PP1-associated responses linked to HMGR-related changes during postharvest squalene accumulation in Camellia oleifera...

Plants synthesize diverse isoprenoids that are essential for growth, development, and adaptation to environmental stress. In Camellia oleifera, squalene is a valuable minor constituent of seed oil, yet the regulatory processes linking postharvest stress to squalene accumulation remain poorly understood. Here, we investigated the relationship between protein phosphatase 1-associated responses and changes related to 3-hydroxy-3-methylglutaryl-coenzyme A reductase during postharvest squalene accumulation in Camellia oleifera seeds exposed to 35 °C and 95% relative humidity. Squalene content increased rapidly and reached approximately 2.6 times its initial level after 24 h. By contrast, the enzyme-linked immunosorbent assay-based immunoreactive signal associated with 3-hydroxy-3-methylglutaryl-coenzyme A reductase increased more gradually and remained approximately 30-40% above the initial level from 24 to 48 h. Transcriptome analysis showed that CoHMGR2, the most abundant 3-hydroxy-3-methylglutaryl-coenzyme A reductase transcript at 0 and 12 h, declined during treatment, whereas several downstream genes in the mevalonate pathway showed treatment-responsive increases. These contrasting patterns indicate that the observed response associated with 3-hydroxy-3-methylglutaryl-coenzyme A reductase could not be explained by transcript abundance alone. Two genes encoding protein phosphatase 1 catalytic subunits also showed increased expression. Recombinant-protein pull-down assays supported an association in vitro between a Camellia oleifera 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein and a protein phosphatase 1 catalytic subunit under the defined assay conditions. Metabolomic profiling revealed extensive metabolic reorganization during the major phase of squalene accumulation. Pull-down proteomic analysis further identified candidate proteins associated with 3-hydroxy-3-methylglutaryl-coenzyme A reductase that were functionally annotated to mitochondrial energy metabolism, redox homeostasis, protein folding, and membrane trafficking. Collectively, these findings support a cautious model in which protein phosphatase 1-associated responses are linked to changes related to 3-hydroxy-3-methylglutaryl-coenzyme A reductase and to reorganization of the mevalonate pathway during postharvest squalene accumulation. Whether protein phosphatase 1 directly regulates 3-hydroxy-3-methylglutaryl-coenzyme A reductase through dephosphorylation remains to be determined by targeted biochemical and phosphoproteomic analyses. This study extends the current protein phosphatase 2A-centered framework of plant 3-hydroxy-3-methylglutaryl-coenzyme A reductase regulation and supports protein phosphatase 1 as a candidate component of a regulatory context associated with sterol and triterpene metabolism in a woody oil crop.