Small non-coding RNAs represent a cohort of different molecules in plants, with a size of 20 – 24 nucleotides. They can be broadly classified into two groups: i) Micro RNAs (miRNA), or ii) Small interfering RNAs (siRNA or sRNA). These two classes differ by the way siRNAs are synthesised and how they operate (Borges and Martienssen, 2015; Kamthan et al., 2015; Singh et al., 2018). In plants, miRNAs are encoded by endogenous genes that are transcribed into long primary miRNAs (pri-miRNAs) that are characteristically folded into a hairpin-like structures. These are then processed by RNase III-like Dicer 1 (DCL1), first into stem-loop structures, known as precursor miRNAs (pre-miRNAs), then into mature miRNA duplexes. The resulting miRNAs can prevent mRNA translation through Post Transcriptional Gene Silencing (PTGS), which ultimately causes gene repression (Borges and Martienssen, 2015) ( Figure 1 ). In comparison, siRNAs are derived from long double-stranded RNA precursors and are grouped based on their function. The first group contains siRNAs with a nucleotide length of 21 - 22 and are derived from the activity of Dicer-like 2 and Dicer-like 4 (DCL2/DCL4) from trans-acting small interfering RNA (TAS) genes, transcripts or other aberrant RNA molecules (Berger et al., 2018). The 21/22-nt siRNAs are known to be involved in short-distance signalling and in the degradation of mRNAs (Tan et al., 2020), as well as in targeted Transcriptional Gene Silencing (TGS) ( Figure 1 ) (Borges and Martienssen, 2015). The second group is known as 24-nt siRNAs which originate from heterochromatic regions by the continuous action of three enzymes: RNA Polymerase IV, RNA Dependent RNA polymerase 2 (RDR2) and Dicer-like 3 (DCL3). The 24-nt siRNAs are renowned for their involvement in the RNA-directed DNA methylation (RdDM) pathway by guiding the Domain rearranged DNA methyltransferases (DRMs) in TGS, but they are also known for their involvement in long-distance signalling (Berger et al., 2018) ( Figure 1 ).

In the model species Arabidopsis thaliana, it has been demonstrated that the systemic movement of siRNAs can direct de novo DNA methylation in tissues distant from their origin (Borges and Martienssen, 2015; Xie and Yu, 2015) ( Figure 2 ). This involves the recruitment of DNA methyltransferases in recipient cells or tissues, and the loading of the siRNA into ARGONAUTEs (AGOs) proteins, which are then recruited into a complex with transcribed scaffold RNA by the action of RNA Polymerase V (Huang et al., 2021). More specifically, AGO1 protein in Arabidopsis has been proposed to remove sRNAs from traversed tissue, allowing their accumulation in recipient cells, a phenomenon defined as “consumption” of the mobile sRNAs (Denvers et al., 2020). Further studies identified in Squash pumpkin (Cucurbita maxima) phloem exudates a set of specific proteins able to bind sRNAs and mediate non-cell autonomous movement of both 21/22- and 24- nt classes (reviewed in Yan and Ham, 2022). Among these, the PHLOEM SMALL RNA-BINDING PROTEIN1 (PSRP1) has being found able to form a ribonucleoprotein complex in the phloem sieve tube system (Ham et al., 2014), while the SMALL RNA-BINDING PROTEIN 1 (SRBP1) homologues in Arabidopsis has been found capable of cell-to-cell movement and has a role in sRNA trafficking into the apoplast (Yang et al., 2019; Karimi et al., 2022). These results suggest the existence of a specific mechanism for the systemic mobilization of sRNA molecules in plants, which is currently subject of research and discussion (Voinnet, 2022; Yan and Ham, 2022).

The translocation of siRNAs across the grafting junction has also been explored in several crops, including eggplant, pumpkin (Cucurbita pepo), cucumber, potato, Nicotiana benthamiana, grapevine (Vitis vinifera) and several bean (Fabaceae) species (Tournier et al., 2006; Bai et al., 2011; Zhang et al., 2014; Cerruti et al., 2021; Li et al., 2021; Davoudi et al., 2022; Rubio et al., 2022; Zhang et al., 2022). In addition, several miRNAs in potato have been demonstrated to be graft-transmissible, including miR156, miR339, miR395 and miR172 (Pant et al., 2008; Buhtz et al., 2010; Kasai et al., 2010). MiR156 has been found to influence plant architecture and tuberization by targeting the regulation of specific genes (Bhogale et al., 2013). The movement of siRNAs was also studied by Kasai et al., who demonstrated that artificial siRNAs in N. benthamiana scions could travel into the potato rootstock to induce TGS via RdDM on both a transgene and an endogenous gene (Kasai et al., 2016). In a recent report, grafting was used to induce editing in scions using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and guide RNA transcripts moved from a transgenic rootstock (Yang et al., 2023). In addition to demonstrating the potential use of grafting in combination with genome editing, this study also showed that mobilised transcripts in the scion can accumulate at a sufficient abundance to introduce a gene editing effect.

In plants, the PTGS pathway falls under RNA interference (RNAi) (Ashfaq et al., 2020). This mechanism involves the suppression of gene expression through degradation of mRNA transcripts via the production of siRNAs (Ashfaq et al., 2020) ( Figure 2 ) (Hamilton and Baulcombe, 1999; Jones et al., 1999; Mette et al., 1999; Dalmay et al., 2000; Marjori et al., 2001). Therefore, it is accepted that both PTGS and DNA methylation function cooperatively or in a dependent manner as initially suggested by Morel et al., 2000 (Morel et al., 2000), and subsequentially proved in multiple experimental conditions (Palauqui et al., 1997; Sonoda and Nishiguchi, 2000; Voinnet et al., 2000; Crété et al., 2001; Mallory et al., 2001; Mallory et al., 2003; García-Pérez et al., 2004; Hewezi et al., 2005; Brosnan et al., 2007; Brumin et al., 2009; Kasai et al., 2011; Taochy et al., 2017; Chen et al., 2018). However, current literature exploring this aspect in grafted plants is limited, and phenotypic changes induced by grafting have only been indirectly linked to DNA methylation alterations (Cerruti et al., 2021).

Unlike siRNAs, mobile mRNAs can directly contribute to the synthesis of proteins into recipient tissues. They have been found to be particularly relevant as transcription factors (TFs), which play a key role in controlling the expression of several genes in a specific pathway. The long-distance mobilisation of mRNAs has been documented in several models using grafting as a tool. For example, in tomato it has been observed that the mRNA transcript PFP-LeT6, composed by a PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE (PFP) and tomato KNOTTED-1–like homeobox (KNOX) domain (LeT6), is able to travel from roots to shoots and induce a change in the leaf morphology (Kim et al., 2001). Similarly, both RT-PCR and in situ hybridization revealed that mRNA transcripts of two partner transcription factors, the potato homeobox1 (POTH1) gene and BEL1-type StBEL5, can move across the graft junction and regulate hormone accumulation, alter leaf architecture, and boost tuber formation (Banerjee et al., 2006; Mahajan et al., 2012; Sharma et al., 2014). Additional studies on pumpkin grafts have also identified other mobile mRNA transcripts that are likely to drive molecular and phenotypic changes, including those encoding transcriptional regulators of auxin signalling (Omid et al., 2007). This was followed by studies on Arabidopsis, which characterised specific auxin mRNA transcripts produced in leaf tissue and translocated to the root system, where they negatively regulate lateral root formation (Omid et al., 2007; Notaguchi et al., 2012). Within the same timeline, researchers have also identified similar hormone-related mRNA transcripts in tomato, cucumber, and pumpkin (Kim et al., 2001; Haywood et al., 2005; Banerjee et al., 2006; Notaguchi et al., 2012; Davoudi et al., 2022). In Arabidopsis, the dwarf phenotype observed in gibberellin (GA)-deficient mutants can be restored if plants are grafted onto WT (Magome et al., 2004), whereas tomato GA-deficient rootstocks can introduce a dwarf-like phenotype in the scion, which is mediated by the movement of transgenic mRNA transcripts up the graft junction (Wang et al., 2012). In tomato has been also found that the mRNA encoding for the GIBBERELLIC ACID INSENSITIVE (GAI) factor produced in a rootstock can transcriptionally regulate gibberellic acid response genes within the scion tissue (Haywood et al., 2005). In addition, there has been evidence that a GAI homologous can be transported in pumpkins by an RNA-protein complex (Ham et al., 2009). In a study investigating drought tolerance in cucumber and pumpkin (Cucumis moschata) grafts, authors identified that the movement of mobile mRNAs correlated with significant accumulation of abscisic acid (ABA) from the pumpkin rootstock in the cucumber scion tissue (Davoudi et al., 2022).

Collectively, it has been well-documented, both in model and crop species, that at least some mRNAs can move across the graft junction and induce phenotypic and developmental changes. However, it remains unclear whether these transcripts are primarily responsible for the observed traits or instead trigger more general epigenetic or post-transcriptional regulation mechanisms responsible for the scion/rootstock physiological response. Hence, further work could be directed to mechanistically associate mobile signalling molecules to an observed epigenetic and/or phenotypic change in grafted plants. In this context, the use of experimental grafting associated with genome-wide sequencing approaches appears to be a powerful tool for correlating phenotypes to mobile mRNA molecules. In experiments where scions and rootstock were from different sequenced genomes, it became possible to compare mRNA sequencing experiments with DNA sequence variants in the two genotypes and identify mobile mRNAs with greater confidence. The methods used to analyse transcriptomic data are well established in plants, but only few analyses or pipelines have been specifically designed for the detection of mobile transcripts (Tomkins et al., 2022). Further development of such tools should be considered to support a better understanding of the molecular mechanisms underlying grafting-induced traits.

The transport mechanism of mobile RNAs is still not well understood and has been predominantly investigated in the model species Arabidopsis thaliana. One of the first studies to identify an association between mRNA abundance and mobility in grafted plants proposed a hypothesis for RNA transport based on passive movement (Thieme et al., 2015). This was supported by a more recent study that found that long-distance movement of mRNA is linked to their local abundance (Calderwood et al., 2016). However, this work could only explain the movement of some mRNAs, and further hypotheses suggest the simultaneous passive and active movement of mobile RNA species (Kim et al., 2014; Notaguchi et al., 2015). The active transportation of RNA molecules has been then proposed in multiple subsequent studies (Zhang et al., 2009; Thieme et al., 2015; Zhang et al., 2016; Garcia-Lozano et al., 2020). Such active movement appears to be associated with the presence of transfer RNA-like structure (TLS) motifs, which can mediate the mobility of some mRNA transcripts to distant locations, as observed in both Arabidopsis and pumpkin (Zhang et al., 2009; Zhang et al., 2016). Experimental work has shown that the addition of a TLS motif, or a specific sequence derived from a TLS motif, is sufficient to introduce transcript mobility, while their removal from an RNA molecule prevents long-distance transportation (Garcia-Lozano et al., 2020; Yang et al., 2023). Collectively, these results suggest that TLSs or related structures may control the translocation of mobile transcripts in graft systems.

In addition to sequences associated with mobility in RNA molecules, also cytosine methylation can play a role in RNA mobility (Yang et al., 2019). Both plant phloem transfer RNAs (tRNAs) and graft-mobile mRNAs contain TLS motifs that have a high probability of being targeted by RNA methyltransferases (Yang et al., 2019). Hence, mobile RNA transcripts are enriched in cytosine methylation, and if such methylation is removed, mobility can be negatively affected (Yang et al., 2019). Much of what is known about the transport of RNA species in plants is credited to research conducted on viroids, which are circular non-coding RNA species often considered plant pathogens, mostly because of their recognition as an ideal model system for molecular transport studies (Capistrán-Barradas et al., 2006; Ding and Itaya, 2007; Wang et al., 2021). For example, research performed on viroids has found that their long-distance mobility is facilitated by selective trafficking based on the presence of RNA motifs or structures (Qi et al., 2004; Zhong et al., 2007; Zhong et al., 2008; Takeda et al., 2018) and a similar concept has been supported by several studies related to plant-derived mobile RNAs (Zhang et al., 2009; Thieme et al., 2015; Zhang et al., 2016; Garcia-Lozano et al., 2020). In addition, experiments performed with grafting in cucumber demonstrated that viroid movement is mediated by specific host proteins (Gómez and Pallás, 2004), suggesting that a similar mechanism exists for endogenous RNA molecules. Interestingly, it was found that a specific plant protein assists the mobility of at least one endogenous mRNA in pear (Pyrus betulaefolia) (Duan et al., 2016), and recent studies have suggested that organelle-assisted movement is also used for the translocation of endogenous sRNAs in herbaceous plants (Luo et al., 2022). This hypothesis suggests that mRNAs move by interacting with organelles that are transported out of the phloem into the destination tissue (Luo et al., 2022). An example of this mechanism was observed with the mRNA of the mobile factor FLOWERING LOCUS T, which has been found to move through the plasmodesmata via microtubules in the endosomes (Luo et al., 2022). A similar finding was observed in maize, where mobilisation of the mRNA of the KNOTTED1 (KN1) TF, a highly conserved protein involved in stem cell production, moves in shoot meristems. This is of particular interest because, although the movement of TFs is widely known, for KN1 functionality it requires trafficking of both its protein and mRNA (Kitagawa et al., 2022).

Collectively, the data indicates the existence of active mechanism for the systemic transport of at least one group of mobile RNA molecules. Considering that distant mobile molecules can trigger effects through a graft junction, these studies are important for clarifying the mechanisms underlying how a rootstock can induce physiological changes in scions or vice versa.

Changes in DNA methylation associated with grafting have been observed in many plant families. The first studies were conducted on Brassicaceae, Cucurbitaceae, and Solanaceae species (Molnar et al., 2010; Avramidou et al., 2015; Lewsey et al., 2016). In Arabidopsis, a siRNA molecule originating from a transgene was found to move across the graft junction with prevalent translocation in the shoot-to-root direction. At specific loci, these molecules have been found to direct DNA methylation, resulting in a silencing signal (Molnar et al., 2010). The use of a transgenic construct has also been employed in tobacco to show that siRNA produced in the rootstock can travel across the graft junction and induce silencing of complementary DNA sequences in the scion by increasing DNA methylation (Bai et al., 2011). It was later revealed that the RdDM pathway has a critical role in triggering silencing by introducing DNA methylation at specific genomic loci (Lewsey et al., 2016). A general pattern of grafting-induced hypermethylation has been observed in Cucurbitaceae species when cucumber and melon were used a scion, grafted onto pumpkin rootstocks (Avramidou et al., 2015). In eggplant, however, hypomethylation in the scions was found to be associated with heterografting and linked to enhanced vigour (Cerruti et al., 2021). Similar studies across Brassicaceae and Cucurbitaceae identified methylation changes, mostly related to the non-CG context, associated with specific grafting combinations, but with different trends of hyper- and hypo-methylation (Avramidou et al., 2015; Lewsey et al., 2016; Cerruti et al., 2021). In these studies, the DNA methyltransferases and DNA demethylase genes in the scions were not found to be substantially differentially expressed, indicating that the epigenetic changes observed cannot be simply explained by altered expression levels of these epigenetic factors. Therefore, most of the DNA methylation changes associated with grafting could indeed be a consequence of a change in the siRNA targeting system or, alternatively, the result of other indirect effects of grafting (e.g. passive de-methylation or carry-over effects from the grafting procedure) (Wu et al., 2013; Cerruti et al., 2021).

Notably, hypomethylation and hypermethylation are associated with opposite effects on gene expression, and different combinations of scion and rootstock genotypes can lead to different phenotypic effects. Therefore, epigenetic changes could potentially explain part of the phenotypic alteration (e.g. vigour) observed in scions grafted onto different rootstock genotypes (Yang et al., 2018). In this context, it would be interesting to test the role of epigenetics in other grafting-induced phenotypes, such as disease resistance, which has been directly associated with epigenetic variation (Wada et al., 2004).

While direct evidence of grafting-induced histone modifications has not yet been observed, there are studies suggesting that epigenetic modification induced by grafting is not limited to the alteration of the DNA methylation landscape ( Figure 2 ). It is well established that there is a crosstalk between DNA methylation, sRNAs accumulation and histone modifications, so that epigenetic changes induced by distant signaling are expected to occur also at level of chromatin structure (Saze et al., 2012).

In a study performed on grapevine heterografts, the vigorous phenotype observed with specific grafting combinations was associated with the overexpression of the core histone genes as well as two nucleosome-associated proteins (Cookson and Ollat, 2013), while on apple tree varieties has been found that the expression of genes encoding histone modification factors was central to control flowering (Fan et al., 2018).

Therefore, histone modifications might play a role in the physiological effects induced by grafting and the extension of specific approaches to study chromatin dynamic (e.g. ATAC-seq or 3C) to grafting experiments could reveal more details on how histone marks can be associated with crop traits.

Epigenetic modifications were originally thought to only affect an individual during their lifespan and were not perceived to impact the offspring. In 1956, these beliefs were challenged and ideas that traits induced by the environment could be inherited began to be discussed (Waddington, 1956). Recently, epigenetic inheritance has shown that environmentally controlled epialleles can be transmitted to progeny, bridging the gap between inheritance by genetic determinants and environmental influences (Van Otterdijk and Michels, 2016; Pang et al., 2017). Hence, determining whether grafting-induced epigenetic alterations are transgenerationally inherited is pivotal to better understand plant developmental mechanisms and to exploit their potential use in breeding schemes to consistently increase crop production. In Brassicaceae models, DNA methylation alterations induced by grafting in the scion was shown to be indeed heritable in the offspring of grafted plants (Cao et al., 2016). The first generation of self-fertilised progenies from tuber mustard (Brassica juncea) grafted onto red cabbage (Brassica oleracea) displayed a genome-wide change in DNA methylation levels by 5.29% to 6.59% (for hypermethylation and hypomethylation, respectively), compared to the parental lines. By the 5^th generation, 31.58% of these changes were still present, while the rest had reverted to the original methylation levels (Cao et al., 2016). Similarly, in tomato and Arabidopsis, the vigorous phenotype induced by a mutation in MutS HOMOLOGUE 1 (MSH1) can be passed through grafting via siRNAs, and is inherited in the offspring of wild-type scions (Kundariya et al., 2020).

Over the years, studies have proven that epialleles can arise naturally and can influence agronomically important traits, such as fruit ripening in tomato (Manning et al., 2006) or the seed size and leaf angle of rice (Zhang et al., 2015). Genome-wide and epigenome-wide association studies performed in maize and wheat have highlighted strong correlations between differentially methylated regions (DMRs) and the expression of many genes responsible for crop traits (Gardiner et al., 2018; Xu et al., 2019). These and other studies indicate the great potential of using epialleles in crop breeding for trait improvement. The identification of epialleles linked to agronomically important traits in the progeny of crop grafts could give rise to novel epi-molecular markers (Noshay and Springer, 2021). The prospects of epigenetic breeding have been illustrated in soybean, where improved yields have been observed in the third generation by suppressing MSH1 (Raju et al., 2018). Similar results were observed when replicating MSH1 suppression in tomatoes and Arabidopsis (Kundariya et al., 2020). A clear benefit to producing heritable traits through grafting is the possibility of efficiently introducing new genetic and epigenetic diversity from wild species into crops, without the necessity to introgress the trait with breeding or by genetic transformation. Introducing stably transmissible epigenetic traits could ultimately represent a viable and unique technique for developing new crop varieties.

For crop species that are popularly grafted commercially, such as cucumber, tomato, and pepper, future endeavours should identify and preserve the biodiversity of natural varieties and species in light of their potential use as rootstock or scions in grafting combinations. As techniques continue to advance (e.g. micrografting), they will allow us to investigate the genetic and epigenetic variations in the genomes of plants improved by grafting. This will enable the development of new resources and drive comparative studies to fully connect the many facets of epigenetic regulation with the final goal of improving modern crop breeding.