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.