Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12β-position by the fungus Alternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12β-hydroxyl cinobufagin, 12β-hydroxyl desacetylcinobufagin, 3-oxo-12β-hydroxyl cinobufagin, 3-oxo-12β-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12α-hydroxyl cinobufagin. The last five products are new compounds. 12β-Hydroxylation of cinobufagin by A. alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12β also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12β-hydroxyl cinobufagin and 3-oxo-12α-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12β-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis

Genome-Wide Identification, Classification, Characterization, and Expression Analysis of the Wall-Associated Kinase Family during Fruit Development and under Wound Stress in Tomato (Solanum lycopersicum L.)

The wall-associated kinase (WAK) and wall-associated kinase like (WAKL) is a subfamily of receptor-like kinases associated with the cell wall, which have been suggested as sensors of the extracellular environment and triggers of intracellular signals. However, these proteins have not yet been comprehensively analyzed in tomato (Solanum lycopersicum L.). In this study, 11 SlWAK and 18 SlWAKL genes were identified in an uneven distribution in 9 of 12 chromosomes. GUB-WAK-bind (wall-associated receptor kinase galacturonan-binding) and epidermal growth factor (EGF) domains appear more often in SlWAK proteins. However, more SlWAKLs (wall-associated kinase like) have a WAK-assoc (wall-associated receptor kinase C-terminal) domain. Based on their phylogenetic relationships, 29 SlWAK-RLKs (wall associated kinase-receptor like kinases) were clustered into three distinct categories analogous to those in Arabidopsis thaliana. High similarities were found in conserved motifs of the genes within each group. Cis-elements in the promoter region of these 29 genes were found mainly in response to methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), anaerobic, light, wound, and MYB transcription factors. Public tomato genome RNA-seq data indicates that multiple SlWAK-RLKs showed different expression patterns under developmental and ripening stages of fruits, such as SlWAK4, SlWAKL11, SlWAKL9, SlWAKL15, SlWAKL14, and SlWAKL1, their RPKM (Reads Per Kilo bases per Million reads) value constantly increases during the fruit expansion period, and decreases as the fruit matures. In tomato leaves, our RNA-seq data showed that nine SlWAK-RLKs transcripts (SlWAK3, SlWAK4, SlWAK10,SlWAKL1, SlWAKL2, SlWAKL3, SlWAKL5, SlWAKL14, and SlWAKL18) were significantly induced (p < 0.001), and three transcripts (SlWAK2, SlWAK5, and SlWAKL15) were significantly inhibited (p < 0.001) under mechanical wounding. The qRT-PCR (Quantitative reverse transcription polymerase chain reaction) of SlWAKL1 and SlWAKL6 verify these results

Additional file 2: of Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis at the transcriptional and post-translational levels

Long-term ethylene production of rin , nor and Cnr tomato fruits after treatment. Tomato fruits were placed in a ventilated and temperature constant room at 25 °C and treated with 1 g/L OGs or the control solution. Ethylene production was detected every day after treatment. Vertical bars indicate the SD (n = 6). (PDF 93 kb

Additional file 3: of Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1-carboxylic acid synthesis at the transcriptional and post-translational levels

Transient ethylene production of AC and mutant fruits pericarp discs after treatment. 1 mL gas was extracted to detect ethylene content. Vertical bars indicate the SD (n = 4), asterisks indicate statistically significant differences compared with control group (*P < 0.05; **P < 0.01, Student’s t-test). (PDF 105 kb

Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants

Tomato is considered as the genetic model for climacteric fruits, in which three major players control the fruit ripening process: ethylene, ripening transcription factors, and DNA methylation. The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species, and H3K27me3, instead of DNA methylation, plays a conserved role in restricting these ripening pathways. In addition, the function of the core tomato ripening transcription factors is now being questioned. We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors, both of which are considered as master regulators in the current tomato ripening model. These plants only displayed delayed or partial non-ripening phenotypes, distinct from the original mutant plants, which categorically failed to ripen, suggesting that they might be gain-of-function mutants. Besides increased DNA methylation genome-wide, the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2, RIN, and TDR4. It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes. Hence, we propose that the field should move beyond these linear and two-dimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels

Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis

Abstract Background Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage. Results The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development. Conclusions Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants

Additional file 2: Figure S1. of Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis

Principal Components Analysis (PCA) bi-plots of metabolite (top) and gene expression level data (bottom) from ‘Granny Smith’ apple peel from fruit stored in air at 1 °C for up to 183 days (6 months). Apples were treated immediately following harvest with 2000 μL L−1 DPA or 1 mL L−1 1-MCP. Gene expression and metabolite datasets were analyzed separately. Shapes represent scores for each observation where symbol size increases with storage duration and symbol color represents scald severity. Gray points represent loadings for individual metabolites or transcripts. (DOCX 2373 kb