The effect of temperature on salt uptake by tomato plants with diurnal and noctural waterlogging of salinized rootzones

Summary Tomato plants were grown at three constant temperatures (10, 20 and 28°C) with drained or waterlogged rootzones and were irrigated with saline solution (0.09M NaCl).Each increase in temperature resulted in an increase in leaf Na-ion and Cl-ion concentrations in plants grown with drained rootzones. However, with plants grown with waterlogged rootzones, maximum leaf concentrations of Na-ions and Cl-ions occurred at 20°C.At 10°C there were no differences between Na-ion and Cl-ion concentrations for drained or waterlogged treatments. At 20 and 28°C, waterlogging of the rootzone resulted in significantly higher concentrations of Na-ions and Cl-ions in leaf and stem tissues than occurred with drained rootzones.There were no differences in Na-ions and Cl-ions and Cl-ions in plant tops if plants were waterlogged with saline solution during the day or night.Transpiration increased significantly with each increase in temperature but showed no other treatment dependent responses.     

Effect of short- and long-term salinity on the activities of antioxidative enzymes and lipid peroxidation in tomato roots

Changes in the antioxidative enzyme activities (SOD, CuZnSOD, GSH-Px, GST), as well as TBARS content in 5-week-old tomato (Lycopersicon esculentum Mill. cv “Perkoz”) roots were examined 1, 3 h (short-term stress) and 1–14 days (long-term stress) after a single application of 50 mM (mild stress) and 150 mM NaCl (severe stress). The severe stress caused an increase in GST, GSH-Px and SODs activities from the beginning of the experiment while mild stress induced enhancement of GST activity from the second day of experiment. The maximum increase in SODs after both NaCl solutions were applied and in GST activity after the higher NaCl dose on the second day of the experiment was observed. Moreover, after 1 h of NaCl treatment with both tested NaCl solutions, the highest induction of GSH-Px activity appeared. TBARS content was elevated from the first hour of salt stress and decreased only 14 days after 50 mM NaCl application which was accompanied by high induction of GSH-Px activity. In conclusion, enhanced activities of tested enzymes indicate their involvement in early and late defence systems under salinity stress. Moreover, the dynamics of the changes in the antioxidant enzymes suggests that the second day following NaCl application is a crucial moment of the experiment with regard to salt-mediated oxidative stress.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

Temperature-induced modifications in the surface features of stamens of a tomato mutant: An SEM study

Summary Stamenless-2 (sl₂/sl₂) is a temperature-sensitive mutant of tomato (Lycopersicon esculentum) which exhibits altered stamen development under different temperatures (Sawhney 1983). By using scanning electron microscopy, this study was conducted to investigate the differentiation of surface features of mutant and normal stamens grown under different temperatures, with the view to further determine the role of temperature in gene expression in stamen development. Mutant stamens grown under intermediate temperatures (23 °C day/18 °C night) differed from the normal in hair production, the shape of epidermal cells and in the pattern of cuticular thickenings. Under low temperatures (18 °C day/15 °C night), all surface features of mutant stamens closely resembled the normal, whereas under high temperatures (28 °C day/23 °C night), the patterns and types of hairs, epidermal cells, stomata, and cuticular thickenings on mutant stamens were similar to that of a gynoecium. The staminal features of normal stamens were not affected by different temperatures. This study shows that the expression of the sl₂/sl₂ allele is influenced by temperature conditions to the extent that the pattern of cellular differentiation characteristic of either the stamens or the carpels can be induced in mutant stamens.     

Totipotency of tomato protoplasts

Summary An efficient and reliable protocol for tomato protoplast isolation, culture, and plant regeneration has been developed. Fourteen diverse cultivars were tested. Fertile plants were regenerated from all 14 cultivars without any modification in the protocol. Plating efficiency (percentage of the protoplasts that formed minicalli) of up to 50% was achieved. Those mini-calli rapidly regenerated shoots at high frequencies. Regenerated shoots can be easily rooted on a basal medium with the appropriate auxin, and have been set to soil within two months after the isolation of the protoplasts.     

Accumulation of cell wall-bound phenolic metabolites and their upliftment in hairy root cultures of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

Alkaline hydrolysis of cell wall material of tomato hairy roots yielded ferulic acid as the major phenolic compound. Other phenolics were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. The content of phenolics was much higher at the early stage of hairy root growth. The ferulic acid content decreased up to 30 days and then sharply increased to 360 microg/g at 60 days of growth. Elicitation of hairy root cultures with Fusarium mat extract (FME) increased ferulic acid content 4-fold after 24 h. As the pathogen-derived elicitors have specific receptors in plants, FME may thus be used for inducing resistance against Fusarium oxysporum f. sp. lycopersici.     

Polyploidy in tomato roots as affected by arbuscular mycorrhizal colonization

Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.     

Ultrastructure of the tapetal cell wall in the stamenless-2 mutant of tomato (Lycopersicon esculentum): correlation between structure and male-sterility

Summary In the stamenless-2 (sl-2) mutant of tomato (Lycopersicon esculentum Mill.), the breakdown in microsporogenesis corresponded with various abnormalities in the ultrastructure of the tapetal cell wall. In some mutant anthers, the inner tangential wall was excessively loosened allowing the passage of tapetal cell wall material and cytoplasmic contents into the anther locule. This presumably altered the osmoticum of the locule and resulted in plasmolysis of the microspores. Membranous fragments commonly observed in the normal tapetal cell wall, and presumed to have a role in transfer of materials from the tapetum to microspores, were absent from thesl-2 mutant. This was associated with reduced transfer of materials, such as lipids, to the developing pollen grains. In addition, a lining of sporopollenin-like deposits that coated the inner tangential wall of the normal tapetum, was discontinuous in the mutant. In mutant anthers where the tapetal cell wall did not loosen, the transfer of all materials was restricted and this resulted in the collapse of sporogenous material.     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.     

Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells

The medium of tomato (Lycopersicon esculentum) cells adapted to grow in the presence of 15 g l^–1 NaCl had a higher peroxidase activity than the medium of an unadapted tomato cell line. When the adapted cells were cultured in a medium without NaCl, the value found for peroxidase activity was intermediate. The increase in peroxidase activity was parallel to an increase of lignin-like compounds in the cell walls, as well as to an increased content or appearance of neutral and basic peroxidase isoenzymes. Apparently, the high values of peroxidase activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process.Abbreviations LO Control tomato cell line unable to grow in the presence of 15 g 1^–1 of NaCl - L15 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in this salt concentration - L15-0 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in the absence of this salt - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PBS phosphate buffer saline     

Comparison of the response to aluminum toxicity in gametophyte and sporophyte of four tomato (Lycopersicon esculentum Mill.) cultivars

Summary We tested pollen from four tomato cultivars differing in sensitivity to aluminum in the sporophyte to determine if Al sensitivity was also expressed in pollen. Pollen sensitivity to Al was measured by the ability to germinate and grow in a control solution after a short period in a high concentration of Al. The response was ranked and compared to the Al sensitivity ranking of the four cultivars based on top growth in Al toxic soil. In addition, seedlings from the most and least sensitive cultivars, based on pollen germination, were compared for Al sensitivity in nutrient solutions. Treatment with Al significantly reduced pollen germination in the two more sensitive cultivars, but not in the two more resistant cultivars. However, the ranking was not the same as that based on the shoot growth of the sporophyte. Root growth as a criterion of sporophytic Al sensitivity produced results similar to pollen germination. The study suggests that although the correspondence is better for some phenotypic responses of the sporophyte than others, Al tolerance appears to be another character expressed in both pollen and sporophyte.     

A genetic analysis of a tomato (Lycopersicon esculentum) genotype with a high frequency of twin spots

Among pale-green tomato plants heterozygous for the xanthophyllic2 (xa-2) mutation that were transformed with a T-DNA harbouring the NPTII and GUS gene, a plant with a high frequency of green/white twin spots was found. The genetic analysis of this plant indicated that the occurrence of these twin spots was caused by a genetic defect located at the distal end of chromosome 10S, where xa-2 also is located. The genetic analysis of green plants regenerated from leaf expiants of this twin-spot plant revealed that the green sectors derive from non-disjunction of the xa-2 ⁺ allele. In an analysis of mitotic chromosome behaviour bridges were observed in approximately 5% of the anaphases, providing arguments that a breakage-fusion-bridge cycle caused by a tissue culture-induced genomic instability is the most likely cause of this aberrant behaviour of chromosome 10.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g^–1 dry wt to 1 mg g^–1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml^–1 for 4 days, the maximum production of artemisinin reached to 13 mg l^–1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.     

A defective pollen-pistil interaction contributes to hamper seed set in the<Emphasis Type="Italic"> parthenocarpic fruit</Emphasis> tomato mutant

In the parthenocarpic fruit (pat) tomato mutant, parthenocarpy is associated with partial aberrations of stamens (shortness and carpelloidy) and ovules (defective integument growth) that contribute to impair seed set. However, these do not seem to be the only reasons for seed infertility because hand-pollination fails to restore seed set in ovaries where a fraction of the ovules are still morphologically normal. Therefore, it is conceivable that other unreported defects occur during the reproductive process in the mutant. In this research, we show that the mutation does not affect pollen or embryo sac development and viability, but generates sporophytic effects that reduce seed production and seed size. While pollen germination and stylar growth were normal in mutant pistils, fertilization does not take place because of abnormalities in the pollen tube-ovary interaction in this genotype. Inside the ovary of pat plants, pollen tubes appeared to be disorientated; they wandered about in the ovarian cavity and often lost their adherence to the placental surface. Interestingly, in pat ovaries fertilization was strongly impaired even in those ovules that appeared normal. It may be that apparently 'normal' ovules cannot guide pollen tubes to their micropyle in the altered pat ovary because adhesion molecules are not properly arrayed on a placenta that is already preparing for cell division or, alternatively, chemotropic signals in the pat ovary may be altered by the presence of aberrant ovules, which are not simply devoid of attractivity, but disrupt pollen tube guidance overall.