Genetic and physiological characterization of tomato cv. Micro-Tom

Based on its compact habit, Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.), has been proposed as a preferred variety to carry out molecular research in tomato. This cultivar, however, is poorly characterized. It is shown here that Micro-Tom has mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. The presence of the self-pruning mutation in Micro-Tom, that generates a determinate phenotype, was confirmed by crossing and sequence analysis. It was also found that Micro-Tom has a mutation in the DWARF gene (d) that leads to mis-splicing and production of at least two shorter mRNAs. The d mutation is predicted to generate truncated DWARF protein. The d sequence defect co-segregates with dark-green and rugose leaves, characteristics of brassinosteroid biosynthesis mutants. Micro-Tom also carries at least another mutation producing internode length reduction that affects plant height but not active gibberellin (GA) levels, which were similar in dwarf and tall Micro-TomxSeverianin segregants. GAs and brassinosteroids act synergistically in Micro-Tom, and the response to GA depends on brassinosteroids because the elongation of internodes was at least six times higher when GA(3) was applied simultaneously with brassinolide. A novel variety, Micro-0 that is fully susceptible to C. fulvum and almost as dwarf as Micro-Tom, has been generated from the cross of Cf0xMicro-Tom. This line represents a valuable resource for future analysis of Cf resistance genes through breeding or transformation.     

Cloning and DNA-binding properties of ethylene response factor,LeERF1 and LeERF2, in tomato

Two new genes, LeERF1 andLeERF2, were isolated from a tomato (Lycopersicon esculentum cv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of the NP24 gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their binding in vitro.Revisions requested 4 January 2005; Revisions received 28 January 2005     

Developmental analysis of carbohydrate metabolism in tomato (Lycopersicon esculentum cv. Micro-Tom) fruits

Carbohydrate metabolism during the development of fruits of the tomato cultivar Micro-Tom was studied. The metabolism of the pericarp and placental tissues was found to be different. Starch was degraded more slowly in the placenta in comparison with the pericarp, whereas soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 days after flowering. Two of these, phosphoenolpyruvate phosphatase and pyruvate kinase, showed a dramatic increase in activity just before this peak, possibly indicating a role in up-regulating glycolysis to generate increased ATP that would be used during climacteric respiration. The expression of plastidial transporters was studied. Both the TPT and Glu6P transporter were expressed greatest in green fruits, before declining. The expression of the triose-phosphate transporter was greater than that of the glucose 6-phosphate transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development.     

Physiological and biochemical responses of photomorphogenic tomato mutants (cv. Micro-Tom) under water withholding

In addition to mediating photomorphogenesis, phytochromes are responsible for many abiotic stress responses, acting upon biochemical and molecular mechanisms of cell signaling. In this work, we measured the physiological and biochemical responses of phytochrome-mutant plants under water stress. In tomato (Solanum lycopersicum L.), the aurea mutant (au) is phytochrome-deficient and the high-pigment-1 mutant (hp1) has exaggerated light responses. We examined the effects of water withholding on water potential, leaf gas exchange, chlorophyll fluorescence, chloroplast pigment content and antioxidant enzyme activity in au and hp1 and their wild-type cultivar Micro-Tom (MT). Initial fluorescence and potential quantum efficiency of photosystem II (PSII) photochemistry were not affected by the treatment, but effective quantum yield of PSII, electron transport rate decreased and non-photochemical quenching increased significantly in MT. Under water withholding conditions, MT had higher malondialdehyde concentration than the mutants, but au had higher activities of catalase and ascorbate peroxidase compared to the other genotypes. The tolerance of mutants to the effects of water withholding may be explained by the higher activity of antioxidant enzymes in au and by a higher concentration of antioxidant compounds, such as carotenoids, in hp1.     

Ectopic expression of apple fruit homogentisate phytyltransferase gene (MdHPT1) increases tocopherol in transgenic tomato (Solanum lycopersicum cv. Micro-Tom) leaves and fruits

Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of tocopherols (vitamin E). Herein, an HPT homolog (MdHPT1) was isolated from apple (Malus domestica Borkh. cv. Fuji) fruits, whose gene expression level gradually decreased during fruit ripening, reaching a background level in ripened apple fruits. The amounts of α- and γ-tocopherols, two major tocopherols in plant organs, were 5- to 14-fold lower in the fruits than in the leaves and flowers of apple plants. Transgenic tomato plants (Solanum lycopersicum cv. Micro-Tom) overexpressing MdHPT1 were next constructed. Transgenic independent T(1) leaves contained ～1.8- to 3.6-fold and ～1.6- to 2.9-fold higher levels of α-tocopherol and γ-tocopherol, respectively, than those in control plants. In addition, the levels of α-tocopherol and γ-tocopherol in 35S:MdHPT1 T(1) fruits increased up to 1.7-fold and 3.1-fold, respectively, as compared to the control fruits, indicating that an increase in α-tocopherol in fruits (maximal 1.7-fold) was less evident than that in leaves (maximal 3.6-fold). This finding suggests that the apple MdHPT1 plays a role in tocopherol production in transgenic tomatoes.     

Vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity and waterlogging

High-density olive orchards are increasing around the world, many of which may be potentially affected by salinity and waterlogging (hypoxia), two important stresses common in irrigated fields in arid and semi-arid climates. However, the response of olive to these stresses under field conditions is not well established. Therefore, our objective was to evaluate the vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) grown in a spatially variable waterlogged, saline-sodic field. We monitored the growth in trunk diameter of 341, 3-year-old olives between September 1999 and September 2000. Field contour maps were developed delineating soil salinity (ECa), relative ground elevation (RGE) and water table depth (WTD). Soil samples were also collected and analyzed for ECe and SARe in order to characterize the salinity and sodicity profiles and develop the ECa-ECe calibration equation. The infiltration rate (IR) of the crusted and uncrusted soil and the penetration resistance (PR) were also measured. The field was characterized by spatially variable ECe (2–15 dS m^–1), SARe (3–40), RGE (–4 to +4 cm) and WTD (0.5–1.9 m, with corresponding ground water EC values between 12 and 6 dS m^–1). Steady-state IR of crusted soil was only 7% of the uncrusted soil. Since the field was heavily irrigated by flooding, waterlogging conditions were related to low RGE values. Soil salinity was negatively correlated (R ² = 0.83, P<0.001) with RGE (ponded water) and WTD (upward flux), due to the evapo-concentration of water and salts at the soil surface. Thus, inverted salinity profiles developed in high salinity areas. Fifty-five percent of the olives were dead 3.5 years after planted, and most of them were located in areas of high ECe (> 10 dS m^–1), low RGE (< – 1.5 cm) and low WTD (< 1.2 m). The surviving trees had vegetative salinity tolerance values of ECe threshold = 4 dS m^–1 and slope = –12% (i.e., percent decline per unit increase in ECe above the treshold), indicating that the Arbequina olive is moderately tolerant to salinity. The RGE and WTD thresholds for olive's survival were > 0.1 cm and > 1.6 m, respectively. Thus, very small changes in ground elevation had a significant effect on olive's survival or death. The coupled effects of salinity and waterlogging (hypoxia) stresses were most detrimental for olive's growth.     

Three-year field response of drip-irrigated grapevine (Vitis vinifera L., cv. Tempranillo) to soil salinity

The aim of this work was to evaluate the effects of Fe deficiency on the activity of several metabolic enzymes (PEPC, PK, PFK, G6PDH and G3PDH), along with the function of the antioxidant enzymes (SK, SDH and PAL) in two lines of Medicago ciliaris, TN11.11 and TN8.7. Plants were grown in a greenhouse under controlled conditions. After germination and pre-treatment, plants were transferred for hydroponic culture. Three treatments were used: 30 μM Fe (+Fe), 0 μM Fe (?Fe) and 30 μM Fe + 10 mM NaHCO₃ (+Bic.). Our results showed that all the enzymatic activities increased in extracts of Fe-deficient roots when compared to the control. The above increases in the activity were particularly evident for the bicarbonate-treated roots of TN11.11. PEPC activity was increased by 277% in TN11.11 plants with the addition of bicarbonate to the nutrient solution. Our results indicate also that, in the two lines of Medic, the activity of SK, SDH and PAL in leaves and roots were increased under Fe deficiency (either direct or induced by bicarbonate), to a greater extent in TN11.11 plants. Furthermore, a considerable increase in lipid peroxidation of roots and leaves of Fe-deficient plants was observed in TN8.7 when compared to TN11.11 plants. Our data suggest that the TN11.11 line is more effective in overcoming Fe deficiency than TN8.7. The tolerance of TN11.11 to Fe deficiency is related to its ability to modulate the carbohydrate metabolism and to increase secondary metabolism pathways.     

Three-year field response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity: Trunk growth and leaf ion accumulation

Irrigated olive is rapidly increasing in arid and semiarid areas, many of which may be negatively affected by soil salinity. We evaluated changes in trunk growth and leaf Cl^–, Na⁺ and K⁺ concentrations in young Arbequina olives (Olea europaea L.) grown in a saline-sodic field over a three-year period. The trunk diameter was measured at the beginning and the end of the 1999 (70 trees), 2000 (59 trees) and 2001 (42 trees) growing periods. Leaves, sampled in August of each year, were analyzed for Cl^–, Na⁺ and K⁺ concentrations. Soil salinity (apparent electrical conductivity, ECa) of each monitored tree was measured 14 times during the 1999–2001 experimental period with an electromagnetic sensor and converted to root zone electrical conductivity of the soil saturation extract (ECe) based on ECa–ECe calibration curves. Salinity tolerance was determined using the Maas and Hoffman threshold–slope response model. Based on salinity thresholds (ECe_thr), the tolerance of olive in terms of trunk growth was high in 1999 (ECe_thr = 6.7 dS m^–1), but declined with age and time of exposure to salts by 30% in 2000 (ECe_thr = 4.7 dS m^–1) and by 55% in 2001 (ECe_thr = 3.0 dS m^–1). Based on the high absolute slopes obtained in all years (values between 16% and 23% dS^–1 m), olive was classified as very sensitive to ECe values above the threshold. Trunk growth thresholds based on leaf ion concentrations varied, depending on years, between 2.6 and 4.0 mg g^–1 (Cl_thr) and between 1.0 and 1.2 mg g^–1 (Na_thr), indicating that Arbequina olive was less sensitive to leaf Cl^– and much more sensitive to leaf Na⁺ than values reported as toxic in greenhouse studies. Leaf K⁺ slightly decreased with increasing salinity, whereas the K⁺/Na⁺ ratio sharply decreased with increasing salinity. We concluded that the initial salinity tolerance of olive was high, but declined sharply with time of exposure to salts and became quite sensitive due primarily to increasing toxic concentrations of Na⁺ in the leaves.     

Effect of Gibberellin and Auxin on Parthenocarpic Fruit Growth Induction in the cv Micro-Tom of Tomato

The effect of applied gibberellin (GA) and auxin on fruit-set and growth has been investigated in tomato (Solanum lycopersicum L.) cv Micro-Tom. It was found that to prevent competition between developing fruits only one fruit per truss should be left on the plant. Unpollinated ovaries responded to GA₃ and to different auxins [indol-3-acetic acid, naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-D)], 2,4-D being the most efficient. GA₃- and 2,4-D-induced fruits had different internal morphology, with poor locular tissue development in the case of GA, and pseudoembryos development in the case of 2,4-D. Also, GA₃ produced larger cells in the internal region of the mesocarp (IM) associated with higher mean C values, whereas 2,4-D produced more cell layers in the pericarp than pollinated fruits. The smaller size of GA₃- compared with 2,4-D-induced fruits was due to them having fewer cells, only partially compensated by the larger size of IM cells. Simultaneous application of GA₃ and 2,4-D produced parthenocarpic fruits similar to pollinated fruits, but for the absence of seeds, suggesting that both kinds of hormones are involved in the induction of fruit development upon pollination. It is concluded that Micro-Tom constitutes a convenient model system, compared to tall cultivars, to investigate the hormonal regulation of fruit development in tomato.     

Metal accumulation and damage in rice (cv. Vialone nano) seedlings exposed to cadmium

The effect of exposure to increasing cadmium concentrations was analyzed in rice seedlings (cv. Vialone nano). The highest Cd accumulation was found in roots, mostly in the apoplastic environment. Cd taken up in cells led to an increase in sulfhydryl groups, the appearance of phytochelatins, and formation of electron-dense vacuolar inclusions. The metal-exposure inhibited root growth and also interfered with correct root morphogenesis, causing disordered division and abnormal and forward enlargement of epidermal and cortical cell layers in the apical region. Cd accumulation in shoots was lower than in roots. In leaf cells, there was neither a substantial increase in sulfhydryl groups nor the appearance of phytochelatins. Shoot growth was reduced and, differently from in roots, leaf cell enlargement was inhibited. Chloroplasts had lowered contents of chlorophyll and a reduced number of thylakoids, but underwent structural alterations only at the highest Cd concentration tested (250 μM). Photosynthetic activity was limited due to the curtailment of CO₂ availability caused by the greater resistance of Cd-exposed leaves. The damage suffered by seedlings worsened with the increase in Cd concentration, but was already evident at the lowest concentration examined (50 μM), showing that the cv. Vialone nano has a Cd-sensitivity higher than other rice cultivars.     

Suitability of mycorrhiza-defective mutant/wildtype plant pairs (Solanum lycopersicum L. cv Micro-Tom) to address questions in mycorrhizal soil ecology

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.     

Involvement of kinetin and spermidine in controlling salinity stress in mulberry (Morus alba L. cv. S1)

In mulberry (Morus alba L.) plants NaCl stress imposed through roots by irrigation during growth period decreased the net photosynthetic rate (NPR), physiological water use efficiency (pWUE), which ultimately reflected on the reduction of growth parameters and leaf yield. Foliar spray of kinetin and spermidine (both at 1 mM) on salinized plants reduced the detrimental effects of saline stress. Kinetin and spermidine sprayed plants increased the total chlorophyll, protein content, as well as leaf yield, but reduced the sugar and proline contents as compared to NaCl treated plants. Kinetin was more effective than spermidine in increasing NPR, pWUE and leaf yield both in nonsalinized and salinized condition.     

Developmental changes in tomato fruit composition in response to water deficit and salinity

Processing tomato ( Lycopersicon esculentum Mill. cv. UC82B) plants were subjected to moderate levels of water deficit and salinity (Na₂SO₄/CaCl₂) in sand culture. Fruit water content and the relative contributions of organic and inorganic constituents to fruit solute potential (_Ψ) and soluble solids content were determined throughout development. Fruit _Ψ averaged –0.63, –0.86 and –0.77 MPa in the control, salinity and water deficit plants, respectively. Reduced net water import and maintenance of solute accumulation, irrespective of water import, accounted for the reductions in _Ψ of stressed fruits. Mineral ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻ and SO^2-₄) contributed –0.31 MPa to _Ψ in salinized fruit, compared with –0.19 MPa in control and water deficit treatments. Changes in net carbon accumulation were not observed among treatments, despite considerable differences in fruit K⁺ status. Starch accumulation in immature fruit was increased and hexose accumulation was decreased by both salinity and water deficit. Maximum starch levels were negatively correlated with total fruit _Ψ, but were independent of fruit K⁺. Organic acid levels were generally higher throughout development in salinized plants, relative to control plants, and correlated with increased inorganic cation rather than anion accumulation in these fruits.     

Enhanced yield of tomato in response to 2-(3,4-dichlorophenoxy)triethylamine (DCPTA)

Application of the bioregulator, 2-(3,4-dichlorophenoxy)triethylamine (DCPTA) to tomato (Lycopersicon esculentum Mill. cv. Pixie) significantly increased the vegetative growth and fruit-yield productivities of mature plants. Compared with controls, application of 30 M (10 ppm) DCPTA as a pre-germination seed treatment increased the mean relative growth rates of leaves, stems, and roots, 37%, 16%, and 20%, respectively, during exponential growth. At fruit harvest, the dry weights of leaves, stems, and roots, and the harvestable yield of 30 M DCPTA-treated plants were doubled when compared with controls. Increases in both the total fruit number per plant and the size of individual fruits contributed to the improved yield of 30 M DCPTA-treated plants. The total soluble solids contents of mature fruits harvested from 30 M DCPTA-treated plants were increased 24%, and the lycopene and beta-carotene contents were doubled as compared with controls. These results indicate that DCPTA treatment does not adversely affect the balanced partitioning of photoassimilate to vegetative and reproductive sinks in tomato.     

The response of lazy-2 tomato seedlings to curvature-inducing magnetic gradients is modulated by light

Shoots of the lazy-2 mutant of tomato (Lycopersicon esculentum Mill., cv. Ailsa Craig) exhibit negative gravitropism in the dark, but respond positively gravitropically in (red) light. In order to test whether high-gradient magnetic fields (HGMFs) exert only ponderomotive effects on amyloplasts or affect other physiological processes, we induced magnetophoretic curvature in wild-type (WT) and lazy-2 mutant seedlings. Straight hypocotyls of 4-d-old plants were selected and the tips of their hooks were placed in an HGMF near the edge of a magnetized ferromagnetic wedge [grad (H²/2) ≈ 10⁹–10¹⁰ Oe²/cm] and mounted on a 1-rpm clinostat. After 4 h in the dark, 85% of WT hypocotyls and 67% of mutant hypocotyls curved toward the wedge. When the seedlings were exposed to red light for 1 h prior to and during the application of the HGMF, 78% of the WT seedlings curved toward the magnetic gradient, but the majority of the lazy-2 seedlings (75%) curved away from the stronger field area. Intracellular amyloplast displacement in the HGMF was similar for both varieties and resembled the displacement after horizontal reorientation. The WT showed a distinct graviresponse pattern depending on the orientation of the hook, even after excision of the apex. Application of HGMFs to decapitated hypocotyls resulted in curvature consistent with that obtained after horizontal reorientation. After light exposure, decapitated lazy-2 seedlings did not respond positively gravitropically. The data imply that the lazy-2 mutants perceive the displacement of amyloplasts in a similar manner to the WT and that the HGMF does not affect the graviresponse mechanism. The study demonstrates that ponderomotive forces due to HGMFs are useful for the analysis of the gravity-sensing mechanism in plants. Received: 31 August 1998 / Accepted: 6 October 1998     

Physiological and biochemical responses resulting from nitrite accumulation in tomato (Lycopersicon esculentum Mill. cv. Ibiza F1)

The sensitivity of hydroponically cultivated tomato (Lycopersicon esculentum Mill. cv. Ibiza F1) submitted to nitrite treatments (0.25-10mM KNO(2)) for 7d was studied. Increasing nitrite levels in the culture medium led to several disruptions of tomato plants, reflected by reductions of both dry matter per plant, chlorophyll concentrations and the appearance of chlorosis symptoms at the leaf surface. This behaviour was accompanied by stimulation of nitrite, nitrate and ammonia ion accumulation, mainly in roots and old leaves. Higher proteolytic and gaiacol peroxidase (GPX, EC. 1.11.1.7) activities and malonyldialdehyde content were also noted. Protein content of the different plant organs was decreased by nitrite treatment. These physiological and biochemical parameters were chosen as they are stress indicators. Taken together, our data partly explain the harmful effects of nitrite ions, when excessive in the culture medium.     

Hormones and young leaves control development of cotyledonary buds in tomato seedlings

Hormonal and plant factors regulating the development of the inhibited cotyledonary buds of Lycopersicon esculentum Mill. cv. `Fireball' seedlings were studied. Excision of the immature plumular leaves of 5- to 20- millimeter length significantly stimulated bud development after 2 to 4 days, but excision of leaves exceeding 20-millimeter length was without effect. Apical application of 20 microliters of 5 millimolar abscisic acid significantly promoted development of the cotyledonary buds after 6 days. A subapical ring of 0.1 millimolar concentration of 2,3,5-triiodobenzoic acid (TIBA) in lanolin significantly promoted cotyledonary bud development after 11 days. Twenty microliters of 0.1 millimolar 6-benzylaminopurine (BAP) applied directly to the cotyledonary bud loci significantly promoted bud development, but 1 micromolar gibberellin A_4/7 was ineffective. Application of 0.1 millimolar BAP in lanolin to the petiole or hypocotyl was ineffective. However, application of 0.1 millimolar TIBA as a ring around the petioles of the cotyledons or 1-centimeter on the hypocotyl below the cotyledons significantly promoted cotyledonary bud development.