Functional characterizations of EFS and LUH in arabidopsis seed germination

Abstract

The Phytochrome-Interacting Factors (PIFs) are basic helix-loop-helix transcription factors that are responsible for repressing phytochrome-mediated light responses. Among them, PIF1 largely governs the inhibition of light-dependent seed germination. The specific function of PIF1 in seed germination is thought to be partly due to its high-level expression in imbibed seeds, but the associated regulatory factors have not yet been identified. Here, I showed that mutation of the EFS gene, which encodes an H3K36 di- and tri-methyltransferase, decreases the deposition of H3K36me2 and H3K36me3 at the PIF1 locus, reduces the targeting of RNA polymerase II to the PIF1 locus, and down-regulates the mRNA expression of PIF1 in imbibed seeds. Consistent with the decreased expression of PIF1 mRNA, the efs mutant geminates at a higher rate under the far-red light treated condition compared to wild type, and the expression profiles of PIF1 target genes in the efs mutant are similar to that of the pif1 mutant in imbibed seeds. Introduction of an EFS transgene into the efs mutant restored the deposition of H3K36me2 and H3K36me3 at the PIF1 locus, the high-level expression of PIF1 mRNA, the expression pattern of PIF1 target genes in imbibed seeds, and the light-dependent germination of these seeds. Conversely, introduction of a PIF1 transgene into the efs mutant restored the expression pattern of PIF1 target genes and light-dependent germination in imbibed seeds, but not the other parameters. Taken together, my results indicate that EFS confers functional specificity to PIF1 by permitting its high-level mRNA expression in imbibed seeds. PIF1 directly binds to its targets mainly through G-box or its variants, and regulates numerous genes involved in hormone signaling and metabolism, and cell wall loosening to inhibit seed germination in the dark. Here, I identified PIF1-interacting factor, LUH. Like the pif1 mutant, the luh mutant germinates even under far-red light treated condition, in which phytochrome B exists as inactive form, indicating LUH inhibits light-dependent seed germination. Microarray analysis showed that luh mutant has a gene expression pattern similar to that of pif1 mutant or red light treated wild type. By chromatin immunoprecipitation assay, I showed that LUH is recruited to the promoter regions defined as PIF1 binding sites. The enrichment of LUH at target genes was reduced in light condition compared with that in dark, suggesting the recruitment of LUH at target genes is dependent on PIF1. ChIP-chip analysis further confirmed that among 946 LUH binding sites, 213 sites are also PIF1 binding sites, and the G-box and its variants are enriched at these sites. Taken together, my results indicate that LUH interacts with PIF1, directly regulates a subset of PIF1-regulated genes in similar pattern with PIF1, and inhibits light-dependent seed germination. Among PIF1 direct target genes, SOMNUS (SOM), which encodes a C3H-type zinc finger protein, is a key negative regulator. However, it was not determined if PIF1 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. Analysis of abi3 mutants and ABI3 overexpression lines showed that ABI3 activates the expression of SOM mRNA collaboratively with PIF1 in imbibed seeds. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assay indicate that ABI3 activates the expression of SOM mRNA by directly binding to two RY motifs in the SOM promoter. I confirmed that ABI3 interacts with the PIF1 protein, however, the interaction between ABI3 and PIF1 does not affect targeting of ABI3 and PIF1 to SOM promoters. Taken together, my results indicate that ABI3 and PIF1 collaboratively activate the expression of SOM mRNA by directly binding to and interacting with each other at the SOM promoter.