Iron-superoxide dismutase and monodehydroascorbate reductase transcripts accumulate in response to internode rubbing in tomato

A cDNA encoding an iron-superoxide dismutase (Fe-SOD) was isolated by RACE-PCR from a Lycopersicon esculentum cDNA library. The Fe-SOD cDNA consists of a 746-bp open reading frame and is predicted to encode a protein of 249 amino acids with a calculated molecular mass of 27.9 kDa. The deduced amino acid sequence was very similar to other plant Fe-SODs and a potential chloroplastic targeting was found. To study the induction of oxidative burst in response to mechanical stimulation, the accumulation of Fe-SOD and monodehydroascorbate reductase (MDHAR) mRNAs was analysed in response to young growing internode rubbing in tomato plants. Northern analyses show that Fe-SOD mRNA and MDHAR mRNA accumulated in tomato internodes 10 min after the mechanical stimulation. These results suggest that reactive oxygen species are early involved in the response of a plant to a mechanical stimulation, such as rubbing. The nucleotide sequence data reported in this paper will appear in the NCBI Nucleotide Sequence Databases under the accession number AY262025.     

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.     

In vitro growth and organogenesis of Prosopis farcta plantlets (Fabaceae, Mimosoideae) in culture medium supplemented with various concentrations of Ca++ and Na+

The objective of this study was to vary the mineral composition of the culture medium of Prosopis farcta seedlings per addition of Na+ and Ca++ ions with the aim to identify the culture media which support the growth and/or the expression of the in vitro plant organogenesis. The Na+ and Ca++ ions were added in the culture medium in various concentrations by taking the Gamborg medium, in which macroelements were diluted 10 times, as the basic one. After two months of culture, parameters relating to the vegetative development of plant seedlings and to the various expressions of organogenesis were measured. Weak concentrations in sodium and calcium ions as well as a weak concentration in Ca++ (0.1 mM) with 50 mM in Na+ support the best vegetative development of the plantlets. The most important percentage of plant seedlings presenting a bud initiation was obtained on a medium containing 0.1 mM of Na+ and 50 mM of Ca++. Our study defined several media likely to support in vitro development of Prosopis farcta plantlets allowing the selection of salt tolerant plants or cellular lines. Some other media were chosen for improving micropropagation of the species without adding growth substances.     

Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption.

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)(2)D(3). Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm(2) bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.     

Geography and host specificity: two forces behind the genetic structure of the freshwater fish parasite Ligula intestinalis (Cestoda: Diphyllobothriidae)

Parasite species with global distributions and complex life cycles offer a rare opportunity to study alternative mechanisms of speciation and evolution in a single model. Here, genealogy and genetic structure, with respect to geography and fish host preference, have been analyzed for Ligula intestinalis, a tapeworm affecting freshwater fish. The data analyzed consisted of 109 tapeworms sampled from 13 fish host species in 18 different localities on a macrogeographic scale. Two mitochondrial genes, cytochrome oxidase subunit I and cytochrome B, and the nuclear sequence of intergenic transcribed spacer 2 (ITS2) were used for the genetic reconstruction. Different evolutionary patterns were found at the local and at the global geographic scales. On a local scale, the flat genetic structure was mainly attributed to contiguous range expansion. Migrating birds are the most likely cause of the homogenisation of the whole population, preventing the creation of significant genetic barriers. By contrast, on a global scale, genetically distant and well-separated clusters are present in different geographic areas. Reproductive isolation was found even between clades living in sympatry and infecting the same definitive host, suggesting the existence of efficient biologically determined genetic barriers, and thus possibly separate species. Although the ITS2 sequences were found to display considerable intragenomic variability, their relationships were generally in good agreement with the topology derived from mitochondrial genes.