Metabolome and Transcriptome Profiling Unveil the Mechanisms of Polyphenol Synthesis in the Developing Endopleura of Walnut (Juglans regia L.).

Walnut (Juglans regia L.) is an important woody nut tree species, and its endopleura (the inner coating of a seed) is rich in many polyphenols. Thus far, the pathways and essential genes involved in polyphenol biosynthesis in developing walnut endopleura remain largely unclear. We compared metabolite differences between endopleura and embryo in mature walnuts, and analyzed the changes of metabolites in endopleura at 35, 63, 91, 119, and 147 days after pollination (DAP). A total of 760 metabolites were detected in the metabolome, and the polyphenol contents in endopleura were higher than those in embryos. A total of 15 types of procyanidins, 10 types of kaempferol glycosides, and 21 types of quercetin glycosides that accumulated during endopleura development were identified. The analysis of the phenylpropane metabolic pathway showed that phenylalanine was gradually transformed into proanthocyanidins and other secondary metabolites with the development of endopleura. A total of 49 unigenes related to polyphenol synthesis were identified by transcriptome analysis of endopleura. The expression patterns of PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, and ANR were similar, and their expression levels were highest in endopleura at maturity. Transcriptome and metabolome analysis showed that endopleura rapidly synthesized and accumulated polyphenols during maturation. Moreover, the transcription factor MYB111 played an important role in synthesizing polyphenols in endopleura, and its expression pattern was positively correlated with the accumulation pattern of quercetin, kaempferol, and proanthocyanidins. MYB111 was co-expressed with NAP, NAC, ATR1, and other genes related to cell senescence and abiotic stress response. Our study analyzed the composition and molecular synthesis mechanism of polyphenols in walnut endopleura, and provided new perspectives and insights regarding the nutritional research of walnut nuts.

Phenotypic Differences and Physiological Responses of Salt Resistance of Walnut with Four Rootstock Types.

Walnut is one of the world's four largest nuts. Currently, the bottleneck in walnut breeding is the production of resistant variants. Soil salinization is a global problem, and the use of salt-tolerant rootstocks is a basic strategy to overcome the challenge of sustained walnut production. Providing a scientific basis for the selection of walnut salt-tolerant rootstocks is possible by studying the physiological and biochemical response characteristics and salt tolerance variations of different walnut genotypes under salt stress. In the present study, seedlings of four genotypes of walnut rootstocks, including J1 (Juglans hindsii), J2 (J. mandshurica), J3 (J. regia × J. mandshurica), and J4 (J. regia × J. hindsii), were employed as test materials to conduct a 28-day pot experiment under NaCl stress with five NaCl concentrations (0, 50, 100, 200, and 300 mmol/L). Under different NaCl treatment concentrations, seedling morphology, growth indices, chlorophyll content, photosynthetic parameters, relative electrical conductivity (REC), malondialdehyde (MDA), proline (Pro), soluble sugar (SS), and the activity of superoxide dismutase (SOD) and peroxidase (POD) in the leaves were examined. Salt stress altered the morphological characteristics and growth indices of seedlings from four genotypes to varying degrees. In addition, according to the analysis of physiological and biochemical data, salt stress had a considerable impact on both the physiological and biochemical processes of seedlings. Salt stress decreased the chlorophyll content and photosynthetic parameters of four genotypes, the REC, MDA content, Pro content, and SS content of each genotype increased by different degrees, and the enzymatic activities showed different trends. The salt tolerance of rootstocks was evaluated thoroughly using principal component analysis and membership function analysis based on the 16 parameters. The results of a comprehensive evaluation of salt tolerance showed that the order of salt tolerance of the four genotypes was J4 > J1 > J3 > J2, which corresponded to the order of the morphological symptoms of salt injury. In conclusion, J4 has strong salt tolerance and is an important germplasm resource for walnut salt-tolerant rootstock breeding.

Intercropping Walnut and Tea: Effects on Soil Nutrients, Enzyme Activity, and Microbial Communities.

The practice of intercropping, which involves growing more than one crop simultaneously during the same growing season, is becoming more important for increasing soil quality, land-use efficiency, and subsequently crop productivity. The present study examined changes in soil physicochemical properties, enzymatic activity, and microbial community composition when walnut (Juglans spp.) was intercropped with tea (Camellia sinensis L.) plants in a forest and compared with a walnut and tea monocropping system. The results showed that walnut-tea intercropping improved the soil nutrient profile and enzymatic activity. The soil available nitrogen (AN), available phosphorus (AP), available potassium (AK), organic matter (OM) content, and sucrase activity were significantly boosted in intercropped walnut and tea than in monocropping forests. The interaction between crops further increased bacterial and fungal diversity when compared to monoculture tea forests. Proteobacteria, Bacteroidetes, Firmicutes, Chlamydiae, Rozellomycota, and Zoopagomycota were found in greater abundance in an intercropping pattern than in monoculture walnut and tea forest plantations. The walnut-tea intercropping system also markedly impacted the abundance of several bacterial and fungal operational taxonomic units (OTUs), which were previously shown to support nutrient cycling, prevent diseases, and ameliorate abiotic stress. The results of this study suggest that intercropping walnut with tea increased host fitness and growth by positively influencing soil microbial populations.

Soil Chemical and Microbiological Properties Are Changed by Long-Term Chemical Fertilizers That Limit Ecosystem Functioning.

Although the effects of fertilization and microbiota on plant growth have been widely studied, our understanding of the chemical fertilizers to alter soil chemical and microbiological properties in woody plants is still limited. The aim of the present study is to investigate the impact of long-term application of chemical fertilizers on chemical and microbiological properties of root-associated soils of walnut trees. The results show that soil organic matter (OM), pHkcl, total nitrogen (TN), nitrate-nitrogen (NO3-), and total phosphorus (TP) contents were significantly higher in non-fertilized soil than after chemical fertilization. The long-term fertilization led to excessive ammonium-nitrogen (NH4+) and available phosphorus (AP) residues in the cultivated soil, among which NH4+ resulted in soil acidification and changes in bacterial community structure, while AP reduced fungal diversity. The naturally grown walnut trees led to an enrichment in beneficial bacteria such as Burkholderia, Nitrospira, Pseudomonas, and Candidatus_Solibacter, as well as fungi, including Trichoderma, Lophiostoma, Phomopsis, Ilyonectria, Purpureocillium, Cylindrocladiella, Hyalorbilia, Chaetomium, and Trichoglossum. The presence of these bacterial and fungal genera that have been associated with nutrient mobilization and plant growth was likely related to the higher soil OM, TN, NO3-, and TP contents in the non-fertilized plots. These findings highlight that reduced chemical fertilizers and organic cultivation with beneficial microbiota could be used to improve economic efficiency and benefit the environment in sustainable agriculture.

Preharvest and postharvest UV radiation affected flavonoid metabolism and antioxidant capacity differently in developing blueberries (Vaccinium corymbosum L.).

Flavonoids can protect plants against UV but the mechanism by which specific flavonoids during fruit development is unclear, especially in blueberries on living plants. We analyzed the gene expression and metabolite profiles of flavonols, proanthocyanidins (PAs), and anthocyanins under preharvest UV-B/-C and postharvest UV-A/-B/-C irradiation in developing blueberries. Both pre- and postharvest UV irradiation significantly increased flavonol accumulation during early fruit development, while increased anthocyanin and PA contents during late fruit development. However, PAs decreased during postharvest but increased during preharvest UV irradiation in green fruit. The antioxidant capacity increased by postharvest UV irradiation, while hardly affected by preharvest UV irradiation. Overall, the gene expression changes paralleled the flavonoid contents after UV irradiation. Notably, VcMYBPA1 was closely related with VcLAR and VcANR under pre- and postharvest UV irradiation, which could relate to PA biosynthesis. During natural fruit maturation and UV conditions, the elevated PA content exhibited higher potential antioxidant activity. Our results show that UV resistance is greater in living plants than detached fruits, the former showing a systemic and moderate response and the latter a non-systemic but strong response. These results might contribute to the development of pre- and postharvest technologies to promote healthier fruit consumption.