Identification and Characterization of Wall-Associated Kinase (WAK) and WAK-like (WAKL) Gene Family in Juglans regia and Its Wild Related Species Juglans mandshurica

Wall-associated kinase (WAK) and WAK-like kinase (WAKL) are receptor-like kinases (RLKs), which play important roles in signal transduction between the cell wall and the cytoplasm in plants. WAK/WAKLs have been studied in many plants, but were rarely studied in the important economic walnut tree. In this study, 27 and 14 WAK/WAKL genes were identified in Juglans regia and its wild related species Juglans mandshurica, respectively. We found tandem duplication might play a critical role in the expansion of WAK/WAKL gene family in J. regia, and most of the WAK/WAKL homologous pairs underwent purified selection during evolution. All WAK/WAKL proteins have the extracellular WAK domain and the cytoplasmic protein kinase domain, and the latter was more conserved than the former. Cis-acting elements analysis showed that WAK/WAKL might be involved in plant growth and development, plant response to abiotic stress and hormones. Gene expression pattern analysis further indicated that most WAK/WAKL genes in J. regia might play a role in the development of leaves and be involved in plant response to biotic stress. Our study provides a new perspective for the evolutionary analysis of gene families in tree species and also provides potential candidate genes for studying WAK/WAKL gene function in walnuts.

Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Transcriptome-based analysis of the hormone regulation mechanism of gender differentiation in Juglans mandshurica Maxim

Juglans mandshurica Maxim is a hermaphroditic plant belonging to the genus Juglans in the family Juglandaceae. The pollination period of female flowers is different from the loose powder period of male flowers on the same tree. In several trees, female flowers bloom first, whereas in others, male flowers bloom first. In this study, male and female flower buds of J. mandshurica at the physiological differentiation stage were used. Illumina-based transcriptome sequencing was performed, and the quality of the sequencing results was evaluated and analyzed. A total of 138,138 unigenes with an average length of 788 bp were obtained. There were 8,116 differentially expressed genes (DEGs); 2,840 genes were upregulated, and 5,276 genes were downregulated. The DEGs were classified by Gene Ontology and analyzed by Kyoto Encyclopedia of Genes and Genomes. The signal transduction factors involved in phytohormone synthesis were selected. The results displayed that ARF and SAUR were expressed differently in the auxin signaling pathway. Additionally, DELLA protein (a negative regulator of gibberellin), the cytokinin synthesis pathway, and A-ARR were downregulated. On April 2nd, the contents of IAA, GA, CTK, ETH and SA in male and female flower buds of two types of J. mandshurica were opposite, and there were obvious genes regulating gender differentiation. Overall, we found that the sex differentiation of J. mandshurica was related to various hormone signal transduction pathways, and hormone signal transduction plays a leading role in regulation.

Different Stand types Alter Soil Properties and Microbial Community in Warm Temperate Forest

Soil microorganism play an important role in maintaining the structure and function in warm temperate forest ecosystem. In order to explore the characteristics of soil microbial community under different stand types in in warm temperate zone, Illunima Miseq High-throughput Sequencing was used to assess the soil bacteria (16S rRNA) and fungi (ITS rRNA) communities of five forest stands (Pinus tabulaeformis [PT], Juglans mandshurica [JM], Betula platyphylla [BP], Betula dahurica [BD] and Quercus mongolica [QM]) in Songshan Nature Reserve. The results showed that the bacterial diversity under Juglans mandshurica forest was higher than other types, the fungal diversity under Pinus tabulaeformis forest was higher than other types. The dominant phyla and gene of soil bacteria were similar in different stand types, but there were significant differences in abundance and dominant gene of fungal community. VPA analysis showed that soil explained 49.1% of the variance in bacterial community composition and 70.6% of the variance in fungal community composition. RDA analysis showed that the dominant phyla were significantly correlated with soil pH, SOM, TN and AN. Based on our results, there are significant differences in soil microbial community structure among different stand types. Consequently, our results have important implications for understanding the driving mechanisms that control the soil microbial community during warm temperate forest.

Pharmacognostic Identification of the Green Peel of Juglans mandshurica Maxim. from Different Regions and Harvest Times

Background: The green peel of Juglans mandshurica Maxim. (QLY) can be used as medicine, also known as Qinglongyi[1]. The aim of this study was to identify QLY from the different regions and harvest times by pharmacognostic identification.Methods: In this study, the morphological character, microscopic character and juglone’s content of QLY from different regions and harvest times were compared by morphological identification, paraffin section and powder section, physico-chemical identification and High Performance Liquid Chromatography (HPLC).Results: Morphological identification results that the colour of outer surfaces of peels which harvested from Tangyuan county of Heilongjiang Province in July, August and September are respectively yellow brown, tan and black brown , and the color of the solution graded gradually from light brown yellow to brownish black by water-based test (WBT). The colour of outer surfaces of peels which harvested from Changbai county of Jilin Province in July, August and September are respectively yellowish white, brownish yellow and pale brown, and the color of the solution gradually changed from light brown to dark brown by WBT. As well as the colour of outer surfaces of peels which harvested from Qingyuan county of Liaoning Province in July, August and respectively September are Light yellowish green, light brownish green and brown green, and the color of the solution gradually changed from light green to dark brown by WBT. With the increase of growth period, the number of microscopic characteristics of different Qinglongyi increased. Through physico-chemical identification, it was found that the yield of juglone in QLY picked on September 21, 2016 was the highest, and the content of juglone, whose order was Heilongjiang > Jilin > Liaoning, was as high as 203.476 μg/g.Conclusions: Through comparison, it is concluded that Heilongjiang Province is one of the high-quality producing areas of QLY, and its best harvest time is in the middle and first ten-day period of September to harvest QLY without softening, yellowing and decay. This experiment provides a theoretical basis for the determination of the best harvest conditions in QLY and the establishment of identification standards for medicinal materials.