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1

Electronic Resource

Structural and physiological changes in the roots of tomato plants over-expressing a basic peroxidase (2003)

Lucena, Miguel A. ; Romero-Aranda, Remedios ; Mercado, Jose A. ; [et al.]

Oxford, UK : Munksgaard International Publishers

Physiologia plantarum 118 (2003), S. 0

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ISSN: 1399-3054

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: Previous studies on the tomato (Lycopersicon esculentum Mill.) peroxidase TPX1, including the development of transgenic tomato over-expressing this gene, supported an involvement of this peroxidase in the synthesis of lignin and suberin. The transgenic plants showed a wilty phenotype at flowering, but the relationship between this role in ligno-suberization and this phenotype was not clear. In the present study a histological approach and the measurement of water-related parameters have been performed in order to obtain an insight into the origin of this phenotype. Clear differences between transgenic and non-transgenic roots were observed in the cross-sections of the basal root zones where secondary growth was evident. The diameter of the xylem vessel was diminished in the transgenic plants. Total area corresponding to xylem in the basal cross-sections decreased 3.9 fold in the transgenic roots. In addition, the radial and outer tangential walls of the exodermis cells were more ligno-suberized in transgenic than in non-transgenic plants. After fruit set, predawn and midday water potentials were lower in transgenic than in-non-transgenic plants. At midday, the stomatal conductance was also lower in the transgenic plants, 494±69 versus 594±60 mmol m−2 s−1. Root hydraulic conductances of the transgenic and non-transgenic plants were 1.4±0.38 and 3.47±0.19 g water min−1 MPa−1, respectively. The results obtained support that the phenotype is caused by the anatomical differences found in the transgenic roots. These differences would be the cause of a increased resistance to water flow in the roots that would negatively affect the water supply to the shoot and, as a consequence, resulted in a decreased water potential in the leaves.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1034/j.1399-3054.2003.00115.x

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2

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Changes in the water binding characteristics of the cell walls from transgenic Nicotiana tabacum leaves with enhanced levels of peroxidase activity (2004)

Mercado, José A. ; Matas, Antonio J. ; Heredia, Antonio ; [et al.]

Oxford, UK; Malden, USA : Munksgaard International Publishers

Physiologia plantarum 122 (2004), S. 0

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ISSN: 1399-3054

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: Pore size in the cell wall matrix may affect cell wall–water relations, particularly under osmotic stress. Cross linkage of plant cell wall matrix polymers is an important step in the formation of this structure and peroxidases have been proposed to catalyse the cross-linking of phenolic constituents. Transgenic tobacco (Nicotiana tabacum) plants expressing a basic tomato peroxidase gene (TPX2) showed increased apoplastic ferulic acid peroxidase activity in mature leaves. This enhanced activity was not associated with a decreased leaf growth. Differential scanning calorimetry (DSC) of control dried cell walls showed a putative glass transition, after Ca2+ removal, that was absent in the transgenic line. This would indicate that transgenic walls were more rigid. DSC analysis of water-hydrated cell wall preparations distinguished two pools of water, freezable and non-freezable water. The amount of non-freezable water, which corresponds to strongly bound water, was higher in the transgenic line (64 versus 55%). DSC thermograms of the transgenic cell wall were displaced to lower temperatures, and this may be interpreted as the result of a stronger interaction between this freezable water and this wall. Water sorption and desorption isotherms, obtained at relative humidity ranging from 5 to 93%, demonstrated the presence of very strongly bound water in the transgenic cell walls that was absent in controls. Water sorption–desorption hysteresis of the isotherms was evident in the control wall but not in the transgenic line. These changes in cell wall–water interaction seem to be relevant at the organ level because leaf discs of transgenic plants maintained higher relative water content than control discs, at water potentials between −1.05 and−2.31 MPa.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1399-3054.2004.00429.x

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3

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Biochemical and phenotypical characterization of transgenic tomato plants overexpressing a basic peroxidase (1999)

El Mansouri, Iman ; Mercado, José A. ; Santiago-Dómenech, Nieves ; [et al.]

Copenhagen : Munksgaard International Publishers

Physiologia plantarum 106 (1999), S. 0

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ISSN: 1399-3054

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: Tomato plants (Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1, under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5-fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild-type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild-type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild-type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild-type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1034/j.1399-3054.1999.106401.x

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4

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Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. (1996)

Mansouri, Iman ; Mercado, José A. ; Valpuesta, Victoriano ; [et al.]

Springer

Plant cell reports 15 (1996), S. 642-646

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ISSN: 1432-203X

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00232469

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5

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Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L. (1996)

El Mansouri, Iman ; Mercado, José A. ; Valpuesta, Victoriano ; [et al.]

Springer

Plant cell reports 15 (1996), S. 642-646

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ISSN: 1432-203X

Keywords: Abbreviations: BA: benzyladenine; IBA: indole-3-butyric acid; MS: Murashige and Skoog basal medium; LSD: least significant difference; NOS: nopaline synthase promoter, NPTII: neomycin phosphotransferase (EC 2.7.2.95); CaMV35S: cauliflower mosaic virus promoter; GUS: beta-glucuronidase (EC 3.2.1.31); LB: Luria Broth base; CTAB: hexadecil trimethyl ammonium bromide; PCR: polymerase chain reaction; X-gluc: 5-bromo-4-chloro-3-indolyl-glucuronide

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary. An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/l kanamycin and successfully acclimatized. The final percentag of transformation obtained based on beta-glucuronidase expression was 6.9%.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002990050090

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6

Electronic Resource

Pollen sporopollenin: degradation and structural elucidation (1999)

Domínguez, E. ; Mercado, José A. ; Quesada, Miguel A. ; [et al.]

Springer

Sexual plant reproduction 12 (1999), S. 171-178

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ISSN: 1432-2145

Keywords: Key words Pollen ; Exine ; Sporopollenin

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract We report the isolation of purified sporopollenin from pollen grains of different species and its complete solubilization. Exine from Pinus pinaster, Betula alba, Ambrosia elatior and Capsicum annuum was extracted by treatment with hydrogen fluoride in pyridine. These exines were purified from their aromatic moieties and from fatty acids linked by ester bonds using acidolysis and saponification treatments. The biopolymer obtained retains almost completely the shape of the original pollen grain. Fourier-transform infrared spectroscopy analysis of the isolated sporopollenin showed the absence of polysaccharide and phenolic material and the presence of carboxylic acid groups joined to unsaturations and ether linkages. Sporopollenin samples were successfully degraded by exhaustive 24-h ozonolysis at room temperature. Gentle ozonolysis (3 h at 0°C) did not completely degrade the biopolymer. The compounds obtained after exhaustive ozonolysis were analysed by gas chromatography-mass spectrometry. Dicarboxylic acids with a low number of carbon atoms were identified as major components of sporopollenin from P. pinaster, A. elatior and C. annuum, representing 28.8%, 63.2% and 88.5%, respectively, of the total compounds obtained. Fatty acids and n-alkanes also were identified in P. pinaster, A. elatior and B. alba sporopollenin. From the data obtained, an hypothesis about the chemical nature and structural arrangement of the sporopollenin is proposed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s004970050189

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7

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Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler (1998)

Barceló, Marta ; El-Mansouri, Iman ; Mercado, José A. ; [et al.]

Springer

Plant cell, tissue and organ culture 54 (1998), S. 29-36

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ISSN: 1573-5044

Keywords: Fragaria × ananassa ; plant transformation ; regeneration ; tissue culture

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1006031527413

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8

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Assessment of in vitro growth of apical stem sections and adventitious organogenesis to evaluate salinity tolerance in cultivated tomato (2000)

Mercado, José A. ; Sancho-Carrascosa, María A. ; Jiménez-Bermúdez, Silvia ; [et al.]

Springer

Plant cell, tissue and organ culture 62 (2000), S. 101-106

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ISSN: 1573-5044

Keywords: adventitious organ formation ; in vitro culture ; Lycopersicon esculentum ; mannitol ; sodium chloride

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The possible use of in vitro shoot morphogenesis and shoot apex culture to evaluate salt tolerance in cultivated tomato (Lycopersicon esculentum Mill.) has been analyzed, using two cultivars with similar salt tolerance, Pera and Hellfrucht frühstamm (HF). The effect of salt on shoot regeneration was studied by culturing leaf explants on media supplemented with 0, 43, 86, 129 and 172 mM NaCl. The presence of NaCl in the regeneration media at 86 mM strongly inhibited shoot regeneration in the cultivar HF, but not in Pera. However, the substitution of NaCl by mannitol, maintaining the same water potential in the culture media, decreased the regeneration percentage in Pera but did not affect HF. Shoot apices of both cultivars were also subcultured at 6-week intervals, for 4 subcultures, at the same NaCl concentrations as used in the previous experiment, and the shoot growth, leaf and root number, rooted shoot and shoot necrosis were recorded at the end of each subculture. Root formation was the parameter most affected by salt in both cultivars, Pera being more sensitive than HF. The substitution of NaCl by mannitol significantly increased the percentage of rooted shoots in Pera after four subcultures, and slightly decreased this percentage in HF. Shoot necrosis was only observed in the last subculture at NaCl higher than 86 mM, the percentage of necrotic shoots being higher in Pera than in HF (75% and 45%, respectively). The lack of agreement between the results obtained with the in vitro tests, e.g., adventitious shoot formation and growth of apical stem sections, suggests that this approach may not be a reliable tool to evaluate salt tolerance in cultivated tomato.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1026503603399

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