Background: WRINKLED1 (WRI1) encodes a transcription factor, belonging to the APETALA2 (AP2) family, and plays a key role in regulating plant oil biosynthesis. As a newly woody oil crop, tree peony (Paeonia rockii) was notable for the abundant unsaturated fatty acids in its seed oil. However, the role of WRI1 during the accumulation of P. rockii seeds oil remains largely unknown. Results: In this study, a new member of the WRI1 family was isolated from P. rockii and was named PrWRI1. The ORF of PrWRI1 consisted of 1269 nucleotides, encoding a putative protein of 422 amino acids, and was highly expressed in immature seeds. Subcellular localization analysis in onion inner epidermal cells showed that PrWRI1 was located at the nucleolus. Ectopic overexpression of PrWRI1 could significantly increase the total fatty acid content in Nicotiana benthamiana leaf tissue and even PUFAs in transgenic Arabidopsis thaliana seeds. Furthermore, the transcript levels of most genes related to fatty acids (FA) synthesis and triacylglycerol (TAG) assembly were also up-regulated in transgenic Arabidopsis seeds. Conclusions: Together, PrWRI1 could push carbon flow to FA biosynthesis and further enhance the TAG amount in seeds with a high proportion of PUFAs.

The tree peony, a novel woody oil crop extensively cultivated in China, necessitates further investigation into artificial pollination technology to enhance seed yield. In this study, we conducted artificial pollination experiments with 6-year-old Paeonia ostii ‘Feng Dan’ seedings for suitable pollen sources, pollen concentration, pollination timing, and pollination frequency. By evaluating seed yields, active ingredients, and oil quality, we derived the following significant conclusions. Firstly, compared to natural pollination, artificial pollination could significantly increase the fruit diameter by 13.94–27.58%, seed yields by 35.17–58.99%, and oil content by 6.45–7.52% in tree peonies. In active ingredients, seeds produced by pollen from Hantai County significantly enhanced starch content (by 48.64%), total phenols (by 41.18%) and antioxidant capacity (by 54.39%). In oil quality, seeds produced by pollen from Heyang County exhibited the highest α-linolenic acid and total fatty acid content with enhancements of 1.68%, 7.41%, and 8.48%. Secondly, hand pollination with pure pollen significantly increased seed yield by 58.99%, total phenol content by 40.97%, antioxidant capacity by 54.39%, and oil content by 1.53% compared to natural pollination. Thirdly, pollination at 2/3 bloom range significantly increased seed number by 63.08% and yield by 45.61% compared to natural pollination. Finally, the effect of one, two, and three pollination events had no difference in seed yield. So, to summarize, applying a 100% concentration of allochthonous pollen once is recommended when the bloom range is more than two thirds.