Gene expression
To determine the molecular basis of the physiological changes, the same graft combinations were grown for 200 days and samples of roots taken for whole gene transcriptome profiling using microarrays. We also used RT-qPCR to confirm the expression of selected genes. More than 1300 transcripts were differentially expressed in NCED OE rootstocks, compared to WT. From this set, more than 850 were down-regulated, while almost 500 were up-regulated. A common set of 365 and 237 genes were down- and up-regulated in SP rootstocks, compared to WT grafts (Figure 5a, b; Table S2-S5). To interpret the gene expression data in a physiological context, we focused on analyzing DEG related to hormone pathways. We started by focusing on ABA-related genes because of the known role of NCED. Both PCR and transcriptomic data showed thatSlNCED1 gene expression was higher in SP5 than SP12 (Figure 6a; Table S2 and S3), confirming previous results (Thompson et al.2007a; Martínez-Andújar et al. 2020b).
Other ABA-metabolic genes were mostly not affected, corroborating their lack of differential regulation in roots of whole plants under control conditions (Martínez-Andújar et al. 2020b). AREB1(Solyc04g078840) and ATHB12 (Solyc01g096320) were induced in SP12 and SP5 rootstocks respectively, while other ABA signalling-related genes WRKY s (e.g. WRKY80/WRKY6 , Solyc03g095770) and ABA-receptor PYL s (e.g., PYL6 , solyc05g052420) were down-regulated in the NCED OE grafts, indicating a reduced response/or sensitivity to ABA compared to the WT (Figure 6a). Thus, increasedSlNCED1 gene expression altered some ABA signalling components, but there was no widespread effect on ABA signalling responses.
Regarding stress-related genes (Figure 6b; Table S2 and S3), theTAS14 (Solyc02g084850), KIN2 (Solyc03g095510) ,LEA (Solyc03g116390), MYB49 (Solyc10g008700) andMYB62 (Solyc03g119370) were upregulated in SP12 rootstocks, while most of those and other MYB genes were not affected or down-regulated in SP5 rootstocks (Figure 6a). Most aquaporin PIPgenes analyzed were down-regulated in NCED OE rootstocks (Figure 6c), while PIP1.7 (Solyc03g096290) in SP5 and NIP6.1(Solyc03g117050) in SP12 were upregulated (Figure 6c). Thus, increasedSlNCED1 gene expression either directly or indirectly generally decreased genes associated with response to stress and water transport.
Rootstock NCED overexpression seems to interact with other hormone-related genes in the roots. These rootstocks downregulatedIPT7 (Solyc01g080150), and a beta-glucosidase gene (Solyc03g119080) involved in biosynthesis of bioactive CKs (Figure 7a). The ACC synthase genes (ACC2 , Solyc01g095080; ACS1a , Solyc08g081540) and most ethylene response factors (ERF s) genes were upregulated in both SP lines (Figure 7d). NCED OE rootstocks showed considerable changes in the ACC oxidase gene family, with 2 and 1 ACC oxidase genes upregulated in SP12 and SP5, respectively, while 13 ACC oxidase genes were downregulated in SP5 and 6 in SP12 (Figure 7d; Table S2 and S3). Thus, these results are consistent with NCED OE rootstocks having enhanced ACC synthesis, but with less conversion through to ethylene due to the majority of ACC oxidase genes being down-regulated, and more active ethylene signalling pathways. The rootstocks should have diminished CK biosynthesis.
SP12 rootstocks showed increased expression of genes involved in IAA conjugation (IAAsGH3, Solyc02g064830) but decreased expression of genes involved in IAA flux (PIN9, Solyc10g078370), along with the downregulation of most auxin responsive proteins (Figure 7e; Table S2 and S3). A gene involved in GA-deactivation (GA2ox3 , Solyc01g079200 -qRT-PCR data) was downregulated in both NCED OE rootstocks. However, while a GA biosynthesis gene was upregulated only in SP12 (GA20ox-2 , Solyc01g108870), four GA2oxidases (involved in GA deactivation) were induced only in SP5 (Figure 7b; Table S2 and S3). Thus, SlNCED1 gene expression decreased auxin activity in the roots, while SP5 rootstocks showed greater changes in GA-related gene expression than SP12 rootstocks.
Transcriptomic data revealed that many JA-related genes in SP lines (LOX, JA1 , MJE , JAZ ) were downregulated, particularly in SP5 (Figure 7c; Table S2 and S3). RT-qPCR analysis revealed that JA2 was also downregulated in SP5, but up-regulated in SP12, confirming the data obtained in the roots of whole NCED OE plants (Martínez-Andújar et al. 2020b).
Overall, the SP5 and SP12 lines showed some differential gene expression besides NCED1 and NCED2 genes, indicating differential ABA sensitivity and GA activity (reduced in SP5), and ethylene signalling and auxin activity (reduced in SP12). Those differences could potentially explain the reduced root growth of SP5 rootstocks compared to plants grafted on SP12 and WT rootstocks.