Mono-carboxylic acids and their salts inhibit wound-induced phenolic accumulation in excised lettuce (Lactuca sativa) leaf tissue

Wounding, as during excision and preparation of lettuce ( Lactuca sativa L.) leaf tissue for salads, induces the synthesis and accumulation of phenolic compounds that participate in subsequent reactions that cause tissue browning. Exposure of excised 5-mm mid-rib segments of romaine lettuce leaf tissue to vapors of mono-carboxylic acids or aqueous solutions of mono-carboxylic acids or their salts inhibited wound-induced phenolic accumulation (WIPA) and subsequent tissue browning. The decline in phenolic content followed a quadratic curve with increasing concentration, reaching a maximum inhibition after 60 min of 74 ± 8% for 50 m M sodium acetate (2 carbons, C2) and 91 ± 4% for 20 m M sodium decanoate (capric acid, C10). Respiration (i.e. carbon dioxide production) was unaffected by concentrations of formic, acetic, or propionic acids that reduced wound-induced phenolic content or that increase ion leakage from the tissue into an isotonic mannitol solution. However, WIPA was suppressed up to 70% at concentrations (20 m M acetate) that did not increase ion leakage over that of water controls. Various acetate salts (i.e. ammonium, calcium, magnesium, and sodium) all produced the same level of inhibition. The effectiveness of the compounds increased with increasing number of carbons in the molecule from 1 to 10, and was unaffected by whether the carbons were a straight chain or branched or whether the treatment was delayed by up to 6 h. The effectiveness of butyrate (C4) in reducing WIPA (27% reduction at 20 m M ) was less than that predicted from the response of the two adjacent mono-carboxylates similarly applied: propionate (C3) (62%) and valerate (C5) (73%). It appears that, unlike the n-alcohols, mono-carboxylates are not interfering with the synthesis or propagation of a wound signal but are interfering with subsequent steps in the production and accumulation of wound-induced phenolic compounds.     

Direct isolation of vacuoles from leaf tissue of lettuce (Lactuca sativa) retaining protoplasts within the leaves

A procedure is described in which vacuoles are isolated from leaf tissue of lettuce ( Lactuca sativa L.). After incubation in an enzyme solution, the vacuoles are directly extracted from the leaf tissue by osmotic shock using a phosphate buffer. In this method no protoplasts are released from the leaf tissue. This procedure avoids the problems of separating vacuoles from protoplasts with similar density. To evaluate the purity of the vacuoles, the activity of glucan synthetase 11 (EC 2.4.1.34), NAD(P) H-cytochrome c reductase (EC 1.6.99.3) and malate dehydrogenase (EC 1.1.1.37) was measured. To measure vanadate- and nitrate-sensitive ATPase activity (EC 3.6.1.8) vesicles were prepared from the vacuoles and ATP-dependent vesicle acidification was measured as acridine orange quenching. Nitrate inhibited the quenching, while addition of vanadate had no effect. It was concluded that the vacuoles were not contaminated with plasma membranes. To evaluate the viability of the vacuoles [¹⁴C]-malate uptake was measured. The vacuoles showed a constant rate of [¹⁴C]-malate uptake during 45 min. This rate was maximal at pH 6.8.     

Propagation of lettuce (Lactuca sativa) breeding material by tissue culture

A tissue culture method using Murashige and Skoog's (MS) medium was devised to propagate healthy plants from field grown lettuce plants selected for seed production. Explants (2–3 mm long) from axillary buds were successfully grown on MS + 1.0 or 2.0 mg litre^-1 kinetin and 6.4 mg litre^-1 IAA to promote shoot growth. Concentrations of 0.5 and 4.0 mg litre^-1 kinetin gave poor shoot growth. The cultures were successfully rooted after 3–4 wk on MS + 6.4 mg litres^-1 IAA after transfer from MS + 1.0 mg litre^-1 kinetin and on MS + 4.8 mg litre^-1 IAA after transfer from MS + 2.0 mg litre^-1 kinetin. Concentrations of 3.2 and 8.0 mg litre^-1 IAA gave poor root initiation. Root initiation was more successful when cultures were grown at 40 Wm^-2 than in cultures grown at 5 Wm^-2. Rooted cultures were established in compost with a 90–95% success rate and the regenerated plants flowered c. 18 wk after the cultures were initiated.     

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R₂ seedlings on media containing G418.Abbreviations IAA indole acetic acid - KIN kinetin - BA benzyladenine - NOS nopaline synthase - NPTII neomycin phosphotransferase II - RMNO tobacco nutrient medium (Marton and Maliga, 1975) - SH Shenk & Hildebrandt nutrient medium (Shenk & Hildebrandt, 1972; Michelmore and Eash, 1985) Present address: Agriculture Canada, P.O. Box 457, St. Jean-sur-Richelieu, Quebec, Canada, J3B 6Z8     

Transactivation of Ds elements in plants of lettuce (Lactuca sativa)

The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R₁ plants containing Ds and three independent R₁ plants expressing transposase. The excision of Ds in F₁ progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F₁ families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F₁ families suggests that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F₁ plants containing both Ds and the transposase were grown to maturity and the F₂ seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F₂ progeny; however, evidence for germinal excision was observed in only one F₂ family.     

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

Temperature sensitive phases during the germination of lettuce (Lactuca sativa) seeds

Lettuce seeds cvs Hilde, Feltham King and Avoncrisp were subjected, at different phases during imbibition at 22°C, to a high temperature (33°C) inhibitory for germination, for periods ranging from 4 to 144 h, before returning them to 22°C. The results showed, that the first 4h of imbibition and also the phase between the commencement of mitosis and the onset of radicle emergence were more sensitive to the effects of high temperature than other phases in the germination process. Short exposures (8–24 h) to 33°C commencing at the latter phase delayed germination by up to 4 days, and at the earlier by up to 8 days. Percentage germination was unaffectd except after prolonged exposures (> 48 h) from the beginning of imbibition, which reduced it. Seedling emergence from moist sieved soil was both delayed and reduced when imbibing seeds were exposed for a short period from the beginning of imbibition to 33°C compared with seeds imbibing continuously at 19°C. Germination was delayed and not reduced when seed was exposed to 33°C at the phase between commencement of mitosis and the onset of radicle emergence.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.     

Germination and growth of lettuce (Lactuca sativa) at low atmospheric pressure

The response of lettuce ( Lactuca sativa L. cv. Waldmann's Green) to low atmospheric pressure was examined during the initial 5 days of germination and emergence, and also during subsequent growth to vegetative maturity at 30 days. Growth took place inside a 66-l-volume low pressure chamber maintained at 70 kPa, and plant response was compared to that of plants in a second, matching chamber that was at ambient pressure (approximately 101 kPa) as a control. In other experiments, to determine short-term effects of low pressure transients, plants were grown at ambient pressure until maturity and then subjected to alternating periods of 24 h of low and ambient atmospheric pressures. In all treatments the partial pressure of O₂ was maintained at 21 kPa (approximately the partial pressure in air at normal pressure), and the partial pressure of CO₂ was in the range 66.5–73.5 Pa (about twice that in normal air) in both chambers, with the addition of CO₂ during the light phase. With continuous exposure to low pressure, shoot and root growth was at least as rapid as at ambient pressure, with an overall trend towards slightly greater performance at the lower pressure. Dark respiration rates were greater at low pressure. Transient periods at low pressure decreased transpiration and increased dark respiration but only during the period of exposure to low pressure. We conclude that long-term or short-term exposure to subambient pressure (70 kPa) was without detectable detriment to vegetative growth and development.     

Intracellular localization of sucrose-Phosphate Phosphate in photosynthetic cells of lettuce (Lactuca sativa)

The intracellular localization of sucrose-phosphate phosphatase (SPP) in photosynthetic cells of lettuce ( Lactuca sativa ) was investigated by using protoplasts isolated directly from leaf tissue. No SPP activity was detected in either vacuolar, chloroplast or mitochondrial preparations. The recovered activity of SPP was found in the cytosolic fractions at proportions parallel with those of the cytoplasmic marker alcohol dehydrogenase. Maximal activity was measured between pH 6. 0 and 7. 0. The data demonstrate a cytosolic location for SPP. Neither sucrose nor any other saccharide tested at 100 m M was a strong inhibitor of the enzyme, which was inactive in the presence of 35 m M ethylenediammetetraacetic acid.     

Vesicular-arbuscular mycorrhizal infection in lettuce (Lactuca sativa) in relation to calcium supply

Summary Infection of lettuce roots (Lactuca sativa L.) by the vesicular-arbuscular mycorrhizal fungiGlomus caledonium andGlomus mosseae was dependent on the amount of calcium (supplied as CaCl₂·2H₂O or CaSO₄·2H₂O) in the nutrient solution; those plants growing at low calcium concentrations being poorly infected.     

Infrequent transposition of Ac in lettuce,Lactuca sativa

The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R₁ plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2 promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.     

Leaf traits as indicators of limiting growing conditions for lettuce (Lactuca sativa)

Lettuce growth under unstressed conditions was compared to growth under four limiting conditions, i.e. no phosphorus fertilization (0_P), no nitrogen fertilization (0_N), low light (LR) and water stress (WR) over two different growing periods. We investigated the adaptive changes in terms of the morphological and physiological leaf traits, identifying stress‐specific and ‘stable’ indicators suitable for use in breeding programmes. The plants subjected to the WR treatments had lower leaf expansion and specific leaf area (SLA), as well as lower soil–plant analysis development (SPAD) values, stomatal conductance (POR), water index (WI) and leaf temperature (TIR) compared with plants in the unstressed CONTROL. Low light increased the leaf area (LA), SLA and leaf mass ratio (LMR). The 0_N treatment induced a general reduction in the normalised difference vegetation index (NDVI) values, as well as strong changes in LMR and SLA. In general, 0_P induced less pronounced effects than the other treatments. Principal component analysis indicated that the stable and suitable selection indicators of adaptive changes for low nitrogen and low light conditions were LA, SLA, leaf area per unit total plant mass (LAR), LMR, SPAD and POR, while SPAD, POR, TIR and WI were suitable indicators for drought.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

Microsatellite fingerprinting in lettuce (Lactuca sativa L.) and wild relatives

Southern hybridisation with a single microsatellite probe, (TCT)₁₀, sufficed to discriminate between a representative set of cultivars/accessions of lettuce, Lactuca sativa L., and its wild relatives L. serriola, L. saligna and L. virosa. Variability within cultivars was tested in a relatively modern cultivar (Hector), where no variation was found, and in an older and morphologically more variable cultivar (Madrilene), where heterogeneity was observed in the TCT fingerprint. (TCT)₁₀ fingerprinting should be useful for variety identification and homogeneity testing in lettuce. Received: 25 July 1997 / Revision received: 5 August 1997 / Accepted: 30 August 1997     

Identification of a phenylalanine ammonia-Iyase inactivating factor in harvested head lettuce (Lactuca sativa)

Exposing head lettuce ( Lactuca sativa L., crisphead or Iceberg type) leaf tissue to hormonal levels of ethylene (10 μl l⁻¹) at 5°C promotes the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and an increase in its activity. It also promotes the appearance of the postharvest physiological disorder called russet spotting (RS). Discontinuing ethylene exposure after 4 days resulted in a rapid decline in PAL activity which was delayed by treating excised midrib leaf tissue with actinornycin D or cycloheximide at 5°C. Only cycloheximide delayed the loss of PAL activity in tissue that was transferred from 5 to 15°C. Activity of PAL from Rhodolorula glutinis was. slowly lost during incubation in buffer alone, but there was a logarithmic decline in its activity over time when it was incubated with aliquots of the resuspended 10000 g pellet from homogenized, lettuce tissue affected with RS. The in vitro loss in PAL activity was 9–fold higher in extracts from lettuce showing RS symptoms than from control lettuce, boiled samples or the buffer control. The PAL-inactivating factor isolated from lettuce affected with RS had a pH optimum around 8.0. It appears that the rapid loss in PAL activity after the discontinuation of exposure to ethylene is dependent on the de novo synthesis of a PAL-inactivating factor.     

The effect of sowing pre-germinated seeds of lettuce (Lactuca sativa) on seedling emergence

Glasshouse and field experiments were conducted to investigate the effects of sowing both untreated and pre-germinated seeds, with or without a gel carrier, and also pelleted seeds into soils of different moisture contents and at different temperatures on seedling emergence of lettuce cvs Cobham Green and Avondefiance. Pre-germinated seeds with radicles 1–2 mm long gave, on average, 15 and 4% higher final percentage emergence than untreated and pelleted seeds respectively. Sowing pre-germinated seeds having radicles longer than 2 mm gave more variable levels of emergence and often reduced emergence, particularly when they were sown into dry or drying seedbeds. Increasing the rate of gel carrier from 0 to 30 ml m^_1 of row reduced emergence progressively from 67 to 30%. Pre-germinated seeds emerged 2–5 and 3-0 days earlier than untreated and pelleted seeds, respectively, at a soil temperature of 10 ^oC; at 15 ^oC the corresponding figures were 0–5 and 3–5 days. Sowing pre-germinated seeds reduced the spread (i.e. variability) of emergence by an average of 37 and 47% compared with untreated and pelleted seeds, respectively.     

Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa)

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.