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

doi:10.21203/rs.3.rs-1320607/v1

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

https://doi.org/10.21203/rs.3.rs-1320607/v1

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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 profile, 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.

Prunus dulcis

transcriptome

tree architecture

root development

In modern orchards, rootstocks are used both to select specific 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 [1,3–6]. 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 [14–17]. Strigolactones (SLs) act in consonance with auxin, controlling lateral root formation and root-hair elongation, while mediating root responses to environment changes [18–20]. Cytokinins (CKs) promote root cell differentiation and cell division in various root tissues and inhibits lateral root formation in opposition to auxin [17,21–23]. Gibberellic acid (GA) is involved in maintaining root cell proliferation and cell elongation in the meristem while arresting lateral root formation [24–26]. 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 flowering [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 flux, changing the direction and activity of the plant growth [34–38].

In this study, we have analyzed both the rootstock influence on the scion and the scion influence 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.

‘Isabelona’ and ‘Lauranne’ vigor was influenced 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 difficulties 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 first 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 dwarfing rootstock ‘GN-8’ (Table 1, Fig. 1).

Table 1

Analysis of architectural traits related to vigor in one-year-old scion/rootstock combinations.

Cultivar	Rootstock	Length (mm)	d_Scion (mm)	d_Rootstock (mm)
‘Isabelona’	‘GN-8’	210 a	2.63 a	4.25 a
‘Isabelona’	Garnem^®	260 b	3.25 ab	4.36 a
‘Lauranne’	‘GN-8’	310 c	2.97 ab	4.50 a
‘Lauranne’	Garnem^®	400 d	3.32 b	4.56 a

Assessed with Tukey’s test. Values within columns followed by the same letter were not significantly different (p < 0.05).

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 significant 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 specific tree architecture of each combination are already developed and their phenotypic effects are not yet visible in these one-year-old plants.

Rootstock only influenced 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 influence rootstock transcriptome. In addition, we analyzed the gene expression in the scion in order to determine if the rootstock influences 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 first (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 influenced 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 significant 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^® influence 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 dwarfing 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 profile 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 profile

The cultivar effect of commercial almond cultivars ‘Lauranne’ and ‘Isabelona’ on the rootstock development was analyzed in a vigorous rootstock like Garnem^®, and a dwarfing 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 first 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 profiles are influenced by the scion that is grafted onto them, responses seem to be specific for each rootstock; at least regarding which specific genes are involved. In any case, that does not mean that the regulatory pathways affected by the scion influence are not similar.

DEGs associated with hormonal regulation were influenced 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 flux 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 [14–17]. 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].

Table 2

Differentially expressed genes (DEGs) associated with hormonal regulation.

logFC 'Isabelona'/Garnem - 'Lauranne'/Garnem	logFC 'Isabelona'/'GN8' - 'Lauranne'/'GN-8'	*P. dulcis* ID	Gene	GO term	Biological process
0.920	1.003	Prudul26A011001	ACO	GO:0009693	ethylene biosynthetic process
-0.416	-1.243	Prudul26A007830	ACO	GO:0009693	ethylene biosynthetic process
1.404	0.024	Prudul26A030744	BAS1	GO:0055114	oxidation-reduction process
-0.731	-1.197	Prudul26A013026	BUD2	GO:0006557	S-adenosylmethioninamine biosynthetic process
1.228	0.717	Prudul26A008430	bZIP58	GO:0006355	regulation of transcription, DNA-templated
1.755	0.337	Prudul26A017801	CKX5	GO:0009823	cytokinin catabolic process
0.740	1.106	Prudul26A028543	CVIF2	GO:0043086	negative regulation of catalytic activity
0.808	1.014	Prudul26A016230	CVIF2	GO:0043086	negative regulation of catalytic activity
0.221	-1.434	Prudul26A017398	CYP94C1	GO:0009611	response to wounding
0.991	1.295	Prudul26A002650	ERF12	GO:0009873	ethylene-activated signaling pathway
0.423	1.232	Prudul26A022504	ERF12	GO:0009873	ethylene-activated signaling pathway
-2.000	-2.949	Prudul26A000689	GA2OX8	GO:0009686	gibberellin biosynthetic process
0.669	1.358	Prudul26A017626	GH3.6	GO:0010252	auxin homeostasis
-5.950	-0.941	Prudul26A016337	IAR3	GO:0009850	auxin metabolic process
1.007	0.135	Prudul26A016134	LOL1	GO:0034052	positive regulation of plant-type hypersensitive response
1.821	-0.772	Prudul26A022418	MAX1	GO:0016117	carotenoid biosynthetic process
-1.005	-1.005	Prudul26A005107	RCA	GO:0050790	regulation of catalytic activity
1.102	0.681	Prudul26A028381	SPL8	GO:0030154	cell differentiation
-1.640	-0.515	Prudul26A006492	SWEET2	GO:0008643	carbohydrate transport
-1.176	-0.730	Prudul26A023995	ZIFL1	GO:0010540	basipetal auxin transport

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.

ET response was also affected by the scion. 1-Aminocyclopropane-1-Carboxylic Acid Oxidase (ACO) carries out a crucial step in ET biosynthesis, controlling ET production [54,55]. Homologues of this gene (Prudul26A011001, Prudul26A007830) were found both upregulated and downregulated in ‘Isabelona’/‘GN-8’ combinations (Table 2). ERF12 has been described to participate in floral transition and seed dormancy in response to ethylene, being activated by its presence [56,57]. Here, two homologues (Prudul26A002650, Prudul26A022504) where upregulated when ‘Isabelona’ was grafted onto ‘GN-8’ (Table 2). ET acts by opposing auxin effect in lateral root formation [29,30], which matches the reduced auxin response that has been reported in combinations with ‘Isabelona’. BRs have an opposite function to ET, favoring the initiation of lateral roots [28,58]. BAS1 (Prudul26A030744), an enzyme that catalyzes BR inactivation [59], is overexpressed in Garnem^® when ‘Isabelona’ is the scion (Table 2).

Scion also influenced 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 influence 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 influenced 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 profile 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).

Table 3

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

logFC 'Isabelona'/Garnem - 'Lauranne'/Garnem	logFC 'Isabelona'/'GN8' - 'Lauranne'/'GN-8'	*P. dulcis* ID	Gene	GO term	Biological process
-1.023	0.073	Prudul26A020211	4CLL6	GO:0006744	ubiquinone biosynthetic process
0.030	-1.627	Prudul26A014215	4CLL9	GO:0000272	polysaccharide catabolic process
1.094	0.291	Prudul26A020939	AGL79	GO:0006355	regulation of transcription, DNA-templated
-0.986	-1.062	Prudul26A005381	ERF3	GO:0072659	protein localization in plasma membrane
-1.265	1.319	Prudul26A009806	EXPL1	GO:0019953	sexual reproduction
3.089	0.646	Prudul26A025382	FIP37	GO:0010073	meristem maintenance
3.072	0.623	Prudul26A011653	FIP37	GO:0010073	meristem maintenance
1.048	1.468	Prudul26A000195	GRF4	GO:0006355	regulation of transcription, DNA-templated
-0.466	-1.032	Prudul26A031613	GUX3	GO:0045492	xylan biosynthetic process
1.180	0.462	Prudul26A024452	IAA4	GO:0009733	response to auxin
1.174	0.195	Prudul26A009950	KING1	GO:0042128	nitrate assimilation
0.401	1.024	Prudul26A023724	LRP1	GO:0048364	root development
1.334	1.832	Prudul26A008528	MYB103	GO:0006355	regulation of transcription, DNA-templated
-1.300	-0.538	Prudul26A012897	MYB20	GO:1901141	regulation of lignin biosynthetic process
-1.074	-0.532	Prudul26A014014	ROL1	GO:0071555	cell wall organization
-0.031	-1.022	Prudul26A008007	SKP2A	GO:0010311	lateral root formation
-1.694	-2.203	Prudul26A014041	SNAK2	GO:0006952	defense response
-0.794	-1.850	Prudul26A015706	SNAK2	GO:0006952	defense response
-1.034	-3.009	Prudul26A007951	TBL19	GO:0045492	xylan biosynthetic process
-0.544	-1.001	Prudul26A014994	TBL29	GO:0045492	xylan biosynthetic process
-1.405	0.011	Prudul26A017201	TCX2	GO:0006355	regulation of transcription, DNA-templated

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 [81]. 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 modification while TBL19 (Prudul26A007951) and TBL29 (Prudul26A014994) participate in xylan acetylation [87–89]. These modifications 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 influenceable by changes that favor growth.

Table 4

Differentially expressed genes (DEGs) associated with light responses and circadian clock regulation

logFC 'Isabelona'/Garnem - 'Lauranne'/Garnem	logFC 'Isabelona'/'GN8' - 'Lauranne'/'GN-8'	*P. dulcis* ID	Gene	GO term	Biological process
1.899	1.208	Prudul26A020068	ABR	GO:0009733	response to auxin
1.596	-0.580	Prudul26A024462	COL6	GO:0006355	regulation of transcription, DNA-templated
-1.751	-1.054	Prudul26A016707	GI	GO:0042752	regulation of circadian rhythm
1.061	0.101	Prudul26A014609	JMJD5	GO:0042752	regulation of circadian rhythm
-1.542	0.905	Prudul26A026608	MDL1	GO:0055114	oxidation-reduction process
1.368	1.015	Prudul26A013341	NPH3	GO:0009638	phototropism
-1.339	0.051	Prudul26A014761	phyE	GO:0009585	red, far-red light phototransduction
-1.364	0.042	Prudul26A002019	phyE	GO:0009585	red, far-red light phototransduction
-1.453	-0.886	Prudul26A027917	PRR7	GO:0007623	circadian rhythm
1.009	0.772	Prudul26A019438	RVE7	GO:0007623	circadian rhythm
-1.078	-0.643	Prudul26A018495	UVR8	GO:0009649	entrainment of circadian clock
-1.078	-1.143	Prudul26A003343	UVR8	GO:0009649	entrainment of circadian clock
-1.798	-1.042	Prudul26A011979	UVR8	GO:0009649	entrainment of circadian clock

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 [31–33]. We detected a mixed pattern of expression profiles 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 flowering 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 [105–107]. While we do not observe any clear trend in the influence of the scion in the circadian clock regulation, it seems clear that these processes can be affected by the interaction between scion and rootstock.

Interaction between scion and rootstock in almond trees occur in both directions, influencing both the scion and the rootstock development. Here, we identified 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 influence seems to have a feedback effect in the development of the scion. We report that cultivars displaying more vigor like ‘Lauranne’ influence 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 influence 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 quantified using a caliper, measuring from 20 mm above and 20 mm below of the graft union respectively.

RNA-Seq analysis

Samples from the four combinations mentioned were collected from 50 mm below and above the graft union of three different individuals per combination during summer 2020. RNA extraction was performed from these samples using the CTAB method described previously [108] with some modifications [109–111]. Stranded mRNA-Seq analysis was carried out at Centro Nacional de Análisis Genómico (CNAG-CRG) in Barcelona, Spain. Sequencing was performed by an Illumina NovaSeq 6000 System - with > 30 M PE reads per sample and a read length of 2×50bp. FASTQ files were converted with FASTQ Groomer (Galaxy Version 1.1.1) [112]. Adapter sequences were removed by processing the reads sequences of the 27 individual datasets with Trimmomatic (Galaxy Version 0.38.0) [113]. RNA-Seq data alignment was carried out by TopHat (Galaxy Version 2.1.1), with a maximum intron length of 1,000 bp, [114] on the P. dulcis ‘Texas’ Genome v2.0 [115]. Duplicated molecules were located and mate-pairs were confirmed using the MarkDuplicates (Galaxy Version 2.18.2.2) and FixMateInformation (Galaxy Version 2.18.2.1) Picard tools respectively (http://broadinstitute.github.io/picard). featureCounts (Galaxy Version 1.6.4+galaxy2) was used to measure gene expression [116] using the gene annotation P. dulcis ‘Texas’ Genome v2.0 containing 27044 genes (https://www.rosaceae.org/analysis/295). Differential analysis of count data was performed by edgeR (Galaxy Version 3.24.1) with default settings [117]. All procedures were carried out using the Galaxy platform.

Statistical analysis

All statistical analyses were carried out in the R platform (https://cran.r-project.org/). Significant differences in phenotypic data were evaluated using an ANOVA test to find. 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.

ABA: Abscisic acid

BR: Brassinosteroid

CK: Cytokinin

DEG: Differentially expressed gene

ET: Ethylene

GA: Gibberellic acid

PCA: Principal component analysis

R:FR: Red:far red ratio

SL: Strigolactone

Ethics approval and consent to participate

Experimental research has been carried out in compliance with relevant national and international guidelines, and legislation regarding plant research.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/home) and are accessible through the accession number PRJEB50411.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the Spanish Research State Agency (AEI), grant numbers RTI- 2018-507 094210-R-100 and FPI-INIA CPD2016-0056.

Authors' contributions

AM, MJRC and JG designed the experiment. AM measured the phenotypical data, collected the plant tissues and performed the RNA extraction. AM and JG analyzed and interpreted the data obtained from the RNA-Seq. AM and JG drafted the manuscript. MJRC substantively revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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

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Abstract

Figures

Background

Results and discussion

‘Isabelona’ and ‘Lauranne’ vigor was influenced by the rootstock

Rootstock only influenced gene expression in combinations with ‘Isabelona’

Scion/rootstock interaction in almond affected rootstock molecular profile

DEGs associated with hormonal regulation were influenced by the cultivar in rootstock tissue

Root development and root cell wall reorganization are negatively influenced by ‘Isabelona’

DEGs associated with light responses are affected by cultivar in rootstock tissue

Conclusions

Methods

Plant material and growth conditions

Phenotypic data collection

RNA-Seq analysis

Statistical analysis

abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

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