Low ethylene production in the root(stock) alleviates salt stress in tomato

Low ethylene production in the root(stock) alleviates salt stress in tomato | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 27 October 2025 V1 Latest version Share on Low ethylene production in the root(stock) alleviates salt stress in tomato Authors : Jose A. Martín-Rodríguez 0000-0002-5768-3864 [email protected] , Purificación A Martínez-Melgarejo , Jesús Guillamón , Ángela S. Prudencio , Juan J. Guerrero , Eduardo Larriba , Jose Manuel Perez-Perez 0000-0003-2848-4919 , Enrique Olmos , Lazaro Peres , Cristina Martínez Andújar 0000-0002-3684-9765 , and FRANCISCO PEREZ ALFOCEA 0000-0003-1057-4924 Authors Info & Affiliations https://doi.org/10.22541/au.176155600.06834638/v1 252 views 153 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Ethylene is a key plant hormone regulating plant development processes, but is often associated with growth inhibition and senescence, particularly under abiotic stress conditions such as salinity. While ethylene is synthesized in different plant tissues, root-derived ethylene plays a critical role as a stress sensor and signalling molecule, mediating roots-to-shoots communication. To examine whether root-sourced ethylene influences salinity responses, a tomato variety ( Solanum lycopersicum L. cv. UniDarkwin) was grafted onto three rootstocks differing in ethylene production: an ethylene-overproducing mutant (epinastic, epi ), a root-specific ACC-deaminase overexpressing transgenic line with reduced ethylene synthesis ( ACCD ), and the wild-type cv. Micro-Tom (WT). After 100 days of salt treatment (75 mM NaCl), ACCD grafts exhibited reduced concentrations of the ethylene-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and enhanced plant growth and yield stability, whereas epi and WT grafts were most adversely affected. ACCD rootstocks modulated both local and systemic mechanisms, promoting a larger and more branched root system, altered leaf gene expression and hormone metabolism, diminished Na + toxicity, and improved leaf P, Ca, and S nutrition, photosynthesis, and source-sink relations. Reduced root-sourced ethylene also decreased stress sensitivity by lowering leaf abscisic acid (ABA) concentration through altering its metabolism, limiting jasmonic acid (JA) and Ca translocation from leaves to flowers, and favouring the activity of growth-promoting and anti-senescing hormones cytokinins (CKs) and gibberellins (GAs) under salinity. These findings highlight the role of root-derived ethylene in modulating local and systemic responses to stress and its potential for improving crop yield stability under salinity. Low ethylene production in the root(stock) alleviates salt stress in tomato Jose A. Martín-Rodríguez 1 *, Purificación A. Martínez-Melgarejo 1 , Jesús Guillamón 1 , Ángela S. Prudencio 1 , Juan J. Guerrero 1 , Eduardo Larriba 2 , Jose Manuel Pérez-Pérez 2 , Enrique Olmos 3 , Lázaro E. Pereira Peres 4 , Cristina Martínez-Andújar 1 *, and Francisco Pérez-Alfocea 1 . 1 Group of Plant Hormones, CEBAS-CSIC, Murcia, Spain; 2 Institute of Bioengineering, Miguel Hernández University, Elche, Spain; 3 Department of Stress Biology, CEBAS-CSIC, Murcia, Spain; 4 Laboratory of Hormonal Control of Plant Development, Escola Superior de Agricultura ”Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, Brazil. * Corresponding author email: [email protected] ; [email protected] Word count: 7928 Number of tables: 4 Number of figures: 5 Supplementary data: 3 (figures) + 11 (tables) Abstract (243 word) Ethylene is a key plant hormone regulating plant development processes, but is often associated with growth inhibition and senescence, particularly under abiotic stress conditions such as salinity. While ethylene is synthesized in different plant tissues, root-derived ethylene plays a critical role as a stress sensor and signalling molecule, mediating roots-to-shoots communication. To examine whether root-sourced ethylene influences salinity responses, a tomato variety ( Solanum lycopersicum L. cv. UniDarkwin) was grafted onto three rootstocks differing in ethylene production: an ethylene-overproducing mutant (epinastic, epi ), a root-specific ACC-deaminase overexpressing transgenic line with reduced ethylene synthesis ( ACCD ), and the wild-type cv. Micro-Tom (WT). After 100 days of salt treatment (75 mM NaCl), ACCD grafts exhibited reduced concentrations of the ethylene-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and enhanced plant growth and yield stability, whereas epi and WT grafts were most adversely affected. ACCD rootstocks modulated both local and systemic mechanisms, promoting a larger and more branched root system, altered leaf gene expression and hormone metabolism, diminished Na + toxicity, and improved leaf P, Ca, and S nutrition, photosynthesis, and source-sink relations. Reduced root-sourced ethylene also decreased stress sensitivity by lowering leaf abscisic acid (ABA) concentration through altering its metabolism, limiting jasmonic acid (JA) and Ca translocation from leaves to flowers, and favouring the activity of growth-promoting and anti-senescing hormones cytokinins (CKs) and gibberellins (GAs) under salinity. These findings highlight the role of root-derived ethylene in modulating local and systemic responses to stress and its potential for improving crop yield stability under salinity. Keywords ABA metabolism, 1-aminocyclopropane-1-carboxylic acid, leaf transcriptomics, plant hormones, root-shoot communication, root system architecture, Solanum lycopersicum, source-sink. Introduction Excess salinization in the soil can occur naturally or result from anthropogenic factors such as poor irrigation and excessive fertilizer use (Guida-Johnson, Abraham & Cony 2017; Velmurugan, Swarnam, Subramani, Meena & Kaledhonkar 2020). Currently, over 20% of irrigated soils worldwide are considered too salinized for agriculture (Otlewska et al. 2020; Velmurugan et al. 2020). Salinity reduces crop growth through a combination of osmotic stress and ionic toxicity caused by Na + and Cl - accumulation, which also disrupts nutrient uptake (Munns 2002; Munns & Tester 2008). The root system is responsible for water and nutrient acquisition, as well as other important regulatory processes under salinity, such as Na + exclusion. Furthermore, root-to-shoot hormonal communication is a key factor that affects shoot physiology, plant growth, and yield (Dodd 2005; Albacete et al. 2015; Julkowska et al. 2017). Indeed, root-sourced hormones play a crucial role in the physiological and agronomical adaptation to salinity by regulating local and systemic responses in the plants, opening a biotechnological strategy to mitigate the effect of salinity on crops through altering root hormone metabolism (Pérez-Alfocea, Albacete, Ghanem & Dodd 2010; Ghanem et al. 2011; Martínez‐Andújar et al. 2021). Root development must be flexible to cope with changes in the soil or root zone properties (Rellán-Álvarez, Lobet & Dinneny 2016). The root system architecture (RSA) varies significantly in response to salt depending on plant species and salt concentration, although in general, salinity inhibits both primary root (PR) and lateral root (LR) growth (Zou, Zhang & Testerink 2022). Furthermore, several plant hormones regulate plant responses to salt stress by modulating RSA. For instance, ABA mediates the induction of a quiescent phase in LR growth, leading to reduced PR length and LR density in ABA mutants (Duan et al. 2013; Kawa et al. 2020). Ethylene signalling is involved in RSA changes and whole plant adaptation to nutrient deficiency, and has been related to reduced PR (Jung, Shin & Schachtman 2009) and LR growth (Lewis, Negi, Sukumar & Muday 2011). Interestingly, studies applying the ethylene precursor ACC have shown that it reduces both root elongation and LR. In contrast, the application of ethylene inhibitors induces PR elongation and increases both the number and size of LR (Belimov et al. 2022). Moreover, ethylene interacts with other hormones such as ABA in shaping RSA, while excessive ethylene usually has a negative impact on root growth (Liu et al. 2024). The role of ethylene and its precursor ACC in the response to abiotic stresses, particularly salinity, remains controversial. In tomato and other species, salinity increases ethylene and ACC levels (Riyazuddin et al. 2020), which can exacerbate stress responses by promoting oxidative damage and leaf senescence (Albacete et al. 2008; Cao, Chen & Zhang 2008; Ghanem et al. 2008). Conversely, in Arabidopsis, exogenous application of ethylene or ACC eliminates ROS, increasing tolerance to salinity (Peng et al. 2014). Several works have shown that ethylene plays an important role in the regulation of nutrient uptake, such as N, P, K, and Fe, under salinity and their homeostasis with Na + (Tao et al. 2015). Reducing ACC content through the action of growth-promoting bacteria (PGPB) that produce ACC deaminase ( ACCD ), which breaks ACC into α-ketobutyrate and ammonia, decreases ethylene production and mitigates its potential adverse effects under salt stress (Win, Tanaka, Okazaki & Ohwaki 2018; Naing, Maung & Kim 2021). Moreover, transgenic plants overexpressing the ACCD gene have been generated in the past decades, demonstrating improved tolerance to multiple abiotic stresses (Zhang, Zhao, Wang, Yang & Chen 2008; Jung, Ali, Kim & Kim 2018). Ethylene interacts with multiple phytohormones during abiotic stress responses. Under salinity, ABA regulates several ethylene biosynthetic genes (Tao et al. 2015). Salinity also down-regulates EIN2 , a key component of ethylene signalling, resulting in increased ABA production (Wang et al. 2007). Furthermore, under salinity, ethylene stabilizes DELLA proteins, repressors of GAs signalling, causing growth arrest (Iqbal, Masood & Khan 2012; Bhardwaj et al. 2022). Finally, an antagonistic interaction between ethylene and CKs under salt stress is characterized by decreased CK levels (Riyazuddin et al. 2020). Tomato ( Solanum lycopersicum L.) is one of the most important crops worldwide, with about 5 million cultivated ha (FAOSTAT 2023). Many of the cultivated areas are affected by salinity, such as the South-Eastern Spain, where grafting onto vigorous rootstocks is a common commercial practice to alleviate the effect of biotic and abiotic stresses on yield without compromising fruit quality (Bletsos & Olympios 2008; Martínez-Andújar, Albacete & Pérez-Alfocea 2018). Grafting also serves as a valuable technique to study how root-targeted genetic modifications influence plant physiology and performance under diverse environments, particularly thorough hormonal-regulation of local and systemic responses mediated by the roots (Pérez-Alfocea et al. 2010; Pérez-Alfocea, Yeboah & Dodd 2021; Ghanem et al. 2011; Albacete et al. 2015). Previous studies in tomato have demonstrated that ACC exhibits a dynamic increase in roots, xylem sap and shoots in response to salinity and has been correlated with the onset of salt-induced leaf senescence (Ghanem et al. 2008) and growth reduction (Albacete et al. 2008). Moreover, increased tolerance to abiotic stresses, through rootstock genetic variability or root-specific alteration in hormone biosynthesis (e.g. CKs and ABA), has been consistently associated with reduced ACC evolution (Albacete et al. 2009; Albacete, Ghanem, Dodd & Pérez-Alfocea 2010; Albacete et al. 2014a; Albacete, Martínez-Andújar & Pérez-Alfocea 2014b; Albacete et al. 2015; Martínez-Andújar et al. 2016; Martínez-Andújar, Ruiz-Lozano, Dodd, Albacete & Pérez-Alfocea 2017; Martínez-Andújar et al. 2021). Therefore, a direct long-term (whole crop cycle) functional analysis of root-specific alteration in ethylene production could provide new insights into the specific role of this hormone in the local and systemic responses to salinity. By using rootstock variability in ethylene production, this study aims to demonstrate that this hormone acts as a negative factor in the tolerance to salinity in an important crop species, such as tomato. Materials and methods Plant material To study the effects of ethylene production in the roots on the plant response to salinity, the ethylene overproducer mutant epinastic ( epi ) and transgenic plants overexpressing the gene encoding for the enzyme ACCD, both on the Micro-Tom (MT) background, were used as rootstocks of the commercial tomato cherry variety UniDarkwin (UD; Unigenia Semillas, Murcia, Spain). The ACCD enzyme catalyzes the cleavage of the ethylene precursor ACC into α-ketobutyrate and ammonia (Glick, Penrose & Li 1998). The transgenic Micro-Tom plants that overexpress ACCD are under the control of the root-specific rolD promoter ( roID::ACCD ) of Agrobacterium rhizogenes, and its overexpression has been observed to reduce ethylene production by more than 90% compared to wild type plants (Klee, Hayford, Kretzmer, Barry & Kishore 1991). The mutant epi is an introgressed LA2089 (cv. VFN8) harbouring the mutated allele leading to ethylene overproduction (Barry et al. 2001). Seeds were germinated in commercial vermiculite, watered with deionized water, and maintained under controlled conditions of 26–28°C temperature and 80–90% relative humidity in the dark. Grafting was performed using the splicing method at the two to three true leaf stages (3–4 weeks after sowing). The scion was attached to the rootstock at the first node (Savvas et al. 2011). Grafting with epi mutant and ACCD transgenic line, along with the wild type MT (WT) used as a control, resulted in three graft combinations: UD/ epi , UD/ ACCD , and UD/WT. Forty days after grafting, once the plants were well established, they were transferred to commercial-like conventional plastic greenhouse conditions and cultivated using a sand substrate during the spring-summer season, in the area of south-eastern Spain (Martínez-Andújar et al. 2023). Six plants per graft combination were randomly distributed in three blocks, and water and fertilizers were applied by drip fertigation. From 30 days after transplanting (DAT), a salinity treatment was applied to 50% of the plants by adding 75mM NaCl to the nutrient solution, resulting in an electrical conductivity (EC) of 8.9 dS m -1 . This treatment was maintained for a period of 100 days (saline treatment). The remained half of the plants received the nutrient solution (EC= 3 dS m -1 ) for the whole period (control treatment). Plant Phenotyping Total yield was determined by collecting all fruits from each plant throughout the harvest period. Fully ripe fruits were harvested weekly for 2 months, and fruit number (FN), fruit weight (FW) and truss length (TL) were recorded. At the end of harvest (130 DAT), shoot (SFW) and root (RFW) biomasses were determined, and stem diameter (SD) at the second node was measured using an electronic digital Vernier calliper (0 - 150 mm). Additionally, young fully expanded leaves, roots and flowers were sampled and immediately frozen in liquid nitrogen, and stored at -80°C for subsequent hormonal and gene expression analysis. After 70 days of salt treatment (DST), photosynthesis ( A N ), stomatal conductance ( g s ), and evapotranspiration (E) were measured on fully expanded young leaves using a CIRAS-2 (PP Systems, Massachusetts, USA) between 09.00 and 12.00 hr (sunrise was around 7.00 hr). During the measurements, CO 2 and photosynthetic active radiation (PAR) were maintained at 400 ppm and 1,500 μmol m -2 s -1 , respectively. Chlorophyll fluorescence was recorded on the same dark-adapted (15min) leaves using a chlorophyll fluorometer OS-30 (OptiSciences, Herts, UK) with an excitation intensity of 3000 μmol m –2 s –1 . Chlorophyll content (SPAD) was determined using a portable chlorophyll meter SPAD-502 (Konica Minolta, Inc., Tokyo, Japan) prior to sampling. For each genotype and treatment, three readings were taken from the same fully expanded leaf on three different plants, and the mean was used for analysis. At 95 DST, the second fully expanded mature leaf over the fourth truss (with actively growing fruits) was collected from 5 plants per graft combination to determine leaf fresh weight (LFW) and leaf area (LA) using an LI-3100 AC area meter (LI-Cor, Lincoln, NE, USA). Pollinator preference quantification Flower pollination was promoted by using managed bumblebees ( Bombus terrestris ), following established commercial standards. Each hive with approximately 50 workers can pollinate between 300 and 500 plants over a period of 8 to 12 weeks. Pollinators’ preferences were monitored weekly across the plant’s entire reproductive phase, with previously assessed flower trusses clearly marked for tracking. The percentage of flowers visited by bumblebees (%FVB, Table 1) was recorded as described in Martínez-Andujar et al., (2023). Ionome analyses The same leaves collected for LA and LFW analysis at 95 DST, along with flowers sampled during the same period, were dried for 48 h at 80 °C, milled to a powder, and 50 mg (flower) or 200 mg (leaf) of dry tissue were digested using a HNO 3 :HClO (2:1, vol/vol) solution. Samples were analysed by inductively coupled plasma spectrometry (ICP-OES, Thermo ICAP 6000 Series). Total C and N contents were determined in 50 mg and 200 mg of dry leaf and flower material, respectively, by the combustion method using an elemental analyser (LECO TRUSPEC, The Netherlands). Hormones, soluble carbohydrates, amino acids and flavonoids quantification The major classes of plant hormones, including CKs [trans-zeatin (t-Z), zeatin riboside (ZR) and isopentenyladenine (iP)], gibberellin A1 (GA1), A3(GA3) and A4 (GA4), jasmonic acid (JA), salicylic acid (SA) and the ethylene precursor ACC, ABA and it derivative ABA-aldehyde and catabolites (ABA glucosyl ester (ABA-GE), neophaseic acid (neoPA), phaseic acid (PA), dihydrophaseic acid (DPA) and DPA glucoside (DPAG) were extracted and analysed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) following the protocol described previously in Albacete et al., (2008) and Martínez-Andujar et al., (2021). Additionally, amino acids (ser, asn, gln, tyr, val, leu, ile, pro, hyp, phe, trp, asp, lys, arg, his) and soluble sugars (sucrose, fructose, and glucose) were also analysed using this method as described in Martínez‐Melgarejo (2022) and Ormazabal et al., (2024). Ten microliters of each filtered extract (leaf, root, or flower) were injected into a UHPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA) coupled to an Orbitrap Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA). To quantify plant hormones, sugars and amino acids, calibration curves were constructed for each analysed component using standar solutions: hormones (0, 1, 10, 50 and 100 μg L −1 ), amino acids (10, 100, 500 and 1000 μg L − 1), and sugars (10, 50, 100 and 400 μg L −1 ) from the companies Olchemim (Olomouc, Czech Republic) and Sigma-Aldrich (Merck Group, Darmstadt, Germany). ABA precursors and catabolites were identified by extracting the exact mass of the target catabolite from the full scan chromatogram obtained in the negative mode, adjusting a mass tolerance of ≤1 ppm. Concentrations were semi-quantitatively determined from the extracted peaks using the calibration curve of ABA (precursors and catabolites). Starch analysis Starch concentration was analysed in the same leaves used for ionomic analysis and collected at 95 DST, following the protocol described by Nicolás et al. , (2015), with some modifications. Microscopic leaf morphometric analysis To identify phenotypic differences in the leaves of different graft combinations grown under salinity, ten square sections (1 mm 2 ) were taken from the youngest fully expanded leaves of 3 plants per graft combination after 90 DST, avoiding the central region. Samples were fixed and post-fixed according to the method described by Fernández-García et al. , (2014). After embedding in Spurr resin to form blocks, they were sectioned into slices 0.5-0.7 μm thick using a Leica EM UC6 ultra-microtome. These sections were stained with 0.5 % toluidine blue, mounted in DPX, and observed with a Leica CTR 6000 fluorescence microscope. The percentages of area occupied by palisade parenchyma (PP), spongy parenchyma (SP), and intercellular spaces (IS) in leaves from grafted plants were measured and processed by the LAS AF Lite software. RNA isolation and RNA-Seq analysis To study the impact of different ethylene-producing rootstocks on leaf gene expression under control and saline conditions of 100 DST, total RNA was extracted from approximately 100 mg of powdered leaf and root tissue using NBS Spin Column All-in-one Miniprep (NBS Biologicals, UK) following the manufacturer’s protocol. The extracted RNA was subsequently stored at −80 °C. RNA integrity was confirmed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). In the extracted RNA from leaf tissue, next-generation sequencing (NGS) was conducted using the BGISEQ-500 pipeline (BGI-Tech, Shenzhen, China), in pair-end mode with 100 sequencing cycles. Clean reads were obtained using SOAPnuke (https://github.com/BGI-flexlab/SOAPnuke) and subsequently evaluated using FastQvalidator (https://github.com/statgen/fastQValidator). For bioinformatics analysis, the workflow described in (Larriba, Sánchez-García, Martínez-Andújar, Albacete & Pérez-Pérez 2021) was followed. Briefly, clean RNA-Seq reads were mapped to the S. lycopersicum genome build SL4.0 (Larriba et al. 2021) using STAR 2.7 (Hosmani et al. 2019). Gene count was performed using feature Counts from the Subread package (Liao, Smyth & Shi 2019) with the ITAG4 gene models from SolGenomics (https://solgenomics.net/). After the statistical treatment, the web-tool ShinyGO 0.8 (Ge, Jung & Yao 2020) was used to perform GO enrichment analysis. Putative tomato orthologs from A. thaliana were identified using Proteinortho software (Lechner et al. 2011). The analyses for heatmap and hierarchical clustering were performed using Morpheus (https://software.broadinstitute.org/morpheus). First-Strand cDNA Synthesis and qRT-PCR Retrotranscription was performed using 5 μg of previously obtained RNA with the RevertAid First Strand cDNA Synthesis Kit (Thermofisher), following the manufacturer’s recommendations. For quantitative PCR (qPCR) amplification, 20 ng of synthesized cDNA was used along with the PyroTaq EvaGreen qPCR Master Mix kit (CMB). qPCR was performed to assess the transcript abundance of genes TAS14 , PIP1.2 , CYP707A1 , ACO1, and ACO2 . Primer names and corresponding sequences are provided in supplemental table S1. The reaction was carried out in a final volume of 10 μL using a StepOne Real-Time PCR System (Thermofihser), with ROX included as a passive reference. The data obtained were analysed using the methodology outlined by Pfaffl (2001), normalized to the expression levels of the housekeeping genes actin , EF and GADPH . Statistical analysis Data from phenotypic, ionomic, and metabolomic analyses were subjected to 2-WAY ANOVA to assess the effects of genotype, treatment, and their interaction. Genotypic means were compared using Tukey’s test (p < 0.05). Correlation among plant variables was performed using SPSS v22.0 (SPSS Inc., Chicago, IL, USA). For the RNAseq data, read count normalization and differential gene expression analysis were carried out using DESeq2, integrated into the Differential Expression and Pathway analysis (iDEP 96) web application (Ge, Son & Yao 2018). Differentially expressed genes (DEGs) were identified using a False Discovery Rate (FDR) > 0.05 and fold change > and unique genes response to salinity among genotypes. Gene-metabolite relationships in selected pathways were carried out through a Network using the GENIE3 package from R, where transcription factors were considered candidate regulators (main nodes). Significant interactions were defined as those within the top 5% of edge weights, represented by edge thickness in the network (Fig. 5). Plant growth and yield At the end of the growth cycle, no significant differences in shoot fresh weight (SFW) and total plant biomass (TB) were observed between graft combinations under optimal conditions (Table 1). However, after receiving saline irrigation (75 mM NaCl, EC= 8.9 dSm -1 ) for 12 weeks, the SFW was negatively affected in all plants; however, the epi grafted plants showed the greatest reduction (Table 1). Other shoot vigour indicators such as, SD, LFW, and LA, were also reduced by salinity, but to a lower extent in the plants grafted onto ACCD rootstocks (Table 1). Indeed, LFW was the most differential parameter in ACCD grafted plants under salinity compared to other graft combinations, suggesting differences in leaf water content and thickness. Contrary to the aerial part, salinity increased RFW in WT and ACCD , but not in epi grafts (Table 1; Fig. 1C). While no differences in yield were observed among graft combinations under control conditions, ACCD grafts were unaffected by salinity and produced significantly more yield than WT and epi plants under stress. This increase in yield was attributed to the stability of both FN and FW relative to control conditions (Table 1). epi grafts were the most affected by salinity in the FW compared to control conditions (Table 1), while the plants grafted onto WT rootstocks registered the lowest FN (Table 1) and the shortest trusses, resulting in a lower fruit per truss ratio (Table 1). Notably, the presence of multiple trusses under salt stress conditions was observed exclusively in ACCD grafts (Table 1). Overall, the ACCD rootstocks maintained yield stability under salinity, while epi rootstocks were the most affected, mainly due to the impact of salt stress on fruit weight. Additionally, during the flowering period, observations of stamen marks indicated a clear distinction between non-visited (no mark) and flowers visited by pollinators (brown marks). Curiously, ACCD -grafted plants exhibited about 20% more flowers visited (%FVB) compared to other grafts (Table 1), suggesting a higher preference of bumblebees for those plants, which could positively influence fruit setting and development under stress. Gas-exchange parameters Plants grafted onto ACCD and WT rootstocks registered the highest and lowest E , g s and A N value under salinity, respectively (Table 1). Indeed, the WT grafted plants were the most negatively impacted in terms of A N , E, g s , and Fv/Fm with respect to control conditions, while the SPAD parameter, which reflects the chlorophyll content, was more affected in plants grafted onto epi rootstocks under salinity conditions (Table 1). Overall, ACCD -grafted plants maintained the highest values of E, gs, A N , and SPAD under salinity, whereas WT (E, g S , A N ) and epi (SPAD) grafts suffered the greatest reductions in these parameters Leaf and flower nutrient status Under control conditions, the advantage of plants grafted onto ACCD over epi rootstocks on leaf nutrient status was observed for Ca, Mg, B, and Fe (Table 2). Indeed, under salinity, ACCD grafts registered the highest macro (P, S, Ca) and micro (B, Fe, Mn, Cu) concentrations in the mature leaves compared with the other grafts (Table 2). However, epi grafted plants registered the lowest N, S, Ca, Mg, Mn, and Cu, and the highest Na + concentrations in the leaves. Similarly, under salinity conditions, the flowers of the epi grafts registered the lowest K, S, Ca, Mg, B, Fe, Mn, Cu, and Zn, and the highest Na + concentrations (Table 2). Interestingly, ACCD grafts maintained stable Ca concentration in both leaf and flowers, compared to WT and epi grafts, in which salinity decreased this nutrient in the leaves and increased it in the flowers (Table 2). Overall, under salinity conditions, low ACC production (through ACCD grafts) in the roots enhances the macro- and micro nutrient uptake, their accumulation in the leaves and their translocation to the flowers. In contrast, high ethylene production (through epi grafts ) only favours the uptake and accumulation of the toxic ion Na + in both source and sink tissues, while limiting the uptake of most macro- and micro-nutrients. Hormonal profiling For a better understanding of rootstock-mediated ACC/ethylene production on local and systemic effects of the plants, a comprehensive analysis of main plant hormones and some ABA derivatives were analysed in root, leaf and flower tissues. 1-aminocyclopropane-1-carboxylic and abscisic acids Under control conditions, the ethylene precursor ACC was undetectable in the leaves across all graft combinations, and similar low concentrations were registered in flowers (Fig. 1, 2; Table S2). However, although not significant, root ACC concentration in epi grafted plants was 3-times higher than in WT and ACCD ones in the absence of stress (Fig. 1, 2; Table S2). Irrespective of genotype, salinity increased ACC levels in roots, leaves, and flowers. Notably, plants grafted onto epi rootstocks displayed a 3.5 and 110-fold rise in ACC in the roots, compared to WT and ACCD grafted plants, respectively, and registered a 2-fold increase in leaves and flowers compared to the other grafts. Indeed, ACCD rootstocks consistently registered the lowest ACC concentrations across all organs, especially in roots, which appear to be insensitive to saline stress. Overall, ACCD rootstocks decreased ACC evolution in both roots and aerial part of the plant, particularly in the flowers, potentially influencing ethylene-dependent local (roots) and systemic (leaves and fruits) growth responses. No significant differences were found between graft combinations in leaf ABA concentration. However, within ACCD grafts, salinity caused a significant decrease in leaf ABA concentration compared to control conditions (Fig. 2; Table 3). In roots, only plants grafted onto WT rootstocks showed a significant increase in ABA levels under salinity relative to control conditions. Therefore, the roots of ACCD grafts registered the highest ABA/ACC ratio under salinity compared to the other grafts’ combinations, while epi rootstocks registered the lowest values (Fig. 2; Table S2). ABA derivatives Given the previously reported interaction found between ethylene and ABA in the roots in response to salinity (Martínez-Andújar et al. 2020; Martínez‐Andújar et al. 2021) and to further explore the basis of ABA changes in ACCD -grafts, the main ABA precursors, catabolites, and conjugates were analysed. The immediate ABA precursor ABA-aldehyde was 2-fold higher in the leaves of plants grafted onto ACCD rootstocks compared to WT and epi ones under control conditions, decreasing by 50% under salinity relative to control conditions, while the opposite trend was found in WT and epi rootstocks (Table 3). This genotypic effect was not observed in flowers, where this ABA precursor was detected only in the WT grafted plants under salinity (Table 3). These results are consistent with those obtained for leaf ABA concentrations, since a reduction was observed in ACCD grafts under salinity (Fig. 2; Table 3; Table S2). Genotypic differences were also observed in some metabolites related to ABA catabolism and conjugation. Regarding the main ABA catabolic pathway through C-8’ hydroxylation, no genotypic differences in PA concentration were observed in leaves under control conditions, while it dropped in plants grafted onto WT and ACCD rootstocks under salinity, remaining constant in epi ones (Table 3). In contrast, DPA and its glycosylated form (DPAG) accumulated across all genotypes under salinity conditions. DPAG consistently exhibited strong increases in the three rootstocks, whereas the increase in DPA was less pronounced in plants grafted onto WT compared to those grafted onto epi and ACCD (Table 3). Finally, neo-PA decreased with salinity; significant differences were found in all genotypes. With respect to ABA conjugation, levels of ABA-GE were similar in the leaves of all three graft combinations under control conditions (Table 3). However, salinity caused a 2-3 times increase in all plants, with the highest concentration detected in leaves of epi grafts. In flowers, no genotypic differences were osberved for the analysed ABA-related metabolites. Salinity decreased PA in ACCD grafts and increased ABA-GE in all genotypes, but to a higher extent in WT and epi ones (Table 3). Overall, the results suggest that ethylene production in roots modulates ABA metabolism in shoots through a putative root-to-shoot mediated mechanism. Gibberellins Under control conditions, leaf GA1 concentrations were similar among graft combinations, GA3 was not detected in the leaves of ACCD grafted plants, and the highest GA4 concentrations were recorded in ACCD rootstocks (Fig. 2; Table S2). Salinity increased gibberellin levels in the leaves in a genotype-dependent way: GA1 in plants grafted onto WT rootstocks, GA3 in ACCD rootstocks and GA4 in the three grafts. No effect of genotype or treatment was observed in the flowers GA1 and GA4 concentrations, while the flower GA3 levels increased in response to salinity across all graft combinations. In roots, GA1 was detected only in ACCD grafts irrespective of the treatment, while GA3 increased under salinity in all graft combinations, but the increase was significant only in WT and epi rootstocks. Additionally, GA4 was specifically salt-induced in the roots of epi grafts (Fig. 2; Table S2). Overall, root ethylene production interacts with GAs and salinity, especially in roots and leaves. Thus, low ethylene production ( ACCD grafts) enhanced GA3 and GA4 in the salinized leaves and GA1 in the roots, while high ethylene ( epi grafts) reduces GA4 in the leaves while promoted it in salinized roots. Cytokinins In leaves, no genotypic differences in tZ, ZR, and iP were observed under control conditions. However, salinity increased leaf tZ and iP in WT and epi grafts, respectively, and decreased ZR in the leaves of the ethylene overproducer epi rootstocks (Fig. 2; Table S2). Indeed, total bioactive CKs (tZ+ZR+iP) increased under salinity in the leaves of plants grafted onto WT, decreased in epi rootstocks and were not altered in ACCD ones, relative to control conditions. In the flowers, no genotypic differences in CKs levels were observed under control conditions, while ACCD grafts registered the highest and lowest ZR and iP concentrations under salinity, respectively. In roots, ZR levels remain stable across all genotypes and treatments. Root tZ concentrations were not affected by salinity in epi and ACCD grafts, while they increased in WT rootstocks. No genotypic differences in root iP levels were detected under control conditions, but were induced under salinity, especially in WT grafts (Fig. 2; Table S2). Overall, plants grafted onto epi rootstocks decreased total CKs and promoted the shift between tZ-type to iP-type in leaves under salinity. In contrast, the levels of analysed bioactive CKs were insensitive to salinity in plants grafted onto low ethylene ACCD rootstocks, which resulted in higher ZR-type CKs relative to iP- and tZ-types, presumably delaying salt-induced leaf senescence and recovering sink activity in the salinized reproductive structures. Salicylic and jasmonic acids SA concentrations were 3-6 times higher in leaves than in flowers and roots, but no genotypic differences were found under control conditions. Salinity decreased SA in leaves and increased in roots of epi grafts, while increasing flower SA in WT grafted plants (Fig. 2; Table S2). Contrary to SA, JA concentration was 10-20 times higher in flowers than in leaves and roots. Salinity increased JA in leaves and roots of epi and WT grafts, while this hormone remained unaltered in all organs of ACCD plants (Fig. 2; Table S2). Carbohydrates Under saline conditions, plants grafted onto ACCD rootstocks exhibited higher hexose (leaves and flowers) and sucrose (flowers) concentrations than WT and epi grafts. Additionally, salinity reduced hexose (leaves and flowers) and sucrose (flowers) levels only in WT and epi rootstocks, compared to control conditions (Fig. 3A; Table S2). In contrast, salinity significantly reduced leaf starch concentration only in WT grafts, while the highest concentrations were found in epi grafts under salinity (Fig. 3A; Table S2). The differences in leaf starch contents were microscopically confirmed (Fig. 3B). Overall, salinity reduced primary carbohydrates in both source and sink organs in normal to high ethylene-producing grafts. In contrast, reduced ethylene ( ACCD grafts) promoted carbohydrate stability, while high-ethylene ( epi grafts) induced starch accumulation in source leaves under stress. Amino acids content in flowers Along with sugars, transport of amino acids to sink organs is an indicator of C/N balance and smooth source-sink relations. Under salinity, flowers of plants grafted onto the low ethylene rootstocks ( ACCD ) accumulated Ile, Pro, and Arg in response to salinity and exhibited significantly higher levels of several amino acids, including Tyr, Ile, Pro, Hyp, Trp, and Lys, compared to others rootstocks (Table S3). Leaf anatomy Changes in leaf morphological structure are an important adaptive response to salinity stress (Acosta-Motos et al. 2017). Indeed, leaf anatomy revealed some rootstock-dependent particularities under salinity conditions (Fig. 3B). Leaves from plants grafted onto WT rootstocks were thinner than in epi and ACCD grafts (Fig. 3C), while the leaf epidermis of ACCD grafts was thicker compared to that from WT and epi rootstocks (Figs. 3D, G). The percentage of palisade parenchyma (PP) relative to the total leaf area in epi grafts was 5% lower than in others graft combinations (Figs. 3E,H). Additionally, the spongy parenchyma (SP) in the leaf of plants grafted onto epi rootstocks appeared more disorganised, compared to other rootstock genotypes (Figs. 3B, F, I). Leaf transcriptomic analysis To characterize the systemic responses mediated by the ethylene-related rootstock genotypes, RNA-seq analyses were performed in leaves sampled on 130-day-old plants cultivated under control and saline conditions (100 DST). RNA-seq of leaves from grafted plants showed that salinity was the main source of transcriptomic variation (Fig. S1A). Over 79% of the RNA-Seq was mapped to the SL4 genome, observing a total of 20,808 transcripts after normalization under salinity. Each cluster comprised over 1,400 genes that exhibited upregulation exclusively within each genotype between treatments (Fig. S1B). Regarding the enrichment analysis, genes in the clusters were mainly associated with biological processes (GO BP), such as redox metabolism and response to abiotic stress, among others. In cluster blue, an enrichment in several GO BP terms related to photosynthesis, carotenoids, and chloroplast organization was found (Fig. S1C). The violet cluster exhibited enrichment in terms linked to protein folding, response to oxidative stresses (ROS), and RNA modification (Fig. S1D). Finally, green cluster enriched terms were related to sterol and steroid biosynthetic and metabolic processes (Figs. S1E-F). Differential expression analysis identified 1,385 differentially expressed genes (DEGs) across genotypes and treatment, with most associated with genotypic responses to salinity, as shown in the C7-C9 contrasts (Fig. 4B). Notably, ACCD grafts (C9 contrast) exhibited the highest number of deregulated genes in response to salt treatment, while WT rootstocks (C7 contrast) had the fewest DEGs (Fig. 4B-D). GO enrichment highlighted stress-related processes across all genotypes, with distinct differences among them (Fig. 4E-G). Specifically, epi grafts (C8) showed enrichment in carbohydrate metabolic processes and transmembrane transport (Fig. 4F). In contrast, ACCD grafts (C9) harbored numerous BP genes associated with both carbohydrate and sterol metabolic processes (Fig. 4G). With regard to sterol metabolism, 8 tomato orthologs from A. thaliana exhibited higher expression in the leaf of ACCD rootstocks under salt treatment (Figs. 4J, S1F). The ACCD vs WT comparison (C4) highlighted four tomato phosphatases orthologs of Arabidopsis genes ( SPX3, PAP17 and PEPC1 / PS2 , respectively) associated with phosphate starvation (Fig. 4I). Additionally, ACCD grafts under salinity (C9), exhibited upregulation of three cytochrome P450 genes (Solyc06g074420, Solyc01g109150, Solyc05g021390) (Tables S4, S10, S11), along with hormone-related genes, including those involved in ethylene signalling ERF (Solyc04g051360) (Tables S4, S5, S11). To validate RNA-seq analysis, the expression of five representative genes ( ACO1, ACO2 , CYP707A1, TAS14, and PIP1.2) was analysed by qRT-PCR. These genes are involved in different biological process, including ethylene biosynthesis ( ACO1 , ACO2 ) , ABA biosynthesis ( CYP707A1 ) , stress responses ( TAS14 ) , and aquaporin function ( PIP1.2 ), which were confirmed through qRT-PCR analysis (del Mar Parra, del Pozo, Luna, Godoy & Pintor-Toro 1996; Reuscher et al. 2013). qRT-PCR validation revealed an expression pattern consistent with those identified through RNA-seq, supporting the reliability of RNA-seq analysis (Fig. S2). Expression of ACC-to-ethylene forming genes in roots and leaves (qRT-PCR) To further support the higher ethylene production in the plants grafted onto epi rootstocks, as suggested by the increased ACC levels (Figs. 1A, 2), the expression of two ACO genes, ACO1 and ACO2 , responsible for converting ACC into ethylene, was measured in both roots and leaves (Fig. 1B). In roots, both genes showed a clear genotype-dependent pattern, which was further modulated by salinity. Specifically, in epi rootstocks, these genes were significantly upregulated under both control and saline conditions, consistent with the increased ACC levels and enhanced ethylene biosynthesis in this genotype. In contrast, ACO1 and ACO2 expression was reduced in ACCD roots under salinity, compared to others, aligning with the lower ACC concentrations and diminished ethylene production in these rootstocks (Fig. 1B). In leaves, ACO1 expression was influenced by salinity, although the effect was not statistically significant , while ACO2 expression was influenced by both, genotype and treatment significantly (Figs. 1A, B, S2; Tables 4, S5). Leaf expression of hormone-related genes (RNA-seq) To place the transcriptomic data in a physiological context, the specific expression of genes related with major hormones ( CK, ABA, JA, SA, GA and ethylene) metabolism and signalling was analysed in the leaves (Jetha, Theißen & Melzer 2014; Larriba et al. 2021) . Over 400 hormone-related genes, we focused on the most representative and highly salt-induced genes in the favourable phenotype produced by ACCD rootstocks (C9 contrast) compared to the other grafts (C7 and C8 contrasts) (Fig. 4A; Tables 4, S5-9). Among sixty-two genes related to ethylene biosynthesis and signalling (Table S5), ACO1 and ACO2 biosynthesis genes (Solyc07g049530, Solyc12g005940) were upregulated by salinity regardless of the graft combination (Fig. 1B, Tables 4, S5), as mentioned above. In contrast, ACS12 and ACS10 genes (Solyc08g079750, Solyc03g007070) were strongly induced by salinity, especially in the leaves of epi grafts (Tables 4, S5). Concerning ABA metabolism , salinity downregulated several NCED isoforms (Solyc09g016720, Solyc07g056570), particularly in ACCD grafts (Tables 4, S6; Fig. 5), while the BG1 gene (Solyc10g079860), responsible for the alternative biosynthesis of ABA from ABA-GE, was down-regulated only in epi rootstocks under saline conditions (Tables 4, S6; Fig. 5). In contrast, two genes associated with ABA catabolism ( CYP707A1/A3 ; Solyc04g078900, Solyc08g005610) were significantly upregulated in response to salt, especially in ACCD rootstocks. For ABA signalling, PYL5 (Solyc09g015380) was salt-induced in ACCD plants, down-regulated in epi , while PP2C (Solyc03g096670), a negative regulator of PYL5, was induced in WT and epi grafts. Additionally, SnRK2 , which is negatively regulated by PP2C , comprises several isoforms, most of which were down-regulated under salt treatment, especially in the epi grafts, except for 2 isoforms (Solyc05g056550 and Solyc02g090390) that were up-regulated in ACCD grafts (Tables 4, S6; Fig. 5). As previously stated, ABA metabolism seems to play a key role in the resilience of plants grafted onto ACCD rootstocks under salt stress. To further explore gene-metabolism relationships within this pathway, we conducted a network analysis integrating expression and metabolite levels from leaves of ACCD grafted plants (Fig. S3), which identified the transcription factors E2L, WRKY40b, ABF1, ABF2 and ABF3 as key regulators of the responses of ACCD grafts to salt stress. For GAs, a significant upregulation was observed in one isoform of GA2ox3 (Solyc07g061730) and two isoforms of GID1 genes (Solyc09g074270, Solyc06g008870), involved in GA catabolism and signal transduction, respectively, across all graft combinations ( Table S7). Furthermore, three isoforms of DXS1 (Solyc11g010850, Solyc01g067890, Solyc08g066950), one isoform of GA20ox2 (Solyc01g093980) and GA3ox2 (Solyc06g066820) displayed a strong upregulation mainly in the ACCD rootstocks. Moreover, more than 15% of CK-related genes exhibited a significant deregulation under salinity ( Tables 4, S8), and their expression varied among rootstock genotypes in response to salinity. LOG3 (Solyc04g081290) and LOG5 (Solyc08g062820), both involved in CK biosynthesis, were upregulated in epi grafts under salinity, whereas IPT5 (Solyc11g066960), another CK biosynthesis gene, together with several glycosyltransferase genes ( ZOGs ) involved in CK homeostasis, were more highly expressed in ACCD grafts under salinity ( Tables 4, S8). Likewise, several CK-signalling genes, including AHPT1L and type-B response regulators ( ARR-B ), were induced in plants grafted onto ACCD rootstocks, whereas several type-A response regulators ( ARR-A) were down-regulated ( Tables 4, S8). Finally, in ACCD grafts exposed to salinity, leaf expression of JA biosynthetic genes, including LOX6, LOX2, and AOC1/2/3/4 ( Solyc08g014000, Solyc03g122340, Solyc02g085730 ), as well as the signalling regulator JAZ1 ( Solyc07g042170 ), was markedly enhanced (Tables 4, S9). By contrast, the JA biosynthetic gene JAR1 ( Solyc07g054580 ) and the JA receptor JAZ2 ( Solyc12g009220 ) had elevated expression in WT and ACCD , but not in epi grafts (Tables 4, S9). Discussion (1845 words) ACCD overexpression in the rootstock-reduces ACC evolution and alleviates salt stress in the scion Rootstock specific production of the ethylene-precursor ACC significantly affected its concentration in the scion (Fig. 1A), particularly under salinity, when root-derived ACC is transported to the shoot (Albacete et al. 2008), influencing both physiological and agronomic responses to stress (Table 1). Consistent with ACC levels, expression of ethylene biosynthetic genes ACO1 and ACO2 was constitutively higher and stress-inducible in epi roots, but lowest in ACCD rootstocks, while leaf expression remained similar among grafts (Fig. 1B). Since ACC synthesis is the rate-limiting step in ethylene biosynthesis (Park et al. 2018), these results support a role for the rootstock-to-scion ACC transport in the ethylene-mediated responses to stress (Dodd, 2005; Albacete et al. 2008; Pérez-Alfocea et al. 2010). However, a direct role of ACC as stress sensor and root-to-shoot signal cannot be excluded (Ghanem et al. 2008; Martínez-Andújar et al. 2016, 2017; Vanderstraeten, Depaepe, Bertrand & Van Der Straeten 2019; Mou et al. 2020). Reduced ACC production in roots correlated with improved plant vigour and yield under salinity, highlighting its negative role in stress responses (Albacete et al. 2008, 2014a b; Bhise & Dandge 2019). The rootstock-sourced ACC/ethylene altered local (RSA) and systemic (through root-to-shoot communication) developmental, nutritional and hormonal responses (Dodd 2005; Pérez-Alfocea et al. 2010; Albacete et al. 2014b; Martínez‐Andújar et al. 2021). Salinity increased the number, length and thickness of LR in WT and ACCD plants, but no in epi grafts (Fig. 1C). These RSA patterns were consistent with ACC levels in the roots and the leaf and flower ionomes, as the ACCD and epi grafted plants registered the highest and lowest nutrient concentrations under salinity, respectively (Table 2). Systemic responses to salinity also depended on the rootstock genotype. ACCD grafts exhibited stronger vegetative growth and higher and stable yield under salt stress, whereas epi rootstocks were the most affected by the stress (Table 1). Enhanced performance of ACCD grafts under salinity was associated with improved photosynthetic parameters, higher fruit number and weight, and increased truss branching (Table 1), linked to SEP4 gene up-regulation (Fig. 4I), involved in inflorescence meristem identity (Jetha et al. 2014; Burgess 2017). Under salinity, ACCD grafted plants also developed thicker leaves, epidermis, and PP (Figs. 3B-H), supporting photosynthetic efficiency, stable stomatal conductance (Table 1), and improved leaf water status (Geissler, Hussin & Koyro 2009; Acosta-Motos et al. 2017; Liu, Wang & Chang 2022). Low root-sourced ACC benefits leaf nutritional status and decreases Na under salinity ACCD grafts enhanced nutrient uptake and accumulation in the scion, particularly P, Ca and S (Table 2). Under salinity, leaf P decreased by 20% in epi and 50% in WT grafts but increased by 20% in ACCD grafts, consistent with induced cellular responses to P starvation (Figs. 4H, I). Despite similar RSA between ACCD and WT plants, the higher shoot P concentrations in ACCD likely reflect enhanced P transport or metabolism rather than RSA traits. Three tomato phosphatases ( SPX3 , PAP17 and PEPC1 ) were significantly upregulated in salinized ACCD leaves (Fig. 4I), supporting adaptative regulation of P uptake, transport, storage, and homeostasis, driving essential adaptations to phosphorus availability (Hanchi et al. 2018; Bhadouria & Giri 2022; Wang et al. 2024). This agrees with earlier findings where low-ACC rootstocks promoted shoot and root growth, and nutrients and bioactive cytokinin ( t -Z) transport to the scion under low K (Martínez-Andújar et al. 2016) and P (Martínez-Andújar et al. 2017) nutrition, compared to high ACC-sourced rootstocks. Moreover, ACCD rootstocks decreased Na + transport to the scion (Table 2), mitigating salt toxicity in the leaves (Pérez-Alfocea et al., 2010; Martínez-Andújar et al., 2016, 2021). Overall, reduced rootstock-sourced ACC under salinity simultaneously improved P, Ca, S, Na + homeostasis in leaves and flowers, contributing to yield stability under salinity through both local (RSA) and systemic (responses to P starvation) mechanisms. Rootstock ACCD overexpression enhances photosynthesis under salinity Upregulation of several steroid biosynthesis-related genes in ACCD grafts under salinity (Figs. S1E, F) likely improved chlorophyll status (SPAD and Fv/Fm) and photosynthesis (A N , g S , E) by reinforcing membranes (Kumar, Ali, Dahuja & Tyagi 2015), preventing water loss (Rogowska & Szakiel 2020), and protecting the photosynthetic apparatus (Guo, Liu & Barkla 2019). Reduced leaf ABA levels in salinized ACCD grafts (Fig. 2; Table 3) also matched higher g S and A N , resulting from decreased biosynthesis and enhanced catabolism (see below). Leaf carbon metabolism was also altered in both epi and ACCD grafts (Figs. 4F, G; S1D, E). ACCD -grafted plants maintained active source-sink relationships under salinity, with stable sucrose and hexoses in leaves and flowers, while these sugars decreased in WT and epi grafts (Fig. 3A). Leaf starch decreased in WT and ACCD , while increased in epi grafts (Fig. 3A), consistent with starch synthase gene induction in epi leaves (Table S4). Starch remobilization supplies carbon and energy when photosynthesis is limited, to sustain growth and protect against stresses (Thalmann & Santelia 2017). This response was operative in WT, impaired in epi, and unnecessary in ACCD grafts, aligning with their distinct leaf anatomy (Figs. 3B-I). Besides carbohydrates, ACCD grafts also showed higher (Tyr, Ile, Pro, Hyp, Lys, Trp) or more stable (Asn, Gln, Tyr, Hyp, Phe, Lys) levels of amino acid concentrations in flowers under salinity, compared to control conditions and other grafts (Table S3). Amino acids support flower development (e.g. Trp and Tyr) favouring yield (Borghi & Fernie 2017), maintaining cell turgor (Pro and Hyp) and withstanding stressful conditions (Kahlaoui, Hachicha, Misle, Fidalgo & Teixeira 2018), and attracting pollinators (Murray et al. 2025), favouring flower pollination and fruit set, as observed in ACCD grafts (Table 1). Rootstock-sourced ACC alters hormone metabolism and signalling in the scion The drop in leaf ABA levels in salinized ACCD grafts is supported by reduced ABA precursor (ABA-aldehyde) and increased ABA catabolites and conjugated forms (DPA, DPAG, ABA-GE), although increases were likewise observed in the other rootstock genotypes (Table 3). These results agree with the strong downregulation of ABA biosynthesis NCED3 gene and the induction of catabolism-related CYP707A1/3 (ABA 8′-hydroxylase) genes (Kushiro et al. 2004), in salt-treated ACCD grafts (Fig. 5; Table S6). In tomato, low ACC and high ABA/ACC ratio in the leaves have been associated with plant growth and fruit yield under salinity (Albacete et al. , 2008, 2009; Ghanem et al. , 2008; Cohen et al. , 2010; this study). Besides, several ABA signalling (e.g., WRKY40 , PYL5) and ABC transporters, were up-regulated in salt-treated ACCD leaves, whereas the negative regulator PP2C (Li et al. 2020a; Bai, Huang & Shen 2021) was up-regulated in the leaves of WT and epi grafts (Tables 4, S6). Up-regulation of WRKY40, linked to salt tolerance in Arabidopsis (Li et al. 2020a), likely contributes to stress adaptation in ACCD grafts. The reduced root-to-shoot ABA transport could also account for the lower leaf ABA in ACCD grafts under salinity, supported by higher DPAG and the lack of ABA accumulation in the roots (Table 3). Overall, while leaf ABA metabolism is altered in ACCD grafts to reduce the level of the hormone, ABA signalling is induced, contributing to stress adaptation (Martínez-Andújar et al. 2020; Rehman et al. 2021), while preserving stomatal conductance and photosynthesis. The increased GA3 concentration in leaves and flowers of salinized ACCD grafts (Fig. 2) matched the upregulation of GA biosynthesis genes ( GA20ox and GA3ox) (Table S7). GA3 promotes growth under salinity by inducing pigment and limiting Na + concentrations (Achard et al. 2006; Shahzad et al. 2021), as observed in ACCD grafts. CKs also mediate stress responses to stress through interaction with ethylene (Liu, Zhang, Meng, Wang & Chen 2020; Nguyen, Nguyen, Kisiala & Emery 2021). Leaf CK levels (Fig. 2) and CK-related gene expression (e.g., LOG5 and IPT5 ) (Tables 4, S8) varied with the rootstock genotype under salinity. In epi grafts, t Z, ZR and iP bioactive CKs shifted in response to salinity, correlating with LOG3 induction (Tables 4, S8), whereas CKs remained stable in ACCD grafts, associated with upregulation of ZOG and AHPT1L , involved in CK homeostasis and signalling. These genes regulate CK through glycosylation into active CK-glucosides (Šmehilová, Dobrůšková, Novák, Takáč & Galuszka 2016; Hallmark, Černý, Brzobohatý & Rashotte 2020) and phosphorylation of ARR-B response regulators, respectively (Argyros et al. , 2008; Ishida et al. , 2008) and have been linked to drought and salt tolerance in rice (Sun et al. 2014; Li et al. 2020b). ARR-B5 and ARR-B12 genes were upregulated in ACCD leaves, while ARR-B11 was induced in epi , suggesting ACC/ethylene modulation of CK signalling, as reported in under cold stress (Jeon et al. 2010; Shi et al. 2012), likely enhancing salinity tolerance. Moreover, Cytokinin Response Factors (CRF2, CRF4) were induced in ACCD grafts under salinity, while CRF4 was downregulated and CRF6 upregulated in epi leaves (Tables 4, S8) . CRFs are involved in osmotic regulation, antioxidant and PSII efficiency under salinity (Qin et al. 2017; Keshishian 2018). Additionally, ARR1-12 regulate AtHKT1;1 and Na accumulation in Arabidopsis shoots (Mason et al. 2010). Indeed, the K transporter SlHAK7 was specifically induced in ACCD leaves under salinity (Table S4), which contributes to K/Na homeostasis and salt tolerance (Wang et al. 2020). Overall, reduced ACC production appears to interact with CK pathway, favouring CK synthesis ( ZOGs ) and signalling ( AHPT1L, ARR-B5,12, CFR2,4 ) under salinity, thereby promoting growth and adaptation to salinity. Salt-induced JA accumulation occurred only in roots and leaves of WT and epi grafts (Fig. 2), while biosynthetic ( LOX6, LOX2 , AOC1/2/3/4 ) and signalling ( JAZ1 ) genes were strongly induced in leaves of ACCD grafts (Tables 4, S9). Conversely, the receptor JAZ2 and the biosynthetic JAR1, essential for JA signalling and stress protection (Staswick & Tiryaki 2004), were unaltered or suppressed in epi leaves, respectively, while both were induced in the other grafts. Interestingly, the lack of JA response to salinity in ACCD roots and leaves agrees with the stability in leaf and flower Ca concentrations, compared to WT and epi grafts (Fig. 2; Table 2). Ca translocation to sink organs is typically restricted (de Bang, Husted, Laursen, Persson & Schjoerring 2021), but stress-induced Ca waves from roots or leaves toward flowers can act as systemic signals under heat or osmotic stresses, mediated by JA and ethylene (Grenzi et al. 2023; Bisht et al. 2025). Therefore, Ca translocation from leaves to flowers may signal stress sensitivity, influenced by the rootstock genotype and its ACC/ethylene production capacity. Overall, rootstock-mediated ethylene production influences JA responses, which are related to systemic (Ca signalling) responses to salinity. In conclusion, reduced ACC production in ACCD rootstocks improved plant vigour and yield under salinity in tomato, highlighting the negative role of ethylene and the importance of root-to-shoot ACC transport in the response to this stress (Albacete et al. 2008, 2014a b; Bhise & Dandge 2019). ACCD rootstocks influenced both local and systemic mechanisms, resulting in a larger and more branched root system, diminished Na + toxicity in shoots, and improved leaf P, Ca and S nutrition, photosynthesis and source-sink relations, which was related to reduced leaf ABA and increased activity of growth-promoting and anti-senescing hormones CKs and GAs. The promotion of resilient physiology and stable yields in a tomato variety grown under salinity through grafting onto low-ethylene producing rootstocks is of particular relevance for rootstock breeding. Acknowledgments This work was funded by the Spanish Ministry of Science, Innovation and Universities through the State Research Agency (AEI, MICIU/AEI/10.13039/501100011033), and by the European Union, through FEDER, UE for project PID2021-128979OB-I00 (SALPTOM), and NextGenerationEU/PRTR for project CNS2023-144700 (ABADAPT). The authors are grateful to the Fundación Séneca, the Science and Technology Agency of the Region of Murcia (FS/10.13039/100007801), for supporting this research through projects 22613/PI/24 (DORATOM) and 22540/PDC/24, and for funding JAMR through project (Saavedra Fajardo program – 2111/SF/19) and JJGF through a predoctoral fellowship (21796/FPI/22). The authors also acknowledge funding from the European Union through Horizon Europe – EIC PathFinder research and innovation programme under grant agreement no. 101098680 (project DARkWIN). The authors thank Nieves Fernández García for her valuable assistance in the preparation of samples for microscopy analysis. Author contribution statement Francisco Pérez-Alfocea : Conceived and designed the research. José A. Martín-Rodríguez and Cristina Martínez-Andújar : Performed all stress experiments. Eduardo Larriba and Jesús Guillamón : Conducted bioinformatic analyses of transcriptomic data. Jose Manuel Pérez-Pérez: Executed the qPCR validation. Enrique Olmos and José A. Martín-Rodíguez : Performed the microscopy analyses. Lázaro E. Pereira Peres: developed the mutant and transgenic lines and provided the seeds. José A. Martín-Rodríguez, Purificación A. Martínez-Melgarejo, Ángela S. Prudencio, Juan José Guerrero and Cristina Martínez-Andújar : Performed the physiological analysis. José A. 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(C) Photographs showing the roots of UD/WT, UD/ epi and UD/ ACCD rootstocks, after 100 days growing under control and salt (75 mM NaCl) treatment. * indicate significant differences between control and salt treatment within each genotype according to the Mann-Whitney U test (P < 0.05). Results of two-way ANOVA (p values reported) for genotype (G), treatment (T) and their interaction (G x T) are indicated in the top right of the panel. *, ** and *** indicate statistically significant difference at p<0.05, p<0.01 and p<0.001, respectively. Figure 2. Heatmap of plant hormone concentrations: 1-aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA) and ABA/ACC ratio, gibberellins GA1, GA3 and GA4, trans-zeatin (tZ), zeatin riboside (ZR), isopentenyl adenine (iP), total cytokinins (CKs) (tZ+ZR+iP), jasmonic acid (JA) and salycilic acid (SA) concentrations in roots, leaves, and flowers of the tomato cv UniDarkwin (UD) grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks after 100 days growing under control and salt (75 mM NaCl) treatment. Hormone concentrations are presented as ng/g of fresh tissue, with colour gradients ranging from green (lowest concentrations) to red (highest concentrations). Different letters indicate significant differences among graft combinations (n = 6, P < 0.05) within and between treatments. Figure 3. (A) Heatmap of hexoses (fructose + glucose) and sucrose concentrations in leaves and flowers (µg·FW -1 ), as well as starch levels in leaves of the tomato cv UniDarkwin grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks after 100 days growing under control and salt (75 mM NaCl) treatment. Sugar concentrations are presented as ng/g of fresh tissue, with colour gradients ranging from green (lowest concentrations) to red (highest concentrations). (B) Optical microscopy images showing longitudinal sections of leaf anatomy of the commercial tomato cv UniDarkwin plants grafted onto WT, epi and ACCD rootstocks after 90 days of salt treatment (75 mM NaCl), showing the different leaf structures, including the epidermis (E), palisade parenchyma (PP), spongy parenchyma (SP), intercellular space (IS), as well as chlorophyll (ch) and starch granules (sg). (C-I) Leaf mophometric traits of the same leaves, including leaf (C) and epidermis (D) thickness, areas of PP (E) and SP (F), and percentages of epidermis (G) PP (H) and SP (I) relative to the total area, icluding the IS. Bars indicate mean values ± SE. Different letters indicate significant differences among graft combinations ( n = 6, P < 0.05). Figure 4. (A) Diagram illustrating the different contrasts used for differential expression analysis. (B) Number of differentially expressed genes (DEG) identified in each contrast. Venn diagram showing the intersection of upregulated (C) and downregulated (D) DEGs identified in the contrasts C7, C8 and C9. GO biological process enrichment analysis of clusters in contrasts C7 (E), C8 (F), C9 (G) and C4 (H). Expression profiles of tomato orthologes of A. thaliana found in C4 contrast (I, J). Expression values are represented as the average of counts per million (CPMs). Table 1. Biomass parameters (Shoot fresh weight, SFW; root fresh weight, RFW; total biomass, TB; stem diameter, SD; leaf fresh weight, LFW; leaf area, LA), yield-related parameters (Total yield, Yield; fruit number, FN; fruit weight, FW; truss length, TL; % multiple trusses; % flowers visited by bumblebees, %FVB), photosynthesis-related parameters (Photosynthesis rate, A N ; stomatal conductance, g S ; evapotranspiration, E), and chlorophyll-related parameters (maximum quantum yield of open photosystem II, Fv/Fm; cholorophyll diynamics with soil plant analytical division, SPAD)) of the tomato cv UniDarkwin grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks, after 100 days growing under control and salt (75 mM NaCl) treatment. Values indicate mean ± SE. Different letters indicate significant differences among graft combinations ( n = 6, P < 0.05) within and between treatments. Results of two-way ANOVA (p values reported) for genotype (G), treatment (T) and their interaction (G x T) are indicated on the right side of the panel. *, ** and *** indicate statistically significant difference at p<0.05, p<0.01 and p<0.001, respectively). Table 2. Total carbon (C), total nitrogen (N), phosphorus (P), potassium (K), sodium (Na), K/Na ratio, sulphur (S), calcium (Ca), magnesium (Mg), boron (B), iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) concentrations in leaf and flowers of the tomato cv UniDarkwin (UD) grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks after 100 days growing under control and salt (75 mM NaCl) treatment. Values indicate mean ± SE. Different letters indicate significant differences among graft combinations (n = 6, P < 0.05) within and between treatments. Results of two-way ANOVA (p values reported) for genotype (G), treatment (T) and their interaction (G x T) are indicated on the right side of the panel. *, ** and *** indicate statistically significant difference at p<0.05, p<0.01 and p<0.001, respectively). Table 3. Concentrations of metabolites related to ABA biosynthesis (ABA aldehyde), ABA catabolism (phaseic acid, PA; dihydrophaseic acid, DPA; dihydrophaseic acid glucosyl, DPAG; neophaseic acid, neoPA), and ABA conjugates (ABA glucosyl ester, ABA-GE) in flowers, leaves and roots of the tomato cv UniDarkwin (UD) grafted onto MT MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks after 100 days growing under control and salt (75 mM NaCl) treatment. Values indicate mean ± SE. Different letters indicate significant differences among graft combinations ( n = 6, P < 0.05) within and between treatments. Results of two-way ANOVA (p values reported) for genotype (G), treatment (T) and their interaction (G x T) are indicated in the right of the panel. *, ** and *** indicate statistically significant difference at p<0.05, p<0.01 and p<0.001, respectively). Table 4. Hormone-related genes (ethylene, ABA, GA, CK and JA) differentially expressed in leaves of the tomato cv UniDarkwin (UD) grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks after 100 days growing under control and salt (75 mM NaCl) treatment. Expression values are expressed as average counts per million (CPMs). A colour gradient represents the expression level, with deep red indicating the highest values and deep blue indicating the lowest values. Supplemental figure S1. (A) Principal component analysis (PCA) of normalized RNA-Seq expression data. (B) Hierarchical clustering of genes expressed in datasets. Specific expression clusters are highlighted in blue, violet and green for UD/WT, UD/ epi and UD/ ACCD plants under salinity conditions, respectively. GO biological process enrichment analysis of cluster blue (C), violet (D) and green (E). (F) Network analysis of genes involved in sterol and steroid process (in cluster green). Supplemental figure S2. Validation of gene expression by real time PCR quantification (qPCR) for ACO1 (Solyc07g049530), ACO2 (Solyc12g005940), TAS14 (Solyc02g084850) , PIP1.2 (Solyc01g094690) and CYP707A1 (Solyc08g005610). qPCR data are normalized to housekeeping genes. Both qPCR and RNAseq values represent the relative expression of each gene compared to WT/UD under control conditions. Different letters indicate significant differences among graft combinations (n = 6, P < 0.05) assessing within and between treatments. Statistical comparisons were performed separately for qPCR and RNAseq. Supplemental figure S3. Network analysis of genes and metabolites involved in ABA metabolism and signalling in the C9 contrast (UD/ ACCD control vs salinity) in leaves, illustrating significant inferred interactions. The weight of the interactions between the different nodes from the network was calculated using the GENIE3 package in R. Nodes from the same network subset shared a consistent colour (ABA in yellow, ABA biosynthesis precursors in light blue, ABA catabolites in light green, and transcription factor, biosynthesis, catabolism, signalling, and transport genes in red, dark blue, dark green, orange, and purple, respectively). Significant interactions were defined as those whose weights ranked in the top 5% of the overall weight distribution. The thickness of the edges in the chart is based on the weight of the calculated edges. Supplemental Table S1. List of primers used for qRT-PCR analysis. Supplemental Table S2. Two-way-ANOVA results (p value reported) for hormones and sugar levels, as shown in Figures 2 and 3A. *, ** and *** indicate statistically significant difference at p< 0.05, p< 0.01 and p< 0.001, respectively. G=Genotype; T=Treatment and GxT=Genotype x Treatment. Supplemental Table S3. Amino acid concentration (polar neutrals (serine (Ser), asparagine (Asn), glutamine (Gln), tyrosine (Tyr)), nonpolar neutrals (valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), hydroxyproline (Hyp), phenylalanine (Phe), tryptophan (Trp)), acids (aspartic acid (Asp), glutamic acid (Glu)), bases (lysine (Lys), arginine (Arg), histidine (His))) concentrations in flower of the tomato cv UniDarkwin grafted onto MT (UD/WT), epi (high ethylene production, UD/ epi ) and ACCD (low ethylene production, UD/ ACCD ) rootstocks, in peak area, after 100 days growing under control and salt (75 mM NaCl) treatment. Values indicate concentration mean ± SE. Different letters indicate significant differences among graft combinations ( n = 6, P < 0.05) assessing within and between treatments. Results of two-way ANOVA (p values reported) for genotype (G), treatment (T) and their interaction (G x T) are indicated on the right side of the panel. *, ** and *** indicate statistically significant difference at p<0.05, p<0.01 and p<0.001, respectively. Supplemental Table S4. Library of expressed genes after filtering and normalization. Expression levels are reported as counts per million (CPM). Supplemental Table S5. Ethylene related-genes. Values represent mean relative expression (AveExpr) and adjusted p-value (Adj.P.val). Colours indicate a gradient scale, with deep red denoting the highest expression values and deep blue the lowest expression values for each gene. Supplemental Table S6. Abscisic acid related-genes. Values represent mean relative expression (AveExpr) and adjusted p-value (Adj.P.val). Colours indicate a gradient scale, with deep red denoting the highest expression values and deep blue the lowest expression values for each gene. Supplemental Table S7. Giberellins related-genes. Values represent mean relative expression (AveExpr) and adjusted p-value (Adj.P.val). Colours indicate a gradient scale, with deep red denoting the highest expression values and deep blue the lowest expression values for each gene. Supplemental Table S8. Cytokinins related-genes. Values represent mean relative expression (AveExpr) and adjusted p-value (Adj.P.val). Colours indicate a gradient scale, with deep red denoting the highest expression values and deep blue the lowest expression values for each gene. Supplemental Table S9. Jasmonic acid related-genes. Values represent mean relative expression (AveExpr) and adjusted p-value (Adj.P.val). Colours indicate a gradient scale, with deep red denoting the highest expression values and deep blue the lowest expression values for each gene. Supplemental Table S10. Network analysis of the top 20 upregulated and top 20 downregulated genes in the C4 Comparison (UD/ ACCD salinity vs UD/WT salinity), depicting significant inferred interactions. The network was constructed using the GENIE3 package in R. The threshold for establishing edges between nodes was set to 0.1. Supplemental Table S11. Network analysis of the top 20 upregulated and top 20 downregulated genes in the C9 Comparison (UD/ACCD control vs salinity), depicting significant inferred interactions. The network was constructed using the GENIE3 package in R. The threshold for establishing edges between nodes was set to 0.1. Supplementary Material File (table 1.xlsx) Download 12.74 KB File (table 2.xlsx) Download 14.04 KB File (table 3.xlsx) Download 12.92 KB File (table 4.xlsx) Download 15.84 KB Information & Authors Information Version history V1 Version 1 27 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords 1-aminocyclopropane-1-carboxylic acid solanum lycopersicum hormones plant hormones root-shoot communication Authors Affiliations Jose A. Martín-Rodríguez 0000-0002-5768-3864 [email protected] Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Purificación A Martínez-Melgarejo Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Jesús Guillamón Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Ángela S. Prudencio Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Juan J. Guerrero Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Eduardo Larriba Universidad Miguel Hernandez de Elche Instituto de Bioingenieria View all articles by this author Jose Manuel Perez-Perez 0000-0003-2848-4919 Universidad Miguel Hernandez de Elche Instituto de Bioingenieria View all articles by this author Enrique Olmos Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Lazaro Peres Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz View all articles by this author Cristina Martínez Andújar 0000-0002-3684-9765 Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author FRANCISCO PEREZ ALFOCEA 0000-0003-1057-4924 Centro de Edafologia y Biologia Aplicada del Segura View all articles by this author Metrics & Citations Metrics Article Usage 252 views 153 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jose A. Martín-Rodríguez, Purificación A Martínez-Melgarejo, Jesús Guillamón, et al. Low ethylene production in the root(stock) alleviates salt stress in tomato. Authorea . 27 October 2025. DOI: https://doi.org/10.22541/au.176155600.06834638/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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