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. 2026 Apr 17;201(3):kiag187. doi: 10.1093/plphys/kiag187

Molecular insights into avocado grafting: the role of chitinase-like genes

Vikas Dwivedi 1,2, Lalita Pal 3,4, Gal Hadas-Brandwein 5,6, Sela Yechezkel 7, Alexander Klaus 8, Adi Faigenboim-Doron 9, Eduard Belausov 10, Balaji U Solanke 11, Roy Weinstain 12, Einat Sadot 13,b,✉,c
PMCID: PMC13360294  PMID: 41999041

Abstract

Grafting is an agricultural technique used widely to increase yield, enhance stress tolerance, and improve disease resistance. The valuable avocado (Persea americana Mill.) rootstock VC801 provides these benefits but exhibits poor rooting capacity, necessitating a labor-intensive double-grafting procedure that involves a nursing seedling and etiolation. In this study, we found that grafting of the “Hass” cultivar onto VC801 was successful only when the rootstocks were etiolated; by contrast, grafts on green rootstocks largely failed, indicating physiological incompatibility. Transcriptome analysis revealed that graft success was associated with differential expression of cell wall–related genes, among which chitinase-like transcripts were particularly prominent. Repeated RT-qPCR validation highlighted the Ctg0022 chitinase-like transcript. The Arabidopsis (Arabidopsis thaliana) mutant ctl1-1, which is defective in a homologous chitinase-like gene, displays graft incompatibility. Remarkably, heterologous expression of Ctg0022, particularly in the rootstock, restored graft compatibility in these mutants. Complementary analyses, including inhibitor treatments, histology, microscopy, and gene expression profiling, indicated that Ctg0022 modulates cell wall remodeling by influencing cellulose, lignin, and pectin composition. Fluorescence lifetime imaging further showed aberrant cell wall assemblies and impaired cell division and cell adhesion at the graft interface of ctl1-1 mutants; these were restored in Ctg0022-expressing plants. Together, our findings demonstrate that the fine-tuned expression of avocado chitinase-like gene Ctg0022 can promote successful graft union formation, highlighting a conserved role in cell wall remodeling during grafting.

Fine-tuned regulation of chitinase-like gene expression, influencing cell wall properties in the context of rootstock physiology, is important for successful grafting.

Introduction

The avocado (Persea americana Mill.) belongs to the family Lauraceae. A rootstock selection project performed by Avraham Ben-Ya’acov in Israel (Ben-Ya’acov et al. 1992; Ben-Ya’acov and Michelson 1995; Ben-Ya’acov and Zilberstaine 1999a, 1999b) yielded a rich collection of rootstocks. The vegetative propagation of these elite rootstock clones by adventitious root formation from stem cuttings is very difficult. Specifically, they can root almost only by the “Frolich method” when grafted on a seedling and grown in the dark to form an etiolated branch (Frolich and Platt 1972; Brokaw 1975), suggesting a prerequisite contribution from the juvenile seedling and from the physiological change by etiolation, which together provide the permissive conditions for adventitious root differentiation. These etiolated branches are then grafted as rootstocks themselves with the required varieties as scions. Recently, we found that the upper part of the etiolated branches from rootstock VC801, (which is pruned before the second graft) can be rooted despite its detachment from the nurse seedling, and even better after a short de-etiolation (Duman et al. 2020). However, it was found that the rate of graft success on the rooted rootstocks (green by this time) is much lower than that on the etiolated branches, which are being nourished by a seed. This suggested that the physiological conditions provided by the nursing juvenile seedling and by etiolation contribute both to adventitious root formation (Duman et al. 2020) and graft success. The latter issue is the subject of the current work described here.

Graft unification success depends on several parameters. For example, taxonomic distance is crucial (Goldschmidt 2014). Since all avocado varieties belong to the species Persea americana, taxonomic distance should not confer a barrier in rootstock/scion compatibility; nevertheless, due to heterozygosity, differences in graft compatibility might occur.

Graft unification is formed in several steps. Histological analysis of graft areas showed, as one of the first responses, the accumulation of pectin which contributes to cell adhesion between the scion and rootstock, forming a middle lamella–like layer (Asahina et al. 2002; Sala et al. 2019). Cell division (in many cases oriented toward the cut) follows from the cambium, cortical parenchyma, and pith. The callus formed by this cell proliferation fills the rootstock–scion gap and is the source for vasculature differentiation at a later step (Melnyk 2017a, 2017b; Wang et al. 2017; Kragler and Bock 2025).

Accumulated data shed light on the molecular mechanisms underlying these processes of graft unification. When transcriptome profiles were compared between separated or grafted scion and rootstocks of Arabidopsis seedlings, it was found that genes related to vascular formation were specific to the grafted tissue (Melnyk et al. 2018). In addition, more differences were found between grafted and separated rootstocks than scions, suggesting that more changes occur in the rootstocks. Temporal asymmetry was found between the scion and rootstocks characterized by sugar-responsive genes but not by auxin-responsive genes (Melnyk et al. 2018), probably because of differences in their transport mechanisms (sugars by vasculature not yet formed but auxin by cell-to-cell transport which is able to cross the junction) and possibly also due to local synthesis of auxin (Omary et al. 2022). Involvement of cell wall modification–related transcripts was observed both in inter- and intrafamily grafting (Notaguchi et al. 2020; Kragler and Bock 2025). Furthermore, following cell wall damage, DNA binding with one finger (DOF) transcription factors were activated at the grafting zone in Arabidopsis (Zhang et al. 2022). Recently the PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) was found to be a central regulator of graft unification in both Norway spruce (Picea abies) and Arabidopsis (Feng et al. 2024). Taken together, these reports suggested auxin and sugar signaling, as well as cell wall modifications, as major players in graft unification.

In this work, we addressed the question of what the molecular basis is for the high rate of success of grafting the avocado Hass variety on a VC801 rootstock when it is an etiolated branch grafted on a seedling, compared to the low rate of success of grafting the same pairing when the rootstock is a green branch on its roots.

Results

The VC801 rootstock exhibits improved grafting success when etiolated and grafted onto a seedling compared to when it is green and grown on its own roots

Due to its strong performance in various tests—including high yields, resistance to soil pathogens Phytophthora cinnamomi and Verticillium dahliae, as well as tolerance to saline irrigation water (Ben-Ya’acov et al. 1992; Lazare et al. 2019; Haberman et al. 2020)—VC801 was selected for this study.

To compare the grafting success of avocado variety Hass scions on etiolated grafted VC801 versus green-rooted VC801 rootstock, it was first necessary to root branches of VC801. Previous efforts yielded an average rooting success rate of less than 20% with de-etiolated branches of VC801 (Duman et al. 2020). Therefore, enhancing rooting efficiency was crucial before proceeding with grafting. Screening our library of synthetic auxin conjugates (Roth et al. 2024) revealed that using a combination of potassium indole-3-butyric acid (K-IBA) at 1,000 ppm and 2,4-dichlorophenoxyacetic acid (2,4-D)-Ile at 8 µM increased the average rooting success to over 50% (Fig. 1a and 1b).

Figure 1.

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Graft incompatibility between green-rooted avocado VC801 rootstocks and Hass scions. a) Rooted cuttings of VC801. b) Rooting efficiency of VC801 cuttings. Error bars represent the standard deviation (±SD) from 4 biological replicates, each including 20 cuttings per treatment. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. c) Grafting success rate of VC801 rootstocks with Hass scions. Error bars represent ±SD from 2 biological replicates, each consisting of 10 to 20 grafts per specific rootstock. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. d) Histological section of a successful graft on an etiolated rootstock, 2 months post-grafting. The arrow indicates tissue union at the graft junction between the rootstock (Rs) and scion (Sc). Inset: bud sprouting from the grafted scion. Scale bar = 500 µm. e) Histological section of an unsuccessful graft on a green-rooted rootstock, 2 months post-grafting. The arrow indicates tissue separation at the graft junction. Inset: dormant bud on the grafted scion. Scale bar = 500 µm. f) RNA-Seq-based transcript levels of 2 chitinase-like genes (Ctg0022 and Ctg1710):0E: Etiolated rootstock at time 0, 5E: Etiolated rootstock after 5 days, 0G: Green rootstock at time 0, 5G: Green rootstock after 5 days. All grafts were performed with identical Hass scions. Error bars represent ±SD from 3 biological replicates, except for the green rootstock at 5 days, which had 2 replicates. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. g) Quantitative RT-qPCR analysis of Ctg0022 and Ctg1710 expression in rootstocks (r) and scions (S), showing differential gene expression patterns. Hass scions were consistent across all grafts. Etiolated vs green and time 0 vs 5d labels are like in (f). h) Quantitative RT-qPCR analysis of Ctg0022 expression in green VC801 rootstocks under different conditions: T0 w/o graft: No graft at time 0, T5 w/o graft: No graft after 5 days, T5 plastic graft: Grafted with a plastic scion mimic after 5 days, T5 self-graft: Self-grafted after 5 days, T5 Hass-graft: Grafted with Hass scion after 5 days I) RT-qPCR analysis of Ctg0022 expression at 0, 1, 3, and 5 days after grafting of VC801/VC801 and Hass/VC801 combinations. Error bars in G-I represent ±SD from 3 biological replicates, each with 3 technical replicates. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA.

The rooted branches were grafted with Hass scions and compared to the commercial grafting method described by Frolich (Frolich and Platt 1972), which involves grafting onto a nursing seedling followed by etiolation. The Frolich method achieved a grafting success rate of 77.5% ± 11.5%, whereas grafting onto green-rooted rootstock resulted in a significantly lower success rate of 4.5% ± 3.5% (Fig. 1c). Interestingly, the VC51 rootstock performed better as a rooted rootstock, indicating unique challenges associated with VC801. Grafting success was assessed based on bud sprouting from the scion 1 to 2 months after grafting (Fig. 1d and 1e insets).

Histological examination of the graft junction 2 months post-grafting revealed that the Frolich method resulted in a continuous cambium and vascular connection between the rootstock and scion (Fig. 1d, arrow). In contrast, grafting onto green-rooted branches showed limited cell activity and partial cell adhesion only at the joint periphery (Fig. 1e, arrow). Since both rootstock (VC801) and scion (Hass) are clonal selections with similar genetics, the observed graft incompatibility likely stems from the physiological state of the rootstock.

To investigate the molecular mechanisms underlying these differences, RNA sequencing was performed on the graft junction (Hass/VC801 etiolated or green) at time 0 and 5 days post-grafting (Supplementary Dataset S1). Bioinformatics analysis identified more than 1,600 significantly differentially expressed genes (DEG), with significant contribution to cell wall–related transcripts (Supplementary Dataset S2), among them chitinase-related transcripts, particularly Ctg0022 and Ctg1710, in green vs etiolated rootstock-based grafting at 5 days after grafting (5DAG) (Supplementary Dataset S1, tab 5E < 5G, and Fig. 1f). These findings were validated in a repeat experiment using reverse transcription quantitative PCR (RT-qPCR), where rootstocks and scions were separated prior to RNA extraction. Results confirmed a significant induction of Ctg0022 and Ctg1710 transcripts in green-rooted rootstocks compared to etiolated rootstocks, with no significant differences in the scions (Fig. 1g).

To determine whether this induction was related to wound response or grafting per se, an additional experiment was conducted using green-rooted rootstocks that had been grown for 1 year. Treatments included cutting without grafting, grafting with Hass scions, self-grafting with VC801 scions, and control grafting with a 3D-printed plastic scion to simulate mechanical pressure without biological interaction (Figure S1), following our observation that columns of dividing cells formed parallel to the graft plane (Figure S1A), similar to pressure-induced cell division in callus cells (Lintilhac and Vesecky 1984). RNA was extracted from the rootstocks at day 0 and at 5 days post-treatment. Self-grafting achieved 60% ± 14.1% successful unification, while grafting Hass on green-rooted VC801 only seldom succeeded (4.75% ± 5.5%). Expression analysis revealed that Ctg0022 induction was comparable across cut-only, plastic scion-grafted, and Hass scion-grafted treatments, suggesting that wounding specifically induces Ctg0022. However, significantly higher Ctg0022 expression occurred in self-grafted rootstocks (Fig. 1h). While Fig. 1f and 1g indicate that higher expression levels were associated with graft failure, Fig. 1h shows that higher expression of Ctg0022 correlated with graft success. To clarify these contrasting observations, the experiment was repeated (Figure S2), and Ctg0022 expression was monitored over time. As shown in Fig. 1i, Ctg0022 expression gradually increased on days 1, 3, and 5 following self-grafting of VC801. In contrast, when Hass was grafted onto VC801, Ctg0022 expression was slightly induced but remained constant over the same period.

Taken together, these results suggest that graft success involves the precise regulation and optimization of Ctg0022 expression, influenced by the physiological state of the rootstock (etiolated, de-etiolated, or green) and its effects, among other factors, on cell wall properties (Duman et al. 2020). Moreover, cell wall components can influence one another; for example, a mutation in a chitinase-like gene led to increased lignin deposition (Zhong et al. 2002). Finally, the specific scion–rootstock combination may further modulate reciprocal gene expression throughout the grafting process (Goldschmidt 2014).

Involvement of chitinase-like transcripts in graft unification

To investigate whether changes in the expression of chitinase-related genes are generally associated with graft unification, we examined their prevalence among the DEGs in our comprehensive avocado dataset. Figure 2a shows the expression levels of 21 transcripts related to chitinases, comparing the graft domain at day 0 and day 5 on etiolated versus green rootstocks. While some transcripts were significantly upregulated when grafting was performed on green rootstocks compared to etiolated ones, others were downregulated after 5 days or showed higher activation in etiolated grafts than in green ones. Additionally, we analyzed 2 published grafting expression profiles involving potentially incompatible interspecies graft pairings. Figures S3 to S7 highlight that several chitinase-like transcripts are consistently regulated during the grafting process in different plants. Specifically, Figure S3 presents a heat map of chitinase-like transcripts during the grafting of Vitis vinifera cv. “Cabernet-Sauvignon N” onto itself (CS/CS) or onto V. riparia cv. “Riparia Gloire de Montpellier” (CS/RG) (Cookson et al. 2014). Notably, 3 transcripts were exclusively upregulated in the heterograft (CS/RG), suggesting a possible link to interspecies incompatibility. A recent comprehensive study on interspecies grafting examined gene expression profiles from several scion/rootstock combinations (Notaguchi et al. 2020). Figures S4 to S7 illustrate the expression patterns of chitinase-like transcripts during several days of both intra- and interspecies grafting of Arabidopsis thaliana, Nicotiana benthamiana, Glycine max, and Ipomoea nil (Notaguchi et al. 2020). The data reveal that the activation or suppression of various chitinase-related transcripts occurs in both intra-species (At/At, Nb/Nb, Gm/Gm, In/In) and interspecies grafts (Nb/At, Gm/At, In/At). Of note, one of the main conclusions of this study (Notaguchi et al. 2020) was that β-1,4-glucanase from the glycosyl hydrolase 9B (GH9B) family plays a critical role in grafting. A phylogenetic analysis of the differentially expressed chitinase-like transcripts during grafting (Fig. 2b) revealed that these transcripts belong to 2 main groups: glycosyl hydrolase 19 (GH19) and GH18. In summary, the evidence suggests that fine-tuning the expression of chitinases during graft unification is a common phenomenon in several plants.

Figure 2.

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Abundance of differentially expressed chitinase-related genes during plant grafting. a) Heat map displaying all avocado chitinase-like transcripts that were differentially expressed during grafting, based on RNA-Seq data (FPKM values)generated in this study. Unit variance scaling is applied to rows. Rows were clustered using correlation distance and average linkage. b) Phylogenetic tree of chitinase-related transcripts with grafting-responsive expression across multiple plant species, including avocado (this study), Vitis species (Cookson et al. 2014), Arabidopsis thaliana, Nicotiana benthamiana, Glycine max, and Ipomoea nil (Notaguchi et al. 2020). Avocado sequences are highlighted in red. GH18 and GH19 indicate members of the glycosyl hydrolase families 18 and 19, respectively. The numbers on the branches indicate bootstrap values (%FPKM values).

The avocado Ctg0022 chitinase-like protein

The sequences of Ctg0022 and, Ctg1710 cDNAs (Fig. 3a and Figure S8) encode chitinases from the GH19 group, subclass II. Notably, Ctg0022 contains a signal peptide at the N-terminus, while Ctg1710 does not. Both proteins lack a classical chitin-binding domain (Taira 2010; Grover 2012). Chitinases are enzymes that catalyze the hydrolytic cleavage of the β-1,4-glycosidic bond present in carbohydrate chains of N-acetylglucosamine, primarily found in chitin (Taira 2010; Grover 2012). The closest Arabidopsis homolog to Ctg0022 based on sequence similarity is At3g12500, sharing 39.4% amino acid identity. However, At3g12500 encodes a chitin-binding site that Ctg0022 lacks. Additionally, At3g12500 shows low expression levels in plants and is downregulated during grafting, unlike Ctg0022. Conversely, the most highly expressed Arabidopsis chitinase transcript in the graft domain is At1g05850 (CTL1) (Notaguchi et al. 2020), also a GH19 class II glycosyl hydrolase. Similar to Ctg0022, CTL1 lacks a chitin-binding domain and exhibited a slight increase in expression during self-grafting (Notaguchi et al. 2020). Although CTL1 shares only 20.4% amino acid sequence identity with Ctg0022, they may have comparable functional roles. To investigate whether Ctg0022 functions similarly to CTL1, the cDNAs of both were isolated, fused to red fluorescent protein (RFP-mCherry), and expressed in Nicotiana benthamiana. As shown in Fig. 3b to 3e, both transcripts encode proteins that are secreted to the cell wall, consistent with previous reports for CTL1 (Sánchez-Rodríguez et al. 2012). To assess functional activity, Ctg0022 was expressed in ctl1-1 mutant plants (Sánchez-Rodríguez et al. 2012) under the control of the CTL1 promoter. As shown in Fig. 3f and 3g, while ctl1-1 plants were shorter compared to wild-type plants, expressing Ctg0022 restored normal growth, demonstrating that the construct encodes a functional protein similar to AT1G05850. In contrast, expressing Ctg1710 did not complement the ctl1-1 mutant (Figure S9). No RFP fluorescence was detected in the transgenic plants, indicating that expression levels were below the detectable threshold, although RT-qPCR confirmed expression similar to CTL1 levels in wild-type Arabidopsis (Fig. 3h). To evaluate the effect on graft success, plants were grafted, and the results are shown in Fig. 3i and 3j and Figure S10. While wild-type plants exhibited an 80% graft success rate, ctl1-1 mutants had almost no successful grafts. Expression of CTL1-GFP under the 35S promoter in ctl1-1 plants partially restored graft success to 40%. Remarkably, expressing Ctg0022 under the CTL1 promoter increased graft success to over 60% in the Ctg0022-L1 line and to almost 60% in the Ctg0022-L2 line under similar conditions. To determine whether chitinase-like protein expression is necessary in the rootstock or the scion, reciprocal grafting experiments were performed using wild-type Col, ctl1-1, or Ctg0022-L1, L2 as either rootstock or scion. Consistent with avocado grafting results, we found that the expression of CTL1 or Ctg0022 is essential in the rootstock for successful graft union (Fig. 3j). All reciprocal grafts with ctl1-1 as the rootstock failed, while successful grafting occurred when ctl1-1 was used as the scion, although with reduced efficiency compared to wild-type plants. Importantly, in agreement with the avocado transcriptomics, expression analysis confirmed the upregulation of CTL1 5 days after grafting in the rootstock of Col wild-type plants. Expression of Ctg0022 in Ctg0022-L1, L2 plants was induced at 48 hours and 5 days (Fig. 3k).

Figure 3.

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Functional complementation of ctl1-1 mutant with Ctg0022. a) Schematic comparison of the protein structures of Ctg0022 and AtCTL1. b, d) Transient expression of 35S::Ctg0022-mCherry and 35S::CTL1-mCherry in Nicotiana benthamiana. Cell walls were stained with calcofluor white (blue). c, e) Line scan analyses demonstrate that both proteins localize to the cell wall. The blue graph represents the fluorescence intensity of calcofluor, which stains cellulose, while the red graph corresponds to the fluorescence intensity of red fluorescent protein mCherry fused to Ctg0022 or CTL1 along the line shown in panels (b) and (d). f, g) Expression of Ctg0022 driven by the native CTL1 promoter rescues the growth arrest phenotype of the ctl1-1 mutant. L1 and L2 represent 2 independent transgenic lines. Error bars in panel (g) represent ±SD from 9 measured plants for each line. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. h) Quantitative RT-qPCR showing expression of endogenous CTL1 in wild-type plants and Ctg0022 in ctl1-1/Ctg0022 transgenic lines. Error bars represent ±SD from 3 biological replicates, each with 3 technical replicates. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. i, j) Self, and reciprocal grafts between Col wild-type, ctl1-1, 35S::CTL1-GFP, and ctl1-1/Ctg0022 lines L1 and L2 after 7 days. RS- rootstock, Sc- scion, AR- adventitious root. Error bars in panel J represent ±SD from 3 biological replicates, each including 10 grafts per combination. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. k) Induced expression of CTL1 (in Col rootstock) and Ctg0022 (in L1 and L2 lines) 5 days after grafting. Error bars represent ±SD from 3 biological replicates, each with 3 technical replicates. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA.

The role of chitinase AvCtg0022 in plant cells during grafting

Previous studies have demonstrated that CTL1 influences cellulose assembly and participates in cross-linking glucan-based polysaccharides characteristic of cellulose and hemicelluloses. Additionally, the reduced cellulose deposition indirectly led to lignin accumulation (Zhong et al. 2000, 2002; Sánchez-Rodríguez et al. 2012). Furthermore, reduced pectin methyl-esterification has been reported in the arm mutant of CTL1 (Hermans et al. 2011).

Expression of Ctg0022 in ctl1-1 mutants restored lignin levels to normal (Fig. 4a). In ctl1-1 plants, elongating root cells exhibit swelling due to decreased cellulose content, which compromises fiber resistance to turgor pressure in a polar manner. As cellulose fiber orientation is directed by microtubules (Paredez et al. 2006), partial disruption of microtubules with oryzalin is expected to further impact these cells. Consistently, ctl1-1 plants grown in the presence of oryzalin showed increased sensitivity compared to wild-type plants and the recovered Ctg0022-expressing lines, as indicated by root width measurements (Fig. 4b and 4c). Isoxaben, a compound that inhibits cellulose deposition by targeting cellulose synthase subunits (Scheible et al. 2001; Desprez et al. 2002), was used to assess the response of ctl1-1 mutants. When grown on isoxaben, ctl1-1 plants exhibited greater sensitivity compared to wild-type and Ctg0022 recovered lines, as measured by root length (Fig. 4d and 4e). Confirming that Ctg0022 chitinase can recover the ctl1-1 hyper-sensitivity to oryzalin and isoxaben.

Figure 4.

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Impact of Ctg0022 on cell wall response to inhibitors. a) Phloroglucinol staining showing lignin deposition in Col, ctl1-1, and ctl1-1/Ctg0022 (L1 and L2) lines. Scale bar = 50 μm. Arrows show high accumulation of lignin staining in the ctl1-1 mutant. b) Morphological response of Col, ctl1-1, and ctl1-1/Ctg0022 (L1 and L2) lines to 100 nM oryzalin treatment. Scale bar = 100 μm. The black line shows the width of root which increases after oryzalin treatment. c) Quantification of root diameter in plants grown on 100 nM oryzalin. Error bars represent ±SD from measurements of 20 roots. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. d) Root growth response of Col, ctl1-1, and ctl1-1/Ctg0022 (L1 and L2) lines to 1.5 nM isoxaben. Scale bar = 0.5 cm. e) Root length measurements of the plants in (d). Error bars represent ±SD from measurements of 20 roots. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA.

To monitor potential changes in pectin during graft formation, we stained newly dividing cells (Figure S11) from both the rootstock and scion—prior to the establishment of the graft union—using anti-pectin antibodies. Specifically, we used LM19, which preferentially binds to de-methyl-esterified pectin, and LM20, which preferentially binds to methyl-esterified pectin (Verhertbruggen et al. 2009). Consistent with previous findings (Sala et al. 2019), our results show that these cells secrete pectin, likely serving as an adhesive material that facilitates the formation of the graft union (Fig. 5a).

Figure 5.

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Pectin composition in newly dividing cells 5d after excision. a) Immunolabeling using anti-pectin antibodies to visualize pectin distribution at the graft domain. b) Quantification of fluorescence intensity in cells as shown in panel (a). Error bars in panel (b) represent ±SD of fluorescence measurements from 24 to 138 regions of interest (ROIs) per scion or rootstock for each line. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. c, d) Expression analysis of PME3 in scions and rootstocks. e, f) Expression analysis of PMEI in scions and rootstocks. Error bars in (c–f) represent ±SD from 3 biological replicates, each with 3 technical replicates. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA. g) Expression network of all avocado differentially expressed genes (DEGs) encoding chitinase-like and pectin-modifying proteins, constructed using Pearson correlation analysis (r > 0.8). Nodes are color-coded as follows: pink, Ctg0022; yellow, pectin-related DEGs; purple, chitinase-related DEGs. Green and red lines indicate positive and negative correlations, respectively. RS, rootstock; Sc, scion, PME, pectin methyl esterase; PMEI, pectin methyl esterase inhibitor; PL, pectin lyase; PAE, pectin acetyltransferase.

Fluorescence intensity measurements revealed notable differences in the LM19/LM20 staining ratio, with the ctl1-1 scion exhibiting a markedly higher level of de-esterified pectin (Fig. 5b). This observation aligns with an increase in the expression of Pectin Methyl Esterase (PME3, At3g14310) (Fig. 5c) and a decrease of Pectin Methyl Esterase Inhibitor (PMEI, At1g14890) (Fig. 5e) in the ctl1-1 scion. Of note, both specific transcripts were previously shown to be differentially expressed during graft formation and to exhibit high expression compared to other family members of PME and PMEI (Notaguchi et al. 2020). In the scion, PME3 expression was significantly higher in ctl1-1 compared to Col and Ctg0022-L1 after 5 days. Notably, elevated PME3 expression was also detected after 5 days in the rootstock of Ctg0022-L1, in contrast to Col wild-type and ctl1-1 (Fig. 5d) plants, suggesting scion/rootstock a symmetry in a possible chitinase-PME3 co-regulation. In addition, higher expression of PMEI was found in both scion and rootstocks of ctl1-1 at time 0 compared to 5 days (Fig. 5E and 5f), in contrast to no difference in Col and Ctg0022-L1.

Expression network analysis using our avocado transcriptomics data (Fig. 5g), between differential expressed chitinase-like transcripts and pectin modification–like transcripts such as PME, PMEI, pectin lyase (PL), and pectin acetyl transferase (PAE) shows interaction (r ≥ 0.8) between Ctg0022 and 3 PMEI-like transcripts. Furthermore, 3 more chitinase-like transcripts, among which is Ctg1710, are closely associated with the same PMEI-like transcripts. Taken together, our data suggest potential context-dependent co-regulation of specific chitinase-like expression and pectin modification in a possible shared transcriptional network (Sinha et al. 2016).

The pronounced impact of chitinase expression on cell wall composition prompted us to take a broader perspective on cell walls in relation to the different steps during the formation of scion–rootstock connections. To this end, we measured the fluorescence lifetime of cell walls, which serves as a sensitive probe for detecting structural modifications (Chabbert et al. 2018). Imaging of the graft junction was carried out at 0, 24, and 48 hours, as well as 5 days post-grafting, focusing on the autofluorescence lifetime of newly dividing cells (Fig. 6A–6O). Consistent with previous findings (Melnyk 2017a), cell division in the scion preceded that in the rootstock (Fig. 6b, 6j, 6n, and 6o). Notably, in both wild-type and Ctg0022-L1, newly dividing cells in the scion were detectable as early as 24 hours after grafting (Fig. 6b and 6j). In contrast, ctl1-1 plants showed signs of cell division only after 48 hours (Fig. 6f, 6g, 6n, and 6o). Moreover, the cell walls of newly dividing cells in Col and Ctg0022-L1 lines exhibited extremely weak and barely detectable autofluorescence (Fig. 6b, 6c, 6j, 6k, and 6m). In contrast, ctl1-1 plants showed strong autofluorescence signals with higher number of events showing longer lifetime profiles (Fig. 6g, 6h and 6m)—a pattern that markedly differed from the wild-type or recovered lines, and suggesting the unexpected accumulation of phenolic compounds. By 5 days post-grafting, cells from the scion and rootstock had adhered in both Col wild-type and Ctg0022-L1 lines. However, in ctl1-1 plants, such adhesion was rare. Collectively, these findings indicate that altered cell wall properties in ctl1-1 plants—reflected by prolonged autofluorescence lifetimes—are associated with delayed cell division and impaired cell adhesion, both of which are critical for successful grafting and were restored upon Ctg0022 expression.

Figure 6.

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Kinetics of graft union formation. Col, ctl1-1, and ctl1-1/Ctg0022 (L1) plants were grafted and imaged at 0, 24 hours, 48 hours, and 5 days post-grafting. Transmitted light imaging was followed by autofluorescence lifetime acquisition to visualize cell walls. Color scale bars indicate fluorescence lifetime in nanoseconds (ns). In each pair, the upper section corresponds to the scion and the lower section to the rootstock. By day 5, Col and ctl1/Ctg0022 had already formed a union and were imaged together. a–d) Col wild-type plants. e–h) ctl1-1 mutant plants. i–l) ctl1-1/Ctg0022 (L1) complemented plants. Scale bars = 10 μm. Arrowheads indicate autofluorescence of cell walls in newly dividing cells or mark the location of the cell walls in the fluorescence image when no autofluorescence is detected. m) Quantification of events associated with the different fluorescence lifetimes (in nanoseconds; ns) measured in the cell walls of newly dividing cells. Graphs represent the average fluorescence lifetimes obtained from 3 to 5 cells per plant, across 3 plants for each Arabidopsis line. n) Quantitative analysis of scions displaying new cell division (n = 30 per time point). o) Quantitative analysis of rootstocks showing new cell division (n = 30 per time point). Error bars in N–O represent ±SD. Different letters indicate statistically significant differences (P < 0.05), determined by one-way ANOVA.

Discussion

Unlike physiological incompatibility, typically manifested as the delayed collapse of grafted plants (Kragler and Bock 2025), we observed immediate graft failure caused by physiological differences between green and etiolated VC801 rootstocks. Comparative transcriptome analysis, supported by data from other species, pointed to a role for plant chitinases whose expression is modulated during compatible and incompatible graft interactions. Among these, the avocado chitinase-like gene Ctg0022 was highly expressed and functionally complemented the graft-incompatible Arabidopsis ctl1-1 mutant. Notably, overexpression of Arabidopsis CTL1 under the 35S promoter reduced grafting efficiency compared with wild-type or Ctg0022-complemented lines, emphasizing the need for precise regulation of chitinase expression. In contrast to our data, reduction of CTL1 expression was observed 5 days after Arabidopsis grafting compared to day 0 in the experimental conditions previously described by Melnyk et al. (2018), possibly due to differences in growth conditions.

Although their name reflects their hydrolytic activity against chitin—a primary component of fungal cell walls and insect exoskeletons (Vaghela et al. 2022)— it is noteworthy that plants themselves do not produce chitin. This suggests that plant chitinases primarily evolved as part of the plant defense system. While some chitinase transcripts are indeed induced by pathogen attacks (Vaghela et al. 2022), others are not, and some members of this enzyme family lack chitin-hydrolyzing activity altogether (Passarinho and de Vries 2002; Taira 2010). This functional diversity indicates that certain chitinases may have evolved additional or entirely different roles beyond pathogen defense.

The role of chitinases in normal plant development remains incompletely understood. While some are secreted into the apoplast (Sánchez-Rodríguez et al. 2012) and others targeted to the vacuole (Stigliano et al. 2014), secreted chitinases have been implicated in somatic embryogenesis (De Jong et al. 1992; De Jong et al. 1995; Domon et al. 2000). These findings align with our observation that precise regulation of chitinase-like expression is essential for successful graft union formation, a process relying on coordinated cell division and differentiation.

Mutations in CTL1 and related chitinase-like genes cause developmental defects associated with altered cell wall composition in several species, including Arabidopsis (Zhong et al. 2000, 2002; Hermans et al. 2010, 2011; Sánchez-Rodríguez et al. 2012), rice (Wu et al. 2012), maize (Jiao et al. 2019), and cotton (Liu et al. 2024). Although CTL1 lacks chitinase activity (Zhong et al. 2002; Hermans et al. 2010), it binds glucan-based polysaccharides and modulates cellulose organization (Sánchez-Rodríguez et al. 2012). In ctl1-1 mutants, reduced cellulose content and compensatory lignin accumulation (Zhong et al. 2000, 2002) mirror other cellulose-deficient phenotypes (Cano-Delgado et al. 2003; Hamann et al. 2009; Denness et al. 2011). Remarkably, the avocado chitinase-like gene Ctg0022 complemented ctl1-1, restoring normal growth, cell wall integrity, and resistance to cellulose synthesis inhibitors, suggesting that despite low sequence similarity, Ctg0022 and CTL1 share a conserved mechanism of action in cell wall remodeling.

Although CTLs do not bind pectin directly (Sánchez-Rodríguez et al. 2012), reduced pectin methyl-esterification has been reported in the arm mutant (Hermans et al. 2011), suggesting indirect effects on pectin remodeling. Pectin demethylesterification, catalyzed by pectin methylesterases (PMEs) and regulated by PME inhibitors (PMEIs), enhances cell adhesion via Ca2+ cross-linking but also increases susceptibility to degradation (Cosgrove 2024). During grafting, pectins promote adhesion at the graft junction (Sala et al. 2019; Frey et al. 2024). In ctl1-1, impaired cell division and adhesion were associated with low graft success, while grafting ctl1-1 scions onto wild-type or Ctg0022-expressing rootstocks restored partial compatibility. This outcome suggests that, due to differences in cellulose and lignin content between wild-type and ctl1-1 mutant plants, the elevated PME3 expression in ctl1-1 scions may serve as a compensatory response to altered cell wall composition, enhancing scion—but not rootstock—performance. Accordingly, under the cell wall conditions of wild-type plants, this increase is unnecessary for graft success, whereas it provides an advantage in ctl1-1 scions. Consistently, expression network analysis revealed a strong correlation between Ctg0022 and PMEI-like transcripts. However, whether these correlations reflect direct protein–protein interactions and whether functional activation or inhibition occurs remain to be determined.

Fluorescence lifetime imaging further revealed abnormal primary cell wall composition in dividing ctl1-1 cells, consistent with increased phenolic accumulation typical of secondary walls. Similar cell wall autofluorescence was previously linked to disrupted cellulose synthesis (Mélida et al. 2015), supporting the idea that CTL1 deficiency alters cell wall integrity and composition during graft formation.

Although Arabidopsis CTL1 and its cotton homolog lack chitinase activity, their transcription is stress-induced—5 days after wounding in Arabidopsis and 4 days after Verticillium dahliae infection in cotton (Liu et al. 2024). In cotton, CTL1-like mitigates fungal toxicity by interacting with pathogen effectors, highlighting a retained role in defense despite loss of enzymatic function. In this context, it is noteworthy that while as mentioned, in green avocado VC801 rootstocks, PMEI-like transcripts are co-expressed with Ctg0022; PMEIs are generally associated with plant defense responses and can enhance resistance to fungal infection (Lionetti et al. 2007, 2012).

Plant responses to wounding balance defense and regeneration, which often act as competing processes (Hernández-Coronado et al. 2022). Our data suggest that VC801's strong resistance to V. dahliae is driven by robust defense, including high chitinase expression, but this may interfere with the precise regulation of cell division, adhesion, and differentiation required for graft union formation with the Hass scion. Overall, successful grafting appears to depend on fine-tuned chitinase expression coordinated with the rootstock's physiological state and specific scion combination.

Materials and methods

General chemistry methods for the production of 2,4-D-Ile

All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise stated. Anhydrous solvents and reagents (dichloromethane [DCM], dimethylformamide [DMF]) were obtained as SureSeal bottles from Sigma-Aldrich. Ethyl acetate (EtOAc) and n-hexane (Hex) were obtained from BioLab Ltd. (Israel), and utilized without further purification. The progress of the reaction mixture was monitored by thin-layer chromatography, and flash chromatography were performed using EMD-precoated silica gel 60 F-254 plates and silica gel 60 (230 to 400 mesh), respectively. High-resolution ESI mass spectrometry was performed on a Waters SYNAPT system. 1H- and 13C NMR spectra were collected in CDCl3 (Cambridge Isotope Laboratories, Cambridge, MA) at 25 °C using a Bruker Advance III spectrometer at 400 and 100 MHz, respectively, at the Department of Chemistry NMR Facility at Tel-Aviv University (Figures S12 and S13). The residual peaks of CDCl3 (7.28 and 77.0 ppm, respectively) were used as internal references. All chemical shifts are reported in the standard (δ) notation of parts per million using either TMS or residual solvent peak as an internal reference. All the proton spectra are reported as following: δ value (multiplicity, J coupling constant [in Hz], number of nuclei). Multiplicity contractions used: (s)-singlet, (d)-doublet, (dd)-doublet of doublet, (t)-triplet, (q)-quartet, (m)-multiplet, and (br)-broad singlet and coupling constants in Hz.

HPLC-MS analysis conditions

HPLC-MS analysis was performed on Waters HPLC with XBridge C18 column (100 × 3 mm, 5 µm) using a water acetonitrile gradient of 0% to 100% solvent B in 17 minutes then 3 minutes at 100% solvent B at flow rate of 1 mL/min. Mass spectrometry was performed on LC/MS Acquity QDa detector coupled with Waters HPLC. Method A: water containing 0.1% trifluoroacetic acid (TFA) (solvent A) and acetonitrile containing 0.1% TFA (solvent B). Method B: water containing 10 mM NH4HCO3 (solvent A) and acetonitrile/Water (9/1) containing 10 mM NH4HCO3.

Synthesis of 2,4-D-Ile-OMe

graphic file with name kiag187il1.jpg

To a suspension of L-isoleucine methyl ester hydrochloride (785 mg, 5.60 mmol, 1.2 equiv.) in 20 ml dry DCM:DMF (1:1), triethylamine (1.25 mL, 8.92 mmol, 2.0 equiv.) was added under argon atmosphere and stirred for 10 min at room temperature. Then, 2,4-D (1.0 g, 4.5 mmol, 1. equiv.) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCL; 1.2 g, 6.7 mmol, 1.5 equiv.) were added and stirred overnight at room temperature under argon atmosphere. Subsequently, the reaction was quenched with a saturated NaHCO3 solution and extracted twice with EtOAc. The combined organic fractions were washed with water (twice) and brine solution. The solvent was removed on a rotary evaporator, and the residue was purified by column chromatography on a silica gel using Hex/EtOAc (7:3) as the eluent to give the coupled product 2,4-D-Ile-OMe (1.45 g, 92%) as sticky white solid.

1H NMR (400 MHz, CDCl3): δ 7.43 (bs, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 8.7, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.66 (d, J = 8.8, 1H), 4.55 (s, 2H), 3.76 (s, 3H), 1.98 (s, 1H), 1.49 to 1.46 (m, 1H), 1.26 to 1.19 (m, 1H), 0.96 to 0.93 (m, 6H) ppm, 13C NMR (100 MHz, CDCl3): δ 171.6, 166.8, 151.4, 130.0, 127.8, 127.3, 123.8, 114.4, 67.9, 57.9, 52.0, 37.7, 24.9, 15.4, 11.4 ppm, HRMS (ESI): calcd for C15H20Cl2NO4 [M + H]+ m/z 348.0772, found 348.0769.

Plant media and growth conditions, rooting, and grafting

Avocado

Shoots of West Indian VC801 avocado (Persea americana Mill.) rootstock, now registered in the United States under the name “Tami,” US Patent no: PP30,384, were first grafted by the Haskelberg nurseries (Kfar Vitkin, Israel). Shoots were grafted on seedlings of the Reed rootstock (Ben-Ya’acov and Michelson 1995) and grown in the dark for 4 to 8 weeks. When shoots reached a length of 20 to 35 cm they were transferred to the light for 1 to 2 weeks for the de-etiolation process. The upper 15 cm of the green shoots were cut and transferred to a climate controlled greenhouse at the Volcani Institute for rooting. The cuttings bases were dipped for 1 min in 1,000 ppm K-IBA and 8 μM 2,4-D-Ile and planted in rooting medium containing peat, vermiculite and polystyrene flakes at a ratio of 1:2:3, respectively, on a heated (25 °C) rooting table under 90% humidity. Fungicides were applied to the rooting media on a weekly basis. Rooting percentage was measured after 2 or 3 months. Grafting was done either on the etiolated shoots just after the dark period or on the rooted green shoots prepared in advance, at the same time. Avocado branches used as scions (Hass) were collected from mother trees, leaves were removed and a 10 cm portion containing a dormant bud at the top was used after disinfection using 0.05% Switch 62.5WG fungicide (Syngenta). The scion base was cut to a “V” shape and in the rootstock a middle longitudinal cut was made in the stem after the removal of the apical part. After pushing the scion into the rootstock's cut, both parts were wrapped with parafilm which covered the connection point and the whole scion.

Arabidopsis

The seeds of ctl1-1 (SALK_093049) mutant and 35S-CTL1-GFP/ctl1-1 At1g05850 were kindly provided by Clara Sánchez-Rodríguez (Sánchez-Rodríguez et al. 2012). Arabidopsis seeds were surface sterilized for 12 min with a sterilization solution containing 0.5% sodium hypochlorite (NaOCl) and a few drops of 10% Triton X-100, followed by 4 to 5 times washing with sterilized water. Grafting was performed as previously described (Vanderstraeten et al. 2022). The surface sterilized seeds were plated on a plate containing 1/2 MS, 0.5% sucrose, and 0.8% agar with pH 5.8. After 24 to 48 hours at 4 °C in the dark, the plates were transferred to a growth room (16/8 hours photoperiod) 22 °C for 3 days and then 27 °C of 1 day in a vertical position. The healthy and straight-grown seedlings were used for grafting under a sterile hood condition with the help of a stereomicroscope. Sterilized Whatman filter (2 sheets) and 1 sheet of Hybond-N paper were put in a petri plate and then saturated with sterile H2O for grafting. A sharp microsurgical blade was used to make a transverse cut across the upper part of the hypocotyl, the apical part, a scion, was placed in contact to the rootstock, the bottom part. The plates were transferred to the growth chamber at 27 °C in a vertical position. After 7 days, the assessment of successful grafting was done by lifting the scion from the filter and examining the degree of its connection to the rootstock under the stereoscope. For treatment with oryzalin and isoxaben, the surface sterilized seeds of Col-0, ctl1-1, and complemented lines of Ctg0022 were directly germinated on the MS media plate containing the 100 nM oryzalin (Chem Service, West Chester, PA, USA) or 1.5 nM isoxaben (Merck, St. Louis, MO, USA) After 5 days of treatment, the seedlings were imaged and measured.

Plasmid construction and agro infiltration and transformation

All plasmids used in this study are listed in Table S1. Constructs including CTL1pro:Ctg0022-mCherry, CTL1pro:Ctg1017-mCherry, 35Spro:Ctg0022-mCherry, and 35Spro:CTL1-mCherry were generated using the Golden Gate MoClo Plant Tool Kit (Engler et al. 2014). Level 0 plasmids containing domesticated promoter, cDNA, fluorescent protein, and terminator sequences were designed with compatible BsaI sites and assembled into the backbone plasmid pICH47742. Assembly reactions included BsaI and T4 DNA ligase (New England Biolabs) were performed in a thermocycler under the following conditions: 30 minutes at 37 °C, followed by 60 to 65 cycles of 37 °C for 5 minutes and 16 °C for 5 minutes, with a final incubation at 16 °C for 15 minutes. The CTL1 promoter used in these constructs comprises a 1,560-bp fragment upstream of the ATG start codon. All DNA sequences were synthesized by Twist Bioscience. Constructs driven by the 35S promoter (35Spro:Ctg0022-mCherry and 35Spro:AtCTL1-mCherry) used the OCS terminator, while CTL1pro:Ctg0022-mCherry and CTL1pro:Ctg1710-mCherry employed the 35S terminator. Level-1 plasmids were assembled into a Level-2 vector (pAGM4723) along with a Level-1 Basta resistance cassette (p1s033), using BbsI and T4 DNA ligase (NEB) under the same thermocycler conditions. To avoid the need for sequence domestication related to BbsI restriction sites, the Basta resistance gene was cloned into the single ApaI site of pICH47742. The NOS promoter–bar gene–NOS terminator cassette was amplified by PCR from the p1s033 plasmid including the elimination of an internal ApaI site in the Basta coding region and the creation of the appropriate flanking restriction sites, and it was inserted using standard ligation methods. The primers used are in Table S4. All plasmids were verified by restriction enzyme digestion and DNA sequencing. For transient expression, Agrobacterium tumefaciens strain GV3101 carrying 35Spro:Ctg0022-mCherry and 35Spro:AtCTL1-mCherry constructs was infiltrated into Nicotiana benthamiana leaves using infiltration buffer (10 mM MES-KOH, pH 5.6, 10 mM MgCl2, 150 μM acetosyringone). Leaves were imaged 48 hours post-infiltration. Stable transgenic Arabidopsis thaliana ctl1-1 plants expressing AtCTL1pro:Ctg0022-mCherry and AtCTL1pro:Ctg1710-mCherry were generated via the floral dip method (Clough and Bent 1998) using a solution containing 5% sucrose and 0.025% Silwet L-77. Transgenic seedlings were selected on MS medium containing Basta (15 mg/L). Two independent lines (Ctg0022-L1 and Ctg0022-L2) were isolated and propagated to homozygosity over several generations.

Histology

Samples of the avocado graft domain were fixed for 7 days at 4 °C in a solution containing 7.4% formaldehyde, 50% ethanol, 5% acetic acid, 1% Triton X-100 (v/v), 10 mg/mL polyvinylpyrrolidone (PVP), 1 mg/mL dithiothreitol, and 150 μg/mL ascorbic acid. Following fixation, samples were serially dehydrated in ethanol at concentrations of 50%, 70%, 90%, and 2 steps of 100%, each for 24 hours. Subsequently, samples were cleared using increasing concentrations of Histoclear (K-clear—Kaltek T280) in ethanol: 25%, 50%, 75%, and 2 changes of 100%. Paraffin infiltration was performed using Hista-Flex 8810/01 wax, starting at room temperature, followed by saturation at 42 °C, and finalized at 58 °C with 6 paraffin replacements. Paraffin-embedded samples were cast into molds and cooled. Tissue blocks were sectioned into 10- to 14-μm slices using a Leica RM2245 rotary microtome and mounted on microscope slides. Paraffin was removed using 100% Histoclear, and slides were rehydrated through a graded ethanol series (100%, 95%, 70%, and 50%). Staining was performed with 1% safranin for 35 minutes, followed by a rinse in water. Samples were then dehydrated through 50%, 75%, and 2 steps of 95% ethanol and stained with 0.3% Fast Green for 3 minutes. After a brief rinse in 95% ethanol, samples were cleared by dipping twice in clover oil (3 minutes each) and 3 times in Histoclear (3 minutes each). Finally, samples were mounted under coverslips using EUKITT mounting medium (Kaltek).

Whole-mount tissue staining

Five days post-grafting, Arabidopsis plants were fixed in freshly prepared 8% paraformaldehyde in buffer containing 100 mM PIPES (pH 6.89), 5 mM MgSO4, and 0.5 mM EGTA. Samples were rinsed 3 times for 15 minutes each in the same buffer without fixative. For blocking, samples were incubated in phosphate-buffered saline (PBS) with 3% (w/v) bovine serum albumin (BSA; Sigma) for 1 hour. Primary antibodies—LM19 (for de-methylesterified pectin) and LM20 (for methylesterified pectin)—(Paul Knox, LM-series, University of Leeds), were diluted 1:10 in PBS with 3% BSA and applied to the sections overnight in a humidified chamber.

The following day, samples were washed 3 times in PBS (15 minutes each) and incubated for 1 hour with Alexa Fluor 488-conjugated goat anti-rat secondary antibodies (Jackson ImmunoResearch Laboratories, USA). After 3 additional PBS washes, 0.1% calcofluor (Fluka 18909) was added to the third wash as an internal standard. Samples were mounted in 50% glycerol and sealed with nail polish under a coverslip. Fluorescence intensity measurements were done in several cells from 5 plants, total of several dozen from each (scion and rootstock separately) graft combination.

For calcofluor staining of live tissue; calcofluor was diluted (v/v) in 10% potassium hydroxide according to the manufacturing protocol. Working solution was further diluted 1:100 with sterile H20. The solution was infiltrated to the transient expressing tobacco leaves, harboring 35Spro: Ctg0022- mCherry or 35Spro: AtCTL-1- mCherry, with the help of a 1-mL syringe and left for 5 minutes before microscopy.

For lignin staining MS grown 5-day-old seedling were stained according to the protocol (Ziegler et al. 2016). The seedlings were treated with 20% HCl containing an ethanolic solution of phloroglucinol (1% v/v) for 10 min followed by washing with sterile H2O. Phloroglucinol staining and roots were imaged under Olympus DP73 microscope. Root length, and root diameter were analyzed by the ImageJ tool (National Institutes of Health; USA http://imagej.nih.gov/ij).

Microscopy

An upright Olympus BX53F microscope was used to image the histological sections, and whole plants with UplanSApo 10× 0.4 and 4× 0.16 objectives and an Olympus DP73 camera.

Fluorescence of calcofluor, RFP-mCherry, or Alexa 488 was imaged using a confocal microscope Leica SP8 (Leica, Wetzlar, Germany) equipped with solid state lasers of 405, 488, and 552 nm light and HC PL APO CS 63 × /1.2 water immersion objective (Leica, Wetzlar, Germany). Emission signals were detected with HyD (hybrid) detectors. Quantification of LM19/LM20 staining was done by choosing regions of interest (ROIs) from the cell wall. For each point 50 to 100 ROIs were measured from at least 5 plants, 3 to 5 cells of each, 3 to 4 ROIs from each cell. Total fluorescence intensity was measured using ImageJ.

Autofluorescence lifetime of cell walls was imaged using a STELLARIS 8 FALCON (Leica, Germany) FLIM system equipped with a white light laser (WLL, 440 to 790 nm). Imaging was performed using 440 nm excitation and detected with a HyD S counting mode detector operating within 450 to 640 nm range. A HC FLUOTAR L VISIR 25x/0.95 NA water objective was used for image collection and digital images with 512 × 512 pixels (910 nm × 910 nm pixel size and 3.16µs Pixel Dwell Time) and 20 frame repetitions were obtained. Quantification of the different fluorescence lifetime events was done using the Falcon software.

RNA preparation and RT-qPCR analysis

Different tissue samples of avocado and Arabidopsis after grafting were quickly frozen in liquid nitrogen and used for RNA preparation. The frozen avocado samples were first crushed with the help of an IKA A11 grinder in liquid nitrogen and then with mortar and pestle to make fine powder. Total RNA from avocado samples (3 biological repeats for each sample, each containing 5 independent grafted plants) was isolated using Norgen RNA isolation kit (Norgen Biotek Corp, ON, Canada) with several modifications; First, 100 mg was used. Second, a few grains of PVP-K30 were added to the lysis buffer in each tube, and third, after elution, RNA was precipitated in 3 M sodium acetate pH 5.5, and 2 volumes of cold ethanol at −20 °C overnight. Arabidopsis RNA samples were prepared by the TRI reagent (Merck, St. Louis, MO, USA), according to the manufacturer's protocol. First strand-cDNA was prepared using 2 to 3 micrograms of total RNA using the abm All-in-One 5× RT master mix cDNA synthesis kit (Canada) according to the manufacturer's instructions. Real-time quantitative PCR was performed on a StepOnePlus system (Thermo Scientific), and the results were analyzed with StepOne software version 2.2.2 (Thermo Scientific). Relative expression levels were normalized using actin AtActin2 (At3g18780), AtTubulin2 (At5g62690), and for avocado Tubulin alpha-3 (Ctg0211) and Tubulin beta-1 (Ctg0005) as a reference gene and calculated using the comparative delta-delta Ct (−ΔΔCt) method (Livak and Schmittgen 2001). Three independent biological replicates were used, and 3 technical replicates were performed. The primers for RT-qPCR are listed in Table S2.

RNA sequencing and bioinformatics

Three biological repeats of etiolated rootstock time 0 (0E) and after 5 days (5E) or green rootstock time 0 (0G) or after 5 days (5G), total of 12 samples were prepared. RNA integrity number (RIN) analysis was performed by the Weizmann Institute of Science Biological Services and library preparation, as well as RNA sequencing, were performed at the Crown Genomics Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute, using a NovaSeq SP (200 cycles: 100 bp ×2 paired-end data). One sample of 5G did not work.

A total of ∼273 million 100 bp paired-end reads (with an average of 24.8 million per sample) were mapped to the reference genome of P. americana cv. Hass (https://genomevolution.org/coge//GenomeInfo.pl?gid=29302) using STAR software (v2.7.1) (Dobin et al. 2012). Gene abundance was estimated using Cufflinks (v. 2.2) (Trapnell et al. 2010) combined with gene annotations from the CoGo database (https://genomevolution.org/coge/). Principal component analysis (PCA) and heatmap visualization were performed using R Bioconductor (accessed on 15 May 2022) (Gentleman et al. 2004). Gene expression values were computed as FPKM. Differential expression analysis was performed with the DESeq2 R package (Love et al. 2014). Genes ≥2-fold differentially expressed with a false discovery–corrected statistical significance of at most 0.05 were considered differentially expressed (Benjamini and Hochberg 1995). The ClusViz tool was used for a heatmap graphical representation (Metsalu and Vilo 2015). KOBAS 3.0 tool (http://bioinfo.org/kobas/; Xie et al. 2011) was applied to find the statistical enrichment of KEGG pathway and Gene Ontology DEGs. We collected the chitinase family proteins of different species, including Arabidopsis, Ipomoea nil, Nicotiana benthamiana, Grapevine, and Glycine max, based on (Grover 2012) and homology searches using blastp. Multiple sequence alignment was performed by MAFFT alignment program (https://mafft.cbrc.jp/alignment/server/). A phylogenetic tree was constructed using the RAxML-NG phylogenetic tree tool based on the maximum-likelihood (ML) optimality criterion and the LG empirical substitution matrix (Kozlov et al. 2019); a total of 100 bootstrap replications were used. Raw reads from Bioproject accession PRJDB9607 were downloaded from NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJDB9607/) (Notaguchi et al. 2020) including Arabidopsis (3 samples), Ipomoea nil (6 samples), Nicotiana benthamiana (15 samples) and Glycine max (12 samples). Reads were mapped using STAR software (Dobin et al. 2012) vs. the reference genomes (GCF_000001735.4, GCF_001879475.1, https://solgenomics.net/ftp/genomes/Nicotiana_benthamiana/assemblies/, GCF_000004515.6, respectively). Gene abundance (FPKM) was estimated using Cufflinks (v. 2.2) (Trapnell et al. 2010). Differential expression analysis was performed with the edgeR R package (Robinson et al. 2010). Vitis vinifera expression data were downloaded from (Cookson et al. 2014). The expression of the chitinase-like genes of Arabidopsis grafting (Melnyk et al. 2018) was extracted from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107203.

Co-expression networks were inferred using CoNet application within Cytoscape (v.3.7.2) (Franz et al. 2016). The Pearson correlation (0.8), implemented by CoNet (Faust and Raes 2016), was used to detect co-occurrence between chitinase-like and pectin-modifying proteins.

The RNA sequencing data from this study were deposited in NCBI https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1354815.

The sequence of avocado transcripts Ctg0022, Ctg1710, are in Figure S8.

Graphs and statistical analysis

All the graphs were made by GraphPad Prism v10.4.1. Statistical analysis was performed using the same software, using the tests as indicated in the figure legends.

Accession numbers

Sequence data from this article can be found in Figure S8 and the GenBank/EMBL data libraries under accession numbers:

Basic endochitinase-like protein: maker-Ctg0022-augustus-gene-3.7

Class I chitinase: maker-Ctg1710-augustus-gene-0.10

Pectin methylesterase 35: maker-Ctg0111-augustus-gene-2.4

Pectin methylesterase inhibitor superfamily: maker-Ctg0254-augustus-gene-1.12

Supplementary Material

kiag187_Supplementary_Data
kiag187_supplementary_data.zip (21.3MB, zip)

Acknowledgments

We gratefully acknowledge the Haskelberg Nursery in Kfar Vitkin, Israel, for providing the grafted VC801 rootstocks, and Dr. Vered Irihimovitch from the Volcani Institute for her expert advice.

Contributor Information

Vikas Dwivedi, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel; The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, the Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel.

Lalita Pal, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel; The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, the Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel.

Gal Hadas-Brandwein, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel; The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, the Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel.

Sela Yechezkel, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel.

Alexander Klaus, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel.

Adi Faigenboim-Doron, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel.

Eduard Belausov, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel.

Balaji U Solanke, School of Plant Sciences and Food Security, Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 6997801, Israel.

Roy Weinstain, School of Plant Sciences and Food Security, Faculty of Life Sciences, Tel-Aviv University, Tel Aviv 6997801, Israel.

Einat Sadot, The Institute of Plant Sciences, The Volcani Institute, Agricultural Research Organization, 68 HaMaccabim Rd., Rishon LeZion 7528809, Israel.

Author contributions

G.H.B. and S.Y. conducted all experiments related to avocado. V.D. and L.P. carried out all Arabidopsis-related work. A.K., FLIM quantification, A.F.-D. was responsible for the bioinformatics analyses, and E.B. performed the microscopy. B.U.S. and R.W. contributed to the design, synthesis, and investigation of rooting enhancers. E.S. conceived the study, designed the experiments, and wrote the manuscript.

Supplementary material

Supplementary material is available at Plant Physiology online.

Funding

This work was jointly supported by grants awarded to E.S. and R.W. from the Chief Scientist of the Israeli Ministry of Agriculture(grant numbers 20-01-0175, 20-10-0067, 13-37-0005, and 20-01-0270), as well as from the United States–Israel Binational Agricultural Research and Development Fund(BARD), through grants IS-5195-19R and IS-5598-23, in collaboration with C.J. Staiger (Purdue University, IN).

Data availability

All data are available from the corresponding author.

References

  1. Asahina  M  et al.  2002. Gibberellin produced in the cotyledon is required for cell division during tissue reunion in the cortex of cut cucumber and tomato hypocotyls. Plant Physiol. 129:201–210. 10.1104/pp.010886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamini  Y, Hochberg  Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal Royal Statistical Society Series B. 57:289–300. 10.1111/j.2517-6161.1995.tb02031.x. [DOI] [Google Scholar]
  3. Ben-Ya’acov  A, Michelson  E. 1995. Avocado rootstocks. In: Janick  J, editor. Horticultural reviews. Vol. 17. John Wiley and Sons, Inc. p. 381–429. [Google Scholar]
  4. Ben-Ya’acov  A, Michelson  E, Zilberstaine  M, Barkan  Z, Sela  I. 1992. Selection of clonal avocado rootstocks in Israel for high productivity under different soil conditions. In: Proc Second World Avocado Congress:521–526. https://www.avocadosource.com/WAC2/WAC2_p521.pdf [Google Scholar]
  5. Ben-Ya′acov  A, Zilberstaine  M. 1999a. Clonal avocado (Persea americana Mili.) rootstocks in Israel. Rev Chapingo Ser Hortic  5:39–42. [Google Scholar]
  6. Ben-Ya′acov  A, Zilberstaine  M. 1999b. The possible use of avocado (Persea americana mill.) germplasm material as rootstocks for soil stress conditions. Rev Chapingo Ser Hortic. 5:25–28. https://www.avocadosource.com/WAC4/WAC4_p025.pdf [Google Scholar]
  7. Brokaw  WH. 1975. Rootrot resistant avocado clonal rootstocks. Plant Prop. 21:7–8. [Google Scholar]
  8. Cano-Delgado  A, Penfield  S, Smith  C, Catley  M, Bevan  M. 2003. Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. Plant J. 34:351–362. 10.1046/j.1365-313X.2003.01729.x. [DOI] [PubMed] [Google Scholar]
  9. Chabbert  B  et al.  2018. Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass. Ind Crops Prod. 123:84–92. 10.1016/j.indcrop.2018.06.058. [DOI] [Google Scholar]
  10. Clough  SJ, Bent  AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735–743. 10.1046/j.1365-313x.1998.00343.x. [DOI] [PubMed] [Google Scholar]
  11. Cookson  SJ  et al.  2014. Heterografting with nonself rootstocks induces genes involved in stress responses at the graft interface when compared with autografted controls. J Exp Bot. 65:2473–2481. 10.1093/jxb/eru145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cosgrove  DJ. 2024. Structure and growth of plant cell walls. Nat Rev Mol Cell Biol. 25:340–358. 10.1038/s41580-023-00691-y. [DOI] [PubMed] [Google Scholar]
  13. De Jong  AJ  et al.  1992. A carrot somatic embryo mutant is rescued by chitinase. Plant Cell. 4:425–433. 10.1105/tpc.4.4.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. De Jong  AJ  et al.  1995. Transient reduction in secreted 32 kD chitinase prevents somatic embryogenesis in the carrot (Daucus carota L.) variant ts11. Dev Genet. 16:332–343. 10.1002/dvg.1020160406. [DOI] [Google Scholar]
  15. Denness  L  et al.  2011. Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis. Plant Physiol. 156:1364–1374. 10.1104/pp.111.175737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Desprez  T  et al.  2002. Resistance against herbicide isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6. Plant Physiol. 128:482–490. 10.1104/pp.010822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dobin  A  et al.  2012. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29:15–21. 10.1093/bioinformatics/bts635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Domon  J-M, Neutelings  G, Roger  D, David  A, David  H. 2000. A basic chitinase-like protein secreted by embryogenic tissues of pinus caribaea acts on arabinogalactan proteins extracted from the same cell lines. J Plant Physiol. 156:33–39. 10.1016/S0176-1617(00)80269-2. [DOI] [Google Scholar]
  19. Duman  Z  et al.  2020. Short de-etiolation increases the rooting of VC801 avocado rootstock. Plants (Basel). 9:1481. 10.3390/plants9111481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Engler  C  et al.  2014. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 3:839–843. 10.1021/sb4001504. [DOI] [PubMed] [Google Scholar]
  21. Faust  K, Raes  J. 2016. Conet app: inference of biological association networks using cytoscape. F1000Res. 5:1519. 10.12688/f1000research.9050.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Feng  M  et al.  2024. A conserved graft formation process in Norway spruce and Arabidopsis identifies the PAT gene family as central regulators of wound healing. Nat Plants. 10:53–65. 10.1038/s41477-023-01568-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Franz  M  et al.  2016. Cytoscape.js: a graph theory library for visualisation and analysis. Bioinformatics. 32:309–311. 10.1093/bioinformatics/btv557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Frey  C, Saez-Aguayo  S, Encina  A, Acebes  JL. 2024. Deepening the role of pectin in the tissue assembly process during tomato grafting. Plants (Basel). 13:3519. 10.3390/plants13243519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Frolich  EF, Platt  RG. 1972. Use of the etiolation technique in rooting avocado cuttings. California Avocado Society 1971-72 Yearbook. 55:97–109. http://www.avocadosource.com/CAS_Yearbooks/CAS_55_1971/CAS_1971-72_PG_097-109.pdf [Google Scholar]
  26. Gentleman  RC  et al.  2004. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5:R80. 10.1186/gb-2004-5-10-r80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goldschmidt  EE. 2014. Plant grafting: new mechanisms, evolutionary implications. Front Plant Sci. 5:727. 10.3389/fpls.2014.00727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Grover  A. 2012. Plant chitinases: genetic diversity and physiological roles. CRC Crit Rev Plant Sci.  31:57–73. 10.1080/07352689.2011.616043. [DOI] [Google Scholar]
  29. Haberman  A  et al.  2020. Management of verticillium wilt of avocado using tolerant rootstocks. Plants (Basel). 9:531. 10.3390/plants9040531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hamann  T, Bennett  M, Mansfield  J, Somerville  C. 2009. Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses. Plant J. 57:1015–1026. 10.1111/j.1365-313X.2008.03744.x. [DOI] [PubMed] [Google Scholar]
  31. Hermans  C  et al.  2011. Dissecting the role of CHITINASE-LIKE1 in nitrate-dependent changes in root architecture. Plant Physiol. 157:1313–1326. 10.1104/pp.111.181461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hermans  C, Porco  S, Verbruggen  N, Bush  DR. 2010. Chitinase-like protein CTL1 plays a role in altering root system architecture in response to multiple environmental conditions. Plant Physiol. 152:904–917. 10.1104/pp.109.149849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hernández-Coronado  M  et al.  2022. Plant glutamate receptors mediate a bet-hedging strategy between regeneration and defense. Dev Cell. 57:451–465.e6. 10.1016/j.devcel.2022.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jiao  S  et al.  2019. Chitinase-like1 plays a role in stalk tensile strength in maize. Plant Physiol. 181:1127–1147. 10.1104/pp.19.00615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kozlov  AM, Darriba  D, Flouri  T, Morel  B, Stamatakis  A. 2019. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 35:4453–4455. 10.1093/bioinformatics/btz305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kragler  F, Bock  R. 2025. The biology of grafting and its applications in studying information exchange between plants. Nat Plants. 11:955–966. 10.1038/s41477-025-01982-2. [DOI] [PubMed] [Google Scholar]
  37. Lazare  S  et al.  2019. Avocado rootstock influences scion leaf mineral content. Arch Agron Soil Sci. 66:1–11. 10.1080/03650340.2019.1672163. [DOI] [Google Scholar]
  38. Lintilhac  PM, Vesecky  TB. 1984. Stress-induced alignment of division plane in plant tissues grown in vitro. Nature. 307:363–364. 10.1038/307363a0. [DOI] [Google Scholar]
  39. Lionetti  V, Cervone  F, Bellincampi  D. 2012. Methyl esterification of pectin plays a role during plant-pathogen interactions and affects plant resistance to diseases. J Plant Physiol. 169:1623–1630. 10.1016/j.jplph.2012.05.006. [DOI] [PubMed] [Google Scholar]
  40. Lionetti  V  et al.  2007. Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea. Plant Physiol. 143:1871–1880. 10.1104/pp.106.090803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Liu  H  et al.  2024. Re-localization of a repeat-containing fungal effector by apoplastic protein chitinase-like 1 blocks its toxicity. Nat Commun. 15:10122. 10.1038/s41467-024-54470-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Livak  KJ, Schmittgen  TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  43. Love  MI, Huber  W, Anders  S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550. 10.1186/s13059-014-0550-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mélida  H  et al.  2015. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures. J Integr Plant Biol. 57:357–372. 10.1111/jipb.12346. [DOI] [PubMed] [Google Scholar]
  45. Melnyk  CW. 2017a. Connecting the plant vasculature to friend or foe. New Phytol. 213:1611–1617. 10.1111/nph.14218. [DOI] [PubMed] [Google Scholar]
  46. Melnyk  CW. 2017b. Plant grafting: insights into tissue regeneration. Regeneration (Oxf). 4:3–14. 10.1002/reg2.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Melnyk  CW  et al.  2018. Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration. Proc Natl Acad Sci U S A. 115:E2447–E2456. 10.1073/pnas.1718263115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Metsalu  T, Vilo  J. 2015. ClustVis: a web tool for visualizing clustering of multivariate data using principal component analysis and heatmap. Nucleic Acids Res. 43:W566–W570. 10.1093/nar/gkv468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Notaguchi  M  et al.  2020. Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases. Science. 369:698–702. 10.1126/science.abc3710. [DOI] [PubMed] [Google Scholar]
  50. Omary  M, Matosevich  R, Efroni  I. 2022. Systemic control of plant regeneration and wound repair. New Phytol. 237:408–413. 10.1111/nph.18487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Paredez  AR, Somerville  CR, Ehrhardt  DW. 2006. Visualization of cellulose synthase demonstrates functional association with microtubules. Science. 312:1491–1495. 10.1126/science.1126551. [DOI] [PubMed] [Google Scholar]
  52. Passarinho  PA, de Vries  SC. 2002. Arabidopsis chitinases: a genomic survey. Arabidopsis Book. 1:e0023. 10.1199/tab.0023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Robinson  MD, McCarthy  DJ, Smyth  GK. 2010. Edger: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26:139–140. 10.1093/bioinformatics/btp616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Roth  O  et al.  2024. Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants. Nat Biotechnol. 42:1705–1716. 10.1038/s41587-023-02065-3. [DOI] [PubMed] [Google Scholar]
  55. Sala  K, Karcz  J, Rypień  A, Kurczyńska  EU. 2019. Unmethyl-esterified homogalacturonan and extensins seal Arabidopsis graft union. BMC Plant Biol. 19:151. 10.1186/s12870-019-1748-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sánchez-Rodríguez  C  et al.  2012. Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis. Plant Cell. 24:589–607. 10.1105/tpc.111.094672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Scheible  WR, Eshed  R, Richmond  T, Delmer  D, Somerville  C. 2001. Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants. Proc Natl Acad Sci U S A. 98:10079–10084. 10.1073/pnas.191361598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sinha  NR, Rowland  SD, Ichihashi  Y. 2016. Using gene networks in EvoDevo analyses. Curr Opin Plant Biol. 33:133–139. 10.1016/j.pbi.2016.06.016. [DOI] [PubMed] [Google Scholar]
  59. Stigliano  E, Di Sansebastiano  GP, Neuhaus  JM. 2014. Contribution of chitinase A’s C-terminal vacuolar sorting determinant to the study of soluble protein compartmentation. Int J Mol Sci. 15:11030–11039. 10.3390/ijms150611030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Taira  T. 2010. Structures and antifungal activity of plant chitinases. J Appl Glycosci. 57:167–176. 10.5458/jag.57.167. [DOI] [Google Scholar]
  61. Trapnell  C  et al.  2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 28:511–515. 10.1038/nbt.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Vaghela  B, Vashi  R, Rajput  K, Joshi  R. 2022. Plant chitinases and their role in plant defense: a comprehensive review. Enzyme Microb Technol. 159:110055. 10.1016/j.enzmictec.2022.110055. [DOI] [PubMed] [Google Scholar]
  63. Vanderstraeten  L, Sanchez-Muñoz  R, Depaepe  T, Auwelaert  F, Van Der Straeten  D. 2022. Mix-and-match: an improved, fast and accessible protocol for hypocotyl micrografting of Arabidopsis seedlings with systemic ACC responses as a case study. Plant Methods. 18:24. 10.1186/s13007-022-00859-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Verhertbruggen  Y, Marcus  SE, Haeger  A, Ordaz-Ortiz  JJ, Knox  JP. 2009. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr Res. 344:1858–1862. 10.1016/j.carres.2008.11.010. [DOI] [PubMed] [Google Scholar]
  65. Wang  J, Jiang  L, Wu  R. 2017. Plant grafting: how genetic exchange promotes vascular reconnection. New Phytol. 214:56–65. 10.1111/nph.14383. [DOI] [PubMed] [Google Scholar]
  66. Wu  B  et al.  2012. Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice. Plant Physiol. 159:1440–1452. 10.1104/pp.112.195529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Xie  C  et al.  2011. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39:W316–W322. 10.1093/nar/gkr483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhang  A  et al.  2022. Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana. Curr Biol. 32:1883–1894. 10.1016/j.cub.2022.02.069. [DOI] [PubMed] [Google Scholar]
  69. Zhong  R, Kays  SJ, Schroeder  BP, Ye  ZH. 2002. Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene. Plant Cell. 14:165–179. 10.1105/tpc.010278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Zhong  R, Ripperger  A, Ye  ZH. 2000. Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants. Plant Physiol. 123:59–70. 10.1104/pp.123.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Ziegler  J  et al.  2016. Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation. J Exp Bot. 67:1421–1432. 10.1093/jxb/erv539. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

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Data Availability Statement

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