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Pablo Gonzalez and Richard J. Gladon

Methionine (MET) is considered the first committed precursor of ethylene (C2H4), and the pathway has been established as MET → S-adenosylmethionine (SAM) → 1-aminocyclopropane-1-carboxylic acid (ACC) → C2H4. It has been suggested that another pathway to C2H4 may exist, and this pathway has been labeled System II. Our objective was to evaluate several compounds as possible precursors of System II C2H4 production. `White Sim' carnations were placed continuously in 20 mM solutions of MET, ACC, δ-aminolevulinic acid, glutamic acid, α-ketoglutarate, or homocysteine. Deionized water was the control. C2H4 production from the entire flower was measured, and ACC in the basal portion of the petals was quantified. Flowers treated with ACC exhibited the greatest C2H4 production and accumulation of ACC. Homocysteine caused greater production of C2H4 and accumulated more ACC than MET and the other possible precursors. These results suggest that homocysteine may be involved in System II C2H4 production in senescing carnation petals.

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Nicole L. Waterland and Richard J. Gladon

Sowing germinated seeds for bedding plant production can decrease the production time and reduce profit losses from sporadically or poorly germinating seeds. Low concentrations of O2 have been used to control radicle length in Impatiens wallerana Hook. F., but only a brief exposure period could be used (12 to 24 h). The effects of prolonged exposures are unknown. Our first objective was to determine if impatiens seedlings could be acclimated to an extreme hypoxic environment by a preliminary exposure to a less severe hypoxic exposure. Our second objective was to determine the effects of longer-duration (greater than 24 h) treatments at low-O2 concentrations on hypocotyl and radicle length, abnormal seedling development, and subsequent plant growth and development. Our third objective was to provide a commercial recommendation of a low-oxygen treatment or treatments that could be used for temporary storage of unused germinated seeds. Germinated seeds were placed in various combinations of 0.5%, 1.0%, and 1.5% O2 for either 4 days (Expt. 1) or 3 days (Expt. 2) followed by 24 h in air to simulate shipping. Germinated seeds were less tolerant of 0.5% O2 than greater O2 concentrations, especially during the first 2 days of treatment, and more abnormal seedlings developed at 0.5% O2. Germinated seeds were more tolerant of 0.5% O2 during Days 3 and 4 of the treatment period or when days at 0.5% O2 were interspersed between days of 1.0% or 1.5% O2. This indicates that germinated impatiens seeds can tolerate extremely low-O2 for longer durations (greater than 24 h) when first acclimated to lower O2 concentrations. Treatment of 1.5% O2 for Days 1 and 2 followed by 0.5% O2 for Day 3 is recommended for commercial use. Hypocotyl and radicle length was controlled to 1 mm without development of abnormal seedlings. Fully grown plants from seedlings that received low-O2 treatments were not different from control plants with regards to hypocotyl and radicle length, percentage abnormal seedlings, and plant growth and quality, ensuring there were no long-term detrimental effects.

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H.P.V. Rupasinghe and Richard J. Gladon

The ethylene biosynthetic pathway has been established as methionine (MET) to S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, and this pathway has been labeled System I. Another pathway to ethylene may exist during synthesis of massive amounts of ethylene, and this system has been labeled System II. Our objective was to evaluate the efficacy of several compounds as possible precursors of System II ethylene in ripening tomato fruit tissue. Discs of `Rutgers' tomato pericarp tissue at the mature green, pink, and red ripe stages were incubated continuously in 10, 25, or 40 mm solutions of MET, ACC, 5-aminolevulinic acid (ALA), homocysteine, glutamic acid (GLU), alpha-ketoglutarate, or citrate buffer (control). The ethylene production rate at 8-hour intervals during a 32-hour incubation period and free and conjugated ACC content at the end of the incubation period were quantified. Fruit discs at the mature green stage treated with MET and ACC exhibited increased ethylene production and increased free ACC content. These results confirmed the role of MET and ACC as the predominant precursors of ethylene during the early stages of fruit development in tomato (System I). At the pink stage (System II); however, ALA increased ethylene production by 75% and free ACC content by 46% over the control, and MET increased ethylene by 27% and free ACC content by 57% over the control. At the red ripe stage, ALA caused a 35% increase and GLU caused a 31% increase in ethylene production over the control. These results suggest that ALA and GLU may be metabolized to ethylene via an unknown pathway during tomato fruit ripening (System II).

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Gary F. Polkinz, David J. Hannapel, and Richard J. Gladon

Tomato fruit ripening is characterized by a decrease in chlorophyll content and an increase in lycopene synthesis. We are interested in the role of chlorophyll metabolism as it relates to tomato fruit ripening. 5-Aminolevulinic acid dehydratase (ALAD) is the first committed enzyme in the chlorophyll biosynthetic pathway, and it catalyzes the conversion of two 5-aminolevulinic acid molecules into porphobilinogen. We have isolated a full-length tomato ALAD cDNA clone from a tomato fruit library. Sequence analysis showed that this tomato ALAD was highly homologous to ALAD found in spinach and pea, and the analysis predicted a protein of 46.8 kDa. Southern analysis indicated that 1 to 3 copies of the ALAD gene are present in the tomato genome. Northern analysis suggested that the gene is expressed constitutively throughout tomato fruit development. Currently, we are subcloning the fragment into an E. coli expression vector in order to obtain protein for antibody production for Western analysis.

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Marios C. Kyriacou, David J. Hannapel, and Richard J. Gladon

Tomato fruit ripening is characterized by a decrease in chlorophyll content and an increase in lycopene synthesis. 5-Aminolevulinic acid (ALA) dehydratase (ALAD) is the fruit committed enzyme in the chlorophyll and heme biosynthetic pathways, and it catalyzes the dimerization of two ALA molecules into porphobilinogen We have focused our attention on the potential pivotal role of ALAD in the developmental regulation of chlorophyll biosynthesis during tomato fruit growth, development, and ripening. We have standardized an assay procedure for measuring the enzymatic activity of ALAD in tomato fruit tissues. The activity of ALAD was assayed from ten days past anthesis to day 60, when fruits where void of chlorophyll. We observed a several-fold decline in ALAD activity to residual levels during fruit ontogeny. Our data also show greater ALAD activity in chlorophyllous organs (leaves, stems, immature fruits) than in nonchlorophyllous organs (roots, ripe fruits), where heme production is predominant.

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Mikhailo V. Kolomiets, David J. Hannapel, and Richard J. Gladon

Plant lipoxygenases (LOXs) (linoleate:oxygen oxidoreductase, EC catalyze the oxygenation of polyunsaturated fatty acids such as linolenic and linoleic acids. Some of the final products of LOX-catalyzed reactions are traumatin, jasmonic acid (JA), methyl jasmonate (MJ), and C-6 volatile compounds, and they serve hormone-like regulatory and defense-related roles in plants. Recently, it has been proposed that LOXs play a role in potato tuberization processes because JA, MJ, and structurally similar tuberonic acid and tuberonic acid glycoside have been shown to be tuber-inducing substances. In order to study possible lipoxygenase involvement in potato tuberization, we have isolated, sequenced, and characterized the expression pattern of two cDNA clones, designated POTLX-1 and POTLX-2, that represent similar, but distinct, LOX genes. Within the scope of our experiments, northern hybridization studies with mRNA extracted from various organs of `Superior' potato plants indicated that the expression of these two genes is restricted to developing tubers and roots only. Moreover, there is a positive correlation between POTLX-1 and POTLX-2 mRNA accumulation and the stage of potato tuber development, and this implicates LOX in tuberization processes. Accumulation of their transcripts was not detected in leaves, flowers, stems, shoot tips, or axillary buds. These results indicate that the isozyme forms encoded by these two genes are tuber-specific, and they are good candidates to study LOX involvement in potato tuberization processes. Treatment of potato leaves with abscisic acid, MJ, gibberellic acid, auxin (NAA), and cytokinin (BA) did not trigger transcriptional activation of either of these genes.

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Mikhailo V. Kolomiets, Richard J. Gladon, and David J. Hannapel

Due to apparent participation of plant lipoxygenases (LOXs) in the biosynthetic pathways for jasmonic acid, methyl jasmonate, traumatin, and several C-6 volatile compounds, LOXs are believed to have a role in senescence, plant growth and development, and wound- and pathogen-induced defense responses. Multiple functions that are ascribed to this enzyme family are in accordance with the heterogeneity of LOX isozyme forms. It is possible that different LOX isoforms may be involved in different physiological processes. In our search for a gene that encodes a LOX isozyme form specifically involved in potato defense responses against pests and pathogens, we have screened an abscisic acid-induced potato leaf cDNA library, and we have isolated, sequenced, and characterized a cDNA clone that we have designated POTLX-3. The high sequence homology of our cDNA clone to other reported plant LOX genes provided evidence that POTLX-3 is a lipoxygenase. This cDNA clone represents a novel potato LOX gene in that it shares the least nucleotide and amino acid sequence homology to other isolated potato LOX genes. Northern analysis indicated that POTLX-3 transcripts did not accumulate in untreated potato leaves, but it was highly induced by treatment with physiological levels of ethylene. Northern analysis also was performed to study whether the POTLX-3 mRNA accumulation could be induced by other plant hormones that affect expression of the other plant LOX and defense-related genes. Treatment of potato leaves with methyl jasmonate, abscisic acid, gibberellic acid, auxin (NAA), and cytokinin (BA) did not induce POTLX-3 gene expression. Because the pattern of POTLX-3 gene expression is similar to that of pathogenesis-related (PR) proteins, especially the PR-1 and PR-5 groups, we suspect that POTLX-3 may be involved specifically in ethylene-induced defense responses against pathogens.

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Gary F. Polking, David J. Hannapel, and Richard J. Gladon

Our recent research has focused on the control of genes and enzymes involved with the synthesis of chlorophyll, especially as it relates to tomato fruit development and ripening. Glutamate-1-semialdehyde-2,1-aminomutase (GSAAM) is one of the first committed enzymes in the chlorophyll biosynthetic pathway, and it is one of three enzymes that catalyze the conversion of glutamate into 5-aminolevulinic acid. We have isolated a full-length cDNA clone of GSAAM from a tomato fruit library. The tomato primary sequence shows extensive homology to GSAAM sequences found in other plant species. The primary structure also predicts a 46.7-kDa, 437-amino acid, mature protein and a transit peptide of 44 amino acids. Southern analysis indicated that GSAAM was present as a single copy. Northern blot analysis showed that GSAAM was expressed differentially in various tomato organs and that GSAAM transcripts decreased with increased fruit age. Immunoblot analysis also indicated that GSAAM protein decreased dramatically with increased fruit age. These results show that there is developmental regulation of the expression of GSAAM in tomato fruits.

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Khalid N. Al-Redhaiman, Gail R. Nonnecke, and Richard J. Gladon

'Honeoye' (June-bearing) and 'Tristar' (day-neutral) strawberries were harvested, graded, and then stored for 7 days et 2C or 21C in air (control) or each of these 8 modified atmospheres: 1.5% O2, 3.5% O2, 15% CO2, 25% CO2, 1.5% O2 + 15% CO2, 1.5% O2 + 25% CO2, 3.5% O2 + 15% CO2, and 3.5% O2 + 25% CO2; all balance N2. When compared with storage at 21C, storage at 2C reduced weight loss and gray mold growth in all corresponding sets of storage atmosphere treatments. The combination of increased CO2 and decreased O2 controlled weight loss and gray mold growth more effectively than treatment with reduced O2 alone. Storage at 2C (versus 21C) reduced respiration of both cultivars. Respiration decreased as the O2 concentration decreased. 'Tristar' did not produce C2H4 at either temperature, whereas `Honeoye' produced more C2H4 at 21C than it did at 2C. Increased CO2 and/or decreased O2 concentrations in the storage atmosphere are not satisfactory substitutes for proper low-temperature storage of strawberries.

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Gary F. Polking, Richard J. Gladon, and David S. Koranski

Generation of pregerminated seeds at a central facility and subsequent mechanical sowing at a remote facility require that radicle elongation during shipment be minimized. Low-oxygen treatments were applied to pregerminated `Super Elfin Salmon Blush' impatiens seeds to suppress radicle growth during a subsequent one-day simulated shipping period in air. In the first experiment, O2 concentrations of 0, 3, 7, 10, 13, and 20% were applied for 24 and 48hr. The 0% O2/24-hr and the 0% O2/48-hr treatments held the radicle length close to the desired length of 1.0mm, but both of these treatments decreasad 7-day hypocotyl length and percentage normal seedlings, when they were compared with the control treatment (untreated pregerminated seeds) In the second experiment, O2 concentrations of 0, 1, 2, 3, 7, and 20% were applied for 24 and 48hr. Five treatments (0, 1, and 2% O2/24-hr and 0 and 1% O2/48-hr) held the radicle to a length <1.0mm. Of these five treatments, only the 2% O2/24-hr treatment resulted in recovery parameters (7-day hypocotyl length and percentage normal seedlings) that compared with those of the control treatment.