Identification of key genes involved in lignin and flavonoid accumulation during Tilia tuan seed maturation.

KEY MESSAGE: Transcriptomics and phenotypic data analysis identified 24 transcription factors (TFs) that play key roles in regulating the competitive accumulation of lignin and flavonoids. Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts, including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into 3 clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into 2 clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2 and TtWRKY15) were involved in flavonoids and lignin biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between flavonoid and lignin biosynthesis pathway during the development of T. tuan seeds, that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuan seeds.

Transcriptomic and Phenotypic Analyses Reveal the Molecular Mechanism of Dwarfing in Tetraploid Robinia pseudoacacia L.

Polyploid breeding techniques aid in the cultivation of new forestry cultivars, thus expanding the suite of strategies for the improvement of arboreal traits and innovation within the field of forestry. Compared to diploid Robinia pseudoacacia L. (black locust) 'D26-5①' (2×), its dwarfed homologous tetraploid 'D26-5②' (4×) variety has better application prospects in garden vegetation guardrails and urban landscape. However, the molecular mechanism of the generation and growth of this dwarf variety is still unclear. Here, plant growth and development as well as histological differences between the diploid and its autotetraploid were investigated. Levels of endogenous hormones at three different developmental stages (20, 40, and 70 days) of 2× and homologous 4× tissue culture plantlets were assessed, and it was found that the brassinosteroid (BR) contents of the former were significantly higher than the latter. Transcriptome sequencing data analysis of 2× and homologous 4× showed that differentially expressed genes (DEGs) were significantly enriched in plant hormone synthesis and signal transduction, sugar and starch metabolism, and the plant circadian rhythm pathway, which are closely related to plant growth and development. Therefore, these biological pathways may be important regulatory pathways leading to dwarfism and slow growth in tetraploids. Additionally, utilizing weighted gene coexpression network analysis (WGCNA), we identified three crucial differentially expressed genes (DEGs)-PRR5, CYP450, and SPA1-that potentially underlie the observed ploidy variation. This study provides a new reference for the molecular mechanism of dwarfism in dwarfed autotetraploid black locusts. Collectively, our results of metabolite analysis and comparative transcriptomics confirm that plant hormone signaling and the circadian rhythm pathway result in dwarfism in black locusts.

The RpTOE1-RpFT Module Is Involved in Rejuvenation during Root-Based Vegetative Propagation in Robinia pseudoacacia.

Vegetative propagation is an important method of reproduction and rejuvenation in horticulture and forestry plants with a long lifespan. Although substantial juvenile clones have been obtained through the vegetative propagation of ornamental plants, the molecular factors that regulate rejuvenation during vegetative propagation are largely unknown. Here, root sprouting and root cutting of Robinia pseudoacacia were used as two vegetative propagation methods. From two consecutive years of transcriptome data from rejuvenated seedlings and mature trees, one gene module and one miRNA module were found to be specifically associated with rejuvenation during vegetative propagation through weighted gene co-expression network analysis (WGCNA). In the gene module, a transcription factor-encoding gene showed high expression during vegetative propagation, and it was subsequently named RpTOE1 through homology analysis. Heterologous overexpression of RpTOE1 in wild-type Arabidopsis and toe1 toe2 double mutants prolonged the juvenile phase. The qRT-PCR results predicted RpFT to be a downstream gene that was regulated by RpTOE1. Further investigation of the protein-DNA interactions using yeast one-hybrid, electrophoretic mobility shift, and dual luciferase reporter assays confirmed that RpTOE1 negatively regulated RpFT by binding directly to the TOE binding site (TBS)-like motif on its promoter. On the basis of these results, we showed that the high expression of RpTOE1 during vegetative propagation and its inhibition of RpFT played a key role in the phase reversal of R. pseudoacacia.

The newly developed genomic-SSR markers uncover the genetic characteristics and relationships of olive accessions.

BACKGROUND: Olive (Olea europaea L.) is an important oil and fruit crop worldwide, owning a rich germplasm with a large number of cultivars. Simple sequence repeats (SSRs) are excellent markers and have been used for the identification of olive cultivars. However, the limited number of SSR markers and the occurrence of confusion on the names of cultivars, as well as the possible appearance of clonal variation make it difficult to identify cultivars and interpret relationships among olive cultivars. METHOD: SSR markers were designed based on trinucleotide repeat sequences by screening the whole genome of olive, and the polymorphic SSR markers were developed that were applied to the identification of 53 olive accessions. The genetic characteristics and relationships of these olive accessions were evaluated based on the developed SSR markers. RESULTS: Twenty-one highly polymorphic genomic-SSR markers were developed, covering most chromosomes of olive. These SSR markers could well distinguish all 53 olive accessions, confirming their effectiveness. DNA fingerprints of the 53 olive accessions were constructed based on the 21 SSR markers. The dendrogram clearly divided the tested accessions into two main groups, which was also supported by the results of principal coordinate analysis. A total of 31 private alleles were detected in 15 olive accessions, which reflected the genetic diversity within 53 olive accessions to some extent. Six homonymy cases were also clarified by genetic analysis. These results suggest that the newly developed olive SSR markers are informative for the exploitation, preservation and breeding of olive.

Molecular Detection and Genetic Characterization of Toxoplasma gondii from Horses in Three Provinces of China.

Horse meat and milk are an important source of nutrition for many Chinese. Previous studies have shown a fairly high prevalence of Toxoplasma gondii infection in horses. However, there is no data concerning the molecular characterization of T. gondii in horses in China. The present study tested 231 cervical lymph node samples of horses from northern China (97 from Jilin, 54 from Liaoning, and 80 from Xinjiang) for the presence of the T. gondii B1 gene by seminested PCR. The B1-positive samples were genotyped at nine nuclear loci, that is, SAG1, alternative SAG2, SAG3, BTUB, c22-8, GRA6, c29-2, PK1, and an apicoplast locus, using PCR-restriction fragment length polymorphism technique. A total of 14 (6.06%) out of 231 samples were T. gondii positive. The overall prevalence of T. gondii in the horses was 5.15% (5/97) for Jilin Province, 5.55% (3/54) for Liaoning Province, and 7.50% (6/80) for Xinjiang Uygur Autonomous Region. Of the 14 positive samples, only 2 were successfully genotyped at all loci, 5 were successfully genotyped at 5-8 loci, and all typed samples belong to ToxoDB genotype no. 9. To our knowledge, this is the first molecular characterization of the T. gondii isolates from horses in China.

Proteomic analysis of the low mutation rate of diploid male gametes induced by colchicine in Ginkgo biloba L.

Colchicine treatment of G. biloba microsporocytes results in a low mutation rate in the diploid (2n) male gamete. The mutation rate is significantly lower as compared to other tree species and impedes the breeding of new economic varieties. Proteomic analysis was done to identify the proteins that influence the process of 2n gamete formation in G. biloba. The microsporangia of G. biloba were treated with colchicine solution for 48 h and the proteins were analyzed using 2-D gel electrophoresis and compared to protein profiles of untreated microsporangia. A total of 66 proteins showed difference in expression levels. Twenty-seven of these proteins were identified by mass spectrometry. Among the 27 proteins, 14 were found to be up-regulated and the rest 13 were down-regulated. The identified proteins belonged to five different functional classes: ATP generation, transport and carbohydrate metabolism; protein metabolism; ROS scavenging and detoxifying enzymes; cell wall remodeling and metabolism; transcription, cell cycle and signal transduction. The identification of these differentially expressed proteins and their function could help in analysing the mechanism of lower mutation rate of diploid male gamete when the microsporangium of G. biloba was induced by colchicine.

FOXO6 promotes gastric cancer cell tumorigenicity via upregulation of C-myc.

The aberrant regulation of many related genes is involved in the development and progression of gastric carcinoma. In the present study, we show that mRNA and protein levels of FOXO6 are upregulated in gastric cancer tissues. Forced overexpression of FOXO6 promotes gastric cancer cell proliferation, while knockdown of FOXO6 expression inhibits proliferation. We show that ectopic FOXO6 expression induces the expression of C-myc. Furthermore, we found that FOXO6 physically interacts with the transcription factor hepatic nuclear factor 4 (HNF4) in gastric cancer cells. FOXO6 induces C-myc expression by associating to HNF4 and mediating histone acetylation, and the dissociation of HDAC3 from the promoter of the C-myc gene. Therefore, our results suggest a previously unknown FOXO6/HNF4/C-myc molecular network controlling gastric cancer development.

TLR4 expression in normal gallbladder, chronic cholecystitis and gallbladder carcinoma.

BACKGROUND/AIMS: Chronic inflammation is a risk factor for gallbladder carcinoma. The molecular mechanisms linking inflammation and gallbladder carcinogenesis are incompletely understood. Toll-like receptors are involved in inflammatory response and play an important role in the innate immune system by initiating and directing immune response to pathogens. We tested the hypothesis that TLR4 participated in the development of gallbladder carcinoma through investigating the expression of TLR4 in chronic cholecystitis, gallbladder carcinoma and normal gallbladder. METHODOLOGY: The expression of TLR4 in 30 specimens of chronic calculous cholecystitis, 13 specimens of gallbladder adenocarcinoma and 10 specimens of normal gallbladder tissue was determined by immunohistochemistry, western blotting analysis and quantitative RT-PCR. RESULTS: We showed that TLR4 was mostly localized to the glandular and luminal epithelium of gallbladder. TLR4 expression was lower in gallbladder carcinoma tissue than in chronic cholecystitis and normal gallbladder tissue, whereas the difference between chronic cholecystitis tissue and normal gallbladder tissue was not statistically significant. CONCLUSIONS: The expression of TLR4 may be closely associated with the course of gallbladder carcinoma.

Differential expression profiles of the Hedgehog signaling pathway between microsatellite-stable and microsatellite-unstable colorectal cancers.

The present study aimed to investigate the expression of the Hedgehog (Hh) signaling pathway between microsatellite-unstable (MSI) and microsatellite-unstable (MSS) colorectal cancers (CRCs). A total of 61 samples of CRC tissue and corresponding blood samples were obtained from the surgical department of our hospital. The tissue samples were examined by immunohistochemistry using antibodies against Sonic Hh (SHH), Pathed (PTCH) and Gli1, and evaluated independently for protein expression by two pathologists blinded to clinical outcome. Based on the immunohistochemistry results, SHH and PTCH expression varied in terms of histological type. In mucinous adenocarcinoma (MA) Hedgehog signaling was not highly expressed. There were more significant differences in the expression of SHH and PTCH (P<0.05), compared with Gli1. Moreover, significant differences were found in the expression of SHH, Gli1 and PTCH between the MSI and MSS groups (P<0.05). Hedgehog signals were more frequently expressed in the MSI group compared with the MSS group. In conclusion, this study indicates that the expression of the Hh signaling pathway may play a significant role in MSI in colorectal cancer.

[Metabolic pathway of polyamines in plants: a review].

Polyamine is an important physiological regulation substance functioning in a wide variety of biological processes, such as plant growth, development, senescence and adversity stress tolerance, which widely exist in all living organisms. Their biosynthetic pathways have already been revealed, and their physiological roles are being elucidated gradually. Previous work on polyamines biosynthetic deficiency mutants and various transgenic plants facilitates improved understanding of the important roles of polyamines and biosynthetic enzymes in plant growth and development. This paper summarizes researches in the biosynthetic pathways of polyamines in plants, focusing on research advances on functions of genes involved in polyamine metabolism. In addition, the potential research directions, especially the application of the genes in the genetic engineering of plant stress tolerance were also discussed.