The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

Secondary growth in woody plants generates new cells and tissues via the activity of the vascular cambium and drives the radial expansion of stems and roots. It is regulated by a series of endogenous factors, especially transcription factors. Here, we cloned the basic helix–loop–helix (bHLH) transcription factor gene UNFERTILIZED EMBRYO SAC12 (UNE12) from poplar (Populus alba × Populus glandulosa Uyeki) and used biochemical, molecular, and cytological assays to investigate the biological functions and regulatory mechanism of PagUNE12. PagUNE12 mainly localized in the nucleus and possessed transcriptional activation activity. It was widely expressed in vascular tissues, including primary phloem and xylem and secondary phloem and xylem. Poplar plants overexpressing PagUNE12 showed significantly reduced plant height, shorter internodes, and curled leaves compared with wild-type plants. Optical microscopy and transmission electron microscopy revealed that overexpressing PagUNE12 promoted secondary xylem development, with thicker secondary cell walls than wild-type poplar. Fourier transform infrared spectroscopy, confocal Raman microscopy, and 2D Heteronuclear Single Quantum Correlation analysis indicated that these plants also had increased lignin contents, with a lower relative abundance of syringyl lignin units and a higher relative abundance of guaiacyl lignin units. Therefore, overexpressing PagUNE12 promoted secondary xylem development and increased the lignin contents of secondary xylem in poplar, suggesting that this gene could be used to improve wood quality in the future.

Tree peony (Paeonia suffruticosa Andr.) is a famous ornamental and medicinal flowering species. However, few high-efficiency chloroplast microsatellite markers have been developed for it to be employed in taxonomic identifications and evaluation of germplasm resources to date. In the present study, a total of 139 cpSSR loci were identified across eleven tree peony plastomes. Dinucleotide repeat SSRs (97.12%) were most abundantly repeated for the AT motif (58.27%), followed by the TA motif (30.94%) and the TC motif (7.91%). Twenty-one primer pairs were developed, and amplification tests were conducted for nine tree peony individuals. Furthermore, 19 cpSSR markers were amplified on 60 tree peony accessions by a capillary electrophoresis test. Of 19 cpSSR markers, 12 showed polymorphism with different alleles ranging from 1.333 to 3.000. The Shannon’s information index and polymorphism information content values ranged from 0.038 to 0.887 (mean 0.432) and 0.032 to 0.589 (mean 0.268), respectively. The diversity levels for twelve loci ranged from 0.016 (at loci cpSSR-8 and cpSSR-26) to 0.543 (at locus cpSSR-15), averaging 0.268 for all loci. A total of 14 haplotypes (23.33%) were detected in the three populations. The haplotypic richness ranged from 0.949 to 1.751, with a mean of 1.233 per population. The genetic relationship suggested by the neighbor-joining-based dendrogram divided the genotypes into two clusters. The Jiangnan population was allotted to Cluster II, and the other two populations were distributed into both branches. These newly developed cpSSRs can be utilized for future breeding programs, population genetics investigations, unraveling the genetic relationships between related species, and germplasm management.

The tree peony (Paeonia section Moutan DC.) is the candidate flower in China, with abundant germplasm resources and high ornamental value. However, the short and concentrated flowering period severely restricted the improvement of the economic value of tree peonies. Based on the full-length transcriptome database of tree peonies, the PoVIN3 (GenBank ID: OP341879), involved in the flowering regulation of tree peonies were identified and cloned for the first time. The PoVIN3 was also characterized by bioinformatics methods, quantitative real-time PCR (qRT-PCR), and the establishment of a transgenic system. The expression levels of PoVIN3 in seven different petals developmental stages were the highest at the initial flowering stage of the variant cultivar of Paeonia ostii ‘Fengdan,’ the initial decay stage of the normal flowering Paeonia ostii ‘Fengdan,’ and the half opening stage of the late flowering Paeonia suffruticosa ‘Lianhe.’ Tissue-specific expression analysis showed that the relative expression levels of PoVIN3 were the highest in sepals of both normal flowering Paeonia ostii ‘Fengdan’ and the late flowering Paeonia suffruticosa ‘Lianhe,’ and the highest expression was in stamens of early flowering mutant Paeonia ostii ‘Fengdan.’ In addition, the flowering time of pCAMBIA2300-PoVIN3 transgenic plants was significantly earlier than that of the wild-type, indicating that PoVIN3 could promote plant flowering. The results provide a theoretical basis for exploring the role of PoVIN3 in the regulation of flowering in tree peonies.

Tree peony (Paoenia ostii) is a famous Chinese traditional flower well-known in many countries of the world. However, the short and concentrated flowering period of tree peony greatly affects the ornamental and economic value of the flowers. Exogenous brassinosteroid (BR) treatment can delay the flowering period of ostii T. Hong et J. X. Zhang var. lishizhenenii B. A. Shen for 3 days, but the underlying regulatory mechanism remains elusive. Here, full-length transcriptome and transcriptome sequencing were used to mine key genes related to BR-induced delayed flowering in tree peony. The transcriptome sequencing of the petals yielded 21.27 G clean data and 62,229 isoforms. Among them, 58,218 isoforms were annotated in NR, NT, SwissProt, KEGG, KOG, InterPro and GO databases. GO and KEGG analyses showed that 2460 DEGs were related to delayed flowering in response to BR. Additionally, a total of seven genes affecting flowering were annotated from 11 isoforms, which responded to BR through three pathways to delay the flowering of P. ostii var. lishizhenii. BR treatment increased the expression of BRASSINOSTEROID-SIGNALING KINASE3 (BSK3), potentially by promoting BRI1 ASSOCIATED KINASE RECEPTOR 1 (BAK1). Moreover, BR treatment suppressed the expression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 1 (SPL1), possibly to inhibit the expressions of downstream WRKY genes and APETALA 2 (AP2). Meanwhile, BR treatment promoted the expression of DELLA, which might inhibit the expressions of CONSTANS (CO) and SPL. These results suggest a theoretical basis for further analyses of the molecular mechanism of flowering regulation in tree peony.