Functional Analysis of the PoSERK-Interacting Protein PorbcL in the Embryogenic Callus Formation of Tree Peony...

SERK is a marker gene for early somatic embryogenesis. We screened and functionally verified a SERK-interacting protein to gain insights into tree-peony somatic embryogenesis. Using PoSERK as bait, we identified PorbcL (i.e., the large subunit of Rubisco) as a SERK-interacting protein from a yeast two-hybrid (Y2H) library of cDNA from developing tree-peony somatic embryos. The interaction between PorbcL and PoSERK was verified by Y2H and bimolecular fluorescence complementation analyses. PorbcL encodes a 586-amino-acid acidic non-secreted hydrophobic non-transmembrane protein that is mainly localized in the chloroplast and plasma membrane. PorbcL was highly expressed in tree-peony roots and flowers and was up-regulated during zygotic embryo development. PorbcL overexpression caused the up-regulation of PoSERK (encoding somatic embryogenesis receptor-like kinase), PoAGL15 (encoding agamous-like 15), and PoGPT1 (encoding glucose-6-phosphate translocator), while it caused the down-regulation of PoLEC1 (encoding leafy cotyledon 1) in tree-peony callus. PorbcL overexpression led to increased indole-3-acetic acid (IAA) content but decreasing contents of abscisic acid (ABA) and 6-benzyladenosine (BAPR). The changes in gene expression, high IAA levels, and increased ratio of IAA to ABA, BAPR, 1-Aminocyclopropanecarboxylic acid (ACC), 5-Deoxystrigol (5DS), and brassinolide (BL) promoted embryogenesis. These results provide a foundation for establishing a tree-peony embryogenic callus system.