Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analysed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG, and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes always had higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza. DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2, CMK, IDI1, HMGR2, DXR, MDS, CYP76AH1, 2OGD25, and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.

Poplar is an important fast-growing tree, and its photosynthetic capacity directly affects its vegetative growth. Stomatal density is closely related to photosynthetic capacity and growth characteristics in plants. Here, we isolated PagSTOMAGEN from the hybrid poplar (Populus alba × Populus glandulosa) clone 84K and investigated its biological function in vegetative growth. PagSTOMAGEN was expressed predominantly in young tissues and localized in the plasma membrane. Compared with wild-type 84K poplars, PagSTOMAGEN-overexpressing plants displayed an increased plant height, leaf area, internode number, basal diameter, biomass, IAA content, IPR content, and stomatal density. Higher stomatal density improved the net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, and transpiration rate in transgenic poplar. The differential expression of genes related to stomatal development showed a diverged influence of PagSTOMAGEN at different stages of stomatal development. Finally, transcriptomic analysis showed that PagSTOMAGEN affected vegetative growth by affecting the expression of photosynthesis and plant hormone-related genes (such as SAUR75, PQL2, PSBX, ERF1, GNC, GRF5, and ARF11). Taken together, our data indicate that PagSTOMAGEN could positively regulate stomatal density and increase the photosynthetic rate and plant hormone content, thereby promoting vegetative growth in poplar. Our study is of great significance for understanding the relationship between stoma, photosynthesis, and yield breeding in poplar.

Long non-coding RNA (lncRNA) plays an important regulatory role in plant growth and development, but its systematic identification and analysis in Eucalyptus has not yet been reported. Cold stress has a huge impact on the survival and yield of Eucalyptus seedlings, but the regulatory mechanism of lncRNA in Eucalyptus in response to cold stress is still unclear. In this study, the transcriptomes of young leaves of Eucalyptus urophylla S.T.Blake under low-temperature treatment and restoration were analyzed by RNA-seq. A total of 11,394 lncRNAs and 46,276 mRNAs were identified, of which 300 were differentially expressed lncRNAs (DE_lncRNAs) and 5606 were differentially expressed target genes of lncRNAs under cold stress, with the total number of target genes of DE_lncRNAs being 1681. A total of 677 differentially expressed transcription factors (TFs) were also identified, mainly including ERF, MYB and the NAC transcription factor family. Gene ontology (GO) analysis of the differentially expressed genes (DEGs) and target genes of DE_lncRNAs was mostly related to the response to cold stress and external stimuli. Furthermore, lncRNA–miRNA–mRNA regulatory networks were constructed, and 22 DE_lncRNAs were predicted to be targets or targeting mimics of 20 miRNAs. A qRT-PCR was used to verify the relative expression of genes in the regulatory EuGBF3-EUC_00002677-MSTRG.7690 network, and it matched the transcriptome data, indicating that it may play an important role in the response to cold stress in E. urophylla. This study provides a new insight into lncRNA and its regulatory network under abiotic stress, especially cold stress in E. urophylla.

Triploid poplar trees have been shown to have a number of growth advantages, especially much bigger leaves that contribute greatly to the increased biomass. In this study, we focused on the relationships between leaf age and leaf metabolism in triploids. We performed comparative proteomic analysis of the 5th (FDR5), 10th (FDR10), and 25th (FDR25) leaves from the apical meristems in allotriploids originated from first-division restitution (FDR). A total of 1970, 1916, and 1850 proteins were identified in the FDR5, FDR10, and FDR25, respectively. Principle component analysis (PCA) and differentially accumulated protein (DAP) analysis showed that FDR10 and FDR25 displayed higher similarities of protein accumulation patterns as compared to FDR5. MapMan enrichment analysis showed that several primary metabolic pathways or processes were significantly enriched in the DAPs. For example, photosynthesis, major CHO metabolism, glycolysis, N metabolism, redox, C1-metabolism, DNA, and protein turnover were significantly altered in both FDR10 and FDR25 compared with FDR5. In addition, amino acid metabolism and gluconeogenesis/glyoxylate cycle also underwent significant changes in FDR25 compared with FDR5. However, only amino acid metabolism was significantly enriched in the DAPs between FDR25 and FDR10. Further, DAP accumulation pattern analysis implied that FDR5, FDR10, and FDR25 were placed in the young, mature, and primary senescence stages of leaves. The most DAPs involved in the light reaction, photorespiration, Calvin cycle, starch and sucrose metabolism, pentose phosphate pathway (OPP), tricarboxylic acid (TCA) cycle, N metabolism, and C1-metabolism displayed higher accumulation in both FDR10 and FDR25 compared to FDR5. However, the most DAPs that are involved in cell wall and lipid metabolism, tetrapyrrole synthesis, nucleotide metabolism exhibited lower accumulation in both FDR10 and FDR25. Almost all DAPs between FDR-10 and FDR-25 showed a dramatic decrease in FDR25. KEGG enrichment analysis showed that carbon metabolism was altered significantly at different leaf ages. DAPs that are involved in carbon metabolism were predicted as different points in protein–protein interaction (PPI) networks from the STRING database. Finally, inconsistent transcript and protein abundance was found for DAPs, indicating the presence of posttranscriptional regulation during leaf-age progression process.