Hormonal and metabolic regulation of tomato fruit sink activity and yield under salinity

Journal of Experimental Botany, Vol. 65, No. 20, pp. 6081–6095, 2014 doi:10.1093/jxb/eru347 Advance Access publication 28 August, 2014 v This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) ReseaRch PaPeR Hormonal and metabolic regulation of tomato fruit sink activity and yield under salinity AlfonsoAlbacete1,2, ElenaCantero-Navarro1, María E. Balibrea1, Dominik K.Großkinsky2,3, Maríade la Cruz González4, CristinaMartínez-Andújar1, Ann C.Smigocki5, ThomasRoitsch2,3,6 and FranciscoPérez-Alfocea1,* 1 Department of Plant Nutrition, CEBAS-CSIC, Campus de Espinardo, 30100 Murcia, Spain 2 Institute of Plant Sciences, Department of Plant Physiology, University of Graz, 8010 Graz, Austria 3 Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Taastrup, Denmark 4 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla, CSIC, 41092 Sevilla, Spain 5 Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD 20705, USA 6 Global Change Research Centre, Czech Globe AS CR, v.v.i., Drásov 470, Cz-664 24 Drásov, Czech Republic * To whom correspondence should be addressed. E-mail: [email protected] Received 27 June 2014; Revised 22 July 2014; Accepted 24 July 2014 Abstract Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity. Key words: Cell wall invertase, cytokinins, fruit, salinity, sink activity, tomato. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. 6082 | Albacete et al. Introduction Salinity decreases crop yield by first reducing growth and the number of assimilate-consuming sink organs (both vegetative and reproductive), and then by decreasing assimilate production in photosynthetically active source tissues (Hsiao, 1973; Cramer, 1992; Chazen et al., 1995). Therefore, to sustain crop productivity under unfavourable saline conditions requires the maintenance of assimilate production in source tissues (leaf area and photosynthesis) and also transport to and use within sink and harvestable tissues (Perez-Alfocea et al., 2010; Albacete et al., 2014). Ideally, maintaining or increasing sink activity in the plant (individually or collectively) will help to avoid initial photoinhibition and premature stressinduced senescence, thus maintaining assimilate production and transport in/from the source tissues (Perez-Alfocea et al., 2010; Albacete et al., 2014). For this reason, the discovery of any novel gene involved in stress adaptation should include an investigation of its effect on sink activity and, ultimately, on yield performance. Increasing sink activity in the reproductive organs by either (i) the overexpression of sucrolytic activities, for example under the control of a fruit-/ grain-specific promoter, or (ii) the positive (i.e. cytokinins, gibberellins) or negative (i.e. ethylene) modulation of hormonal factors regulating the sink number and activity, could be useful strategies for increasing yield stability under suboptimal abiotic stress conditions (Albacete etal., 2014). Sink organs of most plant species are supplied with carbon and energy in the form of sucrose which can be used for the biosynthesis of primary metabolites important for tissue growth and development. The growth capacity, evaluated as net accumulation rate of dry matter, is a measure of sink strength (Ho et al., 1989; Marcelis, 1996). Warren-Wilson (1972) proposed that sink strength is the product of multiplying sink size and sink activity. Sink size is a physical factor that includes cell number and cell size, whereas sink activity is a physiological factor that includes multiple components and key enzymes of carbohydrate metabolism and storage, thus maintaining a sucrose gradient and transport between source and sink organs (Ho, 1984). Under salinity, the competition for carbon between different physiological processes and sink organs significantly affects plant growth and crop yield (Munns, 1993; Balibrea et al., 1996, 1999, 2000; Daie, 1996; Perez-Alfocea et al., 2010). The use of sucrose in sink tissues requires cleavage of the glycosidic bond, catalysed by both sucrose synthase (SUS) and invertases. Three types of invertase isoenzymes are distinguished based on their solubility, subcellular localization, pH optima, and isoelectric point: vacuolar invertase (vacInv), cytoplasmic invertase (cytInv), and cell-wall bound or apoplastic invertase (cwInv) (Roitsch and González, 2004). Decreased fruit set and/or tomato fruit weight and, ultimately, crop yield under salinity conditions have been partially explained in terms of sucrose transport and metabolism (Ho, 1996). In this regard, it has been reported that a highly saline treatment (150 mM NaCl for 10 d) decreased pollen viability, inducing tomato flower abortion, which was thought to be due to decreased carbohydrate transport from source leaves to the inflorescence and pollen-producing tissues, as suggested by marked reductions in sucrolytic activities of cwInv and SUS (Ghanem etal., 2009). Thus, cwInv seems essential in maintaining sucrose import to sink tissues by regulating the apoplastic sucrose unloading from the phloem (Roitsch and Ehness, 2000; Roitsch et al., 2003; Koch, 2004) during pollen development (Roitsch and González, 2004), especially under potential source-limiting stress conditions (Albacete etal., 2011). Assuming that the flower is fertilized, fruit or grain filling is also a susceptible process limiting yield. For example, tomato yield reduction by low to moderate salinity levels in irrigation water (25–75 mM NaCl) is due to decreased fruit weight rather than fruit number (Cuartero and Fernández-Muñoz, 1998), and sucrolytic enzymes have been also implicated. In fact, the inhibition of the cytInv and the use of the accumulated sucrose by other sucrolytic enzymes could be considered as a limiting step and an adaptive response in the control of sucrose import and fruit growth under salinity, respectively (Balibrea etal., 1996, 1999). Although the cytoplasmic cleavage of sucrose has been considered an important regulatory step for assimilate import in tomato fruit (Ho, 1996), cytInv has rarely been studied and most published work only addresses vacInv and SUS. Few studies have shown that cwInv has important functions in establishing and maintaining sink metabolism in tomato fruits and also in phloem unloading, carbohydrate partitioning, and sink growth (Godt and Roitsch, 1997; Sturm, 1999; Roitsch, 1999; Roitsch etal., 2000). An inverse relationship between cytoplasmic sucrolytic activities (CSA) and cwInv in fruits of domestic and hybrid (between Solanum lycopersicum and the wild relatives S. cheesmaniae and S. chmielewskii) tomato plants has been reported as a regulatory mechanism for maintaining sink capacity and dry matter accumulation (Balibrea etal., 2003; Balibrea etal., 2006). Plant hormones are directly implicated in tomato fruit set and development (reviewed in Ariizumi et al. (2013)) and sink-related processes (Ehness and Roitsch, 1997; Roitsch and Ehness, 2000; Roitsch etal., 2003). Exogenous hormonal application to fruits at different developmental stages had highlighted their importance in fruit set and growth (reviewed by De Jong etal. (2009)). The effect of 2,4-dichlorophenoxy- acetic acid (2,4-D)- and gibberellic acid (GA3)-induced fruit set on the expression of diverse genes involved in auxin and gibberellin (GA) signalling has been studied in tomato plants (Serrani et al., 2008; De Jong et al., 2009). Furthermore, increases in indoleacetic acid (IAA), cytokinins (CKs), and bioactive GA concentrations have been found during tomato fruit set and early fruit development, whereas abscisic acid (ABA) decreased (Mariotti etal., 2011). Indeed, endogenous levels of CKs have been linked with fruit growth (Gillaspy etal., 1993; Srivastava and Handa, 2005) and are critically involved in the regulation of early fruit growth through the regulation of cell division by D-type cyclin expression (Baldet etal., 2006). Gene expression profiles have suggested that ethylene is also involved in regulating fruit set (Vriezen etal., 2008). Before tomato fruit development, genes associated with ethylene biosynthesis were strongly expressed and associated Hormonal and metabolic regulation of salinized tomato fruits | 6083 with relatively high ethylene concentrations. However, during fruit development, ethylene concentrations decreased owing to an auto-inhibitory effect of LeACS1A,6 and LeACO1,3,4 genes (Cara and Giovannoni, 2008). Information acquired through biochemical, genetic, and molecular studies is now beginning to reveal a possible interlink between hormonal and metabolic factors in the regulation of tomato fruit growth and crop yield under salinity. Elevated levels of CKs have been associated with the upregulation of sink strength and invertase expression (Ehness and Roitsch, 1997). Root CK biosynthesis has been shown to be important in mediating the relationship between decreased shoot CK status and salt-induced changes in growth, senescence, and fruit yield (Albacete et al., 2008; Ghanem et al., 2008; Albacete et al., 2009; Ghanem et al., 2011). Augmenting root-to-shoot CK transport (through grafting CK-overproducing rootstocks) improved vegetative growth and ion homeostasis, delayed leaf senescence, and increased fruit yield of salinized tomato (Albacete etal., 2009; Ghanem etal., 2011). Grafting WT plants onto a constitutively (35S) expressing IPT rootstock increased fruit yield by 30% compared with salinized self-grafted WT/WT plants. Similarly, reduction of ethylene biosynthesis and perception (reviewed by Stearns and Glick, 2003), mainly focused on the ACC deaminase gene, has been shown to regulate carbon metabolism and sink strength. Therefore, the aim of this work was to study the influence of both metabolic (sucrose metabolism) and hormonal (auxins, CKs, ABA, GA3, and ethylene) factors on fruit sink activity and strength, and hence on overall fruit yield, in tomato plants growing under salinity. In this study, different classical and functional physiological approaches were integrated: the classical exogenous hormonal application and biotechnological overexpression of the cwInv gene CIN1 in fruits or the CK biosynthesis gene IPT in roots. Material and methods Plant cultivation Tomato plants (Solanum lycopersicum L.) were sown in trays filled with a perlite–vermiculite mixture (1/3, v/v) moistened regularly with halfstrength Hoagland’s nutrient solution. Forty days after germination, seedlings were transferred to a polyethylene greenhouse using perlite bags as growing medium. Plants were distributed in a planting pattern of 2 m between rows and 0.5 m between plants within rows, and cultivated with one stem, eliminating all axillary buds. Astandard fertilization solution for tomato was applied by a drip irrigation system. Salt treatment started 10 d after the transfer. The fertilization solution was prepared in two 1500-l tanks and the electrical conductivities of the treatments were 1.2 (control) and 8.7 dS m–1 (75 mMNaCl). Flowers were tagged at anthesis 30 d after the beginning of the salt treatment, and actively growing fruits were harvested and weighted 25 d after anthesis (DAA). Pericarp tissue was cut in small pieces, frozen with liquid nitrogen, and stored at –80ºC until analysis. Three replicates were carried out for each fruit harvest and treatment. Hormone applications From an original population of 40 tomato plants (Solanum lycopersicum L.cv. Durinta F1, from Western Seed 2000 SL, Almeria, Spain) growing at the greenhouse under a moderate salinity level (75 mM NaCl), 25 were selected for exogenous hormonal applications. Two hormonal applications (with an interval of two weeks) were done by spraying directly the fruit trusses at the flowering/ early-fruiting stage with four different plant regulators: gibberellic acid (GA3), kinetin (KIN), indolacetic acid (IAA) (10–5 M) (purchased from Sigma-Aldrich, MO, USA), and ethephon (ET, 0.15% v/v, applied as Ethrel® 48 SL, Bayer CropScience, Monheim am Rhein, Germany). Control plants were sprayed with distilled water. Radiolabelled sucrose allocation into developing fruits: sink activity Forty-day old tomato seedlings (Solanum lycopersicum L. cv. Durinta F1) were placed in pots containing peat as substrate. Plants were grown in a growth chamber under 16 h daylight period. The air temperature ranged from 25–28°C during the day and 17–18°C during the night. Relative humidity was maintained at 70 ± 5% during the night and 50 ± 5% during the day. Light intensity at the top of the canopy was 250µmol m–2 s–1. After 5 d of acclimation, seedlings were exposed to 0 mM (control) or 100 mM NaCl added to the nutrient solution. To study the effects of the exogenous hormonal application on sink activity, KIN (10–5 M) and ET (0.15% v/v) were applied to salinized plants and control plants, respectively, 40 d after salt treatment started, by spraying the second fruit truss of each plant. One week later the radioactivity assay was performed. Five plants per treatment were used for this experiment. The application of the radio-labelled sucrose was made as described by Garrido et al. (2002) and Ben Salah et al. (2009). When the plants showed 2 trusses of developing fruits (8–12 fruits per plant), 10µl of a 150 Bq µl–1 [14C(U)]-sucrose (specific activity 625 mCi mmol–1, Nucliber S.A., Madrid, Spain) aqueous solution were applied to a lightly abraded area (about 1 cm2) in the basal zone of the fruit-feeding mature leaf situated just below the second truss. Ten minutes after application the area was covered with lanoline to avoid desiccation. Plants were kept 24 h with solar photoperiod and at room temperature under a cabin of radioactive gas capture. After that, all treated fruits were weighted and cut into small pieces. Total fruit fresh material was separated in 1 g-packages and each one was immersed in a separate vial containing 10 ml of scintillation cocktail (OptiPhase “HiSafe” 3, Wallac-PerkinElmer, MA, USA) and stored in darkness at room temperature for 24 h. Radioactivity was determined in a Wallac 1400 DSA beta liquid scintillation counter (PerkinElmer, Waltham, MA, USA). Using a standard, the efficiency of the 14C counting was calculated as 97.8 ± 1.2%; therefore, the radioactivity measured in the 14C-sucrose solution applied to the leaves was 146.7 ± 2.7 Bq µl–1. Fruit sink activity and fruit strength were determined as the amount of radioactivity accumulated after that period per gram of FW (Bq g–1 FW) or per fruit (Bq fruit–1), respectively. Similar determinations of fruit sink activity and strength were also carried out in two homozygous tomato transgenic lines for the InvLp6g::CIN1 construct (CIN1-91 and CIN1-93) and in the wild type (cv. P-73), cultivated under control (0 mM NaCl) and saline (100 mM NaCl) conditions. Three plants per line were evaluated. Overexpressing the cell wall invertase CIN1 under the control of a fruit-specific promoter The full-length 1.7 kb CIN1 cDNA under the control of a 2.5 kb fragment of the promoter of vacuolar invertase pInvLp6g from Solanum pimpinellifolium (Elliott etal., 1993) (gene bank accession no. Z12028.1) was cloned into the vector pBI101. After transfer into the Agrobacterium tumefaciens strain LBA4404, cotyledons from the semi-indeterminate cv. P-73 of tomato (Solanum lycopersicum L.) were transformed with the CIN1 overexpression construct. T2 plants from five different transgenic lines containing the InvLp6g::CIN1 construct were evaluated in the greenhouse under moderate salinity (75 mM NaCl) for fruit yield-related analyses. Plants were identified as homozygous, heterozygous, or azygous for the T-DNA based on 6084 | Albacete et al. the expression of the marker gene NPTII that confers resistance to the antibiotics kanamycin and neomycin. IPT-transformed cytokinin-overproducing rootstocks Tomato seeds from different genotypes were sown in a controlled conditions culture chamber. When seedlings had developed three or four true leaves, tomato plants from the cultivar P-73 were grafted (as previously described by Santa-Cruz etal. (2002)) onto a determinate commercial cultivar UC-82B (P-73/UC-82B) as well as the same cultivar overexpressing the IPT gene (P-73/IPT) used as rootstocks. This IPT gene codifies for a key enzyme of the CK biosynthesis isopentenyl transferase from Agrobacterium tumefaciens, and was cloned under the control of the constitutive CaMV 35S promoter. Seeds were supplied by Dr Ann Smigocki (ARS-USDA, Beltstville, MD, USA; Ghanem etal., 2011). Four plants per combination were transferred to the greenhouse after the grafts had established and a salinity level of 75 mM NaCl was applied to the irrigation solution. CIN1 expression analysis Fresh tissues from leaves, roots, seedlings, and fruits were used for total RNA isolation, and 1µg of total RNA was used for first strand cDNA synthesis according to standard methods, using oligo(dT) primers. Semi-quantitative RT-PCR using actin to normalize the obtained cDNA amounts was performed as described previously (Großkinsky etal., 2011). For CIN1 expression analyses the primers CIN1-Forward (5'-CCTGGGAGTATAGTGGCTGAACC-3') and CIN1-Reverse (5'-AGGTCTTCTCTGAATCCG-3') were used. Sucrolytic activities and invertase inhibitor enzymeassays Sucrolytic activities were assayed by determining the NADH delivered in a coupled enzymatic reaction using specific substrates/ enzymes depending on the target enzyme (Balibrea et al., 2003; Balibrea et al., 1999). The absorbance was performed at 340 nm. Briefly, 0.5 g of plant material was homogenized in liquid nitrogen in a mortar and re-suspended in 1 ml homogenization buffer (200 mM HEPES, 3 mM MgCl2, 1 mM EDTA, 2% glycerol, 0.1 mM PMSF, 1 mM benzamidine). The homogenate was mixed for 20 min at 4°C and centrifuged for 15 min at 10 500 g and 4°C. The supernatant was used for soluble enzyme preparation. The pellet (cell-wall fraction) was washed three times with distilled water and re-suspended in 200 mM HEPES, 3 mM MgCl2, 15 mM EDTA, 2% glycerol, 0.1 mM PMSF, 1 mM benzamidine, 1 M NaCl. CwInv and vacInv activities were measured at pH 5, and cytInv activity at pH 7.SUS activity was assayed in the same reaction medium than cytInv, but with the addition of UDP. The proteins were analysed with Bradford reagent using BSA as standard. The invertase inhibitor assay was performed as previously described (Bonfig etal., 2010). Sugar determination A total of 100 mg plant material was ground in liquid nitrogen and 0.9 ml water was added. After homogenization with cationic and anionic exchange resins and centrifugation for 10 min at 20 000 g and 4°C, the supernatant was filtered and 10µl were injected in a normal-phase liquid chromatography system (Shimadzu Corporation, Kyoto, Japan), using acetonitrile/water (85/15, v/v) as the mobile phase at a flow rate of 1 ml min–1 (Balibrea etal., 2000, 2003). Hormone extraction and analysis Hormones were analysed as described previously (Albacete etal., 2008). Briefly, 0.5 g of each sample were homogenized, in triplicate, with liquid nitrogen, and extracted with 2.5 ml of methanol:H2O (80:20). Samples were centrifuged and the precipitates were reextracted with another 2.5 ml of the methanol:H2O mixture. The two supernatants were mixed and passed through a SepPak Plus C18 cartridge (SepPak Plus, Waters, Millford, MA, USA). Samples were evaporated and the residues were dissolved in a methanol:H2O (20:80) mixture and filtrated. 8 µl of sample were injected in an Agilent 1100 Series HPLC (AgilentTechnologies, Santa Clara, CA, USA), equipped with a micro-wellplate autosampler and a capillary pump, connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA,USA) using an electrospray interface. Statistics All experiments were repeated three times, and results of one representative experiment are presented in each case. Data were subjected to an analysis of variance (ANOVA) using the SPSS software (Version 19.0, SPSS Inc., Chicago, IL, USA). The statistical significance of the results was analysed by Student-Newman-Keuls test at the 5% level. Results Salt effect on [14C(U)]-sucrose allocation into developing fruits: a measurement of sink activity The capacity of developing fruits to attract [14C(U)]-sucrose from a source leaf, measured as the amount of radioactivity (Bq) accumulated per gram of fresh weight (Bq g–1 FW), has been considered as an estimation of the sink activity of the fruits. The result of multiplying sink activity by the sink size was considered as the fruit sink strength, expressed in Bq fruit–1. As a consequence, sink activity can be regarded as a metabolic parameter strongly determining the fruit sink strength and the capacity of the fruit to attract assimilates from the active fruit-feeding photosynthetic tissues. Results clearly show that 100 mM NaCl applied to the irrigation solution during 40 d provoked a 3-fold decrease in both sink strength (Fig.1A) and sink activity (Fig.1B) in developing fruits up to 5–7 g FW as compared with the control. Both parameters were also lower in the salinized fruits weighing more than 7 g but the reduction was difficult to quantify owing to the high dispersion of data in the control fruit. This dispersion may be explained by the interaction between different fruit sizes, truss position, and fruit position on a truss and the limited availability of the labelled sucrose. Effects of exogenous hormonal application Fruit growth The application of 10–5 M of GA3, KIN, or IAA to a truss of actively growing fruits on salinized plants increased the final fresh weight of mature fruits by 1.3-, 2-, and 1.6-fold, respectively, as compared with the average of the whole population and water-sprayed fruits (Fig. 2A). The ethylene-releasing chemical Ethephon (ET) had no effect on the final fruit weight but it significantly reduced the equatorial diameter of the fruit (Fig.2B). Sucrolytic activities The activity of sucrose-cleaving enzymes significantly increased in the salinized fruit after treatment with GA3, KIN, or IAA, whereas the ET treatment inhibited the enzymes when compared with the water-sprayed fruits (Fig.2C). The Hormonal and metabolic regulation of salinized tomato fruits | 6085 activity of the cwInv enzyme was up to 1.2-fold higher in GA3-, KIN-, and IAA-treated plants, but the activity was inhibited 28% by the ET treatment. KIN and IAA induced SUS activity 1.2-fold and GA3 1.1-fold. Similar differences between treatments were observed for vacInv, but to a lower extent (5–15%). CytInv activity was the least induced by the three hormonal treatments (5–8%). In addition to cwInv, Et also inhibited the cytoplasmic sucrolytic activities of SUS and cytInv (8%), and the vacInv (15%) compared with the water-sprayed fruits (Fig.2C). Regarding the absolute values of cwInv, exogenous applications of KIN (10–5 M) and ET (0.15%) to actively growing salinized fruits provoked a significant increase (KIN) or decrease (ET) of this sucrolytic enzyme compared with the water-treated fruits (Fig.2D), suggesting an important role for cwInv in the regulation of hormonalmediated sink activity in tomato fruits growing under salinity. Those hormones were selected for further experiments as positive and negative regulators of fruit sink activity. Cytokinin (kinetin) and ethylene (ethephon) effects on sink activity Analyses of the endogenous levels of the most bioactive CK in tomato, tZ, and the ethylene precursor ACC showed an important reduction of the tZ concentrations in salinized developing (green stage, 25 d after anthesis; DAA) fruits (50%), whereas ACC concentrations significantly increased (2-fold) (Fig.3A). To test if hormonal factors have an effect on fruit sink activity that was measured as sucrose import, KIN, the best positive effector, and ET, the negative effector, were applied to salt-stressed plants. Application of KIN recovered sink activity (Bq g–1 FW), and thereafter sink strength, in salinized fruits reaching similar slope values (as a function of fruit size) as compared with the non-stressed control fruits (Fig. 3B, C). A similar but negative effect was observed when the ethylene-releasing chemical ET was applied to control fruits, attaining in this case similar slope values as the salinized fruits (Fig.3B, C). Therefore, the exogenous application of CKs mimics the absence of stress in salinized fruits, whereas the ethylene mimics the salt effect in control fruits, thus supporting the suggestion that CK and ethylene are endogenous effectors regulating sink activity in tomato fruit grown under salinity. Effect of the cell wall invertase gene CIN1 overexpression under the control of a putative fruit-specific promoter Fruit-yield related parameters To test the hypothesis that the specific increase of invertase activity in the fruit could recover sink strength and fruit growth under suboptimal conditions imposed by salinity, we generated transgenic tomato plants overexpressing the cwInv gene CIN1 from Chenopodium rubrum under the control of the putative fruit-specific promoter from the vacuolar invertase gene InvLp6g of Solanum pimpinellifolium (Klann etal., 1993). Solanum lycopersicum cv. P-73 plants were transformed with the pInvLp6g::CIN1 construct and a total of 23 T2 segregating plants were analysed under moderate salinity (75 mM NaCl) for parameters related to fruit yield. Of these, 9 plants generated homozygous and 11 plants heterozygous T3 progenies based on kanamycin resistance, whereas 3 plants produced 100% kanamycinsensitive progenies. Fruit yield was strongly increased for several lines (Fig.4A) evident by higher fruit fresh weight (Fig. 4B) and/or higher fruit number (Fig. 4C), whereas yield-related parameters under control conditions were not affected (Fig.4D–F). Fruit yield under moderate salinity correlated stronger with fruit number (r = 0.89, P≤0.01, n=26) than individual fruit fresh weight (r=0.51, P≤0.01, n = 26) (Fig. 4G). Based on these results, salt stress tolerance during fruit development was investigated for two selected homozygous CIN1 lines, which showed strong CIN1 expression (Fig.5A), one with increased fruit number (CIN1-93) and other one with higher fruit weight (CIN1- 91), to distinguish between a more likely systemic effect on the fertilization and fruit set and a more sink-specific effect on fruit growth. Fruit yield (Fig.5B, line), fruit fresh weight (Fig.5B, bars), and fruit number (Fig.5C, line) were higher, whereas flower abortion index was reduced (Fig.5C, bars) in both CIN1 lines under salt stress. Importantly, fruits from CIN1 plants exhibited from 5- up to 15-fold increase in sink activity, measured by the capacity to Fig.1. Linear correlations between fruit sink strength (A, measured as the amount of radioactivity accumulated in Bq) or fruit sink activity (B, expressed as logarithm of the amount of radioactivity accumulated in Bq per gram of fresh weight), and the fruit fresh weight of tomato plants (cv. Durinta F1) cultivated for 40 d in the absence (closed circles) or presence (open circles) of 100 mM NaCl. 6086 | Albacete et al. Fig.3. Trans-Zeatin (tZ) and 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations (A) in actively growing fruits (25 DDA) of tomato plants cultivated for 40 d in the absence (light-grey bars) or presence (dark-grey bars) of 100 mM NaCl. Data are means of 6 plants±SE. Values marked with a same letter are not significantly different at P<0.05 according to the Student-Newman-Keuls test. Linear correlations between fruit sink strength (B, measured as the amount of radioactivity accumulated in Bq) or fruit sink activity (C, expressed as logarithm of the amount of radioactivity accumulated in Bq per gram of fresh weight), and the fruit fresh weight of non-salinized (sprayed with ethephon) and salinized (100 mM NaCl, treated with kinetin) tomato fruits (cv. Durinta F1). Fig.2. Final fruit fresh weight (A) and equatorial diameter (B) of tomato fruit (cv. Durinta F1) sprayed with different hormones and cultivated in the presence of 75 mM NaCl. Sucrolytic activities in percentage relate to the water-treated fruits (C) and cell wall invertase activity (D) of actively growing tomato fruits (25 DAA) sprayed with different hormones and cultivated in the presence of 75 mM NaCl. Data are means of 6 plants±SE. Values marked with a same letter are not significantly different at P<0.05 according to the Student–Newman–Keuls test. OP=original population. Hormonal and metabolic regulation of salinized tomato fruits | 6087 attract [14C(U)]-sucrose from a truss-feeding mature leaf with respect to WT fruits (Fig. 5D). This shows a direct functional correlation between higher cwInv CIN1 gene expression (and activity) and increased sink activity (and strength) in salinized fruits. Sugar concentrations, sucrolytic activities, and invertase inhibitor in the fruits Asignificantly higher cwInv activity was observed in actively growing fruits (25 DDA) of the selected homozygous lines, from 1.6-fold in line CIN1-93 up to 3-fold in line CIN1-91 (Fig.6A), accompanied by an increase (up to 3-fold) in the other sucrolytic activities: vacInv, sucrose synthase, and especially cytInv (Fig. 6B–D). The increased sucrolytic activity resulted in a 1.6- and 1.2-fold increase in hexose concentration in fruits of CIN1-91 and CIN1-93 lines, respectively (Fig.6E). Most studies on sucrose metabolism under abiotic stress have focused only on the transcriptional regulation of invertases and the invertase activity present in extracts. Therefore, we have addressed the possible role of invertase Fig.4. Fruit yield (A), fruit fresh weight (B), and number of fruits (C) of wild-type tomato plants (cv. P-73) and a segregating T2 tomato population containing the InvLp6g::CIN1 construct and cultivated under moderate salinity (75 mM NaCl). Fruit yield (D), fruit fresh weight (E), and number of fruits (F) of wild-type tomato plants (cv. P-73) and the selected CIN1-93 line theInvLp6g::CIN1 construct and cultivated under control conditions. Linear correlations (G) between fruit number (open circles) or weight (closed circles) and fruit yield. Black bars indicate selected lines. Data are presented as means±SE. 6088 | Albacete et al. inhibitors in post-translational regulation of invertase activity. Invertase inhibitor activity was significantly lower (2-fold) in CIN1 fruits (Fig. 6F), thus further contributing to the maintenance of sink strength under saline conditions. Fruit hormonal concentrations To differentiate between the metabolic effect of the transgene on the fruit sink activity from other putative hormonal effects mediated by the transformation events, the concentrations of the active CK tZ, the auxin IAA, the ABA, and the ethylene precursor ACC were determined in actively growing fruits of the selected homozygous lines and WT plants. Concentrations of tZ significantly increased up to 35% in the fruits of the selected lines with respect to WT (Fig.7A). In contrast, the ethylene precursor ACC was 1.5 times higher in WT fruits than in CIN1-91 and CIN1-93 plants (Fig.7B). Auxin and ABA levels were also increased by 20–30% in the transgenic CIN1 fruits, compared with the WT (Fig.7C, D). Effect of IPT-transformed rootstocks on scion fruit yield, sucrose metabolism, and hormonal concentration To test if the fruit growth under salinity could be enhanced by increasing the endogenous supply of CKs from the roots, plants from the cultivar P-73 were grafted onto transgenic UC-82B rootstocks that overexpress the key enzyme for de novo CK biosynthesis (isopentenyl transferase, IPT) from Agrobacterium tumefaciens (WT/IPT) and compared with grafted plants from the same cultivar P-73 grafted onto UC-82B wild-type plants (WT/WT) (Fig. 8). Fruit yield increased by 30% in WT/IPT grafted plants cultivated under moderate salinity (75 mM NaCl). The fruit yield increase Fig.5. CIN1 expression in the fruit (A), fruit fresh weight (bars) and fruit yield (line) (B), flower abortion index (bars) and fruit number (line) (C), and linear correlations between fruit sink activity (logarithmic scale) and fruit fresh weight (D) of wild-type tomato plants (cv. P-73) and two selected homozygous lines expressing the InvLp6g::CIN1 construct and cultivated under moderate salinity (75 mM NaCl). Data are means of 3 plants±SE. Values marked with a same letter are not significantly different at P<0.05 according to the Student–Newman–Keuls test. Hormonal and metabolic regulation of salinized tomato fruits | 6089 was primarily attributed to a 25% increase in fruit number (lower flower abortion) and to a significant 5% increase in individual fruit weight (Fig. 8, re-plotted from Ghanem etal., 2011). The improvement in fruit yield in the chimeric P-73/ UC-82B grafted plants was correlated to an increase in tZ concentration in mature leaves (40%) and developing (20–35 DAA) fruits (80%) (Fig. 8, re-plotted from Ghanem etal., 2011). Additionally, the fruits registered a 40% increase in IAA and a 30% decrease in ACC concentrations as compared with WT/WT plants. Aconcomitant increase in sucrolytic activities was also observed in the developing fruits. CwInv and cytInv activities increased by 40% and SUS by 80% (Fig. 8), registering a similar response to root-sourced CKs as exogenous KIN application (Fig.2C). Discussion Salinity decreases tomato fruit yield by reducing fruit sink activity Under salinity stress with low assimilate availability, fruit yield is affected by a reduction in fruit set as well as cell division in early fruit development, a main limiting factor for fruit growth, and cell enlargement during the latter stages of fruit development (Bertin etal., 2001). Tomato yield reduction by low to moderate salinity levels (25–75mM NaCl, 3–8 dSm–1) is primarily due to decreased fruit weight, whereas at high salinity (>75–100mM NaCl, >8–10 dSm–1) levels, it is the decreased number of fruit that is responsible for reduced yields (Cuartero and Fernández-Muñoz, 1998). Therefore, processes involved in fruit set and development must be responsible for yield reduction under salinity. Fig.6. Cell wall invertase (A), vacuolar invertase (B), cytoplasmic invertase (C), and sucrose synthase (D) activities, hexose (glucose + fructose) concentrations (E), and invertase inhibitor activity (F) in tomato fruits of wild-type tomato plants (cv. P-73) and two selected homozygous lines expressing the InvLp6g::CIN1 construct and cultivated under moderate salinity (75 mM NaCl). Data are means of 3 plants±SE. Values marked with a same letter are not significantly different at P<0.05 according to the Student–Newman–Keuls test.