Lonicera japonica Thunb. is a widely used medicinal plant and is rich in a variety of active ingredients. Flavonoids are one of the important components in L. japonica and their content is an important indicator for evaluating the quality of this herb. To study the regulation of flavonoid biosynthesis in L. japonica, an R2R3-MYB transcription factor gene LjaMYB12 was isolated and characterized. Bioinformatics analysis indicated that LjaMYB12 belonged to the subgroup 7, with a typical R2R3 DNA-binding domain and conserved subgroup 7 motifs. The transcriptional level of LjaMYB12 was proportional to the total flavonoid content during the development of L. japonica flowers. Subcellular localization analysis revealed that LjaMYB12 localized to the nucleus. Transactivation activity assay indicated that LjaMYB12 was a transcriptional activator. Then, ectopic expression of LjaMYB12 in Arabidopsis could increase PAL activity and flavonoid content and promote transcription of a range of flavonoid biosynthetic genes. Interestingly, the fold changes of downstream genes in the flavonoid biosynthetic pathway were significantly higher than that of the upstream genes, which suggested that LjaMYB12 may have different regulatory patterns for the upstream and downstream pathways of flavonoid biosynthesis. The results provided here will effectively facilitate the study of subgroup 7 MYBs and transcriptional regulation of flavonoid biosynthesis in L. japonica.

Mentha canadensis L. has important economic value for its abundance in essential oils. Menthol is the main component of M. canadensis essential oils, which is certainly the best-known monoterpene for its simple structure and wide applications. However, the regulation of menthol biosynthesis remains elusive in M. canadensis. In this study, transcriptome sequencing of M. canadensis with MeJA treatment was applied to illustrate the transcriptional regulation of plant secondary metabolites, especially menthol biosynthesis. Six sequencing libraries were constructed including three replicates for both control check (CK) and methyl jasmonate (MeJA) treatment and at least 8 Gb clean bases was produced for each library. After assembly, a total of 81,843 unigenes were obtained with an average length of 724 bp. Functional annotation indicated that 64.55% of unigenes could be annotated in at least one database. Additionally, 4430 differentially expressed genes (DEGs) with 2383 up-regulated and 2047 down-regulated transcripts were identified under MeJA treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that “Monoterpenoid biosynthesis” was one of the most significantly enriched pathways in metabolism. Subsequently, DEGs involved in JA signal transduction, transcription factors, and monoterpene biosynthesis were analyzed. 9 orthologous genes involved in menthol biosynthesis were also identified. This is the first report of a transcriptome study of M. canadensis and will facilitate the studies of monoterpene biosynthesis in the genus Mentha.

Background: Lonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis. Methods: The recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s. Results: In this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in L. japonica transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in L. japonica. The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica. qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1. Discussion: Our results provide a comprehensive identification and characterization of CYP450s in L. japonica. Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.

C2H2-type zinc finger protein (C2H2-ZFP) transcription factors play evident roles in regulating plant growth and development and abiotic stress responses. However, the role of C2H2-ZFP from Mentha canadensis remains uncertain. We identified the multifunctional C2H2-ZFP gene McZFP1 from M. canadensis based on phylogenetic analysis. The McZFP1 gene was highly expressed in stems, responding to abiotic stress and phytohormone treatments. McZFP1 localized in the nucleus and showed no transcriptional autoactivation activity in yeast. McZFP1 overexpression in Arabidopsis thaliana significantly reduced the number of trichomes and root hairs, root hair length, and salt stress tolerance. Further study revealed that McZFP1 overexpression increased the expression of negative regulator genes and decreased that of positive regulator genes to inhibit plant trichome and root hair development. Malondialdehyde accumulation was promoted, but the proline content and catalase, superoxide dismutase, and peroxidase activities were reduced and the expression of stress response genes was inhibited in McZFP1 overexpression lines under salt treatment, thereby compromising plant salt tolerance. Overall, these results indicate that McZFP1 is a novel C2H2-ZFP transcription factor that plays negative roles in trichome and root hair development and salt stress tolerance.

Lonicera japonica Thunb. (Caprifoliaceae), a perennial shrub, is widely used in traditional Chinese medicine (Zheng et al. 2022). In June 2023, a severe outbreak of anthracnose was observed in honeysuckle plantations in Changzhou (31.666°N, 119.505°E), Jiangsu Province, China. This disease still exists to this day, affecting the growth of honeysuckle and reducing the yield of flower buds. The planting area was approximately 12 ha, and examination of 120 L. japonica plants revealed an anthracnose leaf infection rate above 30% (n = 120). In the initial stages of the disease, the spots are scattered and appear yellowish-brown. As the disease progresses, the spots gradually expanded into a round or polygonal shape. The edges of the leaves desiccate, leading to extensive necrosis that ultimately results in leaf abscission. Five symptomatic leaves were collected from five different plants, and small tissue fragments (5×5 mm2) were excised from the periphery of the lesions. The tissues were surface sterilized with 75% ethanol for 45 s followed by 1% NaClO for 90 s, and then rinsed three times with sterile water. Subsequently, the leaves were cultured on PDA medium at 25°C in the dark. After 3 days, all samples displayed similar culture morphology. A representative strain (LJ4-1) was obtained through single spore isolation from all samples. The mycelium on PDA medium was white and fluffy at first and turned gray after 7 days. Conidia were unicellular, hyaline, cylindrical, measuring 10.52 to 17.63 μm in length and 4.5 to 5.6 μm in width (n = 50). These isolates were preliminarily identified as Colletotrichum fructicola based on morphological and cultural characteristics (Jiang et al. 2024). To confirm the pathogen's identity, total DNA was extracted and the internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), and chitin synthase (CHS-1) regions were amplified using the primer pairs ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, and CHS-79F/CHS-345R, respectively (Weir et al. 2012). The sequences obtained were submitted to GenBank (PQ157567 and PQ179695-PQ179698) and showed 99 to 100% identity to C. fructicola strains (OQ429091 for ITS (572/573 bp), MK949020 for GAPDH (280/280 bp), MZ888803 for CAL (773/773 bp), MZ153138 for ACT (289/289 bp), and MW768831 for CHS-1 (299/299 bp)). Phylogenetic analysis of the concatenated sequences from the five genetic loci, using the maximum-likelihood method, indicated that the isolate was classified as C. fructicola. To assess pathogenicity, the conidial suspension (1 × 105 conidia/ml) of LJ4-1 was introduced onto the leaves of one-year-old L. japonica seedlings without causing any injury, while sterile water served as the control. Six duplicates were conducted for each treatment and all plants were covered with plastic bags to maintain high humidity and placed in a greenhouse at 25°C with a photoperiod of 12 h. After 7 days, all leaves inoculated with the pathogen exhibited typical lesions analogous to those observed in field plants, whereas the control group remained asymptomatic. The morphological and molecular characteristics of the pathogen isolated from infected leaves indicated that both infections were identical. Furthermore, no fungi were recovered from the control plants, thereby supporting Koch's postulates. Previous studies indicated that C. fructicola has been shown to cause anthracnose on more than 110 plants (USDA Fungal Databases, https://fungi.ars.usda.gov/). To the best of our knowledge, this is the first report of C. fructicola causing anthracnose on L. japonica in China. The relevance of our findings lies in their potential to inform the development of strategies aimed at controlling anthracnose on L. japonica.