Unraveling the role of PlARF2 in regulating deed formancy in Paeonia lactiflora.

MAIN CONCLUSION: PlARF2 can positively regulate the seed dormancy in Paeonia lactiflora Pall. and bind the RY cis-element. Auxin, a significant phytohormone influencing seed dormancy, has been demonstrated to be regulated by auxin response factors (ARFs), key transcriptional modulators in the auxin signaling pathway. However, the role of this class of transcription factors (TFs) in perennials with complex seed dormancy mechanisms remains largely unexplored. Here, we cloned and characterized an ARF gene from Paeonia lactiflora, named PlARF2, which exhibited differential expression levels in the seeds during the process of seed dormancy release. The deduced amino acid sequence of PlARF2 had high homology with those of other plants and contained typical conserved Auxin_resp domain of the ARF family. Phylogenetic analysis revealed that PlARF2 was closely related to VvARF3 in Vitis vinifera. The subcellular localization and transcriptional activation assay showed that PlARF2 is a nuclear protein possessing transcriptional activation activity. The expression levels of dormancy-related genes in transgenic callus indicated that PlARF2 was positively correlated with the contents of PlABI3 and PlDOG1. The germination assay showed that PlARF2 promoted seed dormancy. Moreover, TF Centered Yeast one-hybrid assay (TF-Centered Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay analysis (Dual-Luciferase) provided evidence that PlARF2 can bind to the 'CATGCATG' motif. Collectively, our findings suggest that PlARF2, as TF, could be involved in the regulation of seed dormancy and may act as a repressor of germination.

Improvement of Culture Conditions and Plant Growth Regulators for In Vitro Callus Induction and Plant Regeneration in Paeonia lactiflora Pall.

Owing to its high ornamental, medicinal and horticultural values, herbaceous peony (Paeonia lactiflora Pall.) has been widely used as a landscaping and economical plant around the world. However, the lack of an efficient and stable regeneration system in P. lactiflora restricts its rapid propagation and large-scale production. By testing the key factors affecting callus formation, proliferation, adventitious bud induction and rooting, here, we developed an in vitro system for callus induction and regeneration in P. lactiflora. Our results show that callus formation was affected by explant types, culture environment, basal medium and plant growth regulators. Using cotyledons as explants, we established good conditions for P. lactiflora callus induction and callus proliferation. We effectively obtained adventitious buds differentiated from callus in Murashige and Skoog (MS) medium containing kinetin (KT) and thidiazuron (TDZ). Adventitious bud growth can be further promoted by adding gibberellin 3 (GA3), 1-naphthaleneacetic acid (NAA) and 6-benzyleaminopurine (6-BA) into the MS medium. A high percentage of rooting can be achieved by adding indolebutyric acid (IBA) and activated carbon (AC) to ½ MS medium. Overall, our system promotes callus induction and adventitious bud regeneration for P. lactiflora through improved culture conditions and plant growth regulators in the culture media, and lays a foundation for subsequent genetic engineering research.

Elucidating Biological Functions of 9-cis-Epoxycarotenoid Dioxygenase Genes Involved in Seed Dormancy in Paeonia lactiflora.

Abscisic acid (ABA) is a major phytohormone affecting seed dormancy and germination in plants. ABA is synthesized mainly through the C40 carotenoid pathway. In the ABA biosynthesis pathway, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key rate-limiting enzyme that regulates the accumulation and content of ABA. However, the role of the NCED gene in perennial plants with complex seed dormancy remains largely unknown. Here, we cloned two differentially expressed paralogs of herbaceous peony NCED genes, named PlNCED1 and PlNCED2, and further identified their involvement in seed dormancy from perennial herbaceous peony experiencing complex double seed dormancy. The deduced PlNCED amino acid sequences had high sequence homology with NCED sequences from other plants and contained the typical conserved RPE65 domain of the NCED family. Phylogenetic analysis showed that PlNCED1 and PlNCED2 have a close relationship with PoNCED in Paeonia ostii and VvNCED6 in Vitis vinifera, respectively. A subcellular localization assay demonstrated that the PlNCED1 protein resided within the nucleus, while the PlNCED2 protein was located in the cytoplasm, indicating their different roles in the biosynthesis of ABA. Furthermore, the content of endogenous ABA in transgenic calluses showed that PlNCEDs were positively correlated with ABA content. Both PlNCED transgenic Arabidopsis lines and the functional complementation of Arabidopsis NCED mutants found that PlNCEDs promoted seed dormancy and delayed seed germination. These results reveal that PlNCEDs participate in the seed dormancy of herbaceous peony by regulating the accumulation of endogenous ABA.

An efficient Agrobacterium-mediated transient transformation system and its application in gene function elucidation in Paeonia lactiflora Pall.

Paeonia lactiflora Pall. is known as the king of herbaceous flowers with high ornamental and precious medicinal value. However, the lack of a stable genetic transformation system has greatly affected the research of gene function in P. lactiflora. The Agrobacterium-mediated transient gene expression is a powerful tool for the characterization of gene function in plants. In this study, the seedlings of P. lactiflora were used as the transformation receptor materials, and the efficient transient transformation system with a GUS reporter gene was successfully established by Agrobacterium harboring pCAMBIA1301. To optimize the system, we investigated the effects of germination time, Agrobacterium cell density, infection time, acetosyringone (AS) concentration, co-culture time, negative pressure intensity, Tween-20 concentration and different receptor materials on the transient transformation efficiency of P. lactiflora. The results showed that the highest transient transformation efficiency (93.3%) could be obtained when seedlings in 2-3 cm bud length were subjected to 12 h infection of resuspension solution comprising 1.2 OD600 Agrobacterium, 200 µM AS and 0.01% Tween-20 under 10 of negative pressure intensity followed by 3 days of co-culture in darkness condition. This method is more suitable for the study of gene function in P. lactiflora. Subsequently, stress resistance genes PlGPAT, PlDHN2 and PlHD-Zip were used to verify the effectiveness of this transformation system. These results can provide critical information for identification of key genes in non-model plants, such as P. lactiflora, and promote the development of molecular biology research for P. lactiflora.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the ß-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.