Nayoung Lee

ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs Interact to Activate the Expression of SOMNUS and Other High-Temperature-Inducible Genes in Imbibed Seeds in Arabidopsis

The Arabidopsis aba2, abi3, della pentuple, and som mutant seeds germinate even at high temperature. This work shows that ABI3, ABI5, and DELLA target to the SOM promoter and mediate high-temperature signaling to activate the expression of SOM 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.

ABI3 and PIL5 Collaboratively Activate the Expression of SOMNUS by Directly Binding to Its Promoter in Imbibed Arabidopsis Seeds

This study examines the regulation of SOMNUS (SOM), which is a key negative regulator of seed germination. ABI3 was found to regulate SOM expression together with PIL5, a previously identified regulator of SOM. PIL5 and ABI3, which interact to form a complex, regulate SOM expression independently in maturing seeds, but collaboratively in imbibed seeds. 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.

HONSU, a Protein Phosphatase 2C, Regulates Seed Dormancy by Inhibiting ABA Signaling in Arabidopsis

Seed dormancy, a seed status that prohibits germination even in the presence of inductive germination signals, is a poorly understood process. To identify molecular components that regulate seed dormancy, we screened T-DNA insertion lines and identified a mutant designated honsu (hon). HON loss-of-function mutants display deep seed dormancy, whereas HON-overexpressing lines display shallow seed dormancy. HON encodes a seed-specific group A phosphatase 2C (PP2C) and is one of the major negative regulators of seed dormancy among group A PP2Cs. Like other PP2C family members, HON interacts with PYR1/RCAR11 in the presence of ABA. Our analysis indicates that HON inhibits ABA signaling and activates gibberellic acid signaling, and both of these conditions must be satisfied to promote the release of seed dormancy. However, HON mRNA levels are increased in mutants displaying deep seed dormancy or under conditions that deepen seed dormancy, and decreased in mutants displaying shallow seed dormancy or under conditions that promote the release of seed dormancy. Taken together, our results indicate that the expression of HON mRNA is homeostatically regulated by seed dormancy.

PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1

In vivo PIF1 targeting to specific promoter sites is determined by PIF1-interacting transcription factors and their binding to G-box coupling sequence elements. 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.

Characteristics of high-k gate dielectric formed by the oxidation of sputtered Hf'Zr'Hf thin films on the Si substrate

We have investigated the effects of high temperature annealing on the physical and electrical properties of multilayered high-k gate oxide [HfSixOy/HfO2/intermixed-layer(IL)/ZrO2/intermixed-layer(IL)/HfO2] in metal-oxide-semiconductor device. The multilayered high-k films were formed after oxidizing the Hf/Zr/Hf films deposited directly on the Si substrate. The subsequent N2 annealing at high temperature (⩾ 700 °C) not only results in the polycrystallization of the multilayered high-k films, but also causes the diffusion of Zr. The latter transforms the HfSixOy/HfO2/IL/ZrO2/IL/HfO2 film into the Zr-doped HfO2 film, and improves electrical properties in general. However, the thin SiOx interfacial layer starts to form if annealing temperature increases over 700 °C, deteriorating the equivalent oxide thickness.

Phytochrome-interacting factor from Arabidopsis to liverwort

Phytochromes are red and far-red light photoreceptors that regulate the responses of plants to light throughout their life cycles. Phytochromes do this in part by inhibiting the function of a group of basic helix-loop-helix transcription factors called phytochrome-interacting factors (PIFs). Arabidopsis has eight PIFs that function sometimes redundantly and sometimes distinctively depending on their expression patterns and protein stability, as well as on variations in the promoters they target in vivo. PIF-like proteins exist in other seed plants and non-vascular plants where they also regulate light responses. The mechanism by which phytochrome regulates light responses by promoting the degradation of the PIFs is conserved in liverwort, suggesting it must have evolved some time before the last common ancestor shared by seed plants and non-vascular plants.

Current Limiting Characteristics of a Flux-Lock Type SFCL for a Single-Line-to-Ground Fault

We have fabricated an integrated three-phase flux-lock type SFCL, which consists of an YBCO(YBa₂Cu₃O?) thin film and a flux-lock reactor wound around an iron core of each phase. In order to apply the SFCL in a real power system, fault analyses for the three-phase system are essential. The short-circuit currents were effectively limited by adjusting the numbers of winding of each secondary coil and their winding directions. The flux flow generated in the iron core cancelled out under the normal operation due to the parallel connection between primary and secondary windings. However, the flux-lock type SFCL with same iron core was operated just after the fault due to the flux generating in the iron core. To analyze the current limiting characteristics, the additive polarity winding was compared with the subtractive one in the flux lock reactor. Whenever a single line-to-ground fault occurred in any phase, the peak value of the line current of the fault phase in the additive polarity winding increased up to about 12.87 times during the first-half cycle. On the other hand, the peak value in the subtractive polarity winding increased up to about 34.07 times under the same conditions. This is because the current flow between the primary and the secondary windings changed to additive or subtractive status according to the winding direction. We confirmed that the current limiting behavior in the additive polarity winding was more effective for a single-line-to-ground fault

The Transcriptional Coregulator LEUNIG_HOMOLOG Inhibits Light-Dependent Seed Germination in Arabidopsis

LEUNIG_HOMOLOG interacts with the transcription factor PHYTOCHROME-INTERACTING FACTOR1 and acts as a transcriptional coregulator to inhibit light-dependent seed germination in Arabidopsis. PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination.

PIF1 Regulates Plastid Development by Repressing Photosynthetic Genes in the Endodermis.

Mutations in Phytochrome Interacting Factors (PIFs) induce a conversion of the endodermal amyloplasts necessary for gravity sensing to plastids with developed thylakoids accompanied by abnormal activation of photosynthetic genes in the dark. In this study, we investigated how PIFs regulate endodermal plastid development by performing comparative transcriptome analysis. We show that both endodermal expression of PIF1 and global expression of the PIF quartet induce transcriptional changes in genes enriched for nuclear-encoded photosynthetic genes such as LHCA and LHCB. Among the 94 shared differentially expressed genes identified from the comparative transcriptome analysis, only 14 genes are demonstrated to be direct targets of PIF1, and most photosynthetic genes are not. Using a co-expression analysis, we identified a direct target of PIF, whose expression pattern shows a strong negative correlation with many photosynthetic genes. We have named this gene REPRESSOR OF PHOTOSYNTHETIC GENES1 (RPGE1). Endodermal expression of RPGE1 rescued the elevated expression of photosynthetic genes found in the pif quadruple (pifQ) mutant and partly restored amyloplast development and hypocotyl negative gravitropism. Taken together, our results indicate that RPGE1 acts downstream of PIF1 in the endodermis to repress photosynthetic genes and regulate plastid development.

Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions

Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin-antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.