Pollinator-assisted plant phenotyping, selection, and breeding for crop resilience to abiotic stresses.

Food security is threatened by climate change, with heat and drought being the main stresses affecting crop physiology and ecosystem services, such as plant-pollinator interactions. We hypothesize that tracking and ranking pollinators' preferences for flowers under environmental pressure could be used as a marker of plant quality for agricultural breeding to increase crop stress tolerance. Despite increasing relevance of flowers as the most stress sensitive organs, phenotyping platforms aim at identifying traits of resilience by assessing the plant physiological status through remote sensing-assisted vegetative indexes, but find strong bottlenecks in quantifying flower traits and in accurate genotype-to-phenotype prediction. However, as the transport of photoassimilates from leaves (sources) to flowers (sinks) is reduced in low-resilient plants, flowers are better indicators than leaves of plant well-being. Indeed, the chemical composition and amount of pollen and nectar that flowers produce, which ultimately serve as food resources for pollinators, change in response to environmental cues. Therefore, pollinators' preferences could be used as a measure of functional source-to-sink relationships for breeding decisions. To achieve this challenging goal, we propose to develop a pollinator-assisted phenotyping and selection platform for automated quantification of Genotype × Environment × Pollinator interactions through an insect geo-positioning system. Pollinator-assisted selection can be validated by metabolic, transcriptomic, and ionomic traits, and mapping of candidate genes, linking floral and leaf traits, pollinator preferences, plant resilience, and crop productivity. This radical new approach can change the current paradigm of plant phenotyping and find new paths for crop redomestication and breeding assisted by ecological decisions.

Plant salinity stress, sensing, and its mitigation through WRKY.

Salinity or salt stress has deleterious effects on plant growth and development. It imposes osmotic, ionic, and secondary stresses, including oxidative stress on the plants and is responsible for the reduction of overall crop productivity and therefore challenges global food security. Plants respond to salinity, by triggering homoeostatic mechanisms that counter salt-triggered disturbances in the physiology and biochemistry of plants. This involves the activation of many signaling components such as SOS pathway, ABA pathway, and ROS and osmotic stress signaling. These biochemical responses are accompanied by transcriptional modulation of stress-responsive genes, which is mostly mediated by salt-induced transcription factor (TF) activity. Among the TFs, the multifaceted significance of WRKY proteins has been realized in many diverse avenues of plants' life including regulation of plant stress response. Therefore, in this review, we aimed to highlight the significance of salinity in a global perspective, the mechanism of salt sensing in plants, and the contribution of WRKYs in the modulation of plants' response to salinity stress. This review will be a substantial tool to investigate this problem in different perspectives, targeting WRKY and offering directions to better manage salinity stress in the field to ensure food security.

Overexpression of TgERF1, a Transcription Factor from Tectona grandis, Increases Tolerance to Drought and Salt Stress in Tobacco.

Teak (Tectona grandis) is one of the most important wood sources, and it is cultivated in tropical regions with a significant market around the world. Abiotic stresses are an increasingly common and worrying environmental phenomenon because it causes production losses in both agriculture and forestry. Plants adapt to these stress conditions by activation or repression of specific genes, and they synthesize numerous stress proteins to maintain their cellular function. For example, APETALA2/ethylene response factor (AP2/ERF) was found to be involved in stress signal transduction. A search in the teak transcriptome database identified an AP2/ERF gene named TgERF1 with a key AP2/ERF domain. We then verified that the TgERF1 expression is rapidly induced by Polyethylene Glycol (PEG), NaCl, and exogenous phytohormone treatments, suggesting a potential role in drought and salt stress tolerance in teak. The full-length coding sequence of TgERF1 gene was isolated from teak young stems, characterized, cloned, and constitutively overexpressed in tobacco plants. In transgenic tobacco plants, the overexpressed TgERF1 protein was localized exclusively in the cell nucleus, as expected for a transcription factor. Furthermore, functional characterization of TgERF1 provided evidence that TgERF1 is a promising candidate gene to be used as selective marker on plant breeding intending to improve plant stress tolerance.

Increased branching independent of strigolactone in cytokinin oxidase 2-overexpressing tomato is mediated by reduced auxin transport.

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.

Overproduction of ABA in rootstocks alleviates salinity stress in tomato shoots.

To determine whether root-supplied ABA alleviates saline stress, tomato (Solanum lycopersicum L. cv. Sugar Drop) was grafted onto two independent lines (NCED OE) overexpressing the SlNCED1 gene (9-cis-epoxycarotenoid dioxygenase) and wild type rootstocks. After 200 days of saline irrigation (EC = 3.5 dS m-1 ), plants with NCED OE rootstocks had 30% higher fruit yield, but decreased root biomass and lateral root development. Although NCED OE rootstocks upregulated ABA-signalling (AREB, ATHB12), ethylene-related (ACCs, ERFs), aquaporin (PIPs) and stress-related (TAS14, KIN, LEA) genes, downregulation of PYL ABA receptors and signalling components (WRKYs), ethylene synthesis (ACOs) and auxin-responsive factors occurred. Elevated SlNCED1 expression enhanced ABA levels in reproductive tissue while ABA catabolites accumulated in leaf and xylem sap suggesting homeostatic mechanisms. NCED OE also reduced xylem cytokinin transport to the shoot and stimulated foliar 2-isopentenyl adenine (iP) accumulation and phloem transport. Moreover, increased xylem GA3 levels in growing fruit trusses were associated with enhanced reproductive growth. Improved photosynthesis without changes in stomatal conductance was consistent with reduced stress sensitivity and hormone-mediated alteration of leaf growth and mesophyll structure. Combined with increases in leaf nutrients and flavonoids, systemic changes in hormone balance could explain enhanced vigour, reproductive growth and yield under saline stress.

Multi-omics approach reveals the contribution of KLU to leaf longevity and drought tolerance.

KLU, encoded by a cytochrome P450 CYP78A family gene, generates an important-albeit unknown-mobile signal that is distinct from the classical phytohormones. Multiple lines of evidence suggest that KLU/KLU-dependent signaling functions in several vital developmental programs, including leaf initiation, leaf/floral organ growth, and megasporocyte cell fate. However, the interactions between KLU/KLU-dependent signaling and the other classical phytohormones, as well as how KLU influences plant physiological responses, remain poorly understood. Here, we applied in-depth, multi-omics analysis to monitor transcriptome and metabolome dynamics in klu-mutant and KLU-overexpressing Arabidopsis plants. By integrating transcriptome sequencing data and primary metabolite profiling alongside phytohormone measurements, our results showed that cytokinin signaling, with its well-established function in delaying leaf senescence, was activated in KLU-overexpressing plants. Consistently, KLU-overexpressing plants exhibited significantly delayed leaf senescence and increased leaf longevity, whereas the klu-mutant plants showed early leaf senescence. In addition, proline biosynthesis and catabolism were enhanced following KLU overexpression owing to increased expression of genes associated with proline metabolism. Furthermore, KLU-overexpressing plants showed enhanced drought-stress tolerance and reduced water loss. Collectively, our work illustrates a role for KLU in positively regulating leaf longevity and drought tolerance by synergistically activating cytokinin signaling and promoting proline metabolism. These data promote KLU as a potential ideal genetic target to improve plant fitness.

Genetic Analysis of Root-to-Shoot Signaling and Rootstock-Mediated Tolerance to Water Deficit in Tomato.

Developing drought-tolerant crops is an important strategy to mitigate climate change impacts. Modulating root system function provides opportunities to improve crop yield under biotic and abiotic stresses. With this aim, a commercial hybrid tomato variety was grafted on a genotyped population of 123 recombinant inbred lines (RILs) derived from Solanumpimpinellifolium, and compared with self- and non-grafted controls, under contrasting watering treatments (100% vs. 70% of crop evapotranspiration). Drought tolerance was genetically analyzed for vegetative and flowering traits, and root xylem sap phytohormone and nutrient composition. Under water deficit, around 25% of RILs conferred larger total shoot dry weight than controls. Reproductive and vegetative traits under water deficit were highly and positively correlated to the shoot water content. This association was genetically supported by linkage of quantitative trait loci (QTL) controlling these traits within four genomic regions. From a total of 83 significant QTLs, most were irrigation-regime specific. The gene contents of 8 out of 12 genomic regions containing 46 QTLs were found significantly enriched at certain GO terms and some candidate genes from diverse gene families were identified. Thus, grafting commercial varieties onto selected rootstocks derived from S.pimpinellifolium provides a viable strategy to enhance drought tolerance in tomato.

Impact of overexpression of 9-cis-epoxycarotenoid dioxygenase on growth and gene expression under salinity stress.

To better understand abscisic acid (ABA)'s role in the salinity response of tomato (Solanum lycopersicum L.), two independent transgenic lines, sp5 and sp12, constitutively overexpressing the LeNCED1 gene (encoding 9-cis-epoxycarotenoid dioxygenase, a key enzyme in ABA biosynthesis) and the wild type (WT) cv. Ailsa Craig, were cultivated hydroponically with or without the addition of 100 mM NaCl. Independent of salinity, LeNCED1 overexpression (OE) increased ABA concentration in leaves and xylem sap, and salinity interacted with the LeNCED1 transgene to enhance ABA accumulation in xylem sap and roots. Under control conditions, LeNCED1 OE limited root and shoot biomass accumulation, which was correlated with decreased leaf gas exchange. In salinized plants, LeNCED1 OE reduced the percentage loss in shoot and root biomass accumulation, leading to a greater total root length than WT. Root qPCR analysis of the sp12 line under control conditions revealed upregulated genes related to ABA, jasmonic acid and ethylene synthesis and signalling, gibberellin and auxin homeostasis and osmoregulation processes. Under salinity, LeNCED1 OE prevented the induction of genes involved in ABA metabolism and GA and auxin deactivation that occurred in WT, but the induction of ABA signalling and stress-adaptive genes was maintained. Thus, complex changes in phytohormone and stress-related gene expression are associated with constitutive upregulation of a single ABA biosynthesis gene, alleviating salinity-dependent growth limitation.

The influence of grafting on crops' photosynthetic performance.

In a near scenario of climate change where stress-derived limitations on crops' yield by affecting plant gas-exchange are expected, grafting may become a cheap and easy technique to improve crops photosynthetic performance and water-use efficiency. Inconsistent data of the effect of rootstocks over gas-exchange can be found in literature, being necessary an integrative analysis of the effect of grafting over photosynthetic parameters. With this aim, we present a compilation of the effect of graft on the net CO2 assimilation rate (AN) and other photosynthetic parameters across different species with agronomic interest. No differences were observed in any photosynthetic parameter between non-grafted and self-grafted plants under non-stress conditions. However, differences were found depending on the used rootstock, particularly for the intrinsic water-use efficiency (WUEi). We observed that variations in AN induced by rootstocks were related to changes in both diffusive and biochemical parameters. Under drought or salt stress, different photosynthetic performances were observed depending on the rootstock, although the high variability among studies promted to remarkable results. Overall, we observed that grafting can be a useful technique to improve plant photosynthetic performance, and therefore, crop yield and WUE, and that the rootstock selection for a target environment is determinant for the variations in photosynthesis.

The growth impairment of salinized fenugreek (Trigonella foenum-graecum L.) plants is associated to changes in the hormonal balance.

Fenugreek is a legume cultivated for its medicinal value, especially in arid and semi-arid regions, where soil salinity is an increasing problem. In fact, salinity is one of the major environmental factors limiting plant growth and productivity. Plant hormones are known to play vital roles in the ability of the plants to acclimatize to varying environments by mediating growth, development, and nutrient allocation. Thus, to gain insights about the role of plant hormones in the growth responses of salinized fenugreek plants (Trigonella foenum-graecum L.), a medium-term experiment was conducted under moderate (100 mM NaCl) and high (200 mM NaCl) salinity levels. Results showed that moderate, but especially high salinity stress, impaired shoot growth, total leaf area and leaf number. Salinity also provoked a reduction in relative water content, stomatal conductance and photosynthesis-related pigments, but, surprisingly, photosynthetic rate increased in the leaves of fenugreek plants. Na accumulated in the leaves, particularly at high salinity levels, while most mineral nutrients decreased. Furthermore, important changes in the main hormone classes were observed, associated to growth reduction under salinity. The active cytokinin form, trans-zeatin, and active cytokinin and gibberellin concentrations decreased with salinity in the leaves of fenugreek plants, whereas the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, accumulated in the roots of fenugreek plants, especially at high salinity levels. Importantly, leaf abscisic acid concentrations increased under salinity, which could limit leaf transpiration to adapt growth to the stressful conditions. Therefore, plant hormones seem to play a critical role in the growth responses of fenugreek plants under salinity stress and they could have potential interest in salt tolerance programmes for this species.

The interaction between foliar GA3 application and arbuscular mycorrhizal fungi inoculation improves growth in salinized tomato (Solanum lycopersicum L.) plants by modifying the hormonal balance.

The agriculture industry is frequently affected by various abiotic stresses limiting plant productivity. To decrease the negative effect of salinity and improve growth performance, some strategies have been used, such as exogenous application of plant growth regulators (i.e. gibberellic acid, GA3), or arbuscular mycorrhizal fungi (AMF) inoculation. To gain insights about the cross-talk effect of exogenous GA3 application and AMF inoculation on growth under salinity conditions, tomato plants (Solanum lycopersicum, cv. TT-115) were inoculated or not with the AMF Rhizophagus irregularis and exposed to different treatments during two weeks: 0M GA3+0mM NaCl, 10-6M GA3+0mM NaCl, 0M GA3+100mM NaCl and 10-6M GA3+100mM NaCl. Results have revealed that AMF inoculation or GA3 application alone, but especially their interaction, resulted in growth improvement under salinity conditions. The growth improvement observed in AMF-inoculated tomato plants under salinity conditions was mainly associated to ionic factors (higherK concentration and K/Na ratio) while the alleviating effect of GA3 application and its interaction with AMF appear to be due to changes in the hormonal balance. Foliar GA3 application was found to increase the active gibberellins (GAs), resulting in a positive correlation between GA3 and the growth-related parameters. Furthermore, cytokinins, indoleacetic acid and abscisic acid concentrations increased in AMF inoculated or GA3 treated plants but, notably, in AMF plants treated with GA3, which showed improved growth under salinity conditions. This suggests that there is an interactive positive effect between GAs and AMF which alleviates growth impairment under salinity conditions by modifying the hormonal balance of the plant.

Hormonal and Nutritional Features in Contrasting Rootstock-mediated Tomato Growth under Low-phosphorus Nutrition.

Grafting provides a tool aimed to increase low-P stress tolerance of crops, however, little is known about the mechanism (s) by which rootstocks can confer resistance to P deprivation. In this study, 4 contrasting groups of rootstocks from different genetic backgrounds (Solanum lycopersicum var. cerasiforme and introgression and recombinant inbred lines derived from the wild relatives S. pennellii and S. pimpinellifolium) were grafted to a commercial F1 hybrid scion and cultivated under control (1 mM, c) and P deficient (0.1 mM, p) conditions for 30 days, to analyze rootstocks-mediated traits that impart low (L, low shoot dry weight, SDW) or high (H, high SDW) vigor. Xylem sap ionic and hormonal anlyses leaf nutritional status suggested that some physiological traits can explain rootstocks impacts on shoot growth. Although xylem P concentration increased with root biomass under both growing conditions, shoot biomass under low-P was explained by neither changes in root growth nor P transport and assimilation. Indeed, decreased root P export only explained the sensitivity of the HcLp rootstocks, while leaf P status was similarly affected in all graft combinations. Interestingly, most of the nutrients analyzed in the xylem sap correlated with root biomass under standard fertilization but only Ca was consistently related to shoot biomass under both control and low-P, suggesting an important role for this nutrient in rootstock-mediated vigor. Moreover, foliar Ca, S, and Mn concentrations were (i) specifically correlated with shoot growth under low-P and (ii) positively and negatively associated to the root-to-shoot transport of the cytokinin trans-zeatin (t-Z) and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), respectively. Indeed, those hormones seem to play an antagonistic positive (t-Z) and negative (ACC) role in the rootstock-mediated regulation of shoot growth in response to P nutrition. The use of Hp-type rootstocks seems to enhance P use efficiency of a commercial scion variety, therefore could potentially be used for increasing yield and agronomic stability under low P availability.

Red blotch disease alters grape berry development and metabolism by interfering with the transcriptional and hormonal regulation of ripening.

Grapevine red blotch-associated virus (GRBaV) is a major threat to the wine industry in the USA. GRBaV infections (aka red blotch disease) compromise crop yield and berry chemical composition, affecting the flavor and aroma properties of must and wine. In this study, we combined genome-wide transcriptional profiling with targeted metabolite analyses and biochemical assays to characterize the impact of the disease on red-skinned berry ripening and metabolism. Using naturally infected berries collected from two vineyards, we were able to identify consistent berry responses to GRBaV across different environmental and cultural conditions. Specific alterations of both primary and secondary metabolism occurred in GRBaV-infected berries during ripening. Notably, GRBaV infections of post-véraison berries resulted in the induction of primary metabolic pathways normally associated with early berry development (e.g. thylakoid electron transfer and the Calvin cycle), while inhibiting ripening-associated pathways, such as a reduced metabolic flux in the central and peripheral phenylpropanoid pathways. We show that this metabolic reprogramming correlates with perturbations at multiple regulatory levels of berry development. Red blotch caused the abnormal expression of transcription factors (e.g. NACs, MYBs, and AP2-ERFs) and elements of the post-transcriptional machinery that function during red-skinned berry ripening. Abscisic acid, ethylene, and auxin pathways, which control both the initiation of ripening and stress responses, were also compromised. We conclude that GRBaV infections disrupt normal berry development and stress responses by altering transcription factors and hormone networks, which result in the inhibition of ripening pathways involved in the generation of color, flavor, and aroma compounds.

Root-to-Shoot Hormonal Communication in Contrasting Rootstocks Suggests an Important Role for the Ethylene Precursor Aminocyclopropane-1-carboxylic Acid in Mediating Plant Growth under Low-Potassium Nutrition in Tomato.

Selection and breeding of rootstocks that can tolerate low K supply may increase crop productivity in low fertility soils and reduce fertilizer application. However, the underlying physiological traits are still largely unknown. In this study, 16 contrasting recombinant inbred lines (RILs) derived from a cross between domestic and wild tomato species (Solanum lycopersicum × Solanum pimpinellifolium) have been used to analyse traits related to the rootstock-mediated induction of low (L, low shoot fresh weight) or high (H, high shoot fresh weight) vigor to a commercial F1 hybrid grown under control (6 mM, c) and low-K (1 mM, k). Based on hormonal and ionomic composition in the root xylem sap and the leaf nutritional status after long-term (7 weeks) exposure low-K supply, a model can be proposed to explain the rootstocks effects on shoot performance with the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) playing a pivotal negative role. The concentration of this hormone was higher in the low-vigor Lc and Lk rootstocks under both conditions, increased in the sensitive HcLk plants under low-K while it was reduced in the high-vigor Hk ones. Low ACC levels would promote the transport of K vs. Na in the vigorous Hk grafted plants. Along with K, Ca, and S, micronutrient uptake and transport were also activated in the tolerant Hk combinations under low-K. Additionally, an interconversion of trans-zeatin into trans-zeatin riboside would contribute to decrease ACC in the tolerant LcHk plants. The high vigor induced by the Hk plants can also be explained by an interaction of ACC with other hormones (cytokinins and salicylic, abscisic and jasmonic acids). Therefore, Hk rootstocks convert an elite tomato F1 cultivar into a (micro) nutrient-efficient phenotype, improving growth under reduced K fertilization.

Improving agronomic water use efficiency in tomato by rootstock-mediated hormonal regulation of leaf biomass.

Water availability is the most important factor limiting food production, thus developing new scientific strategies to allow crops to more efficiently use water could be crucial in a world with a growing population. Tomato is a highly water consuming crop and improving its water use efficiency (WUE) implies positive economic and environmental effects. This work aimed to study and exploit root-derived hormonal traits to improve WUE in tomato by grafting on selected rootstocks. Firstly, root-related hormonal parameters associated to WUE were identified in a population of recombinant inbred lines (RILs) derived from the wild tomato species Solanum pimpinellifolium. A principal component analysis (PCA) revealed that some hormonal traits were associated with productivity (plant biomass and photosynthesis) and WUE in the RIL population. Leaf ABA concentration was associated to the first component (PC1) of the PCA, which explained a 60% of the variance in WUE, while the ethylene precursor ACC and the ratio ACC/ABA were also associated to PC1 but in the opposite direction. Secondly, we selected RILs according to their extreme biomass (high, B, low, b) and water use (high, W, low, w), and studied the differential effect of shoot and root on WUE by reciprocal grafting. In absence of any imposed stress, there were no rootstock effects on vegetative shoot growth and water relations. Finally, we exploited the previously identified root-related hormonal traits by grafting a commercial tomato variety onto the selected RILs to improve WUE. Interestingly, rootstocks that induced low biomass and water use, 'bw', improved fruit yield and WUE (defined as fruit yield/water use) by up to 40% compared to self-grafted plants. Although other hormonal factors appear implicated in this response, xylem ACC concentration seems an important root-derived trait that inhibits leaf growth but does not limit fruit yield. Thus tomato WUE can be improved exploiting rootstock-derived hormonal signals which control leaf growth.

Identification of novel stress-responsive biomarkers from gene expression datasets in tomato roots.

Abiotic stresses such as heat, drought or salinity have been widely studied individually. Nevertheless, in the nature and in the field, plants and crops are commonly exposed to a different combination of stresses, which often result in a synergistic response mediated by the activation of several molecular pathways that cannot be inferred from the response to each individual stress. By screening microarray data obtained from different plant species and under different stresses, we identified several conserved stress-responsive genes whose expression was differentially regulated in tomato (Solanum lycopersicum L.) roots in response to one or several stresses. We validated 10 of these genes as reliable biomarkers whose expression levels are related to different signalling pathways involved in adaptive stress responses. In addition, the genes identified in this work could be used as general salt-stress biomarkers to rapidly evaluate the response of salt-tolerant cultivars and wild species for which sufficient genetic information is not yet available.

Unravelling rootstock×scion interactions to improve food security.

While much recent science has focused on understanding and exploiting root traits as new opportunities for crop improvement, the use of rootstocks has enhanced productivity of woody perennial crops for centuries. Grafting of vegetable crops has developed very quickly in the last 50 years, mainly to induce shoot vigour and to overcome soil-borne diseases in solanaceous and cucurbitaceous crops. In most cases, such progress has largely been due to empirical interactions between farmers, gardeners, and botanists, with limited insights into the underlying physiological mechanisms. Only during the last 20 years has science realized the potential of this old activity and studied the physiological and molecular mechanisms involved in rootstock×scion interactions, thereby not only explaining old phenomena but also developing new tools for crop improvement. Rootstocks can contribute to food security by: (i) increasing the yield potential of elite varieties; (ii) closing the yield gap under suboptimal growing conditions; (iii) decreasing the amount of chemical (pesticides and fertilizers) contaminants in the soil; (iv) increasing the efficiency of use of natural (water and soil) resources; (v) generating new useful genotypic variability (via epigenetics); and (vi) creating new products with improved quality. The potential of grafting is as broad as the genetic variability able to cross a potential incompatibility barrier between the rootstock and the scion. Therefore, understanding the mechanisms underlying the phenotypic variability resulting from rootstock×scion×environment interactions will certainly contribute to developing and exploiting rootstocks for food security.

Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.