Abstract

This study developed a micrografting method for conifers, enabling efficient grafting between closely and distantly related species. Graft junctions rapidly formed vascular connections and activated thousands of genes, including those involved in auxin signalling and cell wall modification. Comparison of conifer grafting with Arabidopsis grafting revealed shared gene activation patterns related to vascular differentiation. Analyses identified PAT1 as a central regulator of graft healing, with its homologs essential for tissue attachment and callus formation. Overall, the study demonstrates the ability of young tissues to graft across species and identifies the conserved role of the PAT gene family in graft healing and tissue regeneration.

Background

Grafting combines desirable traits from different plants and is commonly utilised for improving stress tolerance, varietal retention, and yield enhancement. While predominantly applied in eudicots, gymnosperms play a crucial role in forestry and horticulture breeding programs yet remain underexplored in grafting studies. Graft incompatibility, particularly evident across genera, poses a significant obstacle, attributed to structural, metabolic, or defence-related factors. However, instances of successful intergenus grafts in specific plant families indicate exceptions to this rule. Recent advances, such as micrografting, facilitate improved grafting efficiency and offer insights into the grafting process itself. Studies in model plants like Arabidopsis highlight a dynamic healing process involving cell expansion, callus formation, and tissue differentiation, with genes such as ERF115 and PAT1 playing pivotal roles. This study delves into graft formation in widespread conifer species, introducing an efficient grafting method using young plants. Characterisation of graft healing unveils a shared pathway between Norway spruce and Arabidopsis, highlighting cell division, vascular differentiation, and the involvement of cell-wall- and auxin-related genes. The investigation identifies PAT1 as a conserved regulator of wound healing across gymnosperms and eudicots, shedding light on the mechanistic underpinnings of graft formation. Overall, the study advances understanding of grafting dynamics in conifers and elucidates conserved pathways essential for successful graft healing.

Sequencing strategy

RNA Extraction: Total RNA was extracted from frozen tissue using a modified CTAB/LiCl method. Ground tissue was incubated with extraction buffer, and RNA was extracted using chloroformisoamyl alcohol. RNA was precipitated overnight with LiCl, washed, and dissolved in RNasefree water. RNA concentration and integrity were assessed using Qubit and Agilent 2100 Bioanalyzer, respectively.

Library Preparation: RNA-seq libraries were prepared using 1 μg of total RNA per sample. For graft junction libraries, mRNA isolation was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module, followed by library construction using NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Library quality was checked using Agilent 2100 Bioanalyzer. Hormone treatment libraries were constructed by Novogene (UK).

Sequencing platform: Sequencing was performed by Novogene (UK) on the Illumina NovaSeq 6000 system.

Bioinformatic Analysis: RNA-seq data were processed using FastQC, SortMeRNA, and fastp. Filtered reads were aligned to the Picea abies genome using STAR. Read counts were quantified using HTSeq, and DEGs were identified using DESeq2. Common gene expression patterns were analysed using the Mfuzz package. GO enrichment analysis was performed, and gene co-expression network analysis was conducted using WGCNA. Orthologs between Picea abies and Arabidopsis were obtained for comparative analysis.

Results

Micrografting Technique:

The micrografting method involved using 10- to 12-day-old spruce and pine seedlings, excising plants in the hypocotyl region, and tightly attaching scions and rootstocks from different plants using a silicon collar. This technique significantly improved grafting efficiency, with one person capable of performing 50 grafts per hour with over 90% success rate, a substantial enhancement over traditional methods.

Monitoring the dynamics of graft healing revealed rapid formation of xylem and phloem connections post-grafting. Successful grafts exhibited transport dynamics consistent with phloem and xylem connectivity within 20-25 days after grafting, leading to normal growth and high survival rates (90-100%) two months post-grafting. Micrografting was also tested for inter-genus grafting between Picea and Pinus species, demonstrating high survival rates (70-100%) even in heterografts. However, long-term observation revealed lower survival rates in some combinations, particularly in heterografted Picea-Pinus combinations.

Gene Expression Analysis:

RNA sequencing analyses provided insight into the molecular mechanisms underlying conifer grafting. Differentially expressed genes (DEGs) associated with various processes were identified in both scion and rootstock tissues at different time points post-grafting. A principal component analysis (PCA) revealed distinct clustering of samples based on tissue type and time point, indicating dynamic changes in gene expression during graft healing.

Utilising the Mfuzz algorithm, DEGs were grouped into 12 clusters, each representing different expression patterns across the grafting process. These clusters encompassed genes involved in wound response, defence mechanisms, cell cycle regulation, and cell wall development, among others. Notably, certain clusters showed both scion and rootstock up-regulation, suggesting coordinated gene expression between the graft partners.

A Gene Ontology (GO) analysis further elucidated the biological processes enriched within these gene clusters. Cluster 2, for instance, exhibited enrichment in wounding and defence-related processes, indicating the activation of stress response pathways post-grafting. In contrast, cluster 10 showed enrichment in cell wall- and xylem-related processes, highlighting the importance of cell wall modifications and vascular differentiation in graft healing.

Among the identified DEGs, several homologs of known grafting-related genes from other plant species were identified. These included early activating genes involved in wound response (e.g., PaWOX13-like), cell cycle regulation (e.g., PaCDKB2;2-like), and vascular differentiation (e.g., PaAPLlike, PaPRX66-like, PaVND4-like). Importantly, comparison with Arabidopsis grafting transcriptomics data revealed conserved expression patterns of certain genes, suggesting a shared regulatory network underlying grafting processes across diverse plant species.

Experiments were conducted to investigate the role of auxin and cytokine in Picea abies using synthetic cytokinin (BAP), auxin (IAA) or both. Focusing on auxin and cytokinin, differential expression analysis revealed thousands of genes responsive to the hormone treatment. Comparing with grafting, auxin-responsive genes were enriched in scions and rootstocks. In addition, many putative genes related to cell wall modifications were affected both by exogenous auxin treatment and grafting, indicating an increased auxin response at the graft junction.

Together, the gene expression analysis provided a comprehensive view of the molecular events driving graft healing in conifers, highlighting the orchestrated regulation of key genes involved in wound response, cell division, and vascular differentiation. These findings deepen our understanding of the grafting process and lay the groundwork for future studies exploring targeted manipulation of gene expression to enhance grafting success in conifers.

Transcriptional analysis identified a conserved regulatory response involving the up regulation of the PAT1 gene family, known for promoting wound healing. Functional analysis in both Arabidopsis and Picea abies confirmed the role of PAT1 genes in callus formation and graft attachment, indicating a conserved mechanism across species.

Conclusion

In conclusion, this study underscores the global significance of grafting in combining plant species to harness desirable traits, emphasising the imperative for developing efficient grafting methodologies across diverse plant species. Through the introduction of a rapid micrografting technique, this study successfully expanded the repertoire of compatible graft combinations among various conifer species, offering unprecedented insights into graft formation dynamics across genera that diverged over millions of years. Despite previous limitations in conifer micrografting, this approach exhibited notably high success rates, particularly in inter-species graft combinations, albeit with some instances of delayed incompatibility.

Transcriptome analyses unveiled dynamic gene activation patterns reminiscent of those observed in Arabidopsis graft junctions, indicative of a conserved grafting process across gymnosperms and angiosperms. Notably, the identification of PAT1 as a crucial regulator of tissue attachment and graft formation in Arabidopsis, along with evidence of its induction by wounding in rice, suggests its broader role in wound healing across plant species. The success of divergent conifer grafts highlights the efficacy of grafting young tissues in overcoming compatibility barriers, offering a promising avenue for expanding the spectrum of successful grafting in seed plants. These findings not only deepen understanding of grafting mechanisms but also present a valuable tool for enhancing plant breeding and horticultural practices. Further research, including functional studies in mutated Picea abies, is warranted to elucidate the endogenous role of PAT1 in wound healing and graft formation, thus paving the way for future advancements in plant grafting technologies.