https://orcid.org/0000-0001-8459-4931
Dear Editor
The application of transgenic and gene editing technologies in perennial woody plants has been largely limited by low transformation efficiency (Hu et al., 2016). For many commercially important fruit tree species, e.g. apple (Malus domestica Bork.), low-shoot regeneration frequency is a major bottleneck for the success of transformation (Chen et al., 2022). GL-3 is a genotype with high regeneration ability screened out from the seedling population of Royal Gala, with a regeneration efficiency substantially higher than its parent (Dai et al., 2013). Here, we integrated the comparative analysis of transcriptome and small RNAs between Royal Gala and GL-3 to mine key genes controlling shoot regeneration and CRISPR/Cas9 technology to enhance shoot regeneration efficiency.
CRISPR/Cas9-mediated editing of MdSPL6 gene to improve regeneration efficiency in apple. A, Comparison of shoot regeneration ability showing higher regeneration efficiency in GL-3 when compared with Royal Gala, scale bar = 2 mm. The individual images were digitally extracted for comparison. B, Heatmap illustrating the opposite expression of mdm-miR156aa between Royal Gala and GL-3 during the shoot regeneration process. C, mdm-miR156aa overexpressed GL-3 transgenic lines exhibiting higher shoot regeneration ability than wild type, scale bar = 2 mm. D, WGCNA of DEGs involved in adventitious shoot formation. The upper panel showing clustering dendrogram of 15 modules based on the dissimilarity topological overlap of genes across all samples; the middle panel showing gene expression of “magenta” module (rows corresponding to genes, columns referring to samples, red indicating high expression, green indicating low expression); the lower panel showing cytoscape of co-expressed genes in the “magenta” module. Six major hub genes were identified and marked. E, F, Overexpressing MdSPL6 in tobacco inhibited its shoot regeneration (E) without affecting the growth phenotype (F), scale bar = 10 mm in (E) and 10 cm in (F). G, The leaves of Nicotiana benthamiana were used to carry the dual-luciferase sensor system, and the targeted degradation capability of mdm-miR156aa on MdSPL6 were visualized by LUC activities. The red color reflects higher LUC activities while purple reflects lower LUC activities. The luminescence intensities were determined by LUC/REN. LUC, luciferase activities; REN, Renilla luciferase activities. H, Schematic presentation of pKSE401-Cas9-MdSPL6 construct with genotypes of selected mutants. Heterozygous: one wild allele and one mutation allele. Homozygous: identical variations for both two alleles, scale bar = 2 mm. The lower panel exhibiting the regeneration ability of selected mutant lines. The individual images were digitally extracted for comparison. I, TA cloning followed by Sanger sequencing to verify homozygous and bi-allelic mutants of Mdspl6 (#5). Data are shown as means ± SE. Asterisks represent significant differences among different samples using two-tailed Student's t test (**P ≤ 0.01).
We collected GL-3 and Royal Gala leaf explants at five time points (0, 1, 7, 14, and 21 days) during shoot regeneration (Figure A). Approximately 125.9 million unique reads were obtained and analyzed (Supplemental Figures 1 and 2, Table 1). We further subjected the identified 721 known miRNAs (belonging to 54 reported families; Supplemental Figure 3) and 1,765 newly identified miRNAs to pairwise comparisons and found 878 differentially expressed miRNAs (Supplemental Figure 4). mdm-miR156aa (microRNA156aa) was observed to be constitutively up-regulated in GL-3 (Figure B). To confirm the role of mdm-miR156aa in adventitious shoot regeneration, we overexpressed the precursor of mdm-miR156aa (MdMIR156aa) in apple and observed significantly enhanced adventitious shoot formation compared with wild type (Figure C). It has been reported that the targets of miR156 belong to a group of SQUAMOSA PROMOTER BINDNG PROTEIN-LIKE (SPL) transcription factors (Gandikota et al., 2007). Therefore, the enhanced regeneration ability in GL-3 might be due to the target degradation of SPL genes, which could be employed as the editing targets for enhancing shoot regeneration ability.
We performed time-course RNA-seq analysis of Royal Gala and GL-3 at similar time points as miRNA sequencing and generated ∼44.15–65.12 million reads for each sample (Supplemental Table 2). A total of 15,994 unique differentially expressed genes (DEGs) were identified. A weighted gene co-expression network analysis (WGCNA) was conducted by employing transcriptome data as well as phenotypes of shoot formation and 15 distinct gene co-expression modules (labeled with different colors) were identified. The genes in the magenta module showed inhibited expression in GL-3 compared with those in Royal Gala during shoot regeneration (Figure D). In this network, we found MD06G1204000 (MdSPL6, SQUAMOSA PROMOTER BINDNG PROTEIN-LIKE 6) is one hub gene (Supplemental Figure 5). MdSPL6 was exclusively localized to the nucleus, suggesting its function as a transcription factor (Supplemental Figure 6). To test its function, we overexpressed MdSPL6 in tobacco (Nicotiana tabacum), a species highly capable of shoot regeneration, and observed markedly reduced shoot regeneration efficiency compared with the wild-type explants (Figure E). The growth and developmental patterns of transgenic tobacco plants overexpressing MdSPL6 were similar to those of the wild type (Figure F). Using Target Finder software, we identified MdSPL6 as a potential target for mdm-miR156aa. Moreover, we verified the target relationship between mdm-miR156aa and MdSPL6 using the dual-luciferase sensor system (Figure G). Additionally, we observed an inhibited expression of MdSPL6 in MdMIR156aa overexpressing transgenic apple compared with the wild type (Supplemental Figure 7). Therefore, the improved regeneration ability of GL-3 is likely caused by mdm-miR156aa-mediated degradation of MdSPL6, and MdSPL6 could be a target for improving regeneration efficiency using CRISPR/Cas9 techniques.
Although GL-3 is a genotype with relative high regeneration ability compared with its parent Royal Gala and other cultivars, the transformation efficiency remains low compared with several easily-transformable dicot species, such as tobacco, tomato (Solanum lycopersicum), or poplar (Populus tomentosa). Therefore, GL-3 was used as the experimental materials to demonstrate the effects of CRISPR/Cas9-mediated knockout of the MdSPL6 gene on shoot regeneration efficiency. Two sites (MdSPL6-T1 and MdSPL6-T2) in the first and third exon of MdSPL6 were chosen to design sgRNAs, and Agrobacterium was used to transform GL-3 (Figure H). We amplified the target regions of MdSPL6-T1 and MdSPL6-T2, and subjected the PCR products to Sanger sequencing, separately. Ten out of 13 transgenic lines had editing events with superimposed sequencing chromatograms in at least one target site (Ma et al., 2015). TA cloning followed by Sanger sequencing was further employed to confirm the editing events for six selected mutants. For the MdSPL6-T1 target site, one plant (#4) was heterozygous (wild-type/single mutation), one plant (#3) was homozygous (two identical variations), and two plants (#2, #5) were bi-allelic (two distinct variations). For the MdSPL6-T2 target site, one plant (#1) was heterozygous, two plants (#3, #5) were homozygous, and two plants (#4, #8) were bi-allelic. Next, we analyzed shoot regeneration efficiency in three lines (#1, #4, #5). Line #1 (no mutation at T1 site, heterozygous mutation with 1-bp deletion at T2 site) exhibited similar shoot regeneration efficiency as the wild type. Lines #4 (heterozygous mutation with 3-bp deletion at T1 site, bi-allelic mutations with 1-bp insertion, and 15-bp deletion at T2 site) exhibited an increase of 109% in shoot regeneration efficiency compared with the wild type. Line #5 (bi-allelic mutation with 1-bp insertion and 4-bp deletion at T1 site, homozygous mutation with 1-bp deletion at T2 site) had shoot regeneration efficiency increased by 122% relative to the wild type (Figure H and I). We have found that knocking out MdSPL6 exhibited no significant effects on overall shoot and root growth (Supplemental Figure 8). Although Mdspl6 knockout mutant can be useful for enhancing shoot regeneration of apple and other woody plants, we cannot exclude any possible undesirable side effects that may be observed at later or mature stages of plant development. Therefore, if carrying functional studies for specific genes using the Mdspl6 mutant, we suggest using the non-transgenic mutant as a control.
Our combined approaches, i.e. small RNA and transcriptome sequencing analyses, a dual-luciferase sensor assay, as well as transgenic studies, indicate that mdm-miR156aa-MdSPL6 plays a role in adventitious shoot formation in apple. Using CRISPR/Cas9 technology, we have shown that mutating MdSPL6 could significantly improve shoot regeneration efficiency. Because shoot regeneration is a key step for genetic transformation, targeted mutations of MdSPL6 may provide a useful approach to facilitate the use of transgenic and gene editing technologies in apple and other rosaceous fruit trees.
Supplemental data
The following materials are available in the online version of this article.
Supplemental Figure S1. The length distribution of total and unique-mapped small RNA reads from high-throughput small RNA sequencing data.
Supplemental Figure S2. The distribution statistics of identified miRNAs groups.
Supplemental Figure S3. Number of known miRNA families identified during shoot regeneration in apple.
Supplemental Figure S4. Pairwise comparisons of miRNAs among different time points during the regeneration process.
Supplemental Figure S5. Phylogenetic analysis verified the closest relationship between MD06G1204000 (MdSPL6) and AtSPL6.
Supplemental Figure S6. Subcellular localization of MdSPL6 in Nicotiana benthamiana.
Supplemental Figure S7. Relative expression levels of the MdSPL6 gene in two representative MdMIR156aa-OE apple lines.
Supplemental Figure S8. Growth and development of apple Mdspl6 mutant.
Supplemental Table S1. The small RNA sequencing data generated for 30 samples using the Illumina sequencing platform.
Supplemental Table S2. Summary of transcriptome sequencing data.
Acknowledgment
The authors thank Dr Qijun Chen for kindly providing the CRISPR plasmids.
Funding
This work was supported by the National Natural Science Foundation of China (32072529, 31801823), the earmarked fund for the China Agricultural Research System (CARS-27), 2115 Talent Development Program of China Agricultural University, and 111 Project (B17043).
References
Author notes
Z.H. and W.L. conceived and designed the project; H.L., H.S., H.D., S.W., and X.F. performed the experiments and analyzed the data; Y.L., L.C., Z.Z., Y.W., X.Z., and X.X. provided suggestions on experiments and manuscript editing; W.L., Z.H., and H.L. wrote and edited the manuscript. All authors read and approved the manuscript.
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) are: Wei Li ([email protected]) and Zhenhai Han ([email protected]).
Conflict of interest statement. None declared.
