FaTT10, a Laccasel-Like Polyphenol Oxidase Involved in the Accumulation of Proanthocyanins Monomerin Strawberry Fruit

Procyanidins (PAs) is the important flavonoids components in strawberry young fruits, whichis synthesizedthrough direct polymerizationof flavanone-3-ol units. Numerous studies have beendone on the synthesis of PAs, while the oxidation/polymerization processes have not been fully explored. AtTT10, a laccasel-like polyphenol oxidase,was recently reported to be participated in the oxidative polymerization of proanthocyanidins. Whether FaTT10hasapositiveeffect onoxidative polymerization of strawberry fruit PAsis unclear. In this study, FaTT10 in strawberries were isolated and analyzed. Transient overexpression of FaTT10 decreased the accumulation of PAs monomer in strawberry fruit,whereas RNA interference increased the level of anthocyanin and PAs monomer. 4-Dimethylaminocinamaldehyde (DMACA)staining results also showed that lighter blue color in the FaTT10-RNAi fruit and concentrated blue color in the FaTT10-overexpression fruit. Meanwhile, PAs monomer showed a drop-and-rise tendency during strawberry fruit development and ripening.Furthermore, the expression of several flavonoids metabolism-related geneswere affected by the altered FaTT10 gene expression levels, such as L-phenylalanine ammonia-lyase(PAL); dihydroflavonol reductase(DFR);chalcone isomerase(CHI); flavanone 3-hydroxylase(F3H); 4-coumarate:CoA ligase(4CL); Cinnamate 4-hydroxylase(C4H); chalcone synthase(CHS); anthocyanin reductase(ANR); lecoanthocyanidins reductase(LAR) andanthocyanin synthase(ANS). In conclusion, these results revealed that FaTT10 is specifically involved in oxidative polymerization of PAsby regulating the expression of flavonoids metabolism-related genes in strawberry fruits. © 2021 Friends Science Publishers

Genome editing of woody perennial trees

This chapter discusses the challenges facing woody perennial trees, namely the heterozygosity of outcrossing species and limited genomic resources. Gene knockouts (KO) represent the predominant applications of the CRISPR technology in woody perennial crops to date and will be the focus of this chapter. The issues considered for gene KO are generally applicable to other CRISPR applications targeting regulatory sequences or non-coding genes. Case studies from the Populus 4-coumarate:CoA ligase (4CL) family are presented to demonstrate the power of CRISPR in elucidating functional redundancy as well as specificity of duplicated genes in a perennial woody species.

4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids

Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.

Cloning and Immunosuppressive Properties of an Acyl-Activating Enzyme from the Venom Apparatus of Tetrastichus brontispae (Hymenoptera: Eulophidae)

Venom injected into the host plays vital roles in facilitating successful parasitization and development for parasitoid wasps, especially those devoid of polydnavirus, and the abundant venom proteins appear to be most likely involved in parasitization success. Previously, we found the four most abundant venom proteins, including 4-coumarate:CoA ligase-like 4 (4CL4-like), in the Tetrastichus brontispae (Hymenoptera: Eulophidae) venom apparatus. In this study, we cloned, expressed T. brontispae 4CL4-like (Tb4CL4-like) in Escherichia coli, and investigated its immunosuppressive properties. The deduced amino acid sequence for Tb4CL4-like shares high identity at conserved amino acids associated with the acyl-activating enzyme (AAE) consensus motif but shows only <40% identity with the members in the AAE superfamily. mRNA abundance analysis indicated that Tb4CL4-like was transcribed mainly in the venom apparatus. Recombinant Tb4CL4-like inhibited Octodonta nipae (Coleoptera: Chrysomelidae) pupal cellular encapsulation and spreading by targeting the hemocyte cytoskeleton and reduced the hemocyte-mediated phagocytosis of E. coli in vivo. Moreover, Tb4CL4-like exhibited greater affinity to palmitic acid and linolenic acid based on the molecular docking assay and is hypothesized to be involved in fatty acid metabolism. In conclusion, our results suggest that Tb4CL4-like may be an immunity-related AAE protein that is involved in the regulation of host immunity through fatty acid metabolism-derived signaling pathways.

Response of Sorghum Enhanced in Monolignol Biosynthesis to Stalk Rot Pathogens

To increase phenylpropanoid constituents and energy content in the versatile C4 grass sorghum (Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3-O-methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum-inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina-inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum. Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60, Bmr2, or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Tomato aspermy virus elimination improves medicinal quality of chrysanthemum

Chrysanthemum morifolium cv. ?Huaihuang?, a medicinal chrysanthemum of China, undergoes long-term asexual reproduction and virus infection that change its quality characteristics. Our previous studies have shown that tomato aspermy virus (TAV) is the main virus infecting ?Huaihuang?. Many studies indicate that plant virus elimination can improve plant growth, but only a few studies have focused on the effects of detoxification on the medicinal components of medicinal plants. In this paper, the content of medicinal components, including chlorogenic acid, luteoloside and 3,5-O-dicaffeoylquinic acid, was compared between the TAV-free and TAV-infected (control) chrysanthemum. In addition, the reason why TAV elimination improves the medicinal components of chrysanthemum is explored. Our results suggest that TAV elimination significantly improves plant growth, enhances the enzyme activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-coumarate:CoA ligase, and increases the levels of CmHCT and CmCHS expression, thereby greatly improving the medicinal quality of chrysanthemum.