The WRKY transcription factor family plays important regulatory roles in multiple biological processes in higher plants. They have been identified and functionally characterized in a number of plant species, but very little is known in Neolamarckia cadamba, a ‘miracle tree’ for its fast growth and potential medicinal resource in Southeast Asia. In this study, a total of 85 WRKY genes were identified in the genome of N. cadamba. They were divided into three groups according to their phylogenetic features, with the support of the characteristics of gene structures and conserved motifs of protein. The NcWRKY genes were unevenly distributed on 22 chromosomes, and there were two pairs of segmentally duplicated events. In addition, a number of putative cis-elements were identified in the promoter regions, of which hormone- and stress-related elements were shared in many NcWRKYs. The transcript levels of NcWRKY were analyzed using the RNA-seq data, revealing distinct expression patterns in various tissues and at different stages of vascular development. Furthermore, 16 and 12 NcWRKY genes were confirmed to respond to various hormone treatments and two different abiotic stress treatments, respectively. Moreover, the content of cadambine, the active metabolite used for the various pharmacological activities found in N. cadamba, significantly increased after Methyl jasmonate treatment. In addition, expression of NcWRKY64/74 was obviously upregulated, suggesting that they may have a potential function of regulating the biosynthesis of cadambine in response to MeJA. Taken together, this study provides clues into the regulatory roles of the WRKY gene family in N. cadamba.

Background: Cymbidium sinense is an orchid that is typically used as a potted plant, given its high-grade ornamental characteristics, and is most frequently distributed in China and SE Asia. The inability to strictly regulate flowering in this economically important potted and cut-flower orchid is a bottleneck that limits its industrial development. Studies on C. sinense flowering time genes would help to elucidate the mechanism regulating flowering. There are very few studies on the genetic regulation of flowering pathways in C. sinense. Photoperiod significantly affects the flowering of C. sinense, but it was unknown how the CONSTANS gene family is involved in regulating flowering. Results: In this study, eight CONSTANS-like genes were identified and cloned. They were divided into three groups based on a phylogenetic analysis. Five representative CsCOL genes (CsCOL3/4/6/8/9) were selected from the three groups to perform expression characterization and functional study. CsCOL3/4/6/8/9 are nucleus-localized proteins, and all five CsCOL genes were expressed in all organs, mainly in leaves followed by sepals. The expression levels of CsCOL3/4 (group I) were higher in all organs than other CsCOL genes. Developmental stage specific expression revealed that the expression of CsCOL3/4/9 peaked at the initial flowering stage. In contrast, the transcript level of CsCOL6/8 was highest at the pedicel development stage. Photoperiodic experiments demonstrated that the transcripts of the five CsCOL genes exhibited distinct diurnal rhythms. Under LD conditions, the overexpression of CsCOL3/4 promoted early flowering, and CsCOL6 had little effect on flowering time, whereas CsCOL8 delayed flowering of Arabidopsis thaliana. However, under SD conditions, overexpression of CsCOL4/6/8 promoted early flowering and the rosette leaves growth, and CsCOL3 induced flower bud formation in transgenic Arabidopsis. Conclusion: The phylogenetic analysis, temporal and spatial expression patterns, photoperiodic rhythms and functional study indicate that CsCOL family members in C. sinense were involved in growth, development and flowering regulation through different photoperiodic pathway. The results will be useful for future research on mechanisms pertaining to photoperiod-dependent flowering, and will also facilitate genetic engineering-based research that uses Cymbidium flowering time genes.

Abelmoschus Manihot is an important medicinal and edible plant known for its functional secondary metabolites. However, little is known about the key genes involved in production of secondary metabolites in A. manihot. This is largely due to the lack of effective gene expression detection systems for A. manihot, making the screening of real-time PCR reference genes a prerequisite. In this study, 11 candidate reference genes were screened and cloned from A. manihot, and their expression stability was evaluated in different tissues under different flowering stages using four algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The expression stability of eIF and PP2A1 was the highest, while that of tubulin alpha (TUA) was the lowest. The combined use of the two most stable reference genes, eIF and PP2A1, met the experimental requirements for normalizing gene expression in A. manihot. Furthermore, the gene expression of transcription factors bHLH147 and bHLH148 was further validated by data normalization. This study identified potential reference genes in different A. manihot tissues, paving the way for functional gene analysis and dissecting metabolite regulation mechanisms in A. manihot.

Compatible fungal partners of orchids can significantly enhance seed germination and increase seedling establishment under both in vitro and in situ conditions. This study isolated 14 Tulasnella isolates from five-year-old potted plants of three D. officinale cultivars. Three phylogenetically representative strains (Dca122, Dca222, and Dca113) and two additional orchid mycorrhizal fungus (OMFs, ML01 and Pi) were selected to evaluate their effects on D. officinale seed germination and seedling development in vitro, and subsequent seedling growth under greenhouse conditions. All five OMFs supported seed germination and seedling development in vitro. Notably, Dca113, Pi, and ML01 exhibited the most pronounced effects, producing protocorms 3–4 times larger in volume than controls. By day 25, 37.54%, 37.34%, and 42.6% of protocorms developed cotyledons with these isolates, respectively. Furthermore, after 120 days, ML01 and Dca113 treatments yielded 35.6% and 30.68% autotrophic seedlings with fully differentiated roots. Under greenhouse, ML01, Pi, and Dca122 significantly enhanced fresh weight accumulation, plant height, and stem node number in potted seedlings. In contrast, Dca222 primarily stimulated sprouting tillers and adventitious root formation. Our results demonstrate that the mycorrhizal effectiveness of OMFs from different hosts varies significantly in D. officinale. ML01 and Dca113 are ideal candidates for reintroduction programs due to their strong promotion of seed germination and rapid formation of rooted seedlings. ML01 proved the most effective OMF for enhancing growth in potted seedlings, while Dca222 demonstrated potential for co-inoculation strategies.