Characterization of almond scion/rootstock communication in cultivar and rootstock tissues through a RNA-Seq approach

Background: Rootstock genotype determines multiple aspects of the scion development, including the scion three-dimensional structure, or tree architecture. Thus, rootstock choice is an important factor in the establishment of new almond (Prunus amygdalus (L.) Batsch, syn P. dulcis (Mill.)) planting systems, which demand cultivars whose vigor and shape adapt to these new requirements. However, if the rootstock genotype is able to alter scion development, it is likely that the scion genotype affects the rootstock performance. Results: We carried out a transcriptomic analysis of the scion/rootstock interaction in young trees, focusing on the scion effect in the rootstock molecular response. Two commercial almond cultivars were grafted onto two hybrid rootstocks, resulting in four combinations, whose gene expression in both scion and rootstock tissue was analyzed via RNA-Seq. We observed that, in fact, the scion genotype has an impact on the rootstock expression pro�le, affecting the expression of genes associated with hormonal regulation, root development and light signaling. Conclusions: Scion/rootstock communication has a pivotal role in the development of both scion and rootstock, accentuating the importance of a correct choice when establishing new almond orchards.


Background
In modern orchards, rootstocks are used both to select speci c root system traits and to confer traits of agronomic interest to trees and fruits [1,2].These effects on scion development have been described in numerous trees species; ranging from tree vigor to yield or fruit quality [1,[3][4][5][6].Recently, molecular approaches have been carried out in woody plant species to describe how these effects happen at the molecular level [7,8].In a recent study where almond commercial cultivars were grafted onto hybrid rootstocks, a differential expression of genes associated to hormones involved in the regulation of apical dominance, branch formation and vigor control was observed, while genes related to cell wall reorganization and formation were also affected.
The analysis of the scion effect on the rootstock has been limited to the graft formation, analyzing the processes that happens in the moment of that vascular union, leading to vascular regeneration and the establishment of the graft junction [9,10].However, little is known about how the scion can modulate the phenotypes displayed by the rootstock, from nutrient assimilation to pathogen resistance or root development [11].These traits might be affected differently depending which scion cultivar is grafted onto them.
Rootstock development is controlled by various phytohormones, which have roles in regulating cell elongation, cell division and cell differentiation [12,13].As it happens with the aerial part of the plant, auxin has an important role in regulating diverse processes in roots, like root patterning, cell division and cell elongation [14][15][16][17].Strigolactones (SLs) act in consonance with auxin, controlling lateral root formation and root-hair elongation, while mediating root responses to environment changes [18][19][20].Cytokinins (CKs) promote root cell differentiation and cell division in various root tissues and inhibits lateral root formation in opposition to auxin [17,[21][22][23].Gibberellic acid (GA) is involved in maintaining root cell proliferation and cell elongation in the meristem while arresting lateral root formation [24][25][26].Brassinosteroids (BRs) play a crucial role in controlling the root meristem activity, also participating in the regulation of lateral root initiation or root cell elongation [27,28].Ethylene (ET) modulates the meristem maintenance, promoting cell division; whilst opposing auxin in lateral root formation [29,30].
Light signaling can also control plant development through different mechanisms.In plants, the circadian clock regulates several developmental processes in response to light changes, from seed germination, to hypocotyl elongation, root growth or owering [31,32].Carbohydrate metabolism and nutrient assimilation are also linked to the regulation of the circadian clock [33].The shade avoidance response also regulates plant growth which is based on the ratio between red light and far red light (R:FR), captured by phytochrome photoreceptors phyA and phyB.Changes in this ratio provoke a redistribution in the auxin ux, changing the direction and activity of the plant growth [34][35][36][37][38].
In this study, we have analyzed both the rootstock in uence on the scion and the scion in uence on the rootstock at the transcriptional response level.We grafted two commercial almond cultivars with opposite architecture and vigor characteristics onto two almond × peach (Prunus amygdalus (L.) Batsch, syn P. dulcis (Mill.).× P. persica (L.) Batsch) hybrid rootstocks for a total of four combinations.Our goal was to identify which biological processes and molecular responses were affected above and below the graft site.

Results And Discussion
'Isabelona' and 'Lauranne' vigor was in uenced by the rootstock Tree architecture data was collected for the four combinations, 'Isabelona'/Garnem ® , 'Isabelona'/'GN-8', 'Lauranne'/Garnem ® and 'Lauranne'/'GN-8' (Fig. 1).Since trees were too young to have developed any branches, only trunk length (Length) and the diameter of both the scion (d_Scion) and the rootstock (d_Rootstock) was measured.Due to the intrinsic di culties of its measurement, no data was collected of the root architecture.
In a previous study with thirty different scion/rootstock combinations [39], we reported that 'Isabelona' displayed reduced vigor paired with strong apical dominance, which resulted in a phenotype with reduced branching and long trunks.On the contrary, 'Lauranne' presented high vigor and weak apical dominance, resulting in numerous branching and a shortening of the trunk.Here, combinations with 'Lauranne' as scion presented higher Length values, and hence, longer trunks (Table 1, Fig. 1).In this case, trees are in their rst year of growth, so there are no branches yet that compete with the main axis growth.As a result, 'Lauranne' more vigor leads to higher Length values.Regarding the rootstocks, Garnem ® effect as a vigorous rootstock was present on both cultivars, presenting higher Length values than when grafted onto the dwar ng rootstock 'GN-8' (Table 1, Fig. 1).Trunk diameter (d_Scion) is typically used as a vigor measure, normally presented as TCSA (Trunk Cross Sectional Area).As it happened with Length values, 'Lauranne' presented higher d_Scion values than 'Isabelona'.Besides, cultivars grafted onto Garnem ® had also higher d_Scion values than when grafted onto 'GN-8' (Table 1).However, we did not observe a signi cant difference in the rootstock diameters (d_Rootstock), though mean values were slightly lower with 'Isabelona' (Table 1).
The observed phenotype differences seem to depend mostly on the vigor that each combination displays.Though is likely that the biological processes that will shape the speci c tree architecture of each combination are already developed and their phenotypic effects are not yet visible in these one-year-old plants.
Rootstock only in uenced gene expression in combinations with 'Isabelona' We reported in a previous experiment that combinations with 'Lauranne' and 'Isabelona' did show little phenotypic differences when grafted onto different rootstocks, which was correlated with a lack of differentially expressed genes (DEGs) [39].However, in the present experiment, 'Lauranne' and 'Isabelona' were selected because of their consistent scion phenotype, expecting that they could in uence rootstock transcriptome.In addition, we analyzed the gene expression in the scion in order to determine if the rootstock in uences gene expression at an early development stage (Supplementary Data 1).
A PCA (Principal Component Analysis) was carried out using expression for each gene as variables for the four combinations, with the rst (PC1; 33.2% of variability explained) and third (PC3; 11.8%) component selected to represent the data (Fig. 2).As we observed previously, combinations with 'Lauranne' as scion were not differentiated according to rootstock, grouping together (Fig. 2).However, we did observe that gene expression in combinations with 'Isabelona' is in uenced by the rootstock.These individuals could be separated in two groups in the PCA, depending on whether they were grafted onto Garnem ® or 'GN-8'.
Looking at the global picture of gene expression by functional categories, we performed a Gene Ontology (GO) enrichment analysis but due to the low number of genes we did not obtain any signi cant categories.However, we found a molecular response similar to what we observed in previous analysis of almond scion-rootstock combinations.When grafted onto the vigor-conferring rootstock Garnem ® , 'Isabelona' displayed several DEGs overexpressed involved in auxin regulation, mostly in a repressive manner.Besides, DEGs promoting CKs or GA activity or repressing abscisic acid (ABA) response were also overexpressed in these combinations (Supplementary Data 2).Therefore, Garnem ® in uence hormonal regulation here in a similar manner to what we observed before, with auxin responses being downregulated, hence reducing apical dominance [40,41].Moreover, as it happened previously, we found overexpression of DEGs involved in processes associated with active growth, like cell proliferation and cell expansion, or promoting nitrogen and sugar assimilation (Supplementary Data 2).
Genes related to ET regulation were overexpressed when 'Isabelona' was grafted onto the dwar ng rootstock 'GN-8' (Supplementary Data 2).Contrary to what happened when grafted onto Garnem ® , DEGs related to low nitrogen or sugar content were upregulated (Supplementary Data 2).However, some genes involved in cell wall reorganization were overexpressed (Supplementary Data 2), while in a previous experiment, these genes were only upregulated in combinations with vigor-conferring rootstocks.
In general, although the effects in the phenotype are not yet visible, we observed a similar expression pro le to what has been previously described, with auxin responses downregulated in combinations with a vigor-inducing rootstock, while branching and growth are upregulated in combinations with Garnem ® .
Scion/rootstock interaction in almond affected rootstock molecular pro le The cultivar effect of commercial almond cultivars 'Lauranne' and 'Isabelona' on the rootstock development was analyzed in a vigorous rootstock like Garnem ® , and a dwar ng rootstock such as 'GN-8' (Supplementary Data 3).We carried out a PCA using the expression of each gene as variables for the four different scion/rootstock combinations.The rst two components explained 50.1% of the variability, while none of the other variables explained more than a 10%.PC1 and PC2 explained 32.6% and 17.6% of the variability respectively.In the PCA, there was a clear separation between the four different combinations (Fig. 3).Combinations with Garnem ® as rootstock are in the lower-left corner while combinations with 'GN-8' are in the upper-right corner.Therefore, there is a clear effect of the rootstock and it can be observed in the gene expression, with individuals clearly segregating depending on which scion, 'Lauranne' or 'Isabelona', is grafted onto them (Fig. 3).
A total of 168 DEGs were overexpressed in combinations with 'Isabelona' as scion respective to those with 'Lauranne', of which 100 appeared in the combination with Garnem ® and 52 in combination with 'GN-8', while only 16 DEGs were in both combinations (Fig. 4a).A similar display was observed with DEGs that were underexpressed when 'Isabelona' was the scion.A total of 71 DEGs appeared only in Garnem ® , while 74 DEGs were found in 'GN-8'.A total of 34 DEGs were present in both rootstocks (Fig. 4b).
Therefore, while both Garnem ® and 'GN-8' expression pro les are in uenced by the scion that is grafted onto them, responses seem to be speci c for each rootstock; at least regarding which speci c genes are involved.In any case, that does not mean that the regulatory pathways affected by the scion in uence are not similar.
DEGs associated with hormonal regulation were in uenced by the cultivar in rootstock tissue We have seen that changes in hormonal response prompted by a different rootstock affect the almond scion architecture, modifying the number of branches or the growth of the main axis.Therefore, it is likely that the grafted scion also has an effect on the rootstocks, triggering different mechanisms that could affect the rootstock properties.This reciprocal effect has been already described in other species regarding different traits (regulation of rootstock responses to low Pi and phloem sap metabolites) [42,43].Here, we reported that hormonal response is affected by the scion, presumably leading to changes in the root architecture.Although samples were collected from the rootstock trunk, we expect that the variation of the dynamics of hormone ux found there affect the rest of the root system.
In contrast to its function in shoots, auxin has been described to promote the formation of lateral roots [14][15][16][17].Various DEGs involved positively in auxin response were downregulated when 'Isabelona' was the scion in Garnem ® (Table 2).BUD2 (Prudul26A013026) is an auxin inducible member of the SAMDC family, playing a part in mechanisms promoted by auxin, like apical dominance and root branching [44,45].IAR3 (Prudul26A016337) releases IAA from its conjugate form, regulating the levels of free auxin [46,47].ZIFL1 (Prudul26A023995) positively regulates polar auxin transport, favoring processes like lateral root development (Remy et al., 2013).On the other hand, GH3.6 (Prudul26A017626), a negative regulator of auxin levels [48,49], appeared overexpressed in combinations with 'Isabelona' as scion (Table 2).Here, the fact that auxin processes are downregulated in combinations with 'Isabelona' as scion suggests that rootstocks with this cultivar may display hormonal conditions required to develop less lateral roots.Whereas, rootstocks with 'Lauranne' as scion could develop an increased number of lateral roots, which would correlate to higher substrate availability and therefore affect their vigor and aerial branching phenotype [39].Only genes with a logFC superior or infertior to 1 (highlighted in bold) were considered as differentially expressed.
GA acts mostly in opposition to the auxin response, inhibiting lateral root formation while promoting cell elongation and proliferation in the central root [25,26].Three genes related positively to GA activity were found to be upregulated in rootstock tissues in combinations with 'Isabelona' as the scion (Table 2).LOL1 (Prudul26A016134) and bZIP58 (Prudul26A008430) modulate GA levels, favoring its activity and acting in numerous pathways regulated by this hormone [50].SPL8 (Prudul26A028381) can act both in a positive or negative manner, although has been described to negatively affect root elongation in Arabidopsis [51].On the other hand, GA2OX8 (Prudul26A000689) is downregulated in combinations with 'Isabelona' (Table 2).GA2OX8 catalyzes the deactivation of active GA, hence reducing its levels and activity [52,53].In general, genes related to increased GA levels are upregulated in rootstocks when 'Isabelona' is the scion.This could lead to the elongation of the central root, in a similar manner of what we observed in the scion, while inferior expression of GA responses in combinations with 'Lauranne' would favor the development of numerous lateral roots.
Scion also in uenced the expression of genes involved in other hormonal responses.CKX5 (Prudul26A017801) was overexpressed in the 'Isabelona'/Garnem ® combination (Table 2).As a CK dehydrogenase, CKX5 participates in degrading CKs [60].MAX1 (Prudul26A022418), which is part of the SL biosynthetic pathway [61,62], is also upregulated when Garnem ® had 'Isabelona' as scion (Table 2).Jasmonic acid (JA) is typically activated in stress responses [63].CYP94C1 (Prudul26A017398) carries out the oxidative inactivation of this hormone [64].This gene was less expressed in the 'Isabelona'/'GN-8' combination too, suggesting a negative regulation of growth in this combination (Table 2).Finally, a couple of genes related to sugar availability were affected by the scion.Two CVIF2 homologues (Prudul26A028543, Prudul26A016230) were overexpressed in the 'Isabelona'/'GN-8' combination (Table 2).CVIF2 might regulate sucrose cleaving, therefore negatively affecting plant sugar levels [65].RCA (Prudul26A005107), which was downregulated with 'Isabelona' as scion (Table 2), promotes RuBisCO activity and therefore sugar production [66].Moreover, the sugar transporter SWEET2 (Prudul26A006492), which is especially active in roots [67], was also less expressed when 'Isabelona' was the scion.Therefore, 'Isabelona' seems to negatively in uence sugar production in roots, which might lead to a reduction in the formation of roots.
In conclusion, the presence of a different scion affects the hormonal response in the rootstock.In this case, we observed that rootstocks with 'Isabelona' as scion present a hormonal framework that should inhibit the formation of lateral roots, while those with 'Lauranne' as scion are prompted to develop more lateral roots.

Root development and root cell wall reorganization are negatively in uenced by 'Isabelona'
Root architecture is regulated by numerous genes that mediate the formation of the primary root and others, like lateral roots or adventitious roots [68].Though samples were collected from below the grafting site in 'GN-8' and Garnem ® rootstocks, we would expect that changes in the expression pro le would condition the behavior of other parts of the rootstock.
Two inhibitors of lateral root formation were overexpressed in combinations with 'Isabelona' (Table 3).AGL79 (Prudul26A020939) acts as a repressor of lateral root development [69].While not affecting lateral root initiation, LRP1 (Prudul26A023724) does affect its progression.Its overexpression in Arabidopsis reduced the number of lateral roots [70].
IAA4 (Prudul26A024452) is also overexpressed in the 'Isabelona'/Garnem ® combination (Table 3).IAA4 acts in opposition to auxin response, inhibiting the formation of adventitious roots [71].Therefore, there is an upregulation of processes that lead to reduce lateral root formation when 'Isabelona' is the scion.Moreover, two homologues of FIP37 (Prudul26A025382, Prudul26A011653) were highly overexpressed in the 'Isabelona'/Garnem ® combination (Table 3).FIP37 effect in meristem development has been mostly described in shoots, but it acts preventing meristem proliferation and therefore bud outgrowth [72].A similar function is carried out by TSO1 [73,74].Here, we found a homologue of this gene, TCX2 (Prudul26A017201), which is downregulated when 'Isabelona' was the scion (Table 3).Only genes with a logFC superior or infertior to 1 (highlighted in bold) were considered as differentially expressed.
'Lauranne' has been proved to be a more vigorous scion than 'Isabelona'.Here, we also observed several genes involved in cell proliferation being downregulated in the rootstock in combinations with 'Isabelona' (Table 3).ERF3 (Prudul26A005381) promotes cell division and cell elongation of the root meristem [75].SKP2A (Prudul26A008007) is a regulator of cell proliferation, promoting cell division in lateral root primordium, whose degradation is stimulated by auxin [76,77].Two homologues of SNAK2 (Prudul26A014041, Prudul26A015706) were found.SNAK1 has been described to promote cell division in response to external stimuli [78,79].SnRK1 is involved in repressing growth in response to low energy supplies Here, a member of its family, KING1 (Prudul26A009950), was upregulated in the 'Isabelona'/Garnem ® combination (Table 3).
The regulation of several components that are part of the cell wall, like lignins, xyloglucans or pectins, is essential in the control of cell wall formation and cell wall reorganization [81-83].Numerous genes associated to their synthesis or transport were downregulated in combinations with 'Isabelona' compared to those with 'Lauranne' as the scion (Table 3).Members of the 4CL family like 4CLL6 (Prudul26A020211) and 4CLL9 (Prudul26A014215) are part of the phenylpropanoid metabolism pathway, participating in lignin biosynthesis [84].The MYB transcription factor, MYB20 (Prudul26A012897), promotes the lignin biosynthesis pathway [85].However, another MYB TF linked to lignin biosynthesis, MYB103 (Prudul26A008528), was overexpressed in combinations with 'Isabelona' as scion [86].GUX3 (Prudul26A031613) is involved in xylan modi cation while TBL19 (Prudul26A007951) and TBL29 (Prudul26A014994) participate in xylan acetylation [87-89].These modi cations are crucial to ensure xylan integrity and cell wall strength.Knockout mutants of ROL1 (Prudul26A014014) produce aberrant pectin structure which leads to reduced elongation growth, highlighting a role for ROL1 in cell wall reorganization [90,91].Nevertheless, some genes associated also to cell wall formation were found to be upregulated when 'Isabelona' was the scion (Table 3).GRF4 (Prudul26A000195) promotes cellulose biosynthesis in a response involving MYB61 transcription factor [92]. EXPL1 (Prudul26A009806) is associated to cell wall remodeling in response to auxin and lateral root initiation [93].Contradictorily, EXPL1 was overexpressed in 'GN-8', while being downregulated in the 'Isabelona'/Garnem ® combination (Table 3).This could mean a differential response for this gene depending on which rootstock is affected by the scion, maybe linked to the fact that 'GN-8' is a prominently less vigorous rootstock than Garnem ® .
In general, processes related to root formation or active tissue growth like cell wall reorganization were downregulated when 'Isabelona' was the scion, expecting that these combinations should present a root system with fewer lateral roots.This response is in line with the hormonal status reported previously, that favored root formation in rootstocks with 'Lauranne' as scion, and not in those with 'Isabelona'.

DEGs associated with light responses are affected by cultivar in rootstock tissue
Light regulates numerous processes related to plant development, and several pathways are involved in growth control [94,95].Light availability mediates the formation of lateral branches, through several responses like shade avoidance [34,35].In the root, we observed an upregulation of genes involved in responses related to reduced light in combinations that had 'Isabelona' as scion, with ABR (Prudul26A020068) being overexpressed and several homologues of phyE (Prudul26A014761, Prudul26A002019) and UVR8 (Prudul26A018495, Prudul26A003343, Prudul26A011979) downregulated (Table 4).ABR is involved in ABA responses and it is induced by light deprivation [96].phyE regulates responses to low R/FR, in consonance with phyB [97].The photoreceptor UVR8 mediates the signal produced by UV-B that inhibits shade avoidance responses [98].Auxin and light responses are tightly integrated, affecting tree architecture [99].Two inhibitors of auxin response affected by light were overexpressed in combinations with 'Isabelona' (Table 4).NPH3 (Prudul26A013341) participates in an auxin feedback response, modifying auxin transport in response to phototropism [100].RVE7 (Prudul26A019438) is a member of the same family of RVE1, which modulates plant growth through repression of auxin levels [101].'Lauranne', which shows numerous branching, is expected not to be affected as acutely by light availability than 'Isabelona', which displays reduced branching.Here, this effect is more prevalent in Garnem ® , while 'GN-8' is less affected by the scion light perception.This could be caused by the higher vigor presented by Garnem ® , which is more in uenceable by changes that favor growth.Only genes with a logFC superior or infertior to 1 (highlighted in bold) were considered as differentially expressed.
The circadian clock, which is controlled by light, among other environmental responses, regulates numerous processes in plant development, including root growth [31][32][33].We detected a mixed pattern of expression pro les of genes involved in circadian clock regulation.COL6 (Prudul26A024462) and JMJD5 (Prudul26A014609) were overexpressed in the 'Isabelona'/Garnem ® combination (Table 4).CO-like genes are light responsive genes under circadian clock control and affecting circadian rhythms [102,103].JMJD5 is integrated in various responses regulated by circadian period, including owering regulation [104].On the other hand, the circadian clock regulator GI (Prudul26A016707) was downregulated in combinations with 'Isabelona' (Table 4).This gene participates in regulating daily CO expression and in activating FT expression, being controlled by light [105][106][107].While we do not observe any clear trend in the in uence of the scion in the circadian clock regulation, it seems clear that these processes can be affected by the interaction between scion and rootstock.

Conclusions
Interaction between scion and rootstock in almond trees occur in both directions, in uencing both the scion and the rootstock development.Here, we identi ed multiple biological processes which were differentially affected according to the grafted almond cultivar.Among the differentially expressed genes , we observed genes involved in hormonal regulation, root development, cell wall reorganization, light perception and circadian clock regulation (Figure 5).This in uence seems to have a feedback effect in the development of the scion.We report that cultivars displaying more vigor like 'Lauranne' in uence positively root development, including lateral root formation.This would favor the capture of nutrients by the radicular system and, in consequence, would promote scion growth, resulting in the vigorous phenotype that 'Lauranne' presents when compared to 'Isabelona'.Therefore, choosing the correct scion/rootstock combination is essential to the success of the orchard.In intensive systems, the rootstock effect in tree vigor depends not only on its genotype, but also their complementarity as the scion is determinant in root development, and hence, tree growth.

Plant material and growth conditions
For the experiment, two almond commercial cultivars, 'Isabelona' and 'Lauranne' were grafted onto two hybrid rootstocks, Garnem ® , a commercial rootstock, and 'GN-8', a new selection, obtaining four different combinations.Both rootstocks are almond × peach (P.amygdalus (L.) Batsch, syn P. dulcis (Mill.).× P. persica (L.) Batsch) hybrid rootstocks.The two cultivars were selected because the weak in uence that the rootstock displays in their apical dominance and branch formation phenotype [39].Grafted plants were supplied by the Agromillora Iberia S.L. nursery in 2020 (Barcelona, Spain).Plants were kept in a nursery shortly until sample collection at the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA), where conventional orchard practices were applied.

Phenotypic data collection
Phenotypic data was collected for ten replicates of each of the four combinations, before sample collection.Three parameters related to vigor were measured: scion axe length (Length), scion trunk diameter (d_Scion) and rootstock trunk diameter (d_Rootstock).Length was determined from the graft union.d_Scion and d_Rootstock were quanti ed using a caliper, measuring from 20 mm above and 20 mm below of the graft union respectively.
Statistical analysis statistical analyses were carried out in the R platform (https://cran.r-project.org/).Signi cant differences in phenotypic data were evaluated using an ANOVA test to nd.These were assessed with a Tukey's test (p < 0.05) using the agricolae R package (https://CRAN.R-project.org/package=agricolae). PCA was carried out using R stats package with default parameters on the gene expression values for the all the genes in the four combinations.

Figures Figure 1
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Figure 2 Principal
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Figure 3 Principal
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Table 1
Analysis of architectural traits related to vigor in one-year-old scion/rootstock combinations.

Table 3
Differentially expressed genes (DEGs) associated with root development and root cell wall reorganization.