The increasing demand for healthy edible oil has generated the need to identify promising oil crops. Tree peony (Paeonia section Moutan DC.) is a woody oil crop with α-linolenic acid (ALA) contributing for 45% of the total fatty acid (FA) content in seeds. Molecular and genetic differences that contribute to varied FA content and composition among the wild peony species are, however, poorly understood. Analyses of FA content and composition during seed development in three tree peony species (Paeonia rockii, P. potaninii, and P. lutea) showed varied FA content among them with highest in P. rockii, followed by P. potaninii, and P. lutea. Total FA content among these species increased with seed development and reached its maximum in its final stage. Seed FA composition analysis of the three species also revealed that ALA (C18:3) was the most abundant, followed by oleic (C18:1) and linoleic (C18:2) acids. Additionally, quantitative real-time RT-PCR analyses of 10 key seed oil synthesis genes in the three tree peony species revealed that FAD3, FAD2, β-PDHC, LPAAT, and Oleosin gene expression levels positively correlate with total FA content and rate of accumulation. Specifically, the abundance of FAD3 transcripts in P. rockii compared with P. potaninii, and P. lutea suggests that FAD3 might play an important role in synthesis of ALA via phosphatidylcholine-derived pathway. Overall, comparative analyses of FA content and composition in three different peony species revealed a correlation between efficient lipid accumulation and lipid gene expression during seed development. Further characterization and metabolic engineering of these key genes from peonies will allow for subsequent improvement of tree peony oil quality and production.

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Tree peony (Paeonia section Moutan DC.) species are woody oil crops with high unsaturated fatty acid content, including α-linolenic acid (ALA/18:3; >40% of the total fatty acid). Comparative transcriptome analyses were carried out to uncover the underlying mechanisms responsible for high and low ALA content in the developing seeds of P. rockii and P. lutea, respectively. Expression analysis of acyl lipid metabolism genes revealed upregulation of select genes involved in plastidial fatty acid synthesis, acyl editing, desaturation, and triacylglycerol assembly in seeds of P. rockii relative to P. lutea. Also, in association with ALA content in seeds, transcript levels for fatty acid desaturases (SAD, FAD2, and FAD3), which encode enzymes necessary for polyunsaturated fatty acid synthesis, were higher in P. rockii compared to P. lutea. Furthermore, the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and ALA content, respectively, and modulated the final ratio 18:2/18:3 in the seed oil. In conclusion, we identified the key steps and validated the necessary desaturases that contribute to efficient ALA synthesis in a woody oil crop. Together, these results will aid to increase essential fatty acid content in seeds of tree peonies and other crops of agronomic interest.

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of aTobacco rattle virus(TRV)-based virus-induced gene silencing (VIGS) in the tree peony speciesPaeonia ostiiis presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized forAgrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more completeAgrobacteriumpenetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter genePoPDSencoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenousPoPDStranscripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

Tree peony (Paeonia rockii) is an excellent woody oilseed crop, known for its high α-linolenic acid (ALA, ~45%) content, which is of great value for human health. However, the mechanisms underlying this high-level ALA accumulation in tree peony seeds are poorly understood. In this study, we evaluated the dynamic changes in the lipidomic profile of P. rockii seeds during development. A total of 760 lipid molecules were identified in P. rockii seeds; triacylglycerol (TAG) lipid molecules showed the highest abundance and diversity, both increasing during seed development. Particularly, ALA was the predominant fatty acid at the TAG sn-3 position. We further characterized two diacylglycerol acyltransferase (DGAT) genes and three phospholipid:diacylglycerol acyltransferase (PDAT) genes involved in the transfer of fatty acids to the TAG sn-3 position. Gene expression and subcellular localization analyses suggested that PrDGATs and PrPDATs may function as endoplasmic reticulum-localized proteins in seed TAG biosynthesis. In vitro functional complementation analysis showed different substrate specificities, with PrPDAT2 having a specific preference for ALA. Multiple biological assays demonstrated that PrDGAT1, PrDGAT2, PrPDAT1-2, and PrPDAT2 promote oil synthesis. Specifically, PrPDAT2 leads to preferential ALA in the oil. Our findings provide novel functional evidence of the roles of PrDGAT1 and PrPDAT2, which are potential targets for increasing the ALA yield in tree peony and other oilseed crops.

In many higher plants, seed oil accumulation is governed by complex multilevel regulatory networks including transcriptional regulation, which primarily affects fatty acid biosynthesis. Tree peony (Paeonia rockii), a perennial deciduous shrub endemic to China is notable for its seed oil that is abundant in unsaturated fatty acids. We discovered that a tree peony trihelix transcription factor, PrASIL1, localized in the nucleus, is expressed predominantly in developing seeds during maturation. Ectopic overexpression of PrASIL1 in Nicotiana benthamiana leaf tissue and Arabidopsis thaliana seeds significantly reduced total fatty acids and altered the fatty acid composition. These changes were in turn associated with the decreased expression of multitudinous genes involved in plastidial fatty acid synthesis and oil accumulation. Thus, we inferred that PrASIL1 is a critical transcription factor that represses oil accumulation by down-regulating numerous key genes during seed oil biosynthesis. In contrary, up-regulation of oil biosynthesis genes and a significant increase in total lipids and several major fatty acids were observed in PrASIL1-silenced tree peony leaves. Together, these results provide insights into the role of trihelix transcription factor PrASIL1 in controlling seed oil accumulation. PrASIL1 can be targeted potentially for oil enhancement in tree peony and other crops through gene manipulation.