Dianyun Hou

In this study, a UDP-glycosyltransferase gene from Forsythia suspensa was systematically identified and functionally characterized. Phylogenetic analysis showed that this gene clustered with members of the UGT74 subfamily, where it clustered closely with members of known function; accordingly, it was provisionally designated as FsUGT74F1. Prokaryotic expression experiments determined that soluble recombinant protein could be obtained under induction conditions of 0.1 mM IPTG at 16 °C for 8 h. In vitro enzyme activity assays suggested that FsUGT74F1 might catalyze the glycosylation of phillygenin using UDP-glucose under the tested conditions, yielding a product whose HPLC retention time was consistent with that of authentic phillyrin. Subcellular localization results indicated that FsUGT74F1 was predominantly associated with the plasma membrane. Transient overexpression of FsUGT74F1 in F. suspensa leaves resulted in upregulation of gene expression by approximately 247.6%, and the phillyrin content increased to 124.7% of that in the control group. Conversely, virus-induced gene silencing (VIGS) of FsUGT74F1 led to a reduction in transcript levels by approximately 35.6%, and the phillyrin content decreased to 72.5% of the control level. Molecular docking suggested that phillygenin could interact with FsUGT74F1, with Leu169 predicted to participate in substrate positioning near the C4′-OH group. Collectively, these results support the involvement of FsUGT74F1 in phillyrin biosynthesis and provide a theoretical basis for its application in metabolic engineering.

Background: Forsythia suspensa (Thunb.) Vahl is a perennial deciduous shrub of the Oleaceae family. Its dried mature fruits are used as medicine and hold an important position in traditional Chinese medicine. Tyrosine decarboxylase (TyDC) is a key enzyme involved in the synthesis of dopamine in Forsythia suspensa. At the same time, it also affects the growth and development of this species under biotic stress. Methods: This study examined the expression and function of FsTyDC1 under drought and salt stress. The TyDC gene identified in F. suspensa, termed FsTyDC1, has an open reading frame (ORF) of 1518 bp. Results: qRT-PCR and subcellular localization analyses indicated that FsTyDC1 is highly expressed in F. suspensa fruit and its protein is located in the cytoplasm. The gene was silenced using a pTRV2-FsPDS/FsTyDC1 vector with virus-induced gene silencing. Following exposure to drought and salt stress, the leaves of FsTyDC1-silenced plants exhibited increased curling and wilting. Conclusions: The results indicate that FsTyDC1 responds to both salt and drought stress, which provides a foundation for further investigation into the function of FsTyDC1.

Indole-3-acetic acid (IAA) glycosyltransferase (IAGLU) plays vital roles in modulating plant development and responses to environmental cues. Here, we elucidate the regulatory mechanism of OsIAGLU in modulating root gravitropism using OsIAGLU-overexpressing (OE) rice (Oryza sativa L.). OsIAGLU upregulation substantially decreases IAA levels, resulting in the impairment of multiple agronomic traits and root gravitropism, as well as nearly complete suppression of starch granule accumulation in rice root tips. Exogenous application of the auxin analog 1-naphthaleneacetic acid (NAA) effectively rescued both starch granule accumulation and root gravitropism. Starch synthesis genes exhibited relatively stable or slightly decreased expression following NAA treatments, whereas all starch degradation genes displayed a consistent downward trend in expression after NAA treatment. This suggests that starch degradation genes may play a more prominent role in regulating starch granule accumulation in rice roots, contrasting sharply with their roles in Arabidopsis. Moreover, decreased auxin levels perturbed the accumulation and distribution of hydrogen peroxide (H₂O₂) in rice root tips, while NAA treatment restored normal H₂O₂ distribution and accumulation in OE roots. This study clearly demonstrates that auxin not only functions in regulating agronomic traits but also plays an essential role in gravity perception by modulating starch granule accumulation in rice root tips.

Solanum nigrum is a cadmium (Cd) and zinc (Zn) accumulator with potential for phytoextraction of soil contaminated with heavy metals. However, how Zn affects Cd accumulation in S. nigrum remains unclear. In this study, S. nigrum seedlings were treated with 100 μmol·L⁻¹ Zn (Zn100), 100 μmol·L⁻¹ Cd (Cd100), and the Zn and Cd combination (Zn100+Cd100) for 10 days under hydroponic culture. Compared with Cd100, the Cd content in stems, leaves, and xylem saps was 1.8, 1.6, and 1.3 times more than that in Zn100+Cd100, respectively. In addition, the production of reactive oxygen species in leaves was significantly upregulated in Cd100 compared with the control, and it was downregulated in Zn100. Comparative analyses of transcriptomes and proteomes were conducted with S. nigrum leaves. Differentially expressed genes (DEGs) were involved in Cd uptake, transport, and sequestration, and the upregulation of some transporter genes of Zn transporters (ZIPs), a natural resistance associated macrophage protein (Nramp1), a metal–nicotianamine transporter (YSL2), ATP-binding cassette transporters (ABCs), oligopeptide transporters (OPTs), and metallothionein (MTs) and glutathione S-transferase (GSTs) genes was higher in Zn100+Cd100 than in Cd100. In addition, differentially expressed proteins (DEPs) involved in electron transport chain, ATP, and chlorophyll biosynthesis, such as malate dehydrogenases (MDHs), ATPases, and chlorophyll a/b binding proteins, were mostly upregulated in Zn100. The results indicate that Zn supplement increases Cd accumulation and tolerance in S. nigrum by upregulating ATP-dependent Cd transport and sequestration pathways.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa ‘Doulv’, notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from ‘Doulv’, nine candidate reference genes—ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)—were selected. Their expression stability was assessed across various tissues and developmental stages of ‘Doulv’ flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of ‘Doulv’, whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from ‘Doulv’ diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for ‘Doulv’ peony. These results provide a solid experimental foundation for further research on ‘Doulv’ peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in ‘Doulv’, identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

Cornus iridoid glycosides (CIGs), including loganin and morroniside, are the main active components of Cornus officinalis. As one of the key enzymes in the biosynthesis of CIGs, geranyl pyrophosphate synthase (GPPS) catalyzes the formation of geranyl pyrophosphate, which is the direct precursor of CIGs. In this study, the C. officinalis geranyl pyrophosphate synthase (CoGPPS) sequence was cloned from C. officinalis and analyzed. The cDNA sequence of the CoGPPS gene was 915 bp (GenBank No. OR725699). Phylogenetic analysis showed that CoGPPS was closely related to the GPPS sequence of Actinidia chinensis and Camellia sinensis, but relatively distantly related to Paeonia lactiflora and Tripterygium wilfordii. Results from the quantitative real-time PCR showed the spatiotemporal expression pattern of CoGPPS; that is, CoGPPS was specifically expressed in the fruits. Subcellular localization assay proved that CoGPPS was specifically found in chloroplasts. Loganin and morroniside contents in the tissues were detected by high-performance liquid chromatography, and both compounds were found to be at higher levels in the fruits than in leaves. Thus, this study laid the foundation for further studies on the synthetic pathway of CIGs.

Corynebacterium glutamicum is an important industrial production strain that is widely used in amino acid fermentation, biopharmaceuticals, and other fields. It is particularly important to develop efficient genome editing methods for the targeted modification of C. glutamicum production strains. Currently, the gene editing system of C. glutamicum is inefficient and time-consuming. In this paper, we reported on a Francisella novicida (Fn) CRISPR-Cpf1-based system for genome editing. The system used linear DNA detached from the plasmid, and, with the assistance of the recombinase RecET, gene deletion was achieved by simultaneous electrotransformation of linear DNA with a plasmid carrying the FnCpf1 and crRNA expression cassette for double-strand breaks of the genome. Compared with previous all-in-one plasmids, this system reduced the time for one round of constructing recombinant plasmids and shortened the editing cycle by about 24 h. Finally, we successfully constructed an engineered strain (X−2) with high L-valine production by using the linear DNA-mediated gene deletion system. This method is of great importance for accelerating the process of metabolic engineering modification of C. glutamicum and its further application in high value-added products.

Forsythia suspensa is a deciduous shrub that belongs to the family Myrtaceae, and its dried fruits are used as medicine. F. suspensa contains several secondary metabolites, which exert pharmacological effects. One of the main active components is forsythin, which exhibits free radical scavenging, antioxidant, anti-inflammatory, and anti-cancer effects. Mitogen-activated protein kinase (MAPKs) can increase the activity of WRKY family transcription factors in a phosphorylated manner, thereby increasing the content of secondary metabolites. However, the mechanism of interaction between MAPKs and WRKYs in F. suspensa remains unclear. In this study, we cloned the genes of FsWRKY4 and FsMAPK3, and performed a bioinformatics analysis. The expression patterns of FsWRKY4 and FsMAPK3 were analyzed in the different developmental stages of leaf and fruit from F. suspensa using real-time fluorescence quantitative PCR (qRT-PCR). Subcellular localization analysis of FsWRKY4 and FsMAPK3 proteins was performed using a laser scanning confocal microscope. The existence of interactions between FsWRKY4 and FsMPAK3 in vitro was verified by yeast two-hybridization. Results showed that the cDNA of FsWRKY4 (GenBank number: OR566682) and FsMAPK3 (GenBank number: OR566683) were 1587 and 522 bp, respectively. The expression of FsWRKY4 was higher in the leaves than in fruits, and the expression of FsMAPK3 was higher in fruits but lower in leaves. The subcellular localization results indicated that FsWRKY4 was localized in the nucleus and FsMAPK3 in the cytoplasm and nucleus. The prey vector pGADT7-FsWRKY4 and bait vector pGBKT7-FsMAPK3 were constructed and co-transferred into Y2H Glod yeast receptor cells. The results indicated that FsWRKY4 and FsMAPK3 proteins interact with each other in vitro. The preliminary study may provide a basis for more precise elucidation of the synthesis of secondary metabolites in F. suspensa.