Dissecting the Roles of Hippo Signalling Pathway in Organ Size Control in Plants

Gu, S (2025) Dissecting the Roles of Hippo Signalling Pathway in Organ Size Control in Plants. Doctoral thesis, Liverpool John Moores University.

Text 2025SiPhD.pdf - Published Version Access Restricted until 23 June 2026. Available under License Creative Commons Attribution Non-commercial. Download (16MB)

Abstract

Organ size is a fundamental characteristic of plants with fruit size being particularly important for the marketability of strawberries. However, the regulation of organ size is a complex process influenced by multiple signalling pathways. In animals, the Hippo signalling pathway serves as a negative regulator of organ size. Its key components include Ste20 (Sterile 20) serine/threonine kinase as upstream regulators, LATS/NDR serine/threonine kinase as downstream components phosphorylated by Ste20 kinases, scaffold proteins such as MOB1, and transcription factors as effectors. Mutations in Ste20, LATS and MOB1 result in excessive cell proliferation and enlarged organs.
Although the Hippo pathway is less well understood in plants, homologues of Ste20 kinase, NDR kinases, and scaffold proteins such as MOB1 have been identified. Previous studies have shown that Arabidopsis thaliana Ste20 kinase 1 (AtSIK1), a homologue of Ste20, regulates organ size by interacting with AtMOB1 within the Hippo signalling pathway. Notably, in plants, the Hippo pathway positively regulates organ size. Mutations in atsik1, atmon1a, or the double mutant atsik1/mob1a lead to fewer cells and smaller organs. However, the precise mechanisms by which the Hippo pathway controls organ size in plants remain unclear.
This project focuses on the kinase activity of AtSIK1 and its role in regulating organ size in Arabidopsis. We found that its kinase activity, dependent on Lys278 and Thr405, is crucial for organ size control. Further investigation showed that AtSIK1 phosphorylated AtMOB1A and AtMOB1B at the conserved Thr35, consistent with the canonical Hippo pathway. Importantly, we identified a new component of the plant Hippo pathway, AtHXK1. It is a sugar sensor, which acts as a positive regulator of organ size in Arabidopsis. AtSIK1 interacts with and phosphorylates AtHXK1 at Ser177. The double mutant sik1-1/hxk1 exhibits a greater reduction in organ size and sensitivity to high glucose than either single mutant, demonstrating their additive roles in organ size regulation. This is likely mediated through cross-talk between the Hippo pathway and glucose signalling.
To explore the conservation of the Hippo pathway in horticultural crop, we identified FvM4K1, a Ste20 kinase from woodland strawberry (Fragaria vesca). FvM4K1 partially restored defects in a yeast Ste20 mutant ste20Δ and fully rescued growth in Arabidopsis mutant atsik1-4, highlighting its functional conservation. RNAi-mediated downregulation of FvM4K1 reduced strawberry plant and fruit size due to smaller cell size and number, while overexpression increased organ size. Kinase activity analysis showed that FvM4K1 autophosphorylates, with Lys269 and Thr396 being essential for its function. Similar to AtSIK1, FvM4K1 interacts with and phosphorylates the two scaffold proteins FvMOB1A and FvMOB1B at the conserved Thr35 and Thr36 residues, respectively.
Taken together, we conducted an in-depth mechanistic study of AtSIK1 and its interaction with AtMOB1, linking its kinase activity to interaction and organ size control in Arabidopsis. A homologous Ste20 kinase, FvM4K1, was identified, and its role in phosphorylating FvMOB1 proteins and regulating plant and fruit size was validated in strawberry, confirming the distinctive role of the Hippo pathway in plants. The discovery of a novel Hippo pathway component, AtHXK1, in Arabidopsis links sugar signalling to the Hippo pathway. This may help elucidate the positive regulatory role of the Hippo signalling pathway in plants, in contrast to its negative role in animals in regulating organ size.
These findings provide new insights into the molecular mechanisms of organ size control in Arabidopsis and fruit size regulation in strawberry, advancing our understanding of the Hippo signalling pathway in higher plants, a pathway that remains largely unexplored. These discoveries also offer new opportunities for biotechnological strategies to improve crop yield and quality.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Hippo pathway; Ste20; kinase; MOB1; HXK1; phosphorylaion; organ size; glucose sigalling; Arabidopsis; strawberry
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Pharmacy and Biomolecular Sciences
Date of acceptance:	1 May 2025
Date Deposited:	24 Jun 2025 11:06
Last Modified:	24 Jun 2025 11:07
DOI or ID number:	10.24377/LJMU.t.00026451
Supervisors:	Qi, B
URI:	https://researchonline.ljmu.ac.uk/id/eprint/26451

View Item