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.

Seeds monitor the environment to germinate at the proper time, but different species respond differently to environmental conditions, particularly light and temperature. In Arabidopsis thaliana, light promotes germination but high temperature suppresses germination. We previously reported that light promotes germination by repressing SOMNUS (SOM). Here, we examined whether high temperature also regulates germination through SOM and found that high temperature activates SOM expression. Consistent with this, som mutants germinated more frequently than the wild type at high temperature. The induction of SOM mRNA at high temperature required abscisic acid (ABA) and gibberellic acid biosynthesis, and ABA-INSENSITIVE3 (ABI3), ABI5, and DELLAs positively regulated SOM expression. Chromatin immunoprecipitation assays indicated that ABI3, ABI5, and DELLAs all target the SOM promoter. At the protein level, ABI3, ABI5, and DELLAs all interact with each other, suggesting that they form a complex on the SOM promoter to activate SOM expression at high temperature. We found that high-temperature-inducible genes frequently have RY motifs and ABA-responsive elements in their promoters, some of which are targeted by ABI3, ABI5, and DELLAs in vivo. Taken together, our data indicate that ABI3, ABI5, and DELLAs mediate high-temperature signaling to activate the expression of SOM and other high-temperature-inducible genes, thereby inhibiting seed germination.

Summary Phytochromes are red and far-red light receptors in plants that mediate critical responses to light throughout the life cycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome-interacting factors (PIFs) for degradation within the nucleus. It is not known, however, if protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use ChIP analysis to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N-terminal fragment of phyB (NG-GUS-NLS; NGB) also inhibits the binding of PIF3 to its target promoters. Unlike the full-length phyB, however, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIFs’ binding to DNA. We further show that Pfr forms of both full-length phyB and NGB inhibit the DNA binding of PIF1 and PIF3 in vitro. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes, sequestration and protein degradation.

A previous study showed that SOMNUS (SOM), which encodes a C3H-type zinc finger protein, is a key negative regulator of seed germination that acts downstream of PHYTOCHROME INTERACTING FACTOR3-LIKE5 (PIL5). However, it was not determined if PIL5 is the sole regulator of SOM expression. Public microarray data suggest that the expression of SOM mRNA is regulated also by ABSCISIC ACID INSENSITIVE3 (ABI3), another key regulator of seed germination. By analyzing abi3 mutants and ABI3 overexpression lines, we show here that ABI3 activates the expression of SOM mRNA collaboratively with PIL5 in imbibed seeds. Chromatin immunoprecipitation analysis coupled with electrophoretic mobility shift assay indicate that ABI3 activates the expression of SOM mRNA by directly binding to two RY motifs present in the SOM promoter in vivo, which is further supported by the greatly decreased expression of a reporter gene driven by a SOM promoter bearing mutated RY motifs. At the protein level, the ABI3 protein interacts with the PIL5 protein. The ABI3-PIL5 interaction, however, does not affect targeting of ABI3 and PIL5 to SOM promoters. Taken together, our results indicate that ABI3 and PIL5 collaboratively activate the expression of SOM mRNA by directly binding to and interacting with each other at the SOM promoter.

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2, in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis.

DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLAinteracting RING domain proteins, previously designated as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collectively referred to as BOIs). Single mutants of each BOI gene failed to significantly alter GA responses, but the boi quadruple mutant (boiQ) showed a higher seed germination frequency in the presence of paclobutrazol, precocious juvenile-to-adult phase transition, and early flowering, all of which are consistent with enhanced GA signaling. By contrast, BOI overexpression lines displayed phenotypes consistent with reduced GA signaling. Analysis of a gai-1 boiQ pentuple mutant further indicated that the GAI protein requires BOIs to inhibit a subset of GA responses. At the molecular level, BOIs did not significantly alter the stability of a DELLA protein. Instead, BOI and DELLA proteins are targeted to the promoters of a subset of GA-responsive genes and repress their expression. Taken together, our results indicate that the DELLA and BOI proteins inhibit GA responses by interacting with each other, binding to the same promoters of GAresponsive genes, and repressing these genes.

The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) binds G-box elements in vitro and inhibits light-dependent germination in Arabidopsis thaliana. A previous genome-wide analysis of PIF1 targeting indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of PIF1 targeting. The targeting of PIF1 to specific sites could be stabilized by PIF1-interacting transcription factors (PTFs) that bind other nearby sequence elements. Here, we report PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements we named G-box coupling elements (GCEs). One of these GCEs possesses an ACGT core and serves as a binding site for group A bZIP transcription factors, including ABSCISIC ACID INSENSITIVE5 (ABI5), which inhibits seed germination in abscisic acid signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors and together they target a subset of PIF1 binding sites in vivo. In vitro single-molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing multiple G-boxes or the GCE alone. Thus, we show in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.