Browsing by Subject "DELLA"

Plant growth and development are strictly regulated by internal hormonal signaling networks, which integrate and coordinate to promote plants’ adaptation and survival in the changing environment. Among the diverse hormones, gibberellins (GAs) are the phytohormones that regulate various processes, from seed germination to fruit development. The conserved plant-specific GRAS family protein DELLAs, key repressors in the GA signaling pathway, serve as the central coordinator of multiple signaling networks through physical interactions with many key transcription factors/regulators in other pathways.

Diverse DELLA-interacting proteins (DIPs) from different signaling pathways and various protein families have been identified in recent years. All the DIPs interact with the C-terminal GRAS domain of DELLA, however, the mechanism of how the GRAS domain interacts with diverse proteins remains a mystery. To solve this problem, I expressed a number of DELLA proteins in E.coli and obtained high-purity protein for biochemical and structural analysis.

As the central coordinator of plant growth and development, DELLA’s activity and stability are regulated by post-translational modifications. Our lab recently showed that SECRET AGENT (SEC) modulates the activity of DELLA through O-linked N-acetylglucosamine (O-GlcNAc) modification in Arabidopsis. Nevertheless, SEC’s paralog SPINDLY (SPY), a putative O-GlcNAc transferase (OGT) identified 20 years ago, does not have OGT activity, and serves as opposite role to SEC in GA signaling with an unknown mechanism.

Our lab made the breakthrough in uncovering the SPY function, and showed it promotes the O-fucosylation of DELLA in planta. I further proved that SPY is a novel protein O-fucosyltransferase through biochemical analysis. SPY specifically transfers O-fucose from GDP-fucose to its substrate peptide, and SPY mutant proteins showed reduced or abolished transferase activity. This is the first work to identify O-fucosylation of nuclear proteins in any organism. O-fucosylation of DELLA activates DELLA by promoting its interaction with DIPs, opposite to repression of interaction with O-GlcNAcylation. Previous studies showed that SPY is involved in multiple cellular pathways such as GA signaling, cytokinin signaling and the circadian clock. Thus, SPY plays an important role in regulating plant growth and development through O-fucosylation of key components in diverse intracellular pathways.

SPY orthologs are conserved in bacteria, protists, algae and plants, while SEC orthologs are also present in fungi and animals. SPY-like and SEC-like proteins share high sequence similarity, except that two key residues important for the OGT activity of SEC is missing in SPY. Structure analysis of SPY (or its orthologs) would greatly facilitate our understanding of its unique substrate specificity. Toward this goal, I expressed Arabidopsis SPY proteins (as well as bacterial SPY orthologs) in E.coli and obtained high-purity protein for structural analysis. I further identified lead conditions that produce needle-cluster crystals. While optimization would be required, these studies will ultimately reveal the structure of SPY and the architecture of the active site, to show how SPY interacts with GDP-fucose for the transferase activity.

Bioactive gibberellins (GAs) are phytohormones with various effects on plant development, from seed germination through fruit development. The signaling pathway of GA is centered on DELLA proteins (DELLAs), a group of growth repressors degraded upon perception of GA. Previous studies demonstrated that DELLAs administrate global regulation of gene expression. However, given that DELLAs do not contain any canonical DNA-binding domain and that DELLAs only have a moderate association to their target promoters, the nuclear-localized DELLAs are believed to interact with transcription factors for function. Indeed, quite a few transcription factors have been identified as DELLA interactors in the plant model Arabidopsis thaliana, some of which are well-known downstream transcription regulators involved in other signaling pathways. Nevertheless, the molecular mechanisms how DELLAs inhibit so many aspects of plant growth cannot be fully explained by the known DELLA interactors.

Recently, our lab discovered that INDETERMINATE DOMAIN 1 (IDD1), a C2H2 zinc-finger protein in Arabidopsis, and its closest homolog IDD2, have a strong physical interaction with DELLAs, revealing the potential involvement of these two IDD genes in GA signaling. Hence, the objectives of my doctoral research are (1) to evaluate the roles of IDD1 and IDD2 in GA-responsive phenotypes, (2) to investigate the genetic interaction between IDD1, IDD2 and DELLAs and (3) to identify the function of IDD1 and IDD2 and the significance of IDD-DELLA interaction in transcriptional regulation of IDD/DELLA targets. First, we found that both IDDs redundantly promote GA-induced hypocotyl elongation, and that they also play a positive role in stem elongation and floral initiation. Secondly, epistasis analyses exhibit that REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI), two DELLAs with a predominant role in vegetative growth, antagonize IDD1 and IDD2 in hypocotyl elongation, and that GAI also opposes IDD2 in stem elongation and floral initiation. These results entail an antagonistic relationship between IDDs and DELLAs via protein-protein interaction. We then showed that IDD1 and IDD2 repress the expression of canonical DELLA direct targets, including GA20ox2, GA3ox1, GID1b and SCL3. IDD1 also inhibits transcription of CAPRICE (CPC) and GLABRA2 (GL2), two crucial regulators of root epidermal cell patterning. Taking into account that IDD1 and RGA associate with the same region in CPC promoter, we conclude that CPC is a direct target of the IDD1/RGA complex. In addition, transient expression assays suggested that IDD1 and RGA counteract each other's effects on expression of GA20ox2, GID1b, SCL3, CPC and GL2, providing evidence that IDDs and DELLAs function antagonistically in transcriptional regulation of downstream genes in general. Although both IDDs bear a putative repression motif GLGLGL in their C termini, mutation of this motif did not affect the repressive activity of IDD1 in our transient expression system. On the other hand, fusion of the viral protein 16 (VP16) transcriptional activation domain to IDD1 seems to override the original repressive activity of IDD1. Together, these results uncover a new branch of GA signaling pathway through IDD1 and IDD2, shed light on the interplay of the two IDDs and DELLAs in GA feedback regulation and give insights into the molecular mechanism underlying IDD-mediated GA repression of root hair development.