THE PHYSIOLOGICAL ROLES OF THE L80 PEPTIDE OF PLANT PHO1

Chun-Yeung Yeung Ng

Plant phosphorylases (Pho1, α-glucan phosphorylases, EC 2.4.1.1), which catalyze the reversible synthesis-phosphorolysis of α-1,4-glucans, play an important role in starch synthesis and plant growth. A unique structural feature of the higher plant Pho1 is the presence of an extra peptide, which varies in length from 50 to 82 amino acids depending on the plant species. This extra peptide, 80 residues in length (L80) in rice Pho1, is an intrinsically disordered region (IDR) that is not required for enzyme catalysis. A recent study discovered that rice Pho1 interacts with the PsaC subunit of photosystem I (PSI). Interestingly, transgenic plants expressing a Pho1 lacking the L80 peptide (Pho1ΔL80) display higher growth rates, biomass, seed yields, and altered PSI redox parameters compared to WT. These properties suggest that rice Pho1 plays a dual role in starch biosynthesis and photosynthesis, acting as a negative regulator via the L80 peptide.In this dissertation, I present findings on the functional dissection of the L80 peptide and the requirement of Pho1 enzymatic activity for its regulatory roles. To determine if Pho1-mediated regulation of photosynthesis requires enzymatic activity, catalytically inactive Pho1 variants (Pho1K823A and Pho1ΔL80K823A) were generated and analyzed. Although the catalytically inactive Pho1ΔL80K823A mutant retained the ability to interact with PsaC and stimulated early seedling growth, it failed to reproduce the distinct PSI redox phenotype observed in the catalytically active Pho1ΔL80. These results demonstrate that, while the L80 peptide acts as a regulatory switch, effective modulation of PSI electron transport requires a catalytically competent enzyme. To further identify the key regulatory motif(s) within the L80 peptide, various deletions of the N-terminal (ΔN), C-terminal (ΔC), middle (ΔM), and partial N and partial C regions (ΔNC) were generated. Phenotypic analysis revealed that negative regulation of starch accumulation mainly localizes to a specific acidic motif within the C-terminal region (residues 42-59), as ΔC and ΔM lines exhibited increased seed weight. In contrast, PSI modulation did not map to a single motif; L80-partial-deletions failed to consistently recapitulate the donor- and acceptor-side alterations seen in the full L80 deletion (Pho1ΔL80). These findings suggest that the L80 peptide encodes mechanistically separable functions: a sequence-specific inhibitory motif controlling starch biosynthesis and a distributed structural element that coordinates photosynthetic redox balance in concert with Pho1 catalytic activity.

THE PHYSIOLOGICAL ROLES OF THE L80 PEPTIDE OF PLANT PHO1

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