Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high‐temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578 Incubation of fruits of tomato (Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening‐associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1‐aminocyclopropane‐l‐carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening‐associated ethylene production by whole fruits.
Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60,26,21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old na, nor, and Nr pericarp were fractionated into poly(A)' and poly(A)-RNAs. Peak levels of total RNA, poly(A)+ RNA, and poly(A)' RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)+ RNAs from normal percarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33,31,29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)+ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.
Molecular cloning of polygalacturonase (PG; EC 3.2.1.15) from fruits of tomato (Lycoperskon esculentum Mill cv Rutgers) was accomplished by constructing a cDNA library from tuming stage poly(A)+ RNA in Xgtll and immunoscreening with polyclonal antibodies raised against purified PG2. Both PG cDNA and antibody probes were used to quantify changes in PG gene expression in pericarp from normal, mutant, and heterozygous genotypes. Results show that PG mRNA, protein, and enzyme activity sequentially peak at the tuming, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. PG gene expression was attenuated greatly (0-15% of normal on a gram fresh weight basis) for PG mRNA, protein, and enzyme activity in five ripening-impaired mutants (rin, nor, Nr, Gr, and Long Keeper) Considerable research has been conducted with tomato at the genetic, physiological, and biochemical levels to determine the mechanisms of fruit softening during ripening (2-12, 14, 15, 17, 18, 20, 21, 23, 25, 26, 28-30). Much attention has been focused on ripening-impaired genotypes (rin, nor, Nr, Gr, and Long Keeper)3 whose single gene mutations exert pleiotropic effects on fruit ripening, including softening (4, 5, 8-12, 14, 17, 23, 26, 28-30) genes will likely remain unknown until molecular probes specific for their expression are obtained. However, their multiple effects can be dissected further with the use of probes to the products of other ripening-related genes whose expression is altered in these mutants.In tomato fruits, PG has been thought to be the enzyme primarily responsible for textural changes during ripening (4, 10, 1 1, 15, 29), with PG activity being directly correlated with fruit softening (15,29). Researchers have characterized changes in PG mRNA in normally ripening fruits (3,11,12,18), but little has been understood about the temporal regulation between the appearance of PG mRNA and PG protein or PG enzyme activity. Since both softening and PG activity are reduced or absent as a result of genetic lesions at the rin, nor, Nr, Gr, and nor-' loci (8,11,14,17,23,26,29), we have undertaken a study of PG gene expression at the mRNA, protein, and enzyme activity levels to further characterize the effects of these mutations on the ripening of mutant fruits. In addition, these studies in normal, mutant, and heterozygous fruits provide additional information in the elucidation of the overall regulation of the PG gene in tomato fruit. MATERIALS AND METHODS Plant MaterialTomato ((Lycopersicon esculentum Mill.) was grown in a greenhouse in the summer of 1986 and pericarp tissue was harvested as previously described (5 Free, bound, and total polyribosomes from turning stage pericarp were isolated as described (1) using polyoxyethylene tridecylether as detergent for bound and total polysome preparations. Total RNA was extracted from polysomes and denatured at 65°C for 15 min in 7.5 x SSC (1.13 M NaCl, 0.11 M sodium citrate [pH 7.0] containing 4.6 M formaldehyde before being dot blotted to nitrocellulose and washed ...
S. 1985. Effect of tunicatnycin on in vitro ripening of tomato periearp tissue. -Physiol. Planl. 63; 417^24.Ripening of pericarp tissue from mature green, early breaker and late breaker stages of tomato (Lycopersicon esculentutti Mill. cv. Dombito) fruit development was inhibited by tunieamyein. Ripening was evaluated by lyeopene accuinulation, chlorophyll degradation, rale of ethylene production and eell wall-bound polygalacturonase (EC 3.2.1.1.5) activity. Maximum inhibition of these ripening parameters occurred at a treatment of 240 \iM tunieamyein for 2 h exeept for eell wall-bound polygalacturonase activity, which was greatly inhibited by concentrations of 12 [iM tunicamycin or higher. Tunicamycin treattnent at 120 [iM for 2 h inhibited the incotporation of |'H]mannose into niaeronioleeules (about 70%) and pronase-sensitive tnaterial (about 65%) and the incotporation of ('H]-leueine into proteins (about 20%). Our results indicate that protein glycosylation plays an important role in the ripening of tomato pericarp tissue.
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