Gene regulatory networks for compatible versus incompatible grafts identify a role for SlWOX4 during junction formation

Abstract

Grafting has been adopted for a wide range of crops to enhance productivity and resilience; for example, grafting of Solanaceous crops couples disease-resistant rootstocks with scions that produce high-quality fruit. However, incompatibility severely limits the application of grafting and graft incompatibility remains poorly understood. In grafts, immediate incompatibility results in rapid death, but delayed incompatibility can take months or even years to manifest, creating a significant economic burden for perennial crop production. To gain insight into the genetic mechanisms underlying this phenomenon, we developed a model system using heterografting of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). These grafted plants express signs of anatomical junction failure within the first week of grafting. By generating a detailed timeline for junction formation, we were able to pinpoint the cellular basis for this delayed incompatibility. Furthermore, we inferred gene regulatory networks for compatible self-grafts and incompatible heterografts based on these key anatomical events, which predict core regulators for grafting. Finally, we examined the role of vascular development in graft formation and uncovered SlWOX4 as a potential regulator of graft compatibility. Following this predicted regulator up with functional analysis, we show that Slwox4 homografts fail to form xylem bridges across the junction, demonstrating that indeed, SlWOX4 is essential for vascular reconnection during grafting, and may function as an early indicator of graft failure.

Integrated PET and confocal imaging informs a functional timeline for the dynamic process of vascular reconnection during grafting

“…Graft junctions were harvested from 3-8 days post-grafting and vacuum infiltrated with ice-cold FAA (4% formaldehyde, 5% glacial acetic acid, 50% ethanol, 35% milliQ H2O). Samples were fixed overnight at 4 ºC, dehydrated through an ethanol series, rehydrated, stained for 1 hour in 0.002% propidium iodide (Thermo Fisher, Waltham, MA, USA), dehydrated again, and cleared for at least 10 days in 100% methyl salicylate (Sigma-Aldrich, Saint Louis, MO, USA) (26). The stained and cleared junctions were imaged on a Leica SP8 laser scanning confocal microscope with a white light laser set to 514-nm wavelength, with the laser intensity ranging from 10%-80% depending on sample depth.…”

Grafting enhances plants drought resistance: Current understanding, mechanisms, and future perspectives

“…Generally, the survival rate of intra-generic grafting is high, while that of inter-genera is low. Recently, studies on the physiological and molecular mechanisms of grafting junction have gradually become a new hotspot, like the key responsive genes in grafted healing sites ( Melnyk et al., 2018 ; Notaguchi et al., 2020 ; Kurotani and Notaguchi, 2021 ; Thomas et al., 2021 ; Chambaud et al., 2022 ) and genetic information exchange between rootstocks and scions ( Thomas and Frank, 2019 ; Yang et al., 2019 ; Cerruti et al., 2021 ; Okamoto et al., 2022 ).…”

Genetic Relationship, SPAD Reading, and Soluble Sugar Content as Indices for Evaluating the Graft Compatibility of Citrus Interstocks

“…The above studies suggest that osmoregulatory substances and antioxidant enzymes are involved in the graft healing process. These studies on stock–scion compatibility focused on the anatomy, physiological and biochemical indicators, and information exchange of the graft interface, which destroyed the plant and prevented continuous observation of samples [ 24 , 25 , 26 ]. Therefore, attention was turned to the physiological and biochemical indicators of the grafted leaves.…”

“…In addition, some genes are directly implicated in junction formation. For example, WOX4 (protein-coding genes, a WUSCHEL-related homeobox gene family member with 65 amino acids in its homeodomain) is a potential regulator of graft compatibility, regulating vascular reconnection [ 26 ]. Although graft compatibility has been studied deeply and comprehensively on the physiological [ 29 ], biochemical, and even molecular levels [ 30 , 31 ], the combined graft compatibility of the stock–scion mediated by citrus interstock is still rarely reported.…”