A dual RNA-seq analyses revealed dynamic arms race during the invasion of walnut by Colletotrichum gloeosporioides.

BACKGROUND: Walnut anthracnose caused by Colletotrichum gloeosporioides seriously endangers the yield and quality of walnut, and has now become a catastrophic disease in the walnut industry. Therefore, understanding both pathogen invasion mechanisms and host response processes is crucial to defense against C. gloeosporioides infection. RESULTS: Here, we investigated the mechanisms of interaction between walnut fruits (anthracnose-resistant F26 fruit bracts and anthracnose-susceptible F423 fruit bracts) and C. gloeosporioides at three infection time points (24hpi, 48hpi, and 72hpi) using a high-resolution time series dual transcriptomic analysis, characterizing the arms race between walnut and C. gloeosporioides. A total of 20,780 and 6670 differentially expressed genes (DEGs) were identified in walnut and C. gloeosporioides against 24hpi, respectively. Generous DEGs in walnut exhibited opposite expression patterns between F26 and F423, which indicated that different resistant materials exhibited different transcriptional responses to C. gloeosporioides during the infection process. KEGG functional enrichment analysis indicated that F26 displayed a broader response to C. gloeosporioides than F423. Meanwhile, the functional analysis of the C. gloeosporioides transcriptome was conducted and found that PHI, SignalP, CAZy, TCDB genes, the Fungal Zn (2)-Cys (6) binuclear cluster domain (PF00172.19) and the Cytochrome P450 (PF00067.23) were largely prominent in F26 fruit. These results suggested that C. gloeosporioides secreted some type of effector proteins in walnut fruit and appeared a different behavior based on the developmental stage of the walnut. CONCLUSIONS: Our present results shed light on the arms race process by which C. gloeosporioides attacked host and walnut against pathogen infection, laying the foundation for the green prevention of walnut anthracnose.

Transcriptomic Analyses Reveal the Effects of Walnut Kernel Cake on Adipose Deposition in Pigs.

With the rising cost of animal feed protein, finding affordable and effective substitutes is crucial. Walnut kernel cake, a polyphenol-, fiber-, protein- and fat-rich byproduct of walnut oil extraction, has been underexplored as a potential protein replacement in pig feed. In this study, we found that feeding large Diqing Tibetan pigs walnut kernel cake promoted adipose deposition and improved pork quality during pig growth. Transcriptome analysis revealed the upregulation of genes ANGPTL8, CCNP, ETV4, and TRIB3, associated with adipose deposition. Pathway analysis highlighted enrichment in adipose deposition-related pathways, including PPAR, insulin, PI3K-Akt, Wnt, and MAPK signaling. Further analysis identified DEGs (differentially expressed genes) positively correlated with adipose-related traits, such as PER2 and PTGES. Single-cell transcriptome data pointed to the specific expression of CD248 and PTGES in adipocyte progenitor/stem cells (APSCs), pivotal for adipocyte differentiation and adipose deposition regulation. This study demonstrates walnut kernel cake's potential to substitute soybean cake in pig feed, providing high-quality protein and promoting adipose deposition. It offers insights into feed protein replacement, human functional food, fat metabolism, and related diseases, with marker genes and pathways supporting pig breeding and pork quality improvement.

Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

Genome-wide identification of walnut (Juglans regia) PME gene family members and expression analysis during infection with Cryptosphaeria pullmanensis pathogens.

Walnuts exhibit a higher resistance to diseases, though they are not completely immune. This study focuses on the Pectin methylesterase (PME) gene family to investigate whether it is involved in disease resistance in walnuts. These 21 genes are distributed across 12 chromosomes, with four pairs demonstrating homology. Variations in conserved motifs and gene structures suggest diverse functions within the gene family. Phylogenetic and collinear gene pairs of the PME family indicate that the gene family has evolved in a relatively stable way. The cis-acting elements and gene ontology enrichment of these genes, underscores their potential role in bolstering walnuts' defense mechanisms. Transcriptomic analyses were conducted under conditions of Cryptosphaeria pullmanensis infestation and verified by RT-qPCR. The results showed that certain JrPME family genes were activated in response, leading to the hypothesis that some members may confer resistance to the disease.

Investigating the potential of Juglans regia phytoconstituents for the treatment of cervical cancer utilizing network biology and molecular docking approach.

The fourth most frequent type of cancer in women and the leading cause of mortality for females worldwide is cervical cancer. Traditionally, medicinal plants have been utilized to treat various illnesses and ailments. The molecular docking method is used in the current study to look into the phytoconstituents of Juglans regia's possible anticancer effects on cervical cancer target proteins. This work uses the microarray dataset analysis of GSE63678 from the NCBI Gene Expression Omnibus database to find differentially expressed genes. Furthermore, protein-protein interactions of differentially expressed genes were constructed using network biology techniques. The top five hub genes (IGF1, FGF2, ESR1, MYL9, and MYH11) are then determined by computing topological parameters with Cytohubba. In addition, molecular docking research was performed on Juglans regia phytocompounds that were extracted from the IMPPAT database versus hub genes that had been identified. Utilizing molecular dynamics, simulation confirmed that prioritized docked complexes with low binding energies were stable.

Morphological and pomological assessments of seedling-originated walnut (Juglans regia L.) trees to select the promising late-leafing genotypes.

BACKGROUND: In many parts of the world, including Iran, walnut (Juglans regia L.) production is limited by late-spring frosts. Therefore, the use of late-leafing walnuts in areas with late-spring frost is the most important method to improve yield. In the present study, the phenotypic diversity of 141 seedling genotypes of walnut available in the Senejan area, Arak region, Markazi province, Iran was studied based on morphological traits to obtain superior late-leafing genotypes in the cropping seasons of 2022 and 2023. RESULTS: Based on the results of the analysis of variance, the studied genotypes showed a significant variation in terms of most of the studied morphological and pomological traits. Therefore, it is possible to choose genotypes for different values ​​of a trait. Kernel weight showed positive and significant correlations with leaf length (r = 0.32), leaf width (r = 0.33), petiole length (r = 0.26), terminal leaflet length (r = 0.34), terminal leaflet width (r = 0.21), nut length (r = 0.48), nut width (r = 0.73), nut weight (r = 0.83), kernel length (r = 0.64), and kernel width (r = 0.89). The 46 out of 141 studied genotypes were late-leafing and were analyzed separately. Among late-leafing genotypes, the length of the nut was in the range of 29.33-48.50 mm, the width of the nut was in the range of 27.51-39.89 mm, and nut weight was in the range of 8.18-16.06 g. The thickness of shell was in the range of 1.11-2.60 mm. Also, kernel length ranged from 21.97-34.84 mm, kernel width ranged from 21.10-31.09 mm, and kernel weight ranged from 3.10-7.97 g. CONCLUSIONS: Based on important and commercial traits in walnut breeding programs, such as nut weight, kernel weight, kernel percentage, kernel color, and ease of kernel removal from nuts, 15 genotypes, including no. 92, 91, 31, 38, 33, 18, 93, 3, 58, 108, 16, 70, 15, 82, and 32 were superior and could be used in walnut breeding programs in line with the introduction of new cultivars and the revival of traditional walnut orchards to commercialize them.

Identification of ARF genes in Juglans Sigillata Dode and analysis of their expression patterns under drought stress.

BACKGROUND: Auxin response factor (ARF), a transcription factors that controls the expression of genes responsive to auxin, plays a key role in the regulation of plant growth and development. Analyses aimed at identifying ARF family genes and characterizing their functions in Juglans sigillata Dode are lacking. METHODS AND RESULTS: We used bioinformatic approaches to identify members of the J. sigillata ARF gene family and analyze their evolutionary relationships, collinearity, cis-acting elements, and tissue-specific expression patterns. The expression patterns of ARF gene family members under natural drought conditions were also analyzed. The J. sigillata ARF gene family contained 31 members, which were unevenly distributed across 16 chromosomes. We constructed a phylogenetic tree of JsARF genes and other plant ARF genes. Cis-acting elements in the promoters of JsARF were predicted. JsARF28 showed higher expressions in both the roots and leaves. A heat map of the transcriptome data of the cluster analysis under drought stress indicated that JsARF3/9/11/17/20/26 are responsive to drought. The expression of the 11 ARF genes varied under PEG treatment and JsARF18 and JsARF20 were significantly up-regulated. CONCLUSIONS: The interactions between abiotic stresses and plant hormones are supported by our cumulative data, which also offers a theoretical groundwork for comprehending the ARF mechanism and drought resistance in J. sigillata.

Development and characterization of nuclear microsatellite markers for the African walnut Coula edulis Baill (Coulaceae).

PREMISE OF THE STUDY: Coula edulis Baill (Coulaceae) is a common tree species in the Guineo-Congolian forests producing an edible fruit known as African walnut, which is an important food and income resource for rural populations. However, the species suffers from a deficit of natural regeneration. We developed here nuclear microsatellite markers for C. edulis to be able to study the genetic structure of its natural populations and gene flow. METHODS AND RESULTS: A genomic library was obtained using the Illumina platform, and 21 polymorphic microsatellite loci were developed. The polymorphic microsatellites displayed eight to 22 alleles per locus (average: 14.2), with a mean expected heterozygosity ranging from 0.33 to 0.72 in five populations from Central and West Africa. CONCLUSIONS: The high polymorphism of the nuclear microsatellite markers developed makes them useful to investigate gene flow and the organization of genetic diversity in C. edulis, and to assess whether particular genetic resources require conservation efforts.

Establishment of virus-induced gene-silencing system in Juglans sigillata Dode and functional analysis of JsFLS2 and JsFLS4.

Juglans sigillata Dode is one of the important tree species in southwest China, and it has significant economic and ecological value. However, there is still a lack of effective methods to identify the functional genes of J. sigillata. By verifying the model plant tobacco, the pTRV2::JsPDS vector was able to cause photobleaching. This study showed that photobleaching occurred 24 and 30 d after the silencing vector was infected with aseptic seedlings and fruits of J. sigillata, respectively. When the OD600 was 0.6, and the injection dose was 500 µL, the gene silencing efficiency of aseptic seedlings was the highest at 16.7 %, significantly better than other treatments. Moreover, when the OD600 was 0.8, and the injection dose was 500 µL, the gene silencing efficiency in the walnut fruit was the highest (20 %). In addition, the VIGS system was successfully used to silence JsFLS2 and JsFLS4 genes in J. sigillata. This study also showed that the flavonol content and gene expression in the treatment group were decreased compared to the control group. In addition, the proteins transcribed and translated from the JsFLS4 gene may have higher catalytic activity for dihydroquercetin. The above results indicate that the TRV-mediated VIGS system can be an ideal tool for studying J. sigillata gene function.

A haplotype-resolved genome provides insight into allele-specific expression in wild walnut (Juglans regia L.).

Wild germplasm resources are crucial for gene mining and molecular breeding because of their special trait performance. Haplotype-resolved genome is an ideal solution for fully understanding the biology of subgenomes in highly heterozygous species. Here, we surveyed the genome of a wild walnut tree from Gongliu County, Xinjiang, China, and generated a haplotype-resolved reference genome of 562.99 Mb (contig N50 = 34.10 Mb) for one haplotype (hap1) and 561.07 Mb (contig N50 = 33.91 Mb) for another haplotype (hap2) using PacBio high-fidelity (HiFi) reads and Hi-C technology. Approximately 527.20 Mb (93.64%) of hap1 and 526.40 Mb (93.82%) of hap2 were assigned to 16 pseudochromosomes. A total of 41039 and 39744 protein-coding gene models were predicted for hap1 and hap2, respectively. Moreover, 123 structural variations (SVs) were identified between the two haplotype genomes. Allele-specific expression genes (ASEGs) that respond to cold stress were ultimately identified. These datasets can be used to study subgenome evolution, for functional elite gene mining and to discover the transcriptional basis of specific traits related to environmental adaptation in wild walnut.

Provenance and family variations in early growth of Manchurian walnut (Juglans mandshurica Maxim.) and selection of superior families.

This study, conducted in China in November 2020, was aimed at exploring the variations in growth traits among different provenances and families as well as to select elite materials of Juglans mandshurica. Thus, seeds of 44 families from six J. mandshurica provenances in Heilongjiang and Jilin provinces were sown in the nursery and then transplanted out in the field. At the age of 5 years, seven growth traits were assessed, and a comprehensive analysis was conducted as well as selection of provenance and families. Analysis of variance revealed statistically significant (P < 0.01) differences in seven growth traits among different provenances and families, thereby justifying the pursuit of further breeding endeavors. The genetic coefficient of variation (GCV) for all traits ranged from 5.44% (branch angle) to 21.95% (tree height) whereas the phenotypic coefficient of variation (PCV) ranged from 13.74% (tapering) to 38.50% (branch number per node), indicating considerable variability across the traits. Further, all the studied traits except stem straightness degree, branch angle and branch number per node, showed high heritability (Tree height, ground diameter, mean crown width and tapering, over 0.7±0.073), indicating that the variation in these traits is primarily driven by genetic factors. Correlation analysis revealed a strong positive correlation (r > 0.8) between tree height and ground diameter (r = 0.86), tree height and mean crown width (r = 0.82), and ground diameter and mean crown width (r = 0.83). This suggests that these relationships can be employed for more precise predictions of the growth and morphological characteristics of trees, as well as the selection of superior materials. There was a strong correlation between temperature factors and growth traits. Based on the comprehensive scores in this study, Sanchazi was selected as elite provenance. Using the top-percentile selection criteria, SC1, SC8, DJC15, and DQ18 were selected as elite families. These selected families exhibit genetic gains of over 10% in tree height, ground diameter and mean crown width, signifying their significant potential in forestry for enhancing timber production and reducing production cycles, thereby contributing to sustainable forest management. In this study, the growth traits of J. mandshurica were found to exhibit stable variation, and there were correlations between these traits. The selected elite provenance and families of J. mandshurica showed faster growth, which is advantageous for the subsequent breeding and promotion of improved J. mandshurica varieties.

Genome-wide identification analysis of the 4-Coumarate: CoA ligase (4CL) gene family expression profiles in Juglans regia and its wild relatives J. Mandshurica resistance and salt stress.

Persian walnut (Juglans regia) and Manchurian walnut (Juglans mandshurica) belong to Juglandaceae, which are vulnerable, temperate deciduous perennial trees with high economical, ecological, and industrial values. 4-Coumarate: CoA ligase (4CL) plays an essential function in plant development, growth, and stress. Walnut production is challenged by diverse stresses, such as salinity, drought, and diseases. However, the characteristics and expression levels of 4CL gene family in Juglans species resistance and under salt stress are unknown. Here, we identified 36 Jr4CL genes and 31 Jm4CL genes, respectively. Based on phylogenetic relationship analysis, all 4CL genes were divided into three branches. WGD was the major duplication mode for 4CLs in two Juglans species. The phylogenic and collinearity analyses showed that the 4CLs were relatively conserved during evolution, but the gene structures varied widely. 4CLs promoter region contained multiply cis-acting elements related to phytohormones and stress responses. We found that Jr4CLs may be participated in the regulation of resistance to anthracnose. The expression level and some physiological of 4CLs were changed significantly after salt treatment. According to qRT-PCR results, positive regulation was found to be the main mode of regulation of 4CL genes after salt stress. Overall, J. mandshurica outperformed J. regia. Therefore, J. mandshurica can be used as a walnut rootstock to improve salt tolerance. Our results provide new understanding the potential functions of 4CL genes in stress tolerance, offer the theoretical genetic basis of walnut varieties adapted to salt stress, and provide an important reference for breeding cultivated walnuts for stress tolerance.

Integrated metabolomic and transcriptomic dynamic profiles of endopleura coloration during fruit maturation in three walnut cultivars.

BACKGROUND: The color of endopleura is a vital factor in determining the economic value and aesthetics appeal of nut. Walnuts (Juglans) are a key source of edible nuts, high in proteins, amino acids, lipids, carbohydrates. Walnut had a variety endopleura color as yellow, red, and purple. However, the regulation of walnut endopleura color remains little known. RESULTS: To understand the process of coloration in endopleura, we performed the integrative analysis of transcriptomes and metabolomes at two developmental stages of walnut endopleura. We obtained total of 4,950 differentially expressed genes (DEGs) and 794 metabolites from walnut endopleura, which are involved in flavonoid and phenolic biosynthesis pathways. The enrichment analysis revealed that the cinnamic acid, coniferyl alcohol, naringenin, and naringenin-7-O-glucoside were important metabolites in the development process of walnut endopleura. Transcriptome and metabolome analyses revealed that the DEGs and differentially regulated metabolites (DRMs) were significantly enriched in flavonoid biosynthesis and phenolic metabolic pathways. Through co-expression analysis, CHS (chalcone synthase), CHI (chalcone isomerase), CCR (cinnamoyl CoA reductase), CAD (cinnamyl alcohol dehydrogenase), COMT (catechol-Omethyl transferase), and 4CL (4-coumaroyl: CoA-ligase) may be the key genes that potentially regulate walnut endopleura color in flavonoid biosynthesis and phenolic metabolic pathways. CONCLUSIONS: This study illuminates the metabolic pathways and candidate genes that underlie the endopleura coloration in walnuts, lay the foundation for further study and provides insights into controlling nut's colour.

Transcriptomic Evidence of a Link between Cell Wall Biogenesis, Pathogenesis, and Vigor in Walnut Root and Trunk Diseases.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut.

Covalent conjugation with polyphenol reduced the sensitization of walnut and ameliorated allergy by enhancing intestinal epithelial barrier in mice.

In order to reduce the sensitization of walnut protein (WP), the effects of the interaction between WP and (-)-Epigallocatechin gallate (EGCG), quercetin, trans-ferulic acid, and resveratrol were investigated. Covalent and non-covalent conjugations were compared. The results suggested that covalent conjugation reduced the free amino acid content, sulfhydryl content, and surface hydrophobicity. When compared to non-covalent conjugation, covalent modification showed a lower IgE binding capacity, accompanied by changes in protein conformation. Moreover, animal experiments revealed that there were up-regulation of transforming growth factor-ß, T-box expressed in t cells, and forkhead transcription factor Foxp3 mRNA expression, and down-regulation of IL-4, IL-17, GATA binding protein 3 and retinoid-related orphan nuclear receptor γt mRNA expression in the conjugate groups. These results suggested that covalent conjugation of polyphenols, especially EGCG, likely ameliorated allergy by promoting Th1/Th2 and Treg/Th17 balance and alleviating allergy-induced intestinal barrier damage, which might be a support in reducing the allergenicity of WP.

Crystal structure and interconversion of monomers and domain-swapped dimers of the walnut tree phytocystatin.

Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.

Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

Molecular Characterization of Local Walnut (Juglans regia) Genotypes in the North-East Parnon Mountain Region of Greece.

Walnut is one of the most important nuts regarding their production and consumption. The available but uncharacterized genetic resources of walnut are important for the development and breeding of local varieties. Greece holds an important number of genetically uncharacterized walnut landraces, especially within the area of Parnon, which is considered to play a significant role as an in situ gene bank, due to its unique location traits. However, the genetic characterization and further use of these resources has been insufficient, due to the absence of genetic studies. In this study, we implemented SSR molecular markers, both to genetically characterize the walnut tree genetic diversity of the Parnon area and to identify its unique genetic structure, which will form the starting material for subsequent breeding programs. Overall, high levels of genetic variation were found among the individual walnut accessions that were collected in the Parnon mountain region.

Genome-wide identification and molecular expression profile analysis of FHY3/FAR1 gene family in walnut (Juglans sigillata L.) development.

BACKGROUND: Juglans sigillata L. (walnut) has a high economic value for nuts and wood and has been widely grown and eaten around the world. Light plays an important role in regulating the development of the walnut embryo and promoting nucleolus enlargement, which is one of the factors affecting the yield and quality of walnut. However, little is known about the effect of light on the growth and quality of walnuts. Studies have shown that far red prolonged hypocotyl 3 (FHY3) and far red damaged response (FAR1) play important roles in plant growth, light response, and resistance. Therefore, FHY3/FAR1 genes were identified in walnuts on a genome-wide basis during their growth and development to reveal the potential regulation mechanisms involved in walnut kernel growth and development. RESULTS: In the present study, a total of 61 FHY3/FAR1 gene family members in walnuts have been identified, ranging in length from 117 aa to 895 aa. These gene family members have FHY3 or FAR1 conserved domains, which are unevenly distributed on the 15 chromosomes (Chr) of the walnut (except for the Chr16). All 61 FHY3/FAR1 genes were divided into five subclasses (I, II, III, IV, and V) by phylogenetic tree analysis. The results indicated that FHY3/FAR1 genes in the same subclasses with similar structures might be involved in regulating the growth and development of walnut. The gene expression profiles were analyzed in different walnut kernel varieties (Q, T, and F). The result showed that some FHY3/FAR1 genes might be involved in the regulation of walnut kernel ripening and seed coat color formation. Seven genes (OF07056-RA, OF09665-RA, OF24282-RA, OF26012-RA, OF28029-RA, OF28030-RA, and OF08124-RA) were predicted to be associated with flavonoid biosynthetic gene regulation cis-acting elements in promoter sequences. RT-PCR was used to verify the expression levels of candidate genes during the development and color change of walnut kernels. In addition, light responsiveness and MeJA responsiveness are important promoter regulatory elements in the FHY3/FAR1 gene family, which are potentially involved in the light response, growth, and development of walnut plants. CONCLUSION: The results of this study provide a valuable reference for supplementing the genomic sequencing results of walnut, and pave the way for further research on the FHY3/FAR1 gene function of walnut.

Overexpression of JsFLS5 in calli improves salinity tolerance by maintaining active oxygen balance and reducing Na+ toxicity in Juglans sigillata.

The escalating global climate change significantly threatens plant growth, development, and production through salinity stress. Flavonoids, a crucial category of secondary metabolites, have been extensively studied for their role in modulating plant growth and development mechanisms in the face of biological and abiotic stress. The flavonol synthetase (FLS) gene plays a key role in the biosynthesis and accumulation of flavonoids. To investigate the correlation between salt tolerance and flavonol synthesis, JsFLS5 was overexpressed in the callus of Juglans sigillata cv. "Qianhe-7." This study shows that the upregulation of JsFLS5 significantly elevates the overall flavonoid content by modulating the expression of genes associated with flavonoid synthesis under salinity stress conditions. Additionally, the overexpressing callus exhibited enhanced resistance to salt stress compared to the wild-type callus, as evidenced by reduced levels of reactive oxygen species accumulation, electrolyte leakage, and malondialdehyde content in the overexpressing callus relative to the wild type (WT). Moreover, the overexpressing callus showed higher antioxidant enzyme activity and a more efficient ascorbic acid-glutathione cycle. Furthermore, the concentration of Na+ in the overexpressing callus was lower than WT, resulting in a decreased Na+ /K+ ratio. These findings suggest that JsFLS5 overexpression in calli effectively mitigates the oxidative damage induced by osmotic stress and reduces Na+ toxicity by enhancing flavonoid synthesis under salt stress conditions. Consequently, this study offers a novel perspective for comprehending the role of JsFLS5 in the response to abiotic stress in J. sigillata.