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.

Complete mitochondrial genome assembly of Juglans regia unveiled its molecular characteristics, genome evolution, and phylogenetic implications.

BACKGROUND: The Persian walnut (Juglans regia), an economically vital species within the Juglandaceae family, has seen its mitochondrial genome sequenced and assembled in the current study using advanced Illumina and Nanopore sequencing technology. RESULTS: The 1,007,576 bp mitogenome of J. regia consisted of three circular chromosomes with a 44.52% GC content encoding 39 PCGs, 47 tRNA, and five rRNA genes. Extensive repetitive sequences, including 320 SSRs, 512 interspersed, and 83 tandem repeats, were identified, contributing to genomic complexity. The protein-coding sequences (PCGs) favored A/T-ending codons, and the codon usage bias was primarily shaped by selective pressure. Intracellular gene transfer occurred among the mitogenome, chloroplast, and nuclear genomes. Comparative genomic analysis unveiled abundant structure and sequence variation among J. regia and related species. The results of selective pressure analysis indicated that most PCGs underwent purifying selection, whereas the atp4 and ccmB genes had experienced positive selection between many species pairs. In addition, the phylogenetic examination, grounded in mitochondrial genome data, precisely delineated the evolutionary and taxonomic relationships of J. regia and its relatives. We identified a total of 539 RNA editing sites, among which 288 were corroborated by transcriptome sequencing data. Furthermore, expression profiling under temperature stress highlighted the complex regulation pattern of 28 differently expressed PCGs, wherein NADH dehydrogenase and ATP synthase genes might be critical in the mitochondria response to cold stress. CONCLUSIONS: Our results provided valuable molecular resources for understanding the genetic characteristics of J. regia and offered novel perspectives for population genetics and evolutionary studies in Juglans and related woody species.

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.

Differential warming at crown scale impacts walnut primary growth onset and secondary growth rate.

Trees are exposed to significant spatio-temporal thermal variations, which can induce intracrown discrepancies in the onset and dynamics of primary and secondary growth. In recent decades, an increase in late winter and early spring temperatures has been observed, potentially accelerating bud break, cambial activation and their potential coordination. Intracrown temperature heterogeneities could lead to asymmetric tree shapes unless there is a compensatory mechanism at the crown level. An original warming experiment was conducted on young Juglans regia trees in a greenhouse. From February to August, the average temperature difference during the day between warmed and control parts was 4°C. The warming treatment advanced the date of budbreak significantly, by up to 14 days. Warming did not alter secondary growth resumption but increased growth rates, leading to higher xylem cell production (twice as many) and to an increase in radial increment (+80% compared to control). Meristems resumptions were asynchronous without coordination in response to temperature. Buds on warmed branches began to swell two weeks prior to cambial division, which was one week earlier than on control branches. A difference in carbon and water remobilisation at the end of bud ecodormancy was noted under warming. Overall, our results argue for a lack of compensatory mechanisms at the crown scale, which may lead to significant changes in tree architecture in response to intra-crown temperature heterogeneities.

Structural elucidation a complex galactosyl and glucosyl-rich pectin from the pericarp of immature fruits of Juglans mandshurica Maxim.

A glucosyl-rich pectin, JMMP-3 (Mw, 2.572 × 104 g/mol, O-methyl % = 3.62%), was isolated and purified from the pericarp of the immature fruit of Juglans mandshurica Maxim. (QingLongYi). The structure of JMMP-3 was studied systematically by infrared spectroscopy, monosaccharide compositions, methylation analysis, partial acid hydrolysis, and 1/2D-NMR. The backbone of JMMP-3 possessed a smooth region (→ 4GalA1 →) and a hairy region (→ 4GalA1 → 2Rha1 →) with a molar ratio of 2: 5. The substitution of four characteristic side chains (R1-R4) occurs at C-4 of → 2,4)-α-Rhap-(1→, where R1 is composed of → 5)-α-Araf-(1→, R2 is composed of → 4)-ß-Galp-(1 → and ß-Galp-(1→, R3 is composed of α-Glcp-(1→, →4)-α-Glcp-(1 → and → 4,6)-α-Glcp-(1→, and R4 is composed of → 5)-α-Araf-(1→, ß-Galp-(1→, → 4)-ß-Galp-(1→, → 3,4)-ß-Galp-(1→, → 4,6)-ß-Galp-(1 → and → 2,4)-ß-Galp-(1 → . In addition, the antitumor activity of JMMP-3 on HepG2 cells was preliminarily investigated.

Advancing fine branch biomass estimation with lidar and structural models.

BACKGROUND AND AIMS: Lidar is a promising tool for fast and accurate measurements of trees. There are several approaches to estimate above-ground woody biomass using lidar point clouds. One of the most widely used methods involves fitting geometric primitives (e.g. cylinders) to the point cloud, thereby reconstructing both the geometry and topology of the tree. However, current algorithms are not suited for accurate estimation of the volume of finer branches, because of the unreliable point dispersions from, for example, beam footprint compared to the structure diameter. METHOD: We propose a new method that couples point cloud-based skeletonization and multi-linear statistical modelling based on structural data to make a model (structural model) that accurately estimates the above-ground woody biomass of trees from high-quality lidar point clouds, including finer branches. The structural model was tested at segment, axis and branch level, and compared to a cylinder fitting algorithm and to the pipe model theory. KEY RESULTS: The model accurately predicted the biomass with 1.6 % normalized root mean square error (nRMSE) at the segment scale from a k-fold cross-validation. It also gave satisfactory results when scaled up to the branch level with a significantly lower error (13 % nRMSE) and bias (-5 %) compared to conventional cylinder fitting to the point cloud (nRMSE: 92 %, bias: 82 %), or using the pipe model theory (nRMSE: 31 %, bias: -27 %). The model was then applied to the whole-tree scale and showed that the sampled trees had more than 1.7 km of structures on average and that 96 % of that length was coming from the twigs (i.e. <5 cm diameter). Our results showed that neglecting twigs can lead to a significant underestimation of tree above-ground woody biomass (-21 %). CONCLUSIONS: The structural model approach is an effective method that allows a more accurate estimation of the volumes of smaller branches from lidar point clouds. This method is versatile but requires manual measurements on branches for calibration. Nevertheless, once the model is calibrated, it can provide unbiased and large-scale estimations of tree structure volumes, making it an excellent choice for accurate 3D reconstruction of trees and estimating standing biomass.

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.

Quantitative determination of specialised metabolites in different parts of Juglans regia Linn. and Carya illinoinensis (Wangenh.) K. Koch by using UHPLC-DAD-QTOF-MS/MS.

INTRODUCTION: Juglans regia Linn. and Carya illinoinensis (Wangenh.) K. Koch are nut-producing plant species of the Juglandaceae family. Bioactive compounds like naphthoquinones, tetralones, and diarylheptanoids are dominant in these species. OBJECTIVE: The study aimed to develop and validate a fast and sensitive analytical method by ultrahigh-performance liquid chromatography diode array detection mass spectrometry (UHPLC-DAD-MS) for quantification and identification of bioactive compounds in fruit pericarps and leaves of J. regia and C. illinoinensis collected from two different states of north India. METHODOLOGY: The dried pericarps of J. regia and C. illinoinensis (500 mg) were extracted with ethyl acetate-methanol (50:50 v/v, 20 mL, 50°C, 30 min) by ultrasonication and analysed by ultrahigh-performance liquid chromatography diode array detection quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-QTOF-MS/MS) for qualitative and quantitative examination of phytoconstituents. The method was validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human use (ICH) guidelines for linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). RESULTS: Here, we report the quantification of dihydrophaseic acid (1), 4,5-dihydroxytetralone (2), 4,8-dihydroxytetralone (3), 5,8-dihydroxy-3-methoxytetralone (4), and juglanin A (5) in the pericarps and leaves of J. regia and C. illinoinensis. Furthermore, using the hyphenated analytical method, a total of 62 compounds were tentatively characterised in different samples. The principal component analysis (PCA) showed diversity between the analysed sample's composition. Also, the study evaluated the variation of bioactive compounds among different parts of J. regia and C. illinoinensis collected from different regions of northern India by UHPLC-DAD-QTOF-MS/MS. CONCLUSION: The developed method is simple, rapid, and selective for the identification and characterisation of bioactive molecules.

Triterpenoids from Juglans mandshurica with anti-hyaluronidase activities.

Phytochemical study on 90% ethanol extract from the green walnut husks of Juglans mandshurica Maxim. resulted into the isolation of three undescribed triterpenoids, juglansmanoids A-C (1-3). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated components were evaluated in vitro for anti-hyaluronidase activities. As a result, triterpenoid 1 exhibited potent anti-hyaluronidase activity (IC50 = 9.78 µg/ml) three times more than the positive control drug oleanolic acid (IC50 = 40.12 µg/ml).

Influence of Cold Stress on Physiological and Phytochemical Characteristics and Secondary Metabolite Accumulation in Microclones of Juglans regia L.

The current study investigated the impact of cold stress on the morphological, physiological, and phytochemical properties of Juglans regia L. (J. regia) using in vitro microclone cultures. The study revealed significant stress-induced changes in the production of secondary antioxidant metabolites. According to gas chromatography-mass spectrometry (GC-MS) analyses, the stress conditions profoundly altered the metabolism of J. regia microclones. Although the overall spectrum of metabolites was reduced, the production of key secondary antioxidant metabolites significantly increased. Notably, there was a sevenfold (7×) increase in juglone concentration. These findings are crucial for advancing walnut metabolomics and enhancing our understanding of plant responses to abiotic stress factors. Additionally, study results aid in identifying the role of individual metabolites in these processes, which is essential for developing strategies to improve plant resilience and tolerance to adverse conditions.

Comprehensive Characterization of Phytochemical Composition, Membrane Permeability, and Antiproliferative Activity of Juglans nigra Polyphenols.

The aim of our study was the detailed polyphenol profiling of Juglans nigra and the characterization of the membrane permeability and antiproliferative properties of its main phenolics. A total of 161 compounds were tentatively identified in J. nigra bark, leaf, and pericarp extracts by ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS). Eight compounds including myricetin-3-O-rhamnoside (86), quercetin-3-O-rhamnoside (106), quercetin-3-O-xyloside (74), juglone (141), 1,2,3,4-tetrahydro-7,8-dihydroxy-4-oxonaphthalen-1-yl-6-O-galloyl-glucoside (92), ellagic acid (143), gallic acid (14), and ethyl gallate (58) were isolated from J. nigra pericarp. The in vitro antiproliferative activity of the isolated compounds was investigated against three human cancer cell lines, confirming that juglone (141) inhibits cell proliferation in all of them, and has similar activity as the clinical standards. The permeability of the isolated compounds across biological membranes was evaluated by the parallel artificial membrane permeability assay (PAMPA). Both juglone (141) and ethyl-gallate (58) showed positive results in the blood-brain-barrier-specific PAMPA-BBB study. Juglone (141) also possesses logPe values which indicates that it may be able to cross both the GI and BBB membranes via passive diffusion.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

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.

Enrichment and Evaluation of Antitumor Properties of Total Flavonoids from Juglans mandshurica Maxim.

Flavonoids are important secondary metabolites found in Juglans mandshurica Maxim., which is a precious reservoir of bioactive substances in China. To explore the antitumor actions of flavonoids (JMFs) from the waste branches of J. mandshurica, the following optimized purification parameters of JMFs by macroporous resins were first obtained. The loading concentration, flow rate, and loading volume of raw flavonoid extracts were 1.4 mg/mL, 2.4 BV/h, and 5 BV, respectively, and for desorption, 60% ethanol (4 BV) was selected to elute JMFs-loaded AB-8 resin at a flow rate of 2.4 BV/h. This adsorption behavior can be explained by the pseudo-second-order kinetic model and Langmuir isotherm model. Subsequently, JMFs were identified using Fourier transform infrared combined with high-performance liquid chromatography and tandem mass spectrometry, and a total of 156 flavonoids were identified. Furthermore, the inhibitory potential of JMFs on the proliferation, migration, and invasion of HepG2 cells was demonstrated. The results also show that exposure to JMFs induced apoptotic cell death, which might be associated with extrinsic and intrinsic pathways. Additionally, flow cytometry detection found that JMFs exposure triggered S phase arrest and the generation of reactive oxygen species in HepG2 cells. These findings suggest that the JMFs purified in this study represent great potential for the treatment of liver cancer.

'Sorrento' and 'Tulare' Walnut Cultivars: Morphological Traits and Phytochemical Enhancement of Their Shell Waste.

Walnut processing generates considerable quantities of by-products that could be reprocessed into value-added products that have food and non-food applications. In this context, the aim of this study is to characterize the 'Sorrento' and 'Tulare' walnut cultivars using the UPOV guidelines and analyze the chemical composition and antioxidant activity of their shells. Insight into the chemical composition of the different granulometric fractions of walnut shell, obtained by sieving, was obtained following ultrasound-assisted extraction by Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS). The total phenolic, flavonoid, and tannin content and antiradical capacity, obtained by DPPH and ABTS assays, and the Fe(III) reducing power of the extracts were also evaluated. The UHPLC-HRMS analysis indicated the presence of thirty-two compounds ascribable to four major classes of specialized metabolites. Furthermore, the extraction efficiency of gallic acid, ellagic acid derivatives, as well as glansreginin A, increased with the decrease in shell matrix particle size in contrast to chlorogenic acids and flavonoid glycosides. This is the first study to highlight new knowledge on the chemical composition of walnut shells. The results obtained demonstrate the feasibility of recovering valuable bioactive components from agro-waste that may be further valorized.

Foliar application of biostimulants improves nutritional and bioactive quality of walnuts.

BACKGROUND: Owing to their health benefits, walnuts are attracting interest as a good option for nutritious meals, thereby promoting their production. Furthermore, the adoption of ecologically and environmentally friendly agriculture strengthens biostimulant use as a sustainable complement to traditional fertilizers. This study evaluated the effects of different foliar-applied biostimulants in walnut tree orchards, in northeastern Portugal, on walnuts' chemical composition and bioactivity. RESULTS: Walnut samples were rich in fat (particularly the polyunsaturated linoleic acid), dietary fiber and protein. Sucrose was the most prevalent soluble sugar, followed by glucose and fructose. Studied samples also showed an antioxidant activity comparable (or superior) to that of Trolox. Some plant biostimulants (e.g. Sprint Plus®) had a positive impact on the nutritional composition of walnuts, more specifically by boosting tocopherol levels, besides improving the bioactivity of walnut extracts against specific bacteria. CONCLUSION: Overall, this research demonstrated that important quality traits of walnuts can be improved using sustainable agricultural bioproducts and practices. © 2024 Society of Chemical Industry.

Characterization and Techno-Functional Properties of High Protein Walnut Flour from an Oil by-Product.

A high protein walnut flour (HPWF) was obtained by defatting walnut flour (WF), which is a by-product of the oil industry. The objective of this study was the chemical and techno-functional characterization of HPWF. Composition, amino acid content, protein secondary structure, protein solubility and thermal transitions were measured. Besides, the techno-functional properties, emulsion activity and stability, and water holding and oil absorption capacities, of HPWF were evaluated. Also, the molecular mass of proteins under denaturing conditions and the microstructure of HPWF were evaluated by electrophoresis and confocal scanning laser microscopy, respectively. HPWF had 55.4% protein content and 21.5% total dietary fibre. In terms of HPWF amino acid composition, the limiting amino acids were the sulphurated cysteine and methionine. By FTIR analysis, the main secondary structures were ß-sheet (49%) followed by α-helix (24%); both structures are considered to be ordered. Likewise, HPWF soluble proteins increased at basic pH and HPWF proteins were separated in 11 bands with molecular masses ranging from 97 kDa to 18 kDa by electrophoresis. With respect to techno-functional properties, HPWF presented good emulsion activity (51%) and high thermal emulsion stability (46%). In addition, HPWF retained 571% and 242% of water and oil by weight, respectively. Finally, the micrograph showed the predominance of protein structures and fibre fragments, and the presence of few lipids mostly trapped. These results showed that HPWF is an interesting source of plant-based proteins and walnut flour can be used to obtain high protein ingredients from non-traditional sources.

JrWRKY21 interacts with JrPTI5L to activate the expression of JrPR5L for resistance to Colletotrichum gloeosporioides in walnut.

Walnut (Juglans regia L.) anthracnose, induced by Colletotrichum gloeosporioides, is a catastrophic disease impacting the walnut industry in China. Although WRKY transcription factors play a key role in plant immunity, the function of the WRKY gene family in walnut resistance to C. gloeosporioides is not clear. Here, through transcriptome sequencing and quantitative real-time polymerase chain reaction (qRT-PCR), we identified a differentially expressed gene, JrWRKY21, that was significantly upregulated upon C. gloeosporioides infection in walnut. JrWRKY21 positively regulated walnut resistance to C. gloeosporioides, as demonstrated by virus-induced gene silencing and transient gene overexpression. Additionally, JrWRKY21 directly interacted with the transcriptional activator of the pathogenesis-related (PR) gene JrPTI5L in vitro and in vivo, and could bind to the W-box in the JrPTI5L promoter for transcriptional activation. Moreover, JrPTI5L could induce the expression of the PR gene JrPR5L through binding to the GCCGAC motif in the promoter. Our data support that JrWRKY21 can indirectly activate the expression of the JrPR5L gene via the WRKY21-PTI5L protein complex to promote resistance against C. gloeosporioides in walnut. The results will enhance our understanding of the mechanism behind walnut disease resistance and facilitate the genetic improvement of walnut by molecular breeding for anthracnose-resistant varieties.

Genetic imprints of grafting in wild iron walnut populations in southwestern China.

BACKGROUND: Anthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations. RESULTS: We sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations. CONCLUSIONS: Our results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ  and ex situ conservation.

Drought- and soil substrate-induced variations in root nonstructural carbohydrates result from fine root morphological and anatomical traits of Juglans mandshurica seedlings.

BACKGROUND: Nonstructural carbohydrates (NSCs) reflect the carbon supply status and affect the construction and development of plants. Previous studies have focused on the dynamics of NSCs among plant organs, however, few studies have paid attention to the synergistic variations between fine root traits and NSCs under drought based on the perspective of branch order roots. This study aims to explore the responses of fine root traits and NSCs among root orders of Juglans mandshurica seedlings under different drought intensities and soil substrates. The 2-year-old J. mandshurica potted seedlings were planted in three different soil substrates (humus, loam and sandy-loam soil) and subjected to four drought intensities (CK, mild drought T1, moderate drought T2 and severe drought T3) for 60 days. RESULTS: The root biomass of seedlings in sandy-loam soil under the same drought intensity was higher than that of seedlings in humus soil. With an increase in drought, the root biomass, average diameter, root tissue density and cortex thickness decreased significantly, and the specific root length, stele diameter and conduit density increased. The root NSC contents in humus soil were higher than those in sandy-loam soil. The fine root soluble sugar content in all soil substrates decreased with increasing drought intensity, while the root starch and total NSC contents varied among the different soil substrates. Compared with transportive roots, the morphological and anatomical traits jointly explained the higher variation in NSC contents of the absorptive roots. The anatomical traits explained the higher variation in the NSC content of first five order roots. CONCLUSION: Our results suggest that coordinated adaptation of the root traits and NSCs of Manchurian walnut seedlings exposed to water gradients in different soil substrates.

Genome-wide identification, characterization, and expression pattern of the late embryogenesis abundant (LEA) gene family in Juglans regia and its wild relatives J. mandshurica.

BACKGROUND: Late Embryogenesis Abundant (LEA) proteins are a class of proteins associated with plant stress resistance. Two Juglans species, Juglans regia and J. mandshurica, are both diploid (2n = 32), monoecious perennial economic tree species with high edible, pharmaceutical, and timber value. The identification, characterization, and expression patterns of LEA proteins in J. regia and its wild relative, J. mandshurica, would not only provide the genetic basis of this gene family, but it would also supply clues for further studies of the evolution and regulating mechanisms of LEA proteins in other tree species. RESULTS: In this study, we identified 25 and 20 members of the LEA gene family in Juglans regia and its wild relative, Juglans mandshurica, respectively. The results of phylogenetic analysis showed that the LEA members were divided into eight main subgroups. Predictions of their physicochemical properties showed the variable characteristics of LEA proteins, and the subcellular localization analysis indicated that most LEA proteins are localized in the nucleus. Chromosomal localization analysis and gene replication pattern prediction indicated that WGD is the predominant duplication mode of LEA genes. The results of the comparative analysis indicated a high level of collinearity between the two Juglans species. Analysis of cis-acting elements indicated that LEA genes had a relatively wide range of responses to abiotic stresses and phytohormonal processes, particularly in two phytohormones, methyl jasmonate and abscisic acid. Transcriptome profiling and qRT-PCR experiments showed that JrLEAs are commonly expressed in leaves, green husks, and male and female flowers, and most JmLEAs are more highly expressed in male flowers. We also hypothesized that JrLEAs are involved in the process of anthracnose resistance. Anthracnose-resistant varieties of JrLEAs presented relatively high expression levels at later stages. CONCLUSION: In this study, we provide a theoretical basis for the functional study of LEA genes in J. regia and J. mandshurica. Analysis of cis-acting elements and gene expression indicated that JrLEAs and JmLEAs play important roles in resistance to biotic stresses in these species.