Identification and validation of reference genes for RT-qPCR analysis in Iris domestica under Cd stress.

Iris domestica is a widely used ornamental garden and important medicinal plant. Our previous studies have shown that it exhibits significant uptake and translocation capacity under Cd stress compared to other Iris species. Gene expression is studied using RT-qPCR; however, there are no reference genes have been found for I. domestica under Cd stress. In this investigation, thirteen possible reference genes from previous studies and our transcriptome were screened using RT-qPCR in the leaves and roots of Cd-stressed plants. The findings revealed that UBC9 and ACT were the best reference genes for roots with and without Cd stress, whereas YLS8 and ACT7 were the best reference genes for leaves. Among the different tissues without Cd stress, UBC9 and UBC28 exhibited the best results, whereas PP2C06 and UBC9 exhibited the best results under Cd stress. The most stable reference genes in the leaves and roots were UBC9 and UBC28, respectively, under and without Cd stress, and GADPH was the most unstable. Finally, three metal ion response genes, NRAMP2, YSL9 and CYP81Q32 were detected using RT-qPCR and compared with the transcriptome data to further confirm the reliability of the chosen genes. This study identified suitable reference genes for I. domestica under Cd-stress conditions.

Identification and Characterisation of the CircRNAs Involved in the Regulation of Leaf Colour in Quercus mongolica.

Circular RNAs (circRNAs) are important regulatory molecules involved in various biological processes. However, the potential function of circRNAs in the turning red process of Quercus mongolica leaves is unclear. This study used RNA-seq data to identify 6228 circRNAs in leaf samples from four different developmental stages and showed that 88 circRNAs were differentially expressed. A correlation analysis was performed between anthocyanins and the circRNAs. A total of 16 circRNAs that may be involved in regulating the colour of Mongolian oak leaves were identified. CircRNAs may affect the colour of Q. mongolica leaves by regulating auxin, cytokinin, gibberellin, ethylene, and abscisic acid. This study revealed the potential role of circRNAs in the colour change of Q. mongolica leaves.

Integrated physiological and metabolomic analyses reveal changes during the natural senescence of Quercus mongolica leaves.

Quercus mongolica is a common landscape, afforestation, and construction timber species in northern China with high ecological, economic, and ornamental value. Leaf senescence is a complex process that has important implications for plant growth and development. To explore changes of metabolites during the ageing of Quercus mongolica leaves, we investigated physiological responses and metabolite composition in ageing leaves harvested from 15-20-year-old Quercus mongolica. Leaf samples of Q. mongolica were collected when they were still green (at maturity) (stage 1), during early senescence (stage 2), and during late senescence (stage 3). These leaves were then subjected to physiological index and metabolome sequencing analyses. The physiological analysis showed that the leaves of Q. mongolica changed from green to yellow during senescence, which induced significant accumulation of soluble sugar and significant reductions in the concentration of soluble protein and chlorophyll. Peroxidase and catalase were the main antioxidant enzymes mitigating leaf senescence. Metabolomic analysis identified 797 metabolites during leaf senescence. Compared to stage 1, 70 differential metabolites were screened in stage 2 and 72 were screened in stage 3. Differential metabolites in the two senescent stages were principally enriched in amino acid metabolism, lipid metabolism and secondary metabolite biosynthesis. The contents of N-oleoylethanolamine and N, N-dimethylglycine were significantly increased only in stage 2, while the contents of trifolin, astragalin, valine, isoleucine, leucine, and citric acid were significantly increased only in stage 3. Histidine, homoserine, tryptophan, tyrosine, phenylalanine, proline, norleucine, N-glycyl-L-leucine, linoleic acid, linolenic acid, gallic acid, 3-indoleacrylic acid, 3-amino-2-naphthoic acid, 3-hydroxy-3-methylpentane-1,5-dioic acid, 2,3,4-trihydroxybenzoic acid, trifolin, astragalin, DL-2-aminoadipic acid, pinoresinol dimethyl ether, dimethylmatairesinol, and lysophosphatidylcholine increased during both stage 2 and stage 3. Increasing contents of these metabolites may constitute the main mechanism by which Q. mongolica leaves adapt to senescence.

Design, Synthesis and Antitumor Activities of Novel Quinazolinone Derivatives as Potential EGFR Inhibitors.

Human epidermal growth factor (EGFR) is an important target for antitumor drug research. A series of novel quinazolinone derivatives were synthesized and developed as potent inhibitors of EGFR. The results showed that most of the aimed compounds had potential anti-tumor cell proliferation activities. Some compounds were tested for their EGFR inhibitory activity. Especially, compound 6d showed the most potent antitumor activity with IC50 values of 1.58 µM against human breast cancer (MCF-7) cell lines and exhibited the most potent EGFR inhibitory activity with IC50 of 0.77 µM. Docking simulation was performed to position compound 6d into the EGFR active site to determine the probable binding conformation.

Integrated physiological and transcriptomic analyses reveal the molecular mechanism behind the response to cultivation in Quercus mongolica.

Quercus mongolica, a common tree species for building and landscaping in northern China, has great commercial and ecological value. The seedlings of Q. mongolica grow poorly and develop chlorosis when introduced from high-altitude mountains to low-altitude plains. Effective cultivation measures are key to improving the quality of seedlings. To investigate the complex responses of Q. mongolica to different cultivation measures, we compared the adaptability of 3-year-old Q. mongolica seedlings to pruning (P), irrigation (W), and fertilization [F (nitro compound fertilizer with 16N-16P-16K)]. Physiological measurements and transcriptome sequencing were performed on leaves collected under the P treatments (control, cutting, removal of all lateral branches, and removal of base branches to one-third of seedling height), the W treatments (0, 1, 2, 3, 4, or 5 times in sequence), and the F treatments (0, 2, 4, and 6 g/plant). Analyses of the physiological data showed that P was more effective than W or F for activating intracellular antioxidant systems. By contrast, W and F were more beneficial than P for inducing the accumulation of soluble sugar. OPLS-DA identified superoxide dismutase, malondialdehyde, and peroxidase as critical physiological indices for the three cultivation measures. Transcriptome analyses revealed 1,012 differentially expressed genes (DEGs) in the P treatment, 1,035 DEGs in the W treatment, and 1,175 DEGs in the F treatment; these DEGs were mainly enriched in Gene Ontology terms related to the stress response and signal transduction. Weighted gene coexpression network analyses indicated that specific gene modules were significantly correlated with MDA (one module) and soluble sugar (four modules). Functional annotation of the hub genes differentially expressed in MDA and soluble sugar-related modules revealed that Q. mongolica responded and adapted to different cultivation measures by altering signal transduction, hormone levels, reactive oxygen species, metabolism, and transcription factors. The hub genes HOP3, CIPK11, WRKY22, and BHLH35 in the coexpression networks may played a central role in responses to the cultivation practices. These results reveal the mechanism behind the response of Q. mongolica to different cultivation measures at the physiological and molecular levels and provide insight into the response of plants to cultivation measures.

Complete sequence and comparative analysis of the mitochondrial genome of the rare and endangered Clematis acerifolia, the first clematis mitogenome to provide new insights into the phylogenetic evolutionary status of the genus.

Clematis is one of the large worldwide genera of the Ranunculaceae Juss. Family, with high ornamental and medicinal value. China is the modern distribution centre of Clematis with abundant natural populations. Due to the complexity and high morphological diversity of Clematis, the genus is difficult to classify systematically, and in particular, the phylogenetic position of the endangered Clematis acerifolia is highly controversial. The use of the mitochondrial complete genome is a powerful molecular method that is frequently used for inferring plants phylogenies. However, studies on Clematis mitogenome are rare, thus limiting our full understanding of its phylogeny and genome evolution. Here, we sequenced and annotated the C. acerifolia mt genome using Illumina short- and Nanopore long-reads, characterized the species first complete mitogenome, and performed a comparative phylogenetic analysis with its close relatives. The total length of the C. acerifolia mitogenome is 698,247 bp and the main structure is multi-branched (linear molecule 1 and circular molecule 2). We annotated 55 genes, including 35 protein-coding, 17 tRNA, and 3 rRNA genes. The C. acerifolia mitogenome has extremely unconserved structurally, with extensive sequence transfer between the chloroplast and mitochondrial organelles, sequence repeats, and RNA editing. The phylogenetic position of C. acerifolia was determined by constructing the species mitogenome with 24 angiosperms. Further, our C. acerifolia mitogenome characteristics investigation included GC contents, codon usage, repeats and synteny analysis. Overall, our results are expected to provide fundamental information for C. acerifolia mitogenome evolution and confirm the validity of mitochondrial analysis in determining the phylogenetic positioning of Clematis plants.

Species Identification of Oaks (Quercus L., Fagaceae) from Gene to Genome.

Species identification of oaks (Quercus) is always a challenge because many species exhibit variable phenotypes that overlap with other species. Oaks are notorious for interspecific hybridization and introgression, and complex speciation patterns involving incomplete lineage sorting. Therefore, accurately identifying Quercus species barcodes has been unsuccessful. In this study, we used chloroplast genome sequence data to identify molecular markers for oak species identification. Using next generation sequencing methods, we sequenced 14 chloroplast genomes of Quercus species in this study and added 10 additional chloroplast genome sequences from GenBank to develop a DNA barcode for oaks. Chloroplast genome sequence divergence was low. We identified four mutation hotspots as candidate Quercus DNA barcodes; two intergenic regions (matK-trnK-rps16 and trnR-atpA) were located in the large single copy region, and two coding regions (ndhF and ycf1b) were located in the small single copy region. The standard plant DNA barcode (rbcL and matK) had lower variability than that of the newly identified markers. Our data provide complete chloroplast genome sequences that improve the phylogenetic resolution and species level discrimination of Quercus. This study demonstrates that the complete chloroplast genome can substantially increase species discriminatory power and resolve phylogenetic relationships in plants.