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The deduced protein sequence of a partial tomato cDNA clone is rich in the amino acid glycine and contains repeats of the amino acid sequence, (Gly)5Tyr(Gly)4-5Tyr(Gly)3ArgArgGlu. This protein sequence has significant similarity to a sorghum glycine-rich protein [S1, Cretin and Puigdomenech (1990) Plant Mol. Biol. 15: 783] and a maize embryo, abscisic acid-induced glycine-rich protein [Gomez et al. (1988) Nature 334: 262]. Tissue printing was used to localize the glycine-rich protein transcript in tomato fruit, stem and hypocotyl sections. The transcript is present throughout the tomato fruit pericarp but is enriched in the vascular bundles. In tomato hypocotyl tissue prints, the glycine-rich protein transcript as well as rRNA were localized within the vascular tissue. This shows that differential loading of RNA may occur when using the tissue printing technique on hypocotyl sections. Direct isolation and comparison of RNA from vascular-containing and non-vascular stem tissue confirmed, however, that the glycine-rich protein transcript is accumulated abundantly in the vascular tissue of tomato stem.

Several chemicals were used to probe the in situ ethylene forming enzyme systems in apple tissue and Penicillium digilatum. 2,4-Dinitrofluorobenzene, a membrane permeant probe, inhibited ethylene production effectively in apples but far less effectively in P. digitatum. In contrast, salicylaldehyde, another membrane permeant probe, effectively inhibited the P. digitatum system but, except at 0.1 mM concentration, little influenced the apple system. l,5-Difluoro-2,4-dinitrobenzene (DFDNB), a membrane permeant probe which cross-links proteins with proteins and with phospholipids, strongly inhibited ethylene biosynthesis in both apple and P. digitatum, whereas dimethyl suberimidate, the protein cross-linking reagent, inhibited slightly the apple system but not P. digitatum system. Picrylsulfonate (TNBS), a non-permeant membrane probe, up to 0.1 mM, did not inhibit any of the two systems studied. However, in the presence of exogenous methionine in the apple system and glutamate in P. digitatum, TNBS at 0.1 and 1 mM caused inhibition of ethylene synthesis. These probes did not affect respiration of apple slices under similar incubating conditions, excepting for DFDNB which on longer incubation did inhibit respiration, but the effect on ethylene synthesis was 15 times greater. Divalent cation ionophores, A23187 and X537 A, had no effect on ethylene synthesis in both the systems. The water soluble iron chelating agent, o-phenanthroline, was a more potent inhibitor of the apple system but minimally affected P. digitatum. In contrast, the lipophilic chelator, bathophenanthroline, was a more potent inhibitor of the P. digitatum system. Assay of the fatty acid composition of polar lipids from crude membrane fractions showed considerably greater linoleic to linolenic ratio in P. digitatum than in apple. We suggest that the ethylene formations in apple and P. digitatum are sensitive to a modification of membrane structure and that specific chelator-sensitive metals (perhaps iron and copper) are involved in ethylene synthesis in both these systems.

Inorganic phosphate regulated ethylene production in shake cultures of Penicillium digitatum. Decreasing the phosphate level of the medium from 100 to 0.01 mM markedly increased, about 100-fold, the rate of ethylene production, in 96 hr, which was confined entirely to the fungal mycelium. Exogenous addition of between 0.01 to 100 mM phosphate, to high ethylene producing, low-phosphate cultures strongly inhibited their ethylene production and increased the ATP content of the mycelium. Phosphate also inhibited ethylene production in apple slices. Addition of calcium ions to the incubation medium stimulated the production of ethylene in apple slices, subhook epicotyl segments of pea and shake cultures of P. digitatum. We suggest that this stimulatory effect was caused by the reduction of inhibitory levels of phosphate, which complexed with calcium. Thus, phosphate in conjunction with calcium may play an important role in regulating ethylene production not only in P. digitatum but also in higher plants.

Changes in protein and RNA levels which occur in tomato (Lycopersicon esculentum cv. Pik-red) fruit tissue upon wounding were analyzed. Antibodies to nine plant proteins were used to identify the related cross-reactive proteins in tomato fruit tissue and quantify their abundance during wounding. Antibodies tested were pathogenesis-related proteins (PR-la, lb, lc; PR-2, N, O; PR-P, Q; basic chitinase), plastid proteins (sorbitol-6-phosphate dehydrogenase; acyl carrier protein; large subunit of Rubisco), cellulase and 33-kD cationic peroxidase. Marked differences in mRNA populations were also detected when the protein translation products following in vitro translation of poly(A)+ RNAs isolated from unwounded and wounded fruit tissue were compared. More direct evidence for the changes in mRNA levels was obtained by using specific cDNA probes, isolated from a cDNA library of wounded tomato pericarp tissue. Two cDNA clones identified RNAs that were induced upon wounding while a third cDNA clone identified a transcript whose abundance decreased.

A complementary DNA clone, designated GMACS1, encoding an mRNA for soybean 1-aminocyclopropane-1-carboxylate (ACC) synthase was isolated from a leaf cDNA library, and its nucleotide sequence was determined. The GMACS1-specific transcript was found to rapidly but transiently accumulate upon wounding in soybean leaf, pod and seed, and was not induced by 2, 4-D, or by ethylene. However, the transcript level increased 37-fold in the presence of cycloheximide.

Low (2.5%) oxygen atmosphere was previously shown to prevent the normal rise in cellulase activity in avocado fruits primed for ripening by pre-exposure to 10/μl/liter ethylene (Kanellis et al. 1987, Kanellis et al. 1989b). Such primed avocado fruits were used in the present study to determine the effect of 2.5% O2 atmosphere on the accumulation of cellulase protein and its mRNA using specific antibody and cDNA probes. Decrease in cellulase activity was paralleled by a decrease in immunogenic cellulase protein in fruits held in 2.5% O2. Under these conditions only 10-20% of total cellulase gene transcripts were expressed. Inclusion of ethylene in the low O2 atmosphere did not significantly alter the expression of the cellulase gene. Accumulation of cellulase mRNA during ripening was not due to polyadenylation of pre-existing mRNAs. Thus, low O2 appears to regulate cellulase gene expression at a pre-translational level.

Arrhenius plots of ethylene-synthesizing systems of apple and tomato showed discontinuities while the plot for Penicillium digitatum was linear. Triton X-100 markedly lowered the activation energies of the apple and tomato systems without altering that of the fungal system. The data presented suggest that the lipid micro-environment of the ethylene-synthesizing enzyme systems in higher plants and P. digitatum are different, and that cell membrane-cell wall complex may be the site of ethylene-synthesizing enzymic systems in higher plants.