transcription Network for Plant Growth Regulation in Arabidopsis

Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here, we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR-signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light-signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways but also coregulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants that reveals a regulatory network that integrates hormonal and light-signaling pathways for plant growth regulation.

Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses

Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growth regulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood at the molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factor phytochrome-interacting factor 4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal and environmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly 2,000 common target genes, and synergistically regulate many of these target genes, including the PRE family helix–loop–helix factors required for promoting cell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR, darkness or heat. These results show that the BZR1–PIF4 interaction controls a core transcription network, enabling plant growth co-regulation by the steroid and environmental signals.

Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis.

Brassinosteroid and gibberellin promote many similar developmental responses in plants; however, their relationship remains unclear. Here we show that BR and GA act interdependently through a direct interaction between the BR-activated BZR1 and GA-inactivated DELLA transcription regulators. GA promotion of cell elongation required BR signalling, whereas BR or active BZR1 suppressed the GA-deficient dwarf phenotype. DELLAs directly interacted with BZR1 and inhibited BZR1–DNA binding both in vitro and in vivo. Genome-wide analysis defined a BZR1-dependent GA-regulated transcriptome, which is enriched with light-regulated genes and genes involved in cell wall synthesis and photosynthesis/chloroplast function. GA promotion of hypocotyl elongation requires both BZR1 and the phytochrome-interacting factors (PIFs), as well as their common downstream targets encoding the PRE-family helix–loop–helix factors. The results demonstrate that GA releases DELLA-mediated inhibition of BZR1, and that the DELLA–BZR1–PIF4 interaction defines a core transcription module that mediates coordinated growth regulation by GA, BR and light signals.

PIL5, a Phytochrome-Interacting Basic Helix-Loop-Helix Protein, Is a Key Negative Regulator of Seed Germination in Arabidopsis thaliana

The first decision made by an angiosperm seed, whether to germinate or not, is based on integration of various environmental signals such as water and light. The phytochromes (Phys) act as red and far-red light (Pfr) photoreceptors to mediate light signaling through yet uncharacterized pathways. We report here that the PIF3-like 5 (PIL5) protein, a basic helix-loop-helix transcription factor, is a key negative regulator of phytochrome-mediated seed germination. PIL5 preferentially interacts with the Pfr forms of Phytochrome A (PhyA) and Phytochrome B (PhyB). Analyses of a pil5 mutant in conjunction with phyA and phyB mutants, a pif3 pil5 double mutant, and PIL5 overexpression lines indicate that PIL5 is a negative factor in Phy-mediated promotion of seed germination, inhibition of hypocotyl negative gravitropism, and inhibition of hypocotyl elongation. Our data identify PIL5 as the first Phy-interacting protein that regulates seed germination.

PIL5, a Phytochrome-Interacting bHLH Protein, Regulates Gibberellin Responsiveness by Binding Directly to the GAI and RGA Promoters in Arabidopsis Seeds

Previous work showed that PHYTOCHROME-INTERACTING FACTOR3-LIKE5 (PIL5), a light-labile basic helix-loop-helix protein, inhibits seed germination by repressing GIBBERELLIN 3β-HYDROXYLASE1 (GA3ox1) and GA3ox2 and activating a gibberellic acid (GA) catabolic gene (GA2ox2). However, we show persistent light-dependent and PIL5-inhibited germination behavior in the absence of both de novo GA biosynthesis and deactivation by GA2ox2, suggesting that PIL5 regulates not only GA metabolism but also GA responsiveness. PIL5 increases the expression of two GA repressor (DELLA) genes, GA-INSENSITIVE (GAI) and REPRESSOR OF GA1-3 (RGA/RGA1), in darkness. The hypersensitivity of gai-t6 rga-28 to red light and the suppression of germination defects of a rga-28 PIL5 overexpression line show the significant role of this transcriptional regulation in seed germination. PIL5 also increases abscisic acid (ABA) levels by activating ABA biosynthetic genes and repressing an ABA catabolic gene. PIL5 binds directly to GAI and RGA promoters but not to GA and ABA metabolic gene promoters. Together, our results show that light signals perceived by phytochromes cause a reduction in the PIL5 protein level, which in turn regulates the transcription of two DELLA genes directly and that of GA and ABA metabolic genes indirectly.

Genome-Wide Analysis of Genes Targeted by PHYTOCHROME INTERACTING FACTOR 3-LIKE5 during Seed Germination in Arabidopsis

PHYTOCHROME INTERACTING FACTOR 3-LIKE5 (PIL5) is a basic helix-loop-helix transcription factor that inhibits seed germination by regulating the expression of gibberellin (GA)- and abscisic acid (ABA)-related genes either directly or indirectly. It is not yet known, however, whether PIL5 regulates seed germination solely through GA and ABA. Here, we used Chromatin immunoprecipitation-chip (ChIP-chip) analysis to identify 748 novel PIL5 binding sites in the Arabidopsis thaliana genome. Consistent with the molecular function of PIL5 as a transcription regulator, most of the identified binding sites are located in gene promoter regions. Binding site analysis shows that PIL5 binds to its target sites mainly through the G-box motif in vivo. Microarray analysis reveals that phytochromes regulate a large number of genes mainly through PIL5 during seed germination. Comparison between the ChIP-chip and microarray data indicates that PIL5 regulates 166 genes by directly binding to their promoters. Many of the identified genes encode transcription regulators involved in hormone signaling, while some encode enzymes involved in cell wall modification. Interestingly, PIL5 directly regulates many transcription regulators of hormone signaling and indirectly regulates many genes involved in hormone metabolism. Taken together, our data indicate that PIL5 inhibits seed germination not just through GA and ABA, but also by coordinating hormone signals and modulating cell wall properties in imbibed seeds.

Brassinosteroid signaling network and regulation of photomorphogenesis.

In plants, the steroidal hormone brassinosteroid (BR) regulates numerous developmental processes, including photomorphogenesis. Genetic, proteomic, and genomic studies in Arabidopsis have illustrated a fully connected BR signal transduction pathway from the cell surface receptor kinase BRI1 to the BZR1 family of transcription factors. Genome-wide analyses of protein-DNA interactions have identified thousands of BZR1 target genes that link BR signaling to various cellular, metabolic, and developmental processes, as well as other signaling pathways. In controlling photomorphogenesis, BR signaling is highly integrated with the light, gibberellin, and auxin pathways through both direct interactions between signaling proteins and transcriptional regulation of key components of these pathways. BR signaling also cross talks with other receptor kinase pathways to modulate stomata development and innate immunity. The molecular connections in the BR signaling network demonstrate a robust steroid signaling system that has evolved in plants to orchestrate signal transduction, genome expression, metabolism, defense, and development.

SOMNUS, a CCCH-Type Zinc Finger Protein in Arabidopsis, Negatively Regulates Light-Dependent Seed Germination Downstream of PIL5

Light absorbed by seed phytochromes of Arabidopsis thaliana modulates abscisic acid (ABA) and gibberellic acid (GA) signaling pathways at least partly via PHYTOCHROME-INTERACTING FACTOR3-LIKE5 (PIL5), a phytochrome-interacting basic helix-loop-helix transcription factor. Here, we report a new mutant, somnus (som), that germinates in darkness, independently of various light regimens. SOM encodes a nucleus-localized CCCH-type zinc finger protein. The som mutant has lower levels of ABA and elevated levels of GA due to expressional changes in ABA and GA metabolic genes. Unlike PIL5, however, SOM does not regulate the expression of GA-INSENSITIVE and REPRESSOR OF GA1 (RGA/RGA1), two DELLA genes encoding GA negative signaling components. Our in vivo analysis shows that PIL5 activates the expression of SOM by binding directly to its promoter, suggesting that PIL5 regulates ABA and GA metabolic genes partly through SOM. In agreement with these results, we also observed that the reduced germination frequency of a PIL5 overexpression line is rescued by the som mutation and that this rescue is accompanied by expressional changes in ABA and GA metabolic genes. Taken together, our results indicate that SOM is a component in the phytochrome signal transduction pathway that regulates hormone metabolic genes downstream of PIL5 during seed germination.

Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl

As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl. DOI: http://dx.doi.org/10.7554/eLife.03031.001

Integration of Light- and Brassinosteroid-Signaling Pathways by a GATA Transcription Factor in Arabidopsis

Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here, we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant cop1. Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses GATA2 transcription through the BR-activated transcription factor BZR1, whereas light causes accumulation of GATA2 protein and feedback inhibition of GATA2 transcription. Dark-induced proteasomal degradation of GATA2 is dependent on the COP1 E3 ubiquitin ligase, and COP1 can ubiquitinate GATA2 in vitro. This study illustrates a molecular framework for antagonistic regulation of gene expression and seedling photomorphogenesis by BR and light.