A conserved protein family in mirid bug Riptortus pedestris plays dual roles in regulating plant immunity.

The mirid bug (Riptortus pedestris), a major soybean pest, migrates into soybean fields during the pod filling stage and causes staygreen syndrome, which leads to substantial yield losses. The mechanism by which R. pedestris elicits soybean (Glycine max) defenses and counter-defenses remains largely unexplored. In this study, we characterized a protein family from R. pedestris, designated Riptortus pedestris HAMP 1 (RPH1) and its putative paralogs (RPH1L1, 2, 3, 4, and 5), whose members exhibit dual roles in triggering and inhibiting plant immunity. RPH1 and RPH1L1 function as herbivore-associated molecular patterns (HAMPs), activating pattern-triggered immunity (PTI) in tobacco (Nicotiana benthamiana) and G. max. Furthermore, RPH1 stimulates jasmonic acid and ethylene biosynthesis in G. max, thereby enhancing its resistance to R. pedestris feeding. Additionally, RPH1 homologs are universally conserved across various herbivorous species, with many homologs also acting as HAMPs that trigger plant immunity. Interestingly, the remaining RPH1 putative paralogs (RPH1L2-5) serve as effectors that counteract RPH1-induced PTI, likely by disrupting the extracellular perception of RPH1. This research uncovers a HAMP whose homologs are conserved in both chewing and piercing-sucking insects. Moreover, it unveils an extracellular evasion mechanism utilized by herbivores to circumvent plant immunity using functionally differentiated paralogs.

Profiling of phenolic composition in camellia oil and its correlative antioxidant properties analysis.

Less research has been conducted on the association between camellia oil's (CO) phenolic composition and antioxidant capability. In this study, the phenolic profile of CO and its connection to antioxidant capacity were examined utilizing a combination of widely-targeted phenolic metabolomics and multivariate statistical analysis. A total of 751 phenolics were discovered. The WGCNA was used to link phenols to antioxidants, yielding 161 antioxidant-related phenols from the blue module. In response to several antioxidant assays, 59 (FRAP), 59 (DPPH), and 53 (ABTS) phenolics were identified as differential phenolic markers (DPMs). Further stepwise multiple linear regression revealed six DPMs that substantially influenced the antioxidant capacities. Nine metabolic pathways and their associated network mechanisms for the most significant phenolics were developed. This study sheds light on the phenolic content of CO, elucidates their role in antioxidant activity, and lays the groundwork for improving extraction techniques and generating improved product.

PcLRR-RK3, an LRR receptor kinase is required for growth and in-planta infection processes in Phytophthora capsici.

Receptor protein kinases (RPKs) critically provide the basic infrastructure to sense, perceive, and conduct the signalling events at the cell surface of organisms. The importance of LRR-RLKs has been well studied in plants, but much less information has been reported in oomycetes. In this work, we have silenced the PcLRR-RK3 and characterised its functional importance in Phytophthora capsici. PcLRR-RK3 was predicted to encode signal peptides, leucine-rich repeats, transmembrane, and kinase domains. PcLRR-RK3-silenced transformants showed impaired colony growth, decreased deformed sporangia, and reduced zoospores count. The mycelium of silenced transformants did not penetrate within the host tissues and showed defects in the pathogenicity of P. capsici. Interestingly, gene silencing also weakens the ability of zoospores germination and penetration into host tissues and fails to produce necrotic lesions. Furthermore, PcLRR-RK3 localisation was found to be the plasma membrane of the cell. Altogether, our results revealed that PcLRR-RK3 was required for the regulation of vegetative growth, zoospores penetration, and establishment into host leaf tissues.

Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.

External browning mechanism in walnut kernel pellicles under different drying conditions based on the combination of widely-targeted and anthocyanin-targeted metabolomics.

There has been limited research on external browning (EB) of walnut. This work discovered 1888 metabolites and 34 anthocyanins in walnut pellicles (WPs) after three drying methods using widely-targeted and anthocyanin-targeted metabolomics. Based on OPLS-DA and correlation analysis, 64 temperature-responsive metabolites (TRMs; 13 anthocyanins and 51 flavonoids) were identified as critical components in relation to EB. Notably, 14 flavonoids exhibited a strong positive correlation (r > 0.9) with the browning index (BI), with upregulation of >60% after browning. Most of the identified anthocyanins were negatively linked with BI because of degradation (>45%), with correlation coefficients ranging from 0.75 to 0.97. Furthermore, anthocyanidin reductase and laccase were the two key enzymes involved in the EB of WPs, with their activities increasing by 10.57-fold and 1.32-fold, respectively, with increasing drying temperature. A metabolic pathway network of the TRM was built to provide insights into the potential mechanisms underlying EB in WPs.

A MYB-related transcription factor regulates effector gene expression in an oomycete pathogen.

Phytophthora pathogens possess hundreds of effector genes that exhibit diverse expression patterns during infection, yet how the expression of effector genes is precisely regulated remains largely elusive. Previous studies have identified a few potential conserved transcription factor binding sites (TFBSs) in the promoters of Phytophthora effector genes. Here, we report a MYB-related protein, PsMyb37, in Phytophthora sojae, the major causal agent of root and stem rot in soybean. Yeast one-hybrid and electrophoretic mobility shift assays showed that PsMyb37 binds to the TACATGTA motif, the most prevalent TFBS in effector gene promoters. The knockout mutant of PsMyb37 exhibited significantly reduced virulence on soybean and was more sensitive to oxidative stress. Consistently, transcriptome analysis showed that numerous effector genes associated with suppressing plant immunity or scavenging reactive oxygen species were down-regulated in the PsMyb37 knockout mutant during infection compared to the wild-type P. sojae. Several promoters of effector genes were confirmed to drive the expression of luciferase in a reporter assay. These results demonstrate that a MYB-related transcription factor contributes to the expression of effector genes in P. sojae.

Evaluation of Lipid Quality in Fruit: Utilizing Lipidomic Approaches for Assessing the Impact of Biotic Stress on Pecans (Carya illinoinensis).

There is a scarcity of data on how the lipid composition of oily seeds changes in response to biotic stress. Yellow peach moth (Conogethes punctiferalis) has caused massive economic losses on the pecan (Carya illinoinensis) industry. Lipidomics is used in this study to determine the lipid composition of pecan and how it changes in response to insect attack. Pecan had 167 lipids, including 34 glycerolipids (GL), 62 glycerophospholipids (GP), 17 fatty acyls (FA), 41 sphingolipids (SP), and 13 saccharolipids (SL). The effects of biotic stress on lipids, particularly GL and GP, were significant. Biotic stress significantly reduced the lipid content of chains longer than 48. Forty-four significantly different lipids were discovered as potential biomarkers for distinguishing non-infected pecans from infested pecans. In addition, we used bioinformatics to identify the five most important metabolic pathways in order to investigate the processes underlying the changes. Our discoveries may offer valuable insights for enhancing pecan production in the future and contribute novel perspectives towards enhancing the nutritional value of pecans.

Phenazine biosynthesis protein MoPhzF regulates appressorium formation and host infection through canonical metabolic and noncanonical signaling function in Magnaporthe oryzae.

Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.

Phytophthora sojae Effector PsCRN108 Targets CAMTA2 to Suppress HSP40 Expression and ROS Burst.

Oomycete pathogens secrete numerous crinkling and necrosis proteins (CRNs) to manipulate plant immunity and promote infection. However, the functional mechanism of CRN effectors is still poorly understood. Previous research has shown that the Phytophthora sojae effector PsCRN108 binds to the promoter of HSP90s and inhibits their expression, resulting in impaired plant immunity. In this study, we found that in addition to HSP90, PsCRN108 also suppressed other Heat Shock Protein (HSP) family genes, including HSP40. Interestingly, PsCRN108 inhibited the expression of NbHSP40 through its promoter, but did not directly bind to its promoter. Instead, PsCRN108 interacted with NbCAMTA2, a negative regulator of plant immunity. NbCAMTA2 was a negative regulator of NbHSP40 expression, and PsCRN108 could promote such inhibition activity of NbCAMTA2. Our results elucidated the multiple roles of PsCRN108 in the suppression of plant immunity and revealed a new mechanism by which the CRN effector hijacked transcription factors to affect immunity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Soil bacterium manipulates antifungal weapons by sensing intracellular type IVA secretion system effectors of a competitor.

Soil beneficial bacteria can effectively inhibit bacterial pathogens by assembling contact-dependent killing weapons, such as the type IVA secretion system (T4ASS). It's not clear whether these antibacterial weapons are involved in biotrophic microbial interactions in soil. Here we showed that an antifungal antibiotic 2,4-DAPG production of the soil bacterium, Pseudomonas protegens can be triggered by another soil bacterium, Lysobacter enzymogenes, via T4ASS by co-culturing on agar plates to mimic cell-to-cell contact. We demonstrated that the induced 2,4-DAPG production of P. protegens is achieved by intracellular detection of the T4ASS effector protein Le1519 translocated from L. enzymogenes. We defined Le1519 as LtaE (Lysobacter T4E triggering antifungal effects), which specifically stimulates the expression of 2,4-DAPG biosynthesis genes in P. protegens, thereby protecting soybean seedlings from infection by the fungus Rhizoctonia solani. We further found that LtaE directly bound to PhlF, a pathway-specific transcriptional repressor of the 2,4-DAPG biosynthesis, then activated the 2,4-DAPG production. Our results highlight a novel pattern of microbial interspecies and interkingdom interactions, providing a unique case for expanding the diversity of soil microbial interactions.

Myxobacteria restrain Phytophthora invasion by scavenging thiamine in soybean rhizosphere via outer membrane vesicle-secreted thiaminase I.

Public metabolites such as vitamins play critical roles in maintaining the ecological functions of microbial community. However, the biochemical and physiological bases for fine-tuning of public metabolites in the microbiome remain poorly understood. Here, we examine the interactions between myxobacteria and Phytophthora sojae, an oomycete pathogen of soybean. We find that host plant and soil microbes complement P. sojae's auxotrophy for thiamine. Whereas, myxobacteria inhibits Phytophthora growth by a thiaminase I CcThi1 secreted into extracellular environment via outer membrane vesicles (OMVs). CcThi1 scavenges the required thiamine and thus arrests the thiamine sharing behavior of P. sojae from the supplier, which interferes with amino acid metabolism and expression of pathogenic effectors, probably leading to impairment of P. sojae growth and pathogenicity. Moreover, myxobacteria and CcThi1 are highly effective in regulating the thiamine levels in soil, which is correlated with the incidence of soybean Phytophthora root rot. Our findings unravel a novel ecological tactic employed by myxobacteria to maintain the interspecific equilibrium in soil microbial community.

Cross-kingdom analyses of transmembrane protein kinases show their functional diversity and distinct origins in protists.

Transmembrane kinases (TMKs) are important mediators of cellular signaling cascades. The kinase domains of most metazoan and plant TMKs belong to the serine/threonine/tyrosine kinase (S/T/Y-kinase) superfamily. They share a common origin with prokaryotic kinases and have diversified into distinct subfamilies. Diverse members of the eukaryotic crown radiation such as amoebae, ciliates, and red and brown algae (grouped here under the umbrella term "protists") have long diverged from higher eukaryotes since their ancient common ancestry, making them ideal organisms for studying TMK evolution. Here, we developed an accurate and high-throughput pipeline to predict TMKomes in cellular organisms. Cross-kingdom analyses revealed distinct features of TMKomes in each grouping. Two-transmembrane histidine kinases constitute the main TMKomes of bacteria, while metazoans, plants, and most protists have a large proportion of single-pass TM S/T/Y-kinases. Phylogenetic analyses classified most protist S/T/Y-kinases into three clades, with clades II and III specifically expanded in amoebae and oomycetes, respectively. In contrast, clade I kinases were widespread in all protists examined here, and likely shared a common origin with other eukaryotic S/T/Y-kinases. Functional annotation further showed that most non-kinase domains were grouping-specific, suggesting that their recombination with the more conserved kinase domains led to the divergence of S/T/Y-kinases. However, we also found that protist leucine-rich repeat (LRR)- and G-protein-coupled receptor (GPCR)-type TMKs shared similar sensory domain architectures with respective plant and animal TMKs, despite that they belong to distinct kinase subfamilies. Collectively, our study revealed the functional diversity of TMKomes and the distinct origins of S/T/Y-kinases in protists.

The Pythium periplocum elicitin PpEli2 confers broad-spectrum disease resistance by triggering a novel receptor-dependent immune pathway in plants.

Elicitins are microbe-associated molecular patterns produced by oomycetes to elicit plant defense. It is still unclear whether elicitins derived from non-pathogenic oomycetes can be used as bioactive molecules for disease control. Here, for the first time we identify and characterize an elicitin named PpEli2 from the soil-borne oomycete Pythium periplocum, which is a non-pathogenic mycoparasite colonizing the root ecosystem of diverse plant species. Perceived by a novel cell surface receptor-like protein, REli, that is conserved in various plants (e.g. tomato, pepper, soybean), PpEli2 can induce hypersensitive response cell death and an immunity response in Nicotiana benthamiana. Meanwhile, PpEli2 enhances the interaction between REli and its co-receptor BAK1. The receptor-dependent immune response triggered by PpEli2 is able to protect various plant species against Phytophthora and fungal infections. Collectively, our work reveals the potential agricultural application of non-pathogenic elicitins and their receptors in conferring broad-spectrum resistance for plant protection.

NLRs derepress MED10b- and MED7-mediated repression of jasmonate-dependent transcription to activate immunity.

Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b, which confers resistance to tospovirus, as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus. MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b-MED7-JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus. Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.

Online data resource for exploring transposon insertion polymorphisms in public soybean germplasm accessions.

Soybean (Glycine max L. Merrill) is one of the most important economical crops. A large number of whole-genome resequencing datasets have been generated and are increasingly expanded for exploring genetic diversity and mining important quantitative trait loci. Most genome-wide association studies have focused on single-nucleotide polymorphisms, short insertions, and deletions. Nevertheless, structure variants mainly caused by transposon element mobilization are not fully considered. To fill this gap, we uniformly processed the publicly available whole-genome resequencing data from 5,521 soybean germplasm accessions and built an online soybean transposon insertion polymorphisms database named Soybean Transposon Insertion Polymorphisms Database (SoyTIPdb) (https://biotec.njau.edu.cn/soytipdb). The collected germplasm accessions derived from more than 45 countries and 160 regions representing the most comprehensive genetic diversity of soybean. SoyTIPdb implements easy-to-use query, analysis, and browse functions to help understand and find meaningful structural variations from TE insertions. In conclusion, SoyTIPdb is a valuable data resource and will help soybean breeders/researchers take advantage of the whole-genome sequencing datasets available in the public depositories.

A Phytophthora receptor-like kinase regulates oospore development and can activate pattern-triggered plant immunity.

Plant cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) and receptor-like proteins (LRR-RLPs) form dynamic complexes to receive a variety of extracellular signals. LRR-RLKs are also widespread in oomycete pathogens, whereas it remains enigmatic whether plant and oomycete LRR-RLKs could mediate cell-to-cell communications between pathogen and host. Here, we report that an LRR-RLK from the soybean root and stem rot pathogen Phytophthora sojae, PsRLK6, can activate typical pattern-triggered immunity in host soybean and nonhost tomato and Nicotiana benthamiana plants. PsRLK6 homologs are conserved in oomycetes and also exhibit immunity-inducing activity. A small region (LRR5-6) in the extracellular domain of PsRLK6 is sufficient to activate BAK1- and SOBIR1-dependent immune responses, suggesting that PsRLK6 is likely recognized by a plant LRR-RLP. Moreover, PsRLK6 is shown to be up-regulated during oospore maturation and essential for the oospore development of P. sojae. Our data provide a novel type of microbe-associated molecular pattern that functions in the sexual reproduction of oomycete, and a scenario in which a pathogen LRR-RLK could be sensed by a plant LRR-RLP to mount plant immunity.

Quorum quenching by a type IVA secretion system effector.

Proteobacteria primarily utilize acyl-homoserine lactones (AHLs) as quorum-sensing signals for intra-/interspecies communication to control pathogen infections. Enzymatic degradation of AHL represents the major quorum-quenching mechanism that has been developed as a promising approach to prevent bacterial infections. Here we identified a novel quorum-quenching mechanism revealed by an effector of the type IVA secretion system (T4ASS) in bacterial interspecies competition. We found that the soil antifungal bacterium Lysobacter enzymogenes OH11 (OH11) could use T4ASS to deliver the effector protein Le1288 into the cytoplasm of another soil microbiome bacterium Pseudomonas fluorescens 2P24 (2P24). Le1288 did not degrade AHL, whereas its delivery to strain 2P24 significantly impaired AHL production through binding to the AHL synthase PcoI. Therefore, we defined Le1288 as LqqE1 (Lysobacter quorum-quenching effector 1). Formation of the LqqE1-PcoI complex enabled LqqE1 to block the ability of PcoI to recognize/bind S-adenosy-L-methionine, a substrate required for AHL synthesis. This LqqE1-triggered interspecies quorum-quenching in bacteria seemed to be of key ecological significance, as it conferred strain OH11 a better competitive advantage in killing strain 2P24 via cell-to-cell contact. This novel quorum-quenching also appeared to be adopted by other T4ASS-production bacteria. Our findings suggest a novel quorum-quenching that occurred naturally in bacterial interspecies interactions within the soil microbiome by effector translocation. Finally, we presented two case studies showing the application potential of LqqE1 to block AHL signaling in the human pathogen Pseudomonas aeruginosa and the plant pathogen Ralstonia solanacearum.

Plant secondary metabolite citral interferes with Phytophthora capsici virulence by manipulating the expression of effector genes.

Phytophthora capsici is a notorious pathogen that infects various economically important plants and causes serious threats to agriculture worldwide. Plants deploy a variety of plant secondary metabolites to fend off pathogen attacks, but the molecular mechanisms are largely unknown. In this study, we screened 11 plant secondary metabolites to evaluate their biofumigation effects against P. capsici, and found that citral, carvacrol, and trans-2-decenal exhibited strong antimicrobial effects. Intriguingly, a low concentration of citral was effective in restricting P. capsici infection in Nicotiana benthamiana, but it was unable to inhibit the mycelial growth. A high concentration of citral affected the mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici. Further investigations showed that citral did not induce expression of tested plant immunity-related genes and reactive oxygen species (ROS) production, suggesting that a low concentration of citral could not trigger plant immunity. Moreover, RNA-Seq analysis showed that citral treatment regulated the expression of some P. capsici effector genes such as RxLR genes and P. cactorum-fragaria (PCF)/small cysteine-rich (SCR)74-like genes during the infection process, which was also verified by reverse transcription-quantitative PCR assay. Five candidate effector genes suppressed by citral significantly facilitated P. capsici infection in N. benthamiana or inhibited ROS triggered by flg22, suggesting that they were virulence factors of P. capsici. Together, our results revealed that plant-derived citral exhibited excellent inhibitory efficacy against P. capsici by suppressing vegetative growth and manipulating expression of effector genes, which provides a promising application of citral for controlling Phytophthora blight.

Lysobacter enzymogenes prevents Phytophthora infection by inhibiting pathogen growth and eliciting plant immune responses.

The Phytophthora pathogen causes enormous damage to important agricultural plants. This group of filamentous pathogens is phylogenetically distant from fungi, making them difficult to control by most chemical fungicides. Lysobacter enzymogenes OH11 (OH11) is a biocontrol bacterium that secretes HSAF (Heat-Stable Antifungal Factor) as a broad-spectrum antifungal weapon. Here, we showed that OH11 could also control a variety of plant Phytophthora diseases caused by three major oomycetes (P. sojae, P. capsici and P. infestans). We provided abundant evidence to prove that OH11 protected host plants from Phytophthora pathogen infection by inhibiting mycelial growth, digesting cysts, suppressing cyst germination, and eliciting plant immune responses. Interestingly, the former two processes required the presence of HSAF, while the latter two did not. This suggested that L. enzymogenes could prevent Phytophthora infection via multiple previously unknown mechanisms. Therefore, this study showed that L. enzymogenes could serve as a promising alternative resource for promoting plant resistance to multiple Phytophthora pathogens.

Identification of Key Antioxidants of Free, Esterified, and Bound Phenolics in Walnut Kernel and Skin.

Walnut is a natural source of antioxidants. Its antioxidant capacity is determined by the distribution and composition of phenolics. The key phenolic antioxidants in various forms (free, esterified, and bound) in walnut kernel (particularly seed skin) are unknown. The phenolic compounds in twelve walnut cultivars were analyzed using ultra-performance liquid chromatography coupled with a triple quadrupole mass spectrometer in this study. A boosted regression tree analysis was used to identify the key antioxidants. Ellagic acid, gallic acid, catechin, ferulic acid, and epicatechin were abundant in the kernel and skin. The majority of phenolic acids were widely distributed in the free, esterified, and bound forms in the kernel but more concentrated in bound phenolics in the skin. The total phenolic levels of the three forms were positively correlated with antioxidant activities (R = 0.76-0.94, p < 0.05). Ellagic acid was the most important antioxidant in the kernel, accounting for more than 20%, 40%, and 15% of antioxidants, respectively. Caffeic acid was responsible for up to 25% of free phenolics and 40% of esterified phenolics in the skin. The differences in the antioxidant activity between the cultivars were explained by the total phenolics and key antioxidants. The identification of key antioxidants is critical for new walnut industrial applications and functional food design in food chemistry.