ABSTRACT
The ability for cut tissues to join together and form a chimeric organism is a remarkable property of many plants, however, grafting is poorly characterized at the molecular level. To better understand this process we monitored genome-wide temporal and spatial gene expression changes in grafted Arabidopsis thaliana hypocotyls. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than the other. This asymmetry correlated with sugar responsive genes and we observed an accumulation of starch above the graft that decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues indicating that a recognition mechanism activated that was independent of functional vascular connections. Auxin which is transported cell-to-cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes were expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, inter-tissue communication and tissue fusion events.
INTRODUCTION
For millennia people have cut and rejoined plants through grafting. Generating such chimeric organisms combines desirable characteristics from two plants, such as disease resistance, dwarfing and high yields, or can propagate plants and avoid the delays entailed by a juvenile state (1). Agriculturally, grafting is becoming more relevant as a greater number of plants and species are grafted to increase productivity and yield (2). However, our mechanistic understanding of grafting and the biological processes involved, including wound healing, tissue fusion and vascular formation, remain limited.
Plants have efficient mechanisms to heal wounds and cuts, in part through the production of wound-induced pluripotent cells termed callus. The callus fills the gap or seals the wound, and later, differentiates to form epidermal, mesophyll and vascular tissues (3). In grafted Arabidopsis hypocotyls, tissues adhere 1-2 days after grafting and the phloem, the tissue that transports sugars and nutrients, connects after three days (4, 5). The xylem, tissue that transports water and minerals, connects after seven days (4). Plant hormones are important regulators of vascular formation, and at the graft junction, both auxin and cytokinin responses increase in the vascular tissue (4–⇓6). Auxin is important for differentiation of vascular tissues whereas cytokinin promotes vascular stem cells, termed cambium, to divide and proliferate in a process known as secondary growth (7, 8). Auxin is produced in the upper parts of a plant and moves towards the roots via cell-to-cell movement. Auxin exporters, including the PIN proteins, transport auxin into the apoplast, whereas auxin importers, such as the AUX and LAX proteins, assist with auxin uptake into adjacent cells (8). Disrupting this transport, such as by mutating PIN1, inhibits healing of a wounded stem (9). Blocking auxin transport with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) in the shoot inhibits vascular formation and cell proliferation at the Arabidopsis graft junction (6). In addition to auxin, other compounds, including sugars, contribute to vascular formation. The localised addition of auxin to callus induces phloem and xylem but requires the presence of sugar (10, 11). In plants, sugars are produced in the leaves and transported through the phloem to the roots (12). The role of sugars in vascular formation and wound healing is not well established, however, sugars promote cell division and cell expansion (13), processes important for development including vascular formation.
The molecular and cellular mechanisms for wound healing, tissue reunion and graft formation remain largely unknown. One emerging theme is that the top and bottom of the cut do not behave similarly. Such tissue asymmetry occurs in other plant tissues, most notably leaves. Developing leaf primordia have an inherent asymmetry that is thought to derive from positional signals from the shoot apical meristem that specifies differences between the top and the bottom of the leaf. The molecular mechanisms that establish asymmetry are not well described, though one hypothesis is that auxin contributes towards or acts on the putative signal (14). Asymmetry also appears in cut Arabidopsis inflorescence stems where the transcription factor RAP2.6L expresses exclusively below the cut whereas the transcription factor ANAC071 expresses exclusively above the cut (9). Both were important for stem healing and ANAC071 and a close homologue, ANAC096, were important for graft formation (6). Asymmetry also exists in genetic requirements, since ALF4 and AXR1, two genes involved in auxin perception, are important below but not above the graft junction for phloem connection (4). However, ANAC071 is expressed symmetrically around the hypocotyl graft junction three days after grafting (6) so the extent of asymmetry and the mechanistic basis for it during wound healing remains largely uncharacterised.
Previous efforts have characterised wound healing and tissue reunion using transcriptomic analyses. Mechanical wounding altered approximately 8% of the Arabidopsis transcriptome and showed a high degree of overlap with transcriptomic changes elicited by pathogen attack and abiotic stress (15). Stem wounding and wound-induced callus formation altered the expression of hundreds or thousands of genes (9, 16, 17), whereas grafting grape vines, lychee trees and hickory trees induced hundreds or thousands of differentially expressed genes involved in hormone response, wound response, metabolism, cell wall synthesis and signal transduction (5, 18–⇓⇓21). These grafting studies provide limited information, as tissues from above and below the graft junction were not isolated to test whether these tissues behaved differently, and controls were not performed to distinguish how grafting and tissue fusion might differ from a response associated with cut tissues that remained separated. Here, we perform an in-depth analysis to describe the spatial and temporal transcriptional dynamics that occur during healing of cut Arabidopsis tissues that are joined (grafted) or left unjoined (separated). We find that the majority of genes differentially expressed are initially asymmetrically expressed at the graft junction and that many of these genes are sugar responsive, which correlates with severing of the phloem tissue and the accumulation of starch above the junction. However, genes associated with cell division and vascular formation activate on both sides of the graft and, similarly, auxin responsiveness activates equally on both sides. We propose that the continuous transport of substances, including auxin, independent of functional vascular connections, promoted division and differentiation, while the enhanced auxin response and blocked of sugar transport provided a unique physiological condition to activate genes specific to graft formation that promote wound healing.
RESULTS
Genes are asymmetrically expressed around the graft
Previous analyses revealed an asymmetry in several genes important for tissue reunion or graft formation and identified a number of genes expressed above a cut that were not expressed below (4, 9). To investigate whether asymmetry was a common feature of grafting and tissue reunion, we generated RNA deep sequencing libraries from Arabidopsis thaliana hypocotyl tissues immediately above and immediately below the graft junction 0, 6, 12, 24, 48, 72, 120, 168 and 240 hours after grafting (HAG)(Figure 1A). Prior to RNA extraction, we separated top and bottom tissues at the graft junction. We found that the strength required to break apart the graft junction increased linearly (Figure S1) similarly to previously reported breaking strength dynamics of grafted Solanum pennellii and Solanum lycopersicum (22, 23). When pulling apart grafts to separate top and bottom for sample preparation, grafts broke cleanly with minimal tissue from one half present in the other half (Figure S1, Movie S1, S2). We measured the amount of tissue from tops adherent to bottoms and vice versa (Figure S1) and found less than 4% cross-contamination. In addition to grafting, we also prepared libraries from ungrafted hypocotyls (“intact” treatment) and cut plants that had not been reattached (“separated” treatment)(Figure 1A). We herein refer to tissues harvested above the graft junction or from the shoot side of separated tissue as “top” and that from below the graft or from the root side of separated tissue as “bottom” (Figure 1A).
(A) Intact, cut and separated, or cut and grafted Arabidopsis tissues were harvested approximately 0.5mm above (top), 0.5mm below (bottom) the cut site or for intact plants 1mm segments spanning the region where cuts were made in grafted and separated plants. (B) Pairwise analysis between the grafted top and grafted bottom identified sets of protein-coding genes symmetrically or asymmetrically expressed at the graft junction, with an FDR < 0.05 and a likelihood of symmetric/asymmetric expression of greater than 50%. Asymmetrically expressed genes were further divided into those whose RNAs were higher in the top (orange dotted line) or higher in the bottom (purple dotted line), again with an FDR < 0.05 and a likelihood of asymmetric expression greater than 50%. (C,D) Expression profiles for transcripts of cambium-associated genes that initially activate symmetrically (HCA) or asymmetrically (ANT) plotted for intact, separated and grafted samples. (E-G) HCA2 transcription upregulates above the graft junction (E) and below the graft junction. (F) pHCA2::RFP was grafted to Col-0 roots (E) or Col-0 shoots (F) to avoid ambiguity of signal origin at the junction. HCA2 was also upregulated in separated shoots but not in intact samples or in high levels in separated roots (G). HAG, hours after grafting. HAS, hours after separation. White triangle denotes initial fluorescent signal, dashed lines denote the graft junction.
RNAs that were differentially expressed as a consequence of grafting (compared to intact hypocotyls) equally in tops and bottoms of grafts (symmetrically expressed) or were more highly expressed in one tissue than the other (asymmetrically expressed), were identified by performing a pairwise analysis of the protein-coding transcriptome datasets that were differentially expressed relative to the intact group. Several thousand RNAs were identified that fit either pattern of expression including the transcript of the cambial markers HCA2 that was induced symmetrically, and ANT that was induced asymmetrically (Figure 1B-G, Figure S2). 6 to 48 hours after grafting, the number of graft-differentially expressed genes that were asymmetrically expressed was roughly 3-fold greater than those symmetrically expressed indicating that tissues above the cut changed their expression dynamics relative to below the cut. However, at 72 hours the numbers were nearly equal, and by 120 hours, the number of symmetrically differentially expressed genes was 3-fold greater than those asymmetrically expressed (Figure 1B). As a second approach, we performed a hierarchical clustering analysis that indicated that the grafted top and grafted bottom became most similar after 72 hours (Figure S3), consistent with the symmetry analysis (Figure 1B). Thus, graft healing and tissue reunion promoted a shift from asymmetry to symmetry (Figure 1B).
Sugar response correlates with asymmetric gene expression
The shift from asymmetry to symmetry could be due to phloem reconnection at 72 hours ((4), Figure 2A) and the resumption of hormone, protein and sugar transport. We tested a role for sugar by grafting in the presence of exogenous sucrose which has previously been reported to affect grafting success (24). Low levels of exogenous sucrose lowered grafting efficiency (Figure 2A), suggesting that differential sugar responses at the graft junction might be important for vascular reconnection. Expression of ApL3, a gene whose expression is induced by sugar (25), was rapidly upregulated in separated tops and grafted tops, whereas expression of DIN6, GDH1 and STP1, genes whose expression is repressed by sugar (25–⇓27), was rapidly upregulated in separated bottoms and grafted bottoms (Figure 2B, Figure S4). These observations were consistent with sugar accumulation in the grafted top and sugar depletion in the grafted bottom. The expression of these genes returned to levels similar to intact samples by 120 hours and, with the exception of ApL3, the grafted samples normalised expression more rapidly than the separated tissues. Genes associated with photosynthesis increase expression in separated bottoms 24 hours after cutting, a common response to starvation (13), but likely too late to affect sugar levels before 24 hours (Figure S4). A transcriptional overlap analysis with RNAs from known glucose-responsive genes (Table S1) revealed a strong overlap with genes differentially expressed by grafting. RNAs from known glucose-induced genes were upregulated in separated tops and grafted tops, whereas transcripts from known glucose-repressed genes were upregulated in separated bottoms and grafted bottoms (Figure 2C, Figure S4). This trend was not observed with genes differentially expressed by mannitol treatment (Figure S4), suggesting the effect was specific to metabolically active sugars. To further investigate this effect, we stained grafted, separated and intact plants with Lugol solution to assay for the presence of starch. Staining above the graft junction increased 48-72 hours after grafting (Figure 2D). By 120 hours, staining was equal on both sides of the graft whereas in separated tops staining became stronger after 72 hours (Figure 2D, Figure S4). We concluded that starch accumulated above the graft junction, but after 72 hours, this asymmetry disappeared. To test whether the accumulation of starch and increased sugar responsiveness could explain the observed transition from asymmetry to symmetry, we compared our datasets to previously published genes that are induced by starvation or are induced by sucrose re-addition (Table S1). In early time points, 20-31% of asymmetrically expressed genes are genes known to respond to sugars compared to 2-5% of symmetrically expressed genes (Table 1). However, at 72 hours, the overlap between asymmetrically expressed genes and sugar responsive genes reduced substantially (Table 1).
(A) pSUC2::GFP-expressing Arabidopsis shoots were grafted to Col-0 wild type roots and GFP movement to the roots was monitored over 7 days for phloem connection in the presence or absence of various concentrations of sucrose. (B) Expression profiles for transcripts of a sugar-repressed gene (GDH1), and a sugar-induced gene (ApL3) were plotted for intact, separated and grafted samples. (C) Transcriptional overlap between previously published glucose-induced or glucose repressed genes and our dataset. The number in brackets represents the number of glucose-responsive genes identified in the previous dataset, and overlap is presented as a ratio out of 1.0 for differentiation expressed genes (DEG) up- or down-regulated in our dataset relative to intact samples. Asterisks represent a significant difference (p < 0.05) between the ratio of up- and-down- regulated genes in a previously published transcriptome dataset compared to the ratio of all up- and-down- regulated genes in our grafting dataset at a certain time point. (D) Lugol staining of grafted plants at various time points revealed dark brown staining associated with starch accumulation. HAG, hours after grafting.
Symmetrically and asymmetrically differentially expressed genes were compared to previously published sugar-responsive genes (28)1 and the percent overlap calculated (* p<.05). HAG, hours after grafting.
Vascular formation and cell division activates on both sides of the graft
Since the grafted bottom samples exhibited a starvation response up to 48 hours after grafting, we reasoned that pathways associated cell division and cell differentiation would be delayed or inhibited. We looked at the expression of markers associated with vascular formation and cell division in the transcriptome datasets. Cambium, phloem and provascular markers activated within 6 hours in grafted top samples, but activation was only delayed 0-24 hours in grafted bottom samples depending on the gene (Figure 1B-C, Figure 3, Figure S5, Figure S6). Expression of phloem markers peaked in both grafted tops and grafted bottoms at 72 hours (Figure 3, Figure S5, Figure S6), the time when phloem reconnections form in grafted Arabidopsis (Figure 1, (4, 5)). Notably, the early phloem marker NAC020 activated before the mid phloem marker NAC086 which activated before the late phloem marker NEN4, consistent with the dynamics of phloem transcriptional activation during primary root development and leaf vascular induction (Figure S5)(29, 30). Certain markers associated with xylem formation, such as VND7 and BFN1, activated early in the grafted top. Other xylem markers, such as IRX3 and CESA4, activated late in grafted samples. By 120 hours after grafting, xylem markers were activated in top and bottom, consistent with when the first xylem strands differentiate at the graft junction (4). Genes associated with cell division were activated by 12 hours in the grafted top and by 24 hours in the grafted bottom (Figure 3, Figure S5). On the other hand, control genes whose expression does not typically vary between tissues and treatments (31) were not differentially expressed in grafted tops or bottoms (Figure S6). The RNAseq expression data appeared to correlate well with transcriptional fluorescent reporters for both activation dynamics and the localisation of expression (Figure 1D-F, Figure S2).
Expression levels were plotted over time for intact, separated and grafted samples.
The similar activation dynamics of vascular differentiation genes in grafted tops and grafted bottoms prompted us to test whether this phenomenon occurred with other known developmental processes. We obtained lists of genes whose expression is associated with various biological processes from previous publications (Table S1) and tested how many of the genes differentially expressed in our transcriptomes overlapped with the previously published lists. Differentially expressed genes in grafted samples and separated tops partially overlapped with those whose expression is associated with phloem, xylem and procambium formation (Figure 4, Figure S7). There was a high overlap between Arabidopsis inflorescence stem healing and grafting, as well as between vascular induction in leaf disk cultures and grafting (Figure 4). Various genes expressed in a cell type-specific manner also showed a high transcriptional overlap with grafting, including phloem, endodermis and protoxylem (Figure 4, Figure S7). In nearly all cases, the separated top, grafted top and grafted bottom samples showed similar activation dynamics. The separated bottom samples were exceptional though since gene expression associated with vascular development and cell-specific processes was downregulated (Figure 4, Figure S7). We also compared our datasets to RNAs expressed in longitudinal cross sections of the Arabidopsis root (32). There was little overlap between grafted bottoms and sections from the root meristemic zone, whereas overlap existed between grafted tops and the root meristemic zone in early time points, and between grafting and the root maturation zone (Figure S8). Our analysis also revealed that two genes expressed in cambium, WOX4 and PXY, were induced by grafting but the primary root marker WOX5 and lateral root marker LBD18 were not (Figure 3, Figure S5, Figure S6).
Genes whose transcripts are associated with various cell types or biological processes were taken from previously published datasets (see Table S1) and compared to the transcriptomic datasets generated here. The number in brackets represents the number of cell type-specific or process-specific genes identified in the previous dataset, and overlap is presented as a ratio out of 1.0 for differentiation expressed genes (DEG) up- or down-regulated in our dataset relative to intact samples. Asterisks represent a significant difference (p < 0.05) between the ratio of up- and down- regulated genes in a previously published transcriptome dataset compared to the ratio of all up- and down- regulated genes in our grafting dataset at a certain time point.
Auxin response is symmetric at the graft
The rapid activation of vascular markers in the grafted bottoms despite the starvation response promoted us to investigated whether other mobile substances such as phytohormones could play a role in gene activation. We compared lists of genes known to respond to cytokinin, ethylene or methyl jasmonate (33) and found no substantial overlap between these lists and genes differentially expressed by grafting (Figure S9)(Table S1). Abscisic acid-responsive and brassinosteroid-responsive genes showed overlap with genes differentially expressed in our datasets, but this overlap was of a similar magnitude in both separated and grafted datasets suggesting the effect was not specific to grafting (Figure S9). Auxin responsive transcripts were exceptional though, as they showed a substantial overlap with RNAs differentially expressed in our data sets that varied depending on the treatment (Figure 5A-B, Figure S9). Auxin-induced genes were upregulated in separated tops, grafted bottoms and grafted tops whereas they were repressed in separated bottoms (Figure 8). Auxin responsive genes such as IAA1 and IAA2 (34) were induced to similar levels in grafted tops and grafted bottoms at 24 hours. To further investigate whether auxin response was uniform between grafted tops and grafted bottoms, we grafted the auxin-responsive fluorescent reporter p35S:DII-Venus whose fluorescent protein is degraded in the presence of auxin (35). DII-Venus fluoresced in the separated bottoms but did not fluoresce in grafted bottoms 14 hours after cutting (Figure 5C) indicating separated bottoms had a low level of auxin response but grafted tops, grafted bottoms and separated tops had a high level of auxin response.
(A,D) Expression profiles for various auxin-responsive genes (IAA1, IAA2) or auxin transporter genes (PIN1 and ABCB1 genes) were plotted for intact, separated and grafted samples. (B) Overlap between previously published auxin-induced or auxin-repressed RNAs and our dataset. The number in brackets represents the number of auxin-responsive genes identified in the previous dataset, and overlap is presented as a ratio out of 1.0 for differentiation expressed genes (DEG) up- or down-regulated in our dataset relative to intact samples. Asterisks represent a significant difference (p < 0.05) between the ratio of up- and down- regulated genes in a previously published transcriptome dataset compared to the ratio of all up- and down- regulated genes in our grafting dataset at a certain time point. (C) Grafted and separated plants expressing the auxin responsive p35S::DII-Venus transgene that is degraded in the presence of auxin reveal a reduction of auxin response in cut bottoms, but not in grafted bottoms. HAG, hours after grafting.
Tissue fusion imparts a unique physiological response that differs from tissue separation
We hypothesized that the symmetric auxin response and asymmetric sugar response at the graft junction could allow a unique transcriptional response since neither separated plants nor intact plants had similar response dynamics to sugars and auxin as measured by genome-wide gene response, starch accumulation and p35S::DII-Venus expression (Figure 2, Figure 5). To uncover protein-coding genes differentially expressed specifically by grafting, we performed an empirical Bayesian analysis (36) where at each time point we computed likelihoods for each gene for each possible pattern of differential expression in the intact, grafted top, grafted bottom, separated top and separated bottom tissues (Figure 6A). These likelihoods were used to define a similarity score between pairs of genes, which was used to cluster genes with similar patterns of gene expression across the five tissues (37). Clusters were then identified by the predominant pattern of gene expression observed within a cluster. This analysis produced 113 clusters containing at least 10 RNAs at a single time point (Table S2; Dataset S1). Approximately 6000 genes were differentially expressed in at least one tissue whereas between 1000 and 4000 genes were not differentially expressed (Figure 6B).
(A) Cartoon depicting the Bayesian segmentation. (B) Analysis of differential behaviour produced 113 categories containing at least 10 genes whose expression was expressed in a specific differential pattern in at least one time point (see Table S2). One group is comprised of genes whose transcript levels are not substantially changed in the five tissues (unchanged) whereas the other group is comprised of the sum of the other 112 groups (genes whose transcript levels changed after treatment in a least one tissue) over the time points tested. (C) Major categories in the segmentation revealed RNAs whose levels changed in all the treatments listed relative to intact samples. Note that a gene can be represented only in one category for a given time point, that category in which the transcript level changes best fit the category.
To simplify the analysis, we considered clusters where gene expression was grouped into patterns consisting of one comparison. At early time points, the cluster containing genes differentially expressed in grafted tops, grafted bottoms, separated tops and separated bottoms had high numbers that decreased with time and could represent an general wound response (Figure 6C). Similarly, clusters containing separated tops and grafted tops and clusters containing separated bottoms and grafted bottoms initially had high numbers that decreased with time. This observation indicated the grafted top was initially transcriptionally similar to the separated top, whereas the grafted bottom was initially transcriptionally similar to the separated bottom. After the 48-hour time point, clusters containing genes only differentially expressed in separated tops or only differentially expressed in separated bottoms increased in numbers, suggesting these tissues gained a unique pattern of gene expression. The cluster containing genes differentially expressed in grafted tops and grafted bottoms increase in numbers throughout the healing process (Figure 6C). We searched for cluster categories that contained genes only differentially expressed by grafting and found very few genes downregulated by grafting or upregulated only in the grafted top (Figure 7A). Instead, there were clusters that contained several hundred differentially expressed genes present either in the grafted bottom only, or present in both grafted bottom and grafted top (Figure 7A). Genes whose expression changed only in the grafted bottom sample were prevalent early during grafting and were most common at 48 hours, whereas genes activated in both top and bottom became prevalent at 48 hours and were most common at 120 hours (Figure 7A). We performed a gene ontology (GO) analysis on the genes whose expression was grafting-specific and found that genes coding for RNAs differentially expressed only in the grafted bottom sample were enriched in the immune response and chitin response biological process categories (Table S3). Previously published chitin-induced RNAs had a high proportion of overlap with differentially expressed graft bottom-specific genes (Figure 7E, Figure S10). Grafting-specific RNAs expressed symmetrically, in both the grafted top and grafted bottom, were enriched in vascular-related biological processes (Table S3). Previously published phloem-enriched, endodermal-enriched, vascular-induction and stem-wounding associated RNAs had a high proportion of overlap with differentially expressed graft-specific genes (Figure 7C-D, Figure S10). Since few genes were grafting-specific and grafted tissues were initially transcriptionally quite similar to separated tissues (Figure 6C, Figure 7A), we reasoned that tissues separated for short periods (<48 hours) could be grafted with similar reconnection dynamics as tissues that had been grafted immediately. To test this hypothesis, plants were cut at day 0, and grafted at 1-5 days after separation. Separated tissues did not speed up vascular reconnection, and instead, it always took three days from the point of tissue attachment for vascular connections to form (Figure S10). Furthermore, the shoot lost competence to graft 2-3 days after separation whereas the root remained competent to graft up to 5 days after separation (Figure S10). Together, it appears the grafted shoot and root have a unique physiological response that differ from separated plants and that tissue attachment is required to activate graft formation and tissue fusion.
(A) Certain genes were only differentially expressed in grafted tops, grafted bottoms or both in grafted tops and grafted bottoms. (B) Expression profiles for a graft bottom-specific (ERF6) or a graft top and bottom differentially regulated gene (RTM2) were plotted for intact, separated and grafted samples. (C) Grafting-specific genes are also expressed in other processes of tissue fusion, such as stem healing. Here, 246 previously published RNAs whose differential expression is associated with stem healing were compared to our dataset to assess transcriptional overlap with the grafting-specific genes. Asterisks represent a significant high overlap (p <0.05) of previously published genesets that are also differentially expressed in the grafted samples at a certain time point. (D-E) Genes differentially expressed in grafted tops and grafted bottoms show high overlap to 324 previously published genes whose transcripts are associated with phloem, whereas genes expressed in grafted bottoms show high overlap to 286 previously published genes whose encoded RNAs are associated with chitin treatment. Asterisks represent a significant high overlap (p <0.05) of previously published genes that are also differentially expressed in the grafted samples at a certain time point.
DISCUSSION
To better understand how plants graft, we analysed in depth an RNA deep sequencing dataset that spatially and temporally distinguished genes activated by cutting followed by tissue attachment or continuous tissue separation. Cutting promoted a similar wound response in both grafted and separated tissues, however, by 72 hours after cutting the grafted and separated tissues became transcriptionally dissimilar (Figure 6C) indicating that tissue fusion was mechanistically different from healing an unattached cut surface. During graft formation, tissues had a very high transcriptional overlap with genes differentially expressed by inflorescence stem healing and by vascular induction in leaves (Figure 4, Figure S7) (9, 29) suggesting grafting is closely related to tissue reunion and vascular formation processes. Graft formation had little transcriptional overlap with lateral root formation (Figure S6)(32) and appeared to follow a pathway similar to secondary root growth (Figure S6, Figure S8) since cambium markers WOX4 and PXY that are specific to secondary growth were expressed during grafting (38)(Figure S5). Grafted tops initially showed a short-lasting and small transcriptional overlap with genes expressed during primary root formation which may be related to the accumulation of substances activating adventitious root formation, a common response in failed grafts or in cut shoots (Figure S4C). Thus, we conclude that grafting proceeds via a pathway involving secondary growth with radial meristems activating in the mature cambium to heal the wound. Vascular formation genes including those specifying cambium and phloem were activated early, followed by an activation of cell division genes, suggesting that the start of cellular differentiation preceded activation of cell division. Xylem identity genes showed an early and a late activation peak (Figure 3, Figure S5). There is no visible xylem differentiation at the graft junction during the first peak of expression (4) and these genes might be suppressed by phloem differentiation genes such as APL and CLE41 that activate early after grafting and are known to suppress protoxylem formation (39, 40)(Figure S6)(Table S4). Alternatively, the first peak could represent programmed-cell death that does not lead to xylem differentiation. The second expression peak of xylem-expressed genes at 120 hours occurred after the differentiation of functional phloem and coincided with the differentiation of xylem strands at the graft junction (4). Previous studies highlighted the importance of callus and pericycle cells during regeneration (16, 41), but we see little evidence that genes expressed in the pericycle or during callus formation have high transcriptional overlap with genes differentially expressed by grafting (Figure S4). Expression profiles for all protein-coding genes can be found in table S4.
We observe only a slight delay in phloem, cambium and cell division activation below the graft junction compared to above it (Figure 3, Figure S5, Figure S6) whereas several genes associated with vascular formation, such as HCA2(42) and TMO6(43), activated equally in both grafted top and grafted bottom at 6 hours after grafting (Figure 1D-G, Figure 3). These data indicate that, at least transcriptionally, the grafted root rapidly responded to the presence of the grafted shoot and this response was independent of functional vascular connections. This response was not present in separated roots, indicating that attachment was key for recognition. Sugars are known activators of cell division and cell elongation (13) and in our datasets, a large proportion of asymmetrically expressed genes are genes that are sugar-responsive. However, sugars are transported in the phloem (12) that is severed upon grafting whereas the grafted root exhibited a sugar starvation response and showed similar sugar-response dynamics as the separated root. Instead, we infer that not sugar but auxin, or some other molecular that is transported in the absence of vascular connections, could be the signal in the grafted bottom that activates HCA2, TMO6 as well as cell division, phloem- and cambium-related genes. Alternatively, separation might cause a build-up of molecules from the root that suppress vascular induction genes such as HCA2 and TMO6.
Given auxin’s role in vascular patterning and formation ((38), it is a strong candidate for the activating signal. Auxin response was largely symmetric, particularly from 12 hours after grafting (Figure 6) consistent with previous findings that the auxin-inducible DR5 and ANAC071 genes are symmetrically expressed around the graft junction (4–⇓6, 44). One idea is that auxin transport was not substantially interrupted by the grafting process, and instead, where opposing tissues adhered, auxin moved regardless of vascular connections since auxin is transported from cell to cell through the apoplast (8). The genes encoding auxin efflux proteins PIN1 and ABCB1 were transcriptionally activated above the graft junction (Figure 5D), similar to putative Pisum sativum PIN1 protein accumulating above a cut stem prior to vascular reconnection (45), and could reflect a role for these proteins in exporting auxin across the cut. Consistent with these observations, adding an auxin transport inhibitor to grafted Arabidopsis shoots prevented the expression of grafting-induced genes below the graft junction (6). Since auxin is fundamental for patterning vasculature (38), it is plausible that auxin movement across the graft occurs prior to sugar movement since the flow of auxin could assist with reconnecting the phloem. Although auxin response was symmetric, our previous work demonstrated that the auxin response factors ALF4 and AXR1 affected grafting only below the graft junction (4). Mutating ALF4 below the graft junction more strongly reduced auxin response than mutating ALF4 above the junction (4). Thus, proteins such as ALF4 or AXR1 might enhance or promote rootstock-specific auxin response and vascular regeneration that could be particularly important when there is incomplete attachment, cellular damage or inefficient transport. All higher plants transport auxin from shoot to root, yet not all plant species can be successfully grafted (3) so the response to auxin rather than the transport itself may be a determining factor in the ability to graft. A role for sugars is not completely ruled out though, since the magnitude of differential expression was often lower in the grafted bottom and exogenous sugar reduced grafting efficiency (Figure 2A). Pericycle cells require auxin to divide and the addition of sugars enhances the rate of divisions (46), suggesting that the presence of sugars in the grafted top enhanced cell division and differentiation. Another hypothesis that warrants testing is whether graft healing activates via a mechanical signal provided by the physical presence of the opposing tissues.
Our analyses identified two groups of genes whose expression changes were unique to graft formation in our experiments (Figure 7). One group activated shortly after grafting below the graft junction and was enriched in immune-responsive and chitin-responsive genes (Table S3, Figure 7, Figure S10). The breakdown products of cell walls are potent elicitors of defence responses (47), so it is possible that the grafted bottom upregulates pathways specific to wound damage response. This group was not upregulated in separated bottoms though, so the unique physiological state of the grafted root, indicated by the presence of auxin response but the absence of sugar response, could have promoted their upregulation. The second group activated both above and below the graft junction and became highly expressed later during graft formation (Figure 7). This group was enriched in RNAs associated with vascular development (Table S3, Figure 7) and we suggest that the products of these genes are involved in the vascular reconnection processes between the two tissues. Despite many transcriptional similarities between separated tops and grafted tissues, tissues had to be attached for at least three days for phloem connections to form, regardless of when cutting occurred (Figure S10). Thus, it appears that RNAs expressed in the separated top or separated bottom are insufficient to drive graft formation. Instead attachment and the genes activated by this recognition process including grafting-specific RNA changes (Figure 7A) or RNAs expressed in the grafted bottom, grafted top and separated top (Figure 6C) are those that contribute to a response that distinguishes attached from separated plant tissues. Future work should focus on these RNAs to identify the pathways required for grafting that could be modified to improve graft formation, wound healing and vascular regeneration. Likewise, the rapid transcriptional changes below the graft indicate a recognition system that promotes tissue regeneration. Identifying the exogenous cue that triggers recognition and understanding how it is perceived should be priorities, as should understanding whether this phenomenon applies more broadly to inter-tissue communication, tissue regeneration or tissue fusion events, such as parasitic plant infections (48), epidermal fusions (49, 50) or petal fusions (51).
METHODS
Plant material and microscopy
Arabidopsis thaliana accession Columbia was used throughout. The p35S::GFP-ER (52), pSUC2::GFP (53), pUBQ10::PM-tdTomato (54), pARR5::GFP (55), pANT::H2B-YFP (56), pLOG4::n3GFP (57), pCASP1::NLS-GFP (58), p35S::DII-Venus (35) lines have been previously published. For the construction of pHCA2::RFP, a 2.9kb 5’ upstream region of the HCA2 gene was cloned into pDONRp4-p1R donor vector, and recombined with tagRFPer into a destination vector by the Multisite Gateway system (59). Arabidopsis thaliana micrografting and grafting assays were performed according to previously published protocols (60, 61). Fluorescent images were taken on a Zeiss LSM-700 or LSM-780 confocal microscope. Black and white fluorescent images of graft junctions were taken on a Zeiss V12 dissecting microscope. FIJI software (Fiji.sc) was used to process images. A micro-extensometer was used for all breaking force graft measurements according to a previously published protocol (62).
RNAseq sample and library preparation
Grafted wild type Arabidopsis thaliana accession Col-0 were harvested at the respective time points and care taken to separate grafts by gently pulling plants apart. Approximately 0.5mm of tissue was taken above or below each cut site and kept separate. Intact plants had 1mm of tissue taken in a similar location on the hypocotyl as separated or grafted plants. Grafted, separated or intact tissues were pooled into groups of approximately 24 tissues. Tissues were ground using a microcentrifuge pestle frozen in liquid nitrogen. RNA was extracted using an RNeasy Kit (Qiagen, UK) following the manufacturer’s instructions. 90-100ng of RNA was used to prepare RNAseq libraries using the TruSeq® Stranded mRNA LT kit (Illumina, UK) according to the manufacturer’s instructions. The final PCR was for 15 cycles and 11-12 barcoded samples were randomly mixed to make a total of 7 mixes for 7 flow lanes, one mix per lane. Samples were sequenced on the HiSeq 4000 platform (Illumina, UK) with Paired End 75bp transcriptome sequencing (BGI Tech Solutions, Shenzhen, China).
Iodine staining
Arabidopsis seedlings were placed in a fixation solution (3.7% formaldehyde, 50% ethanol, 5% acetic acid) for 1 hour at room temperature, then transferred to 70% ethanol for 10 minutes. Afterwards, plants were transferred to 96% ethanol and stored at −20°C for up to a week. Samples were rehydrated in 50% ethanol for 1 hour at room temperature, transferred to distilled water for 30 minutes, then stained for 10 minutes in Lugol solution (Sigma) at room temperature. Plants were rinsed with water and mounted on microscope slides. Images were taken on a Zeiss Axioimager.M2 microscope.
Bioinformatic analyses
The reads acquired through high-throughput sequencing were quality trimmed with sickle (63) and aligned using the eXpress tool to protein-coding gene sequences acquired from TAIR10 using Bowtie2. Library scaling factors were inferred from the sum of the number of reads assigned to the genes in the lowest seventy-five percentiles of expressed genes for each library (64). Analyses of the data were carried out using the R package baySeq (36)and clustering based on the posterior probabilities acquired from this package. The gene ontology analysis (GO) enrichment analysis on grafting-specific genes was done with a customized R script using the package GOstats (65).
ACKNOWLEDGEMENTS
We thank Niko Geldner, Dolf Weijers, Paul Tarr, Yka Helariutta, Ruth Stadler and The Arabidopsis Information Resource for providing seeds. Funding for this work was provided by Gatsby Charitable Trust grants GAT3272/C and GAT3273-PR1, by a Knut and Alice Wallenberg Academy Fellowship KAW2016.0274 (to C.W.M), and by the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation grant GBMF3406 (to E.M.M.)
Footnotes
Contact: charles.melnyk{at}slu.se
REFERENCES
