Phenoloxidases: catechol oxidase - the temporary employer and laccase - the rising star of vascular plants

Phenolics are vital for the adaptation of plants to terrestrial habitats and for species diversity.Phenoloxidases(catechol oxidases,COs,and laccases,LACs)are responsible for the oxidation and polymerization of phenolics.However,their origin,evolution,and differential roles during plant development and land colonization are unclear.We performed the phylogeny,domain,amino acids,compositional biases,and intron analyses to clarify the origin and evolution of COs and LACs,and analysed the structure,selective pressure,and chloroplast targeting to understand the species-dependent distribution of COs.We found that Streptophyta COs were not homologous to the Chlorophyta tyrosinases(TYRs),and might have been acquired by horizontal gene transfer from bacteria.COs expanded in bryophytes.Structural-functionality and selective pressure were partially responsible for the species-dependent retention of COs in embryophytes.LACs emerged in Zygnemaphyceae,having evolved from ascorbate oxidases(AAOs),and prevailed in the vascular plants and strongly expanded in seed plants.COs and LACs coevolved with the phenolic metabolism pathway genes.These results suggested that TYRs and AAOs were the first-stage phenoloxidases in Chlorophyta.COs might be the second key for the early land colonization.LACs were the third one(dominating in the vascular plants)and might be advantageous for diversified phenol substrates and the erect growth of plants.This work provided new insights into how phenoloxidases evolved and were devoted to plant evolution.

Silencing JA hydroxylases in Nicotiana attenuata enhances jasmonic acid-isoleucine-mediated defenses against Spodoptera litura

Jasmonic acid(JA)plays important roles in plant resistance to insect herbivores.One important derivative of JA is 12-OH-JA,which is produced by two independent pathways:direct hydroxylation of JA by jasmonate-induced oxygenases(JOXs)or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown.In this study,we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing(VIGS),and found that all four JOX homologs are involved in JA hydroxylation.Simultaneously silencing the four JA hydroxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33%,but JA and JA-Ile levels increased by 45%and 30%,respectively,compared to those in control plants.Compared to direct hydroxylation from JA,hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OHJA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants,12-OH-JA decreased 34%.Moreover,VIGSNaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura.The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense metabolites in the VIGS-NaJOXs plants.When we simultaneously silenced all four JA hydroxylases in the JAIle-deficient irJAR4/6 background,the enhanced herbivore resistance diminished,demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels.Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels,we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.

Study on the Control of Tobacco Black Shank by Using Dry Mycelium of Penicillium Chrysogenum

The water extract of dry mycelium ofPenicillium chrysogenum （DME） was used to induce resistance in Virginia tobacco plants against Phtophthora parasitica var. nicotianae. Results showed that the efficacy of DME in controlling black shank disease depended on both DME solution concentration and its＇ treatment methodology. Soil application of 1.5-5% DME 72 hr before inoculation with Phtophthora parasitica vat. nicotianae provided highly significant protection against black shank, relative to the control without DME treatment. Optimized tobacco plant treatment with 2.5% DME significantly increased peroxidase （POD） and polyphenol oxidase （PPO） activity levels in the upper leaf sections of the tobacco plants. DME had no direct antifungal activity on the growth of Phtophthora parasitica var. nicotianae in vitro, suggesting that disease control with DME treatment resulted from the induced propagation of natural defense mechanisms in the tobacco plants.

Genomic characterization and expression profiles of stress-associated proteins(SAPs)in castor bean(Ricinus communis)

Stress-associated proteins(SAPs)are known as response factors to multiple abiotic and biotic stresses in plants.However,the potential physiological and molecular functions of SAPs remain largely unclear.Castor bean(Ricinus communis L.)is one of the most economically valuable non-edible woody oilseed crops,able to be widely cultivated in marginal lands worldwide because of its broad adaptive capacity to soil and climate conditions.Whether SAPs in castor bean plays a key role in adapting diverse soil conditions and stresses remains unknown.In this study,we used the castor bean genome to identify and characterize nine castor bean SAP genes(RcSAP).Structural analysis showed that castor bean SAP gene structures and functional domain types vary greatly,differing in intron number,protein sequence,and functional domain type.Notably,the AN1-C2H2eC2H2 zinc finger domain within RcSAP9 has not been often observed in other plant families.High throughput RNA-seq data showed that castor bean SAP gene profiles varied among different tissues.In addition,castor bean SAP gene expression varied in response to different stresses,including salt,drought,heat,cold and ABA and MeJA,suggesting that the transcriptional regulation of castor bean SAP genes might operate independently of each other,and at least partially independent from ABA and MeJA signal pathways.Cis-element analyses for each castor bean SAP gene showed that no common cis-elements are shared across the nine castor bean SAP genes.Castor bean SAPs were localized to different regions of cells,including the cytoplasm,nucleus,and cytomembrane.This study provides a comprehensive profile of castor bean SAP genes that advances our understanding of their potential physiological and molecular functions in regulating growth and development and their responses to different abiotic stresses.

Multi-omics analysis reveals the evolutionary origin of diterpenoid alkaloid biosynthesis pathways in Aconitum

Diterpenoid alkaloids(DAs) have been often utilized in clinical practice due to their analgesic and anti-infammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown.In this study, we successfully assembled a highquality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum(A.vilmorinianum)(5.76 Gb). An A. vilmorinianumspecific whole-genome duplication event was discovered using comparative genomic analysis,which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A.vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.