Search Results (14)

MIKC-type MADS-box transcription factors constitute one of the largest gene families in plants, playing pivotal roles in regulating plant growth and development, hormone signaling transduction, and responses to biotic and abiotic stresses. However, there have been no reports on the systematic identification and characterization of MIKC-type MADS-box proteins in walnuts. In this study, we identified 52 JrMADS genes in the walnut genome and transcriptome, and categorized them into 14 subfamilies through structural domain and phylogenetic tree analysis. It was found that these genes were unevenly distributed across 16 chromosomes. Within the MIKC-type MADS-box gene family, we identified three pairs of tandem-duplicated genes and 40 pairs of segmental duplicated genes, indicating that segmental duplication was the primary mechanism of gene amplification in walnut. Ka/Ks analysis showed that the family genes have undergone purifying selection during evolutionary processes. The promoter was predicted to contain cis-acting elements related to growth, development, plant hormones, and stress response. Expression profile analysis showed that JrMADS genes have different expression patterns in various tissues and developmental stages of male and female flower buds. Notably, an ancient clade of TM8 (JrMADS43) genes was found, which is absent in Arabidopsis but present in other flowering plants. Another gene, TM6 gene (JrMADS4), belongs to the AP3 subfamily and is a clade that has diverged from tomatoes. Through qPCR analysis, we verified the differential expression of JrMADS genes at different developmental stages (MB-1/2/3 and FB-1/2/3), with JrMADS5, JrMADS8, JrMADS14, JrMADS24, JrMADS40, JrMADS46, JrMADS47, JrGA3ox1, and JrGA3ox3 showing significantly higher expression in male than in female flower buds. In summary, our results provide valuable information for further biological functions research on MIKC-type MADS-box genes in walnut, such as flower organ development, and lays a solid foundation for future studies. Full article

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts. Full article

Walnut trees are grown worldwide for their edible fruits, which have high nutritional value. To address climate change, researchers have studied walnut phenology to create cultivars adapted to warmer climates. The objective of this study is to propose a scale for phenological Persian walnut observations using the Biologische Bundesanstalt, Bundessortenamt, und CHemische Industrie (BBCH) codification and alignment with historical alphameric scales. Here, the principal growth stages (PGSs) of Persian walnut (Juglans regia L.) are described using stages from a previously available alphanumeric scale. This standardised phenological scale describes Persian walnut growth from the dormant vegetative state through reproductive budding and senescence. This phenological scale is expected to increase the efficiency of walnut phenological monitoring. Fifty-seven stages are used to describe the life cycle of Persian walnut in this BBCH scale. Of these 57 stages, 3 stages are dedicated to seed germination (PGS-0), 4 stages are dedicated to bud development (PGS-0), 7 stages are dedicated to leaf development (PGS-1), 4 stages are dedicated to stem elongation (PGS-3), 8 stages are dedicated to inflorescence emergence (PGS-5), 5 stages are dedicated to male flowering (PGS-6), 5 stages are dedicated to female flowering (PGS-6), 5 stages are dedicated to fruit development (PGS-7), 12 stages are dedicated to fruit ripening (PGS-8), and 4 stages are dedicated to leaf senescence (PGS-9). Full article

SPL transcription factors affect plant growth and development, including blooming and photoperiod control. The investigation began with transcriptome data screening of 28 JrSPL genes in walnut (Junglans regia L.) ‘Wen185’. These genes were discovered on all chromosomes except 6 and 15. Phylogenetic study divides the 28 JrSPL genes into five groupings. The biggest cluster, cluster IV, has 12 JrSPL genes. The expression of JrSPL genes in different tissues was investigated by qRT-PCR. JrSPL02 gene expression was greater in walnut female and male flower tissues than other genes. Subcellular localization has shown the JrSPL02 gene resides in the nucleus. Jre-miR156 may target JrSPL02’s 3′-UTR region, according to miRNA sequencing, RACE, and BiFC studies. Arabidopsis plants expressing the JrSPL02 gene flowered 3 days faster than the wild type, according to phenotypic observation. Transgenic lines had more stem branches and siliques than the control group but fewer rosette leaves. In summary, this study functionally analyses the metamorphosis of the miR156-SPL module during the blooming stage and the underlying mechanisms that govern early fruiting in early-fruiting walnuts in Xinjiang. Full article

Walnut (Juglans regia) populations are considered a valuable genetic resource for genetic variability conservation and walnut cultivar improvement. In the current study, two walnut selections from Central Greece, “FM3” and “FM6”, were evaluated for their characteristics to determine their perspective as new cultivars. Their main phenological and pomological characteristics were assessed according to IPGR (1994) and UPOV-TG/125/6 (1999) criteria for 10 consecutive years and compared with “Chandler”. Results revealed that “FM3” presents high lateral flowering (70–80%), whereas “FM6” is 30–35%. Although the date of bud break and male blooming of both selections did not differ from “Chandler” (p > 0.05), the female blooming phase of “FM6” occurred earlier (p < 0.05) than “Chandler” and nut harvest was earlier than both “Chandler” and “FM3” (p < 0.05). Nut dimensions of both selections were higher than “Chandler” (p < 0.001). The mean in-shell nut weight and kernel weight of “FM3” were higher than “FM6” and “Chandler”, and those of “FM6” were higher than “Chandler” (p < 0.001). Other positive nut characteristics were their light kernel color, the well kernel fill, and the easy removal of the kernel halves. Thus, the current results indicate that “FM3” and “FM6” could be considered promising gene pools in crossbreeding activities. Full article

The aim of this study is to assess the effect of adding dried and ground walnut male flowers to wheat flour on selected physicochemical and antioxidant properties of the obtained wheat bread. The control sample was bread without the addition of walnut male flowers. In the tests, the addition of flowers was used in the amounts of 0.5%, 1.0%, 1.5%, 2.0% and 2.5% of the weight of the flour used in the control sample. Bread dough was prepared according to the direct method with the use of yeast. The parameters of the baking process of the control bread and of the test bread were calculated. The selected parameters determining the quality of the obtained bread, including the loaf volume, the specific volume, the crumb porosity, the crumb texture and the color were measured. The antioxidant potential and total polyphenol content were tested both in the dough and in the bread. The test results were analyzed statistically. It was found that the partial replacement of wheat flour with walnut male flowers resulted in a significant reduction in the loaves’ volume and in the darkening of both the color of the dough and the crumb. The value of the L* parameter decreased with the increasing addition of walnut flowers. The lowest values of this parameter were found to be 53.87 (crust) and 39.94 (crumb) in the sample with 2.5% addition. The average volume of the loaves ranged from 565 cm³ (0.5%) to 675 cm³ (control). The use of the additive in the amount of 2.5% caused significant changes in the values of most of the examined parameters of the bread crumb texture. The addition of walnut male flowers to the flour had a significant effect on the antioxidant potential and the total polyphenol content of the tested doughs and breads. In the case of bread, the total polyphenol content value ranged from 96.90 mg GAE/g dw to 208.08 mg GAE/g dw. Similarly, the antioxidant potential increased with increasing walnut male flower supplementation. The antioxidant potential determined by the ABTS and FRAP methods ranged from 1.42 mmol TE/g dw to 2.02 mmol TE/g dw and from 0.08 mmol TE/g dw to 0.33 mmol TE/g dw, respectively. The obtained research results prove the application potential of walnut flowers in the design of food, with beneficial pro-health properties, and are an example of the use of plant by-products in the process of food enrichment. Full article

The GRF gene family plays an important role in plant growth and development as regulators involved in plant hormone signaling and metabolism. However, the Juglandaceae GRF gene family remains to be studied. Here, we identified 15, 15, 19, and 20 GRF genes in J. regia, C. illinoinensis, J. sigillata, and J. mandshurica, respectively. The phylogeny shows that the Juglandaceae family GRF is divided into two subfamilies, the ε-group and the non-ε-group, and that selection pressure analysis did not detect amino acid loci subject to positive selection pressure. In addition, we found that the duplications of the Juglandaceae family GRF genes were all segmental duplication events, and a total of 79 orthologous gene pairs and one paralogous homologous gene pair were identified in four Juglandaceae families. The Ka/KS ratios between these homologous gene pairs were further analyzed, and the Ka/KS values were all less than 1, indicating that purifying selection plays an important role in the evolution of the Juglandaceae family GRF genes. The codon bias of genes in the GRF family of Juglandaceae species is weak, and is affected by both natural selection pressure and base mutation, and translation selection plays a dominant role in the mutation pressure in codon usage. Finally, expression analysis showed that GRF genes play important roles in pecan embryo development and walnut male and female flower bud development, but with different expression patterns. In conclusion, this study will serve as a rich genetic resource for exploring the molecular mechanisms of flower bud differentiation and embryo development in Juglandaceae. In addition, this is the first study to report the GRF gene family in the Juglandaceae family; therefore, our study will provide guidance for future comparative and functional genomic studies of the GRF gene family in the Juglandaceae specie. Full article

Pollen is one of the major by-products of the walnut tree, yet it is poorly investigated. Thus, the aim of this study was to investigate the total phenolics, flavonoids, antioxidant, anticancer potential, and polyphenol profile of pollen obtained from male Juglans regia flowers. A total of 24 phenolic compounds were identified in Juglans regia pollen and all of them were reported for the first time for this raw material. The content of polyphenols was 408.03 mg/100 g dry weight (dw) and the most abundant components were quercetin 3-O-sophoroside and 4′,5,7-trihydroxy-3,6-dimethoxyflavone-7-O-beta-D-glucoside. The concentration of these compounds, as well as the total content of polyphenols and flavonoids, strongly determined the antioxidant and cytotoxic activity of Juglans regia pollen. Antioxidant action using the ABTS and CUPRAC methods had the values of 3.35 and 0.32 mmol TE/g dw, respectively. In turn, in the tests of chelating ability of ferrous ion, O₂^•− and OH⁻ radical scavenging activity, of which the results were expressed as IC₅₀, the values were equal to 335.01, 459.31, and 92.89 µg/mL, respectively. Among the six cancer cell lines, the strongest effect was demonstrated for Caco-2 (140.65 µg/mL) and MCF-7 (140.98 µg/mL) cells. The results provide valuable and previously unpublished data on the polyphenol composition and biological potential of Juglans regia pollen. Full article

Juglans regia L., walnut, is a large, long-living tree, cultivated in temperate climates around the world. It is highly appreciated for its nutritional kernels and high-quality timber. Its barks, leaves, and husk are used as dyes and in folk medicine as herbal remedies for several diseases. From a biological and chemical standpoint, relatively little is known about the male flowers of the tree. Therefore, the aim of the study was to evaluate the phenolic profile as well as in vitro antioxidant, antimicrobial, and antiproliferative activity of male Juglans regia L. flowers. Phenolic content was determined by UPLC/PDA/MS/MS analyses; antioxidant activity was assessed by five different methods; antimicrobial activity was evaluated against the six most common pathogenic strains of Gram-positive and Gram-negative bacteria, and antiproliferative properties were assessed against six cell lines. Most of the analyses carried out in this study were performed for the first time for this raw material. J. regia flower extract was characterized by a strong ability to scavenge DPPH˙ free radicals, hydroxyl radicals, and chelating metal ions. Among the examined bacterial strains and neoplastic lines, the strongest antimicrobial activity was shown against S. aureus, L. monocytogenes, and B. cereus, and cytotoxic activity against breast cancer, glioblastoma, and astrocytoma cells. Male J. regia flowers have also been found to be a rich source of phenolic compounds. The content of polyphenols in the extract was 4369.73 mg/100 g d.w., and 24 compounds from the group of flavonoids, phenolic acids, and juglunosides were identified. Additionally, a strong correlation between the content of polyphenols and the antioxidant capacity and cytotoxic activity was observed. This is why the tested J. regia flowers are an excellent source of effective natural antioxidant, antibacterial, and chemopreventive compounds that have potential to be used in the pharmaceutical or food industries. Full article

Ιn an effort to create walnut cultivars (Juglans regia) with high productivity, fruit quality and lateral bearing, a new cultivar, named “Leto”, was created by the cross “Gustine” × “Pedro”. Its main phenological and pomological characteristics were assessed according to the criteria of IPGR (1994) and UPOV-TG/125/6 (1999), for 10 consecutive years and compared with its maternal cultivars and “Chandler”. Observations showed that “Leto” has high lateral bearing habit (90%) and presents satisfactory yield at the full production age. The tree size is smaller than that of its parents and “Chandler”, female flowers bloom from 11 to 22 of April and male from 3 to 6 of April. “Leto” nuts are harvested at the end of September, present easy hull dehiscence and high kernel percentage. Other positive nut characteristics of “Leto” are light kernel color, well kernel filling and easy removal of the kernel halves. “Leto” is a mid-early cultivar of great interest due to its high- quality nuts, suitable for dense plantings, in regions where the last spring frosts occur in late March to early April, thus, making it a promising cultivar for Greece, but also for other regions with similar geomorphological and climatic conditions. Full article

The extraction of bioactive compounds present in walnut (Juglans regia L.) male flowers (WMFs) was performed based on an experimental design using ultrasonic-assisted extraction. Solvent nature, extraction time, and water content were selected as experimental variables, and phenolic, flavonoidic, and condensed tannins contents and antioxidant properties were evaluated. Acetone was the solvent with the highest extraction performance, with the extracts obtained using this solvent displaying an increased concentration of bioactive compounds and increased antioxidant activities. For several extracts with high bioactive content, individual polyphenolic and tocopherolic compounds were evaluated by means of LC-MS and LC-MS/MS. The best extraction conditions for polyphenolic (2.86 mg gallic acid equivalents/g WMF) and tocopherolic compounds (29.4 µg/g WMF) were acetone with 40% water content (N20) and acetone with 20% water content (N15), respectively. Although the total tocopherol concentrations were lower than in other Juglans regia parts, most of the total tocopherol quantity was provided by the highly biologically active δ-tocopherol (84%). Significant quantities of quercetin (101.9 µg/g), hyperoside (2662.9 µg/g), quercitrin (405.7 µg/g), and isoquercitrin (1293.7 µg/g) were determined in WMF (N20). Both extracts inhibited the enzymatic activity of α-glucosidase and tyrosinase; however, an increased inhibition was observed for N20, the extract with the higher polyphenolic content. Conversely, N15 had higher anticancerous activity on the cell lines used, with a moderate selectivity towards the cancerous phenotype being observed for both extracts. At non-cytotoxic concentrations, both extracts displayed good antioxidant activities in cellular cultures, decreasing basal and H₂O₂-induced oxidative stress. This is the first characterization of both hydrophilic and lipophilic phytochemicals in WMF extracts. The outcomes of our study reveal that walnut male flowers have strong biological activities, thus justifying further research to demonstrate their usefulness in the food, pharmaceutical, and/or cosmetic industries. Full article

The common walnut (or Persian walnut), Juglans regia L., is an economically important temperate tree species valued for both its edible nut and high-quality wood. F-box gene family members are involved in plant development, which includes regulating plant development, reproduction, cellular protein degradation, response to biotic and abiotic stresses, and flowering. However, in common walnut (J. regia), there are no reports about the F-box gene family. Here, we report a genome-wide identification of J. regia F-box genes and analyze their phylogeny, duplication, microRNA, pathway, and transcriptional expression profile. In this study, 74 F-box genes were identified and clustered into three groups based on phylogenetic analysis and eight subfamilies based on special domains in common walnut. These common walnut F-box genes are distributed on 31 different pseudo-chromosomes. The gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and microRNA profiles showed that the F-box gene family might play a critical role in the flowering of common walnut. The expressions were significantly higher in female flowers and male flowers compared with leaf and hull tissues at a transcriptome level. The results revealed that the expressions of the F-box gene in female flowers were positively correlated with male flowers, but there was no correlation between any other tissue combinations in common walnut. Our results provided insight into the general characteristics of the F-box genes in common walnut. Full article

Juglans regia L. is an economically important crop cultivated worldwide for its high quality and quantity of wood and nuts. Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway that plays a critical role in plant growth, development, and adaptation, but there have been few reports of the PAL gene family in common walnut. Here, we report a genome-wide study of J. regia PAL genes and analyze their phylogeny, duplication, microRNA, and transcriptional expression. A total of 12 PAL genes were identified in the common walnut and clustered into two subfamilies based on phylogenetic analysis. These common walnut PALs are distributed on eight different pseudo-chromosomes. Seven of the 12 PALs (JrPAL2-3, JrPAL4-2, JrPAL2-1, JrPAL4-1, JrPAL8, JrPAL9, and JrPAL6) were specific found in J. regia, and JrPAL3, JrPAL5, JrPAL1-2, JrPAL7, and JrPAL2-2 were found to be closely associated with the woody plant Populus trichocarpa. Additionally, the expression patterns of JrPAL3, JrPAL7, JrPAL9, and JrPAL2-1 showed that they had high expression in female and male flowers. The miRNA ath-miR830-5p regulates two genes, JrPAL5 and JrPAL1, such that they have low expression in the male and female flowers of the common walnut. Our research provides useful information for further research into the function of PAL genes in common walnut and Juglans. Full article

Persian walnut (Juglans regia L.), valued for both its nut and wood, is an ecologically important temperate tree species native to the mountainous regions of central Asia. Despite its importance, there are still few transcriptomic resources in public databases for J. regia, limiting gene discovery and breeding. Here, more than 49.9 million sequencing reads were generated using Illumina sequencing technology in the characterization of the transcriptome of four J. regia organs (bud, leaf, female flowers, and male flowers). De novo assembly yielded 117,229 unigenes with an N50 of 1955 bp. Based on sequence similarity searches against known proteins, a total of 20,413 (17.41%) genes were identified and annotated. A set of 27,584 unigenes with SSR (simple sequence repeats) motifs were identified as potential molecular markers, and a sample of 77 of these EST-SSRs (express sequence tags) were further evaluated to validate their amplification and assess their polymorphism. Next, we developed 39 polymorphic microsatellite markers to screen 88 Persian walnut individuals collected from 11 populations. These markers and transcriptomic resources will be useful for future studies of population genetic structure, evolutionary ecology, and breeding of Persian walnut and other Juglans species. Full article