Pecan (Carya illinoinensis), as a popular nut tree, has been widely planted in China in recent years. Grafting is an important technique for its cultivation. For a successful grafting, graft union development generally involves the formation of callus and vascular bundles at the graft union. To explore the molecular mechanism of graft union development, we applied high throughput RNA sequencing to investigate the transcriptomic profiles of graft union at four timepoints (0 days, 8 days, 15 days, and 30 days) during the pecan grafting process. After de novo assembly, 83,693 unigenes were obtained, and 40,069 of them were annotated. A total of 12,180 differentially expressed genes were identified between by grafting. Genes involved in hormone signaling, cell proliferation, xylem differentiation, cell elongation, secondary cell wall deposition, programmed cell death, and reactive oxygen species (ROS) scavenging showed significant differential expression during the graft union developmental process. In addition, we found that the content of auxin, cytokinin, and gibberellin were accumulated at the graft unions during the grafting process. These results will aid in our understanding of successful grafting in the future.

Background: Calcium (Ca²⁺) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca²⁺ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca²⁺ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop. Results: In the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT–PCR analysis. Conclusions: The characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

Understanding microbial communities associated with bulk and rhizosphere soils will benefit the maintenance of forest health and productivity and the sustainable development of forest ecosystems. Based on MiSeq sequencing, we explored the differences between the bulk soil and the rhizosphere soil on bacterial and fungal communities of pecan plantation. Results suggested that rhizosphere-associated fungal rather than bacterial community structures differed from bulk soil, and rhizosphere soil had lower fungal diversity than bulk soil. Actinobacteria and Cantharellales were the bacterial and fungal biomarkers of the rhizosphere soil of pecan plantation, respectively. In addition, Pleosporales, which are mainly involved in saprophylaxis and plant pathogenic processes, was identified as one of the most important fungal biomarkers for the bulk soil, and the FunGuild predicted a higher relative abundance of pathogenic fungi in bulk soil compared to rhizosphere soil. The pH, ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3--N), and total carbon (TC) contents drove microbial community structure and composition. The bacterial network was simpler in the rhizosphere soil than in the bulk soil. However, fungi showed the opposite network pattern. Keystone species in bacterial and fungal networks were mostly involved in nutrient cycling and the C cycling, and were found to be enriched in the rhizosphere soil. Overall, in terms of bacterial and fungal communities, the rhizosphere soil behaves more healthily than the bulk soil and has a higher potential for nutrient cycling.

Although ammonia–nitrogen (NH₄⁺–N) and nitrate–nitrogen (NO₃⁻–N) are the two main forms of N absorbed and utilized by plants, the preferences of plants for these forms are still unclear. In this study, we analyzed the growth, photosynthesis, and nutrients of pecan under different NH₄⁺:NO₃⁻ ratios (0/0, 0/100, 25/75, 50/50, 75/25, 100/0) by indoor aerosol incubation. The results showed that additions of different N forms promoted the growth and development of pecan seedlings. When NO₃⁻–N was used as the sole N source, it significantly promoted the ground diameter growth of pecan and increased the leaf pigment content and photosynthetic rate. The NH₄⁺:NO₃⁻ ratio of 75:25 and NH₄⁺–N as the sole N source significantly increased the soluble sugars in stems and roots, starch in leaves, stems and roots, soluble protein in leaves and stems, and soluble phenols in stems and roots. Additionally, the NH₄⁺:NO₃⁻ ratio of 75:25 increased plant height, leaf number, root soluble protein, and leaf soluble phenol contents. In conclusion, regarding the physiological aspects of pecan growth, pecans are more inclined to use NH₄⁺–N. Considering that the NH₄⁺–N as the only N source may lead to nutrient imbalance or even toxicity, the NH₄⁺:NO₃⁻ ratio of 75:25 was most favorable for the growth and development of pecan seedlings.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a high-value fruit tree with a long juvenile period. The fruiting process of pecan seedlings can be largely accelerated through grafting. As non-coding small RNAs, plant miRNAs participate in various biological processes through negative regulation of gene expression. To reveal the roles of miRNAs in the graft union development of pecan, four small RNA libraries were constructed from the graft union at days 0, 8, 15, and 30 after grafting. A total of 47 conserved miRNAs belonging to 31 families and 39 novel miRNAs were identified. For identified miRNAs, 584 target genes were bioinformatically predicted, and 266 of them were annotated; 29 miRNAs (including 16 conserved and 13 novel miRNAs) were differentially expressed during the graft process. The expression profiles of 12 miRNA were further validated by quantitative reverse transcription PCR (qRT-PCR). In addition, qRT-PCR revealed that the expression levels of 3 target genes were negatively correlated with their corresponding miRNAs. We found that miRS26 might be involved in callus formation; miR156, miR160, miR164, miR166, and miRS10 might be associated with vascular bundle formation. These results indicate that the miRNA-mediated gene regulations play important roles in the graft union development of pecan.