Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lycopersicon esculentum Mill.)

The apoplasmic and symplasmic iron pools were determined in roots and leaves of Lycopersicon esculentum Mill. cv. Bonner Beste and its mutant chloronerva. The mutant is auxotrophic for the ubiquitous plant constituent nicotianamine (NA) and exhibits an impaired iron metabolism. Formation of apoplasmic iron pools in roots was dependent on the iron source in the nutrient solution. With Fe-ethylenediaminedi-(2-hydroxyphenylacetate) (FeEDDHA) only a very small apoplasmic iron pool was formed in the roots of both genotypes. Plants grown with FeEDTA increased their apoplasmic iron pool with increasing exogenous iron concentrations in the nutrient solution. The size of the apoplasmic pools in roots did not differ between the wild-type and the mutant (about 85 mol Fe · g^–1 DW). By contrast, the symplasmic iron concentrations in roots and leaves of the mutant were significantly higher when compared to the wild-type. An exogenous NA supply to the leaves of the mutant reduced the high symplasmic iron concentrations to the level of the wild-type. Mutant leaves exhibited a gradient of symplasmic iron concentrations depending on the developmental age of the leaves. The oldest leaves contained considerably more symplasmic iron than the youngest. The results demonstrate that the apparent iron deficiency of the mutant is not the consequence of an impaired iron transport from the apoplasm to the symplasm. Therefore, it is concluded that NA is not required for the transport of Fe(II) through the plasmalemma into the cell.Abbreviations BPDS bathophenanthroline disulfonic acid, Na₂ salt - FeEDDHA ferric N-N-ethylenediaminedi-(2-hydroxy-phenylacetate) - NA nicotianamine Part 40 in the series The normalizing factor for the tomato mutant chloronerva. For part 39 see Pich et al. (1991)The valuable technical assistance of Mrs. Christa Kallas and Mr. Günter Faupel is gratefully acknowledged.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Nuclear replication activities during imbibition of abscisic acid- and gibberellin-deficient tomato (Lycopersicon esculentum Mill.) seeds

The role of cis-abscisic acid (ABA) and gibberellins (GAs) in the induction of cell-cycle activities has been studied during imbibition and subsequent germination of tomato seeds. Using flow cytometry, nuclear replication activity was investigated in embryo root tips isolated from seeds of the ABA-deficient mutant sit ^w , the GA-deficient mutant gib-1, and the wild-type (MM) tomato (Lycopersicon esculentum Mill. cv. Moneymaker) upon imbibition in water, 10 μM GA₄₊₇, 5 μM ABA or 5 μM ABA+10 μM GA₄₊₇. The nuclei of fully matured dry MM, sit ^w and gib-1 seeds predominantly showed 2C DNA signals, indicating that the cell-cycle activity of most root-tip cells had been arrested at the G₁ phase of nuclear division. However, ABA-deficient sit ^w seeds contained a significantly higher amount of G₂ cells (4C DNA) compared with the other genotypes, suggesting that, during maturation, cell-cycle activity in sit ^w seeds is less efficiently arrested in G₁. Upon imbibition in water, an induction of the 4C signal, indicating nuclear replication, was observed in the root tip cells of both MM and sit ^w embroys. The augmentation in the 4C signal occurred before visible germination. Gib-1 seeds did not show cell-cycle activity and did not germinate in water. Upon imbibition in GA₄₊₇, both cell-cycle activity and subsequent germination were enhanced in MM and sit ^w seeds, and were induced in gib-1. In ABA, the germination of MM and sit ^w seeds was inhibited while nuclear replication of these seeds was not affected. It is concluded that GA influences germination by acting upon processes that precede cell-cycle activation, while ABA affects growth by acting upon processes that follow cell-cycle activation.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Physiological response and yield of paclobutrazol treated tomato plants (Lycopersicon esculentum Mill.)

Experiments were conducted to study the physiologicaleffect of the plant growth retardant paclobutrazol(PBZ) and its impact on the yield of tomato plants(cv. Precador). Seedlings were treated at the time of prickingout with soil and foliar applications of PBZ atconcentrations of 1.0 and 25.0 mg l^-1respectively. The results established that:-- The reduced height and the increased thickness ofthe young plant stem, as well as the accelerated rootformation are a significant advantage of the PBZtreatment, contributing to the improvement of seedlingquality at planting.-- Soil treatment (1 mg l^-1) and foliar treatment(25 mg l^-1) with PBZ improves the photosyntheticactivity and water balance of tomato cv. Precador.-- PBZ accelerates fruit formation and increases earlyfruit yield.-- The concentrations of the retardant used and themode of its application ensure the production offruits without any residual retardant and harmless tohuman health from a phytosanitary point of view.     

Principal directions of growth and the generation of cell patterns in wild-type and gib-1 mutant roots of tomato (Lycopersicon esculentum Mill.) grown in vitro

The patterns of cell growth and division characteristic of the apex of tomato roots grown in vitro were simulated by computer using a growth tensor (GT). The GT was used to clarify the basis of the altered cell patterns found within apices of roots whose gibberellin levels had been depressed by mutation (at the GIB-1 locus) or through application of the gibberellin-biosynthesis inhibitor, 2S,3S paclobutrazol. At the pole of wild-type roots, where the cell files of the cortex converge, there are commonly only one or two tiers of cortical cells sandwiched between the pole of the stele and the cap initials. By contrast, root apices of the gib-1 mutant contain additional tiers in this region. The development of these additional tiers is suppressed when roots of the mutant are grown in the presence of gibberellic acid (GA₃), but could be induced in wild-type roots when they are grown in 2S,3S paclobutrazol. The wild-type cell pattern can be simulated using the GT and by the application of appropriate rules that govern cell growth and division. The induced variations in cell pattern are interpreted as being due to displacements, within the apex, of the principal directions of growth (PDGs), which are represented, in part, by the set of periclines and anticlines seen in the cell wall network; these, in turn, are utilized in the specification of the GT. During normal (wild-type) root growth, the PDGs maintain a stable pattern and the corresponding cell pattern is also stable. However, in order to interpret the cellular behaviour found in wild-type roots grown in 2S, 3S paclobutrazol, simulation using the GT shows that, if the pattern of PDGs is destabilized and displaced distally along the root axis, the cell pattern reorganizes into that typical of gib-1 mutant roots. Conversely, the cell pattern of gib-1 roots, which reverts to wild-type upon exposure to GA₃, can be simulated if the PDGs are displaced proximally to the inside of the apex whereupon the number of cortical tiers at the root pole decreases. These results suggest a link between endogenous gibberellin level and the specification of the PDGs in the growing tomato root apex. Furthermore, the evidence of cell patterns from gib-1 roots suggests that, in order to achieve stability of PDGs with concomitant stable cellular patterning, an optimal gibberellin level is necessary. In practice, this can be attained by culturing the mutant roots in medium containing 1 M GA₃.Abbreviations GA₃ gibberellic acid - GT growth tensor - NCS natural coordinate system - PDG principal direction of growth - QC quiescent centre - RERG relative elemental rate of growth We are grateful to the former Agricultural and Food Research Council for financial support under the International Scientific Interchange Scheme to enable J.N. to work at Long Ashton Research Station, and to K. Kurczyski (Silesian University, Katowice, Poland) for help in writing a computer program for cell proliferation. Preparation of the model for growth and division was supported in part by a grant from the Committee for Scientific Research, Poland.     

In vivo and in vitro investigation of salt tolerance in three anthocyaninless mutants in tomato (Lycopersicon esculentum Mill.)

Growth responses of a tomato cultivar Ailsa Craig and the ah-, aw- and bls-isogenic/near isogenic lines (IL/NIL) from it were evaluated and compared at cotyledons stage under salt treatment in vivo and in vitro experiments. No differences in hypocotyl and root growth responses were detected between the anthocyanin-containing and the anthocyaninless lines within the in vivo experiments. The anthocyaninless mutants, (except in some cases the bls mutant), exhibited higher callogenic and shoot-forming capacity on both, control and salinized media. It was concluded that for this reason it would be difficult to determine the relationship between the in vivo and in vitro responses of the lines studied and as well as to evaluate the usefulness of the in vitro method in testing these lines for salt tolerance.     

Functional male sterility in tomato (Lycopersicon esculentum Mill.) and its application in hybrid seed production

Advantages and disadvantages in using functional male sterility (positional sterile — ps, positional sterile 2 — ps 2, and excerted stigma — ex) in tomato hybrid seed production and attempts to elaborate systems for their more efficacious use in breeding were discussed in this review. It was concluded that the application of one of these types of sterility, (ps 2) in practice, although in a limited number of countries, showed the functional male sterility in tomato was a potential not to be underestimated in developing approaches that aimed at reducting the time and cost associated with hybrid seed production.     

The distribution and utilization of calcium by two tomato (Lycopersicon esculentum Mill.) lines differing in calcium efficiency when grown under low-Ca stress

Two lines of tomato (Lycopersicon esculentum Mill.) representing extremes in utilization effciency of absorbed Ca were studied to detect internal differences in Ca transport and distribution and factors responsible for strain differences in susceptibility to low Ca-stress. Differences in efficiency of Ca use were expressed as CaER (mg of dry weight produced for each mg of Ca absorbed by the plant).Ca-efficiency in line 113(E) appeared to be associated with a slow continuous movement of absorbed Ca, allowing for continued growth of the shoot apex and upper lamina under Ca-deficiency conditions. In the inefficient line 67(I), in contrast, Ca was rapidly deposited in the lower leaves with little upward movement in the plant after absorption.Fractionation of tissue Ca into various chemical forms suggested that Ca inefficiency also was associated with higher concentrations of insoluble Ca in the shoot tissue. The efficient line, although sustaining growth at lower levels of Ca, was capable of maintaining a higher ratio of soluble to insoluble Ca in all shoot tissues.Calcium was concentrated in the lower plant tissues of the inefficient strain, limiting its availability for continued shoot growth.Autoradiographs of lines fed⁴⁵Ca during the final 8 days of a 24-day experiment suggested that upward movement was sustained in line 113(E), in spite of vastly reduced transpiration rates and a root system characterized by leakage of K ions from the roots back into the solution.     

Increase in the rate of recombinants in tomato (Lycopersicon esculentum L.) after in vitro regeneration

Summary Modification to the cross-over (C. O.) rate of tomato (Lycopersicon esculentum) was attempted by using in vitro plant regeneration. F1 hybrids with the same genetical homozygous background were compared at two loci: bs-ms32 on chromosome I, and aa-d on chromosome II. For each, the genetic distance separating the two markers was about 20 to 30 map units. One cotyledon of each F2 hybrid seedling was used as in vitro tissue culture material, while the rest of the plantlet was grown as a control. Recombination rates of the selfed progenies from each regenerated and matched control couple were compared. For the first set of markers 59,000 seeds were analysed (5 controls' and 7 regenerated progenies), and for the second, 11,000 (5 controls' and 8 regenerated progenies). There were significant increases in the genetic distance between markers in about half the regenerated individuals. For the first set the increases ranged from 6.07 to 6.91 units out of a control distance of the 19.84 to 25.65, corresponding to lengthenings of 30.59 to 35.29%. For the second set they ranged from 4.92 to 6.04 out of a control distance of 25.05 to 26.57, representing increases of 19.64 to 22.75%. Such a phenomenon can be important either from a fundamental or practical viewpoint, regarding selection efficiency in plants, and potential for gene reassortment.The experiment reported in this paper has been submitted by M. Biglary in partial fulfillment of the requirements for Docteur-Ingénieur degree. (Nov. 1982, Laboratoire d'Amélioration des Plantes, Université Paris-Sud, F-91405 Orsay)     

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.     

Effect of salinity on tomato (Lycopersicon esculentum Mill.) during seed germination stage

A study was conducted using ten genetically diverse genotypes along with their 45F₁ (generated by diallel mating) under normal and salt stress conditions. Although, tomato (Lycopersicon esculentum Mill.) is moderately sensitive to salinity but more attention to salinity is yet to be required in the production of tomato. In present study, germination rate, speed of germination, dry weight ratio and Na⁺/K⁺ ratio in root and shoot, were the parameters assayed on three salinity levels; control, 1.0 % NaCl and 3.0 % NaCl with Hoagland’s solution. Increasing salt stress negatively affected growth and development of tomato. When salt concentration increased, germination of tomato seed was reduced and the time needed to complete germination lengthened, root/shoot dry weight ratio was higher and Na⁺ content increased but K⁺ content decreased. Among the varieties, Sel-7 followed by Arka Vikas and crosses involving them as a parent were found to be the more tolerant genotypes in the present study on the basis of studied parameters.     

New RAPD markers of tomato spotted wilt virus (TSWV) resistance in Lycopersicon esculentum Mill

The selection of TSWV resistant individuals can be facilitated by molecular markers. RAPD analysis was carried out on three forms (Stevens × Rodade — resistant; Rey de los Tempranos — moderately tolerant; Potentat — susceptible) with the use of 271 primers. Out of 271 primers 28 generated stable polymorphism and so they were tested for linkage to resistance gene. Bulk segregant analysis (BSA) was applied to F₂ segregating progeny developed from resistant × susceptible parents. As a result, 5 primers enabling us to distinguish between resistant and susceptible forms were detected. Only one of them had previously been reported by Chague et al. (1996). The analysis should be repeated on a larger population to confirm the results obtained.     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.