Colin J. Brady

Molecular characterization of tomato fruit polygalacturonase

SummaryUsing the expression vector λgt11 and immunological detection, cDNA clones of an endopolygalacturonase gene of tomato (Lycopersicon esculentum Mill.) were isolated and sequenced. The 1.6 kb cDNA sequence predicts a single open reading frame encoding a polypeptide of 457 amino acids. The PG2A isoform of tomato fruit endopolygalacturonase was purified and 80% of the amino acid sequence determined. The amino acid sequence predicted by the cDNA sequence was identical to the amino acid sequence of the PG2A isoform. The position of the codon for the N-terminal amino acid of mature PG2A in the open reading frame indicates the presence of a 71 amino acid N-terminal signal peptide which is post-translationally processed. The C-terminus of purified PG2A occurred 13 amino acids before the stop codon in the cDNA suggesting that C-terminal processing of PG2A may also occur. The nucleotide and amino acid sequence data predict a mature protein of 373 amino acids and a polypeptide molecular weight of 40279. The sequence contains four potential glycosylation sites. Northern analysis detected endopolyga-lacturonase mRNA in stage 3 (turning) and stage 6 (red) ripening fruit, but not in leaves, roots, or green fruit of normal cultivars or in mature fruit of the rin mutant.

Silver Ions Interrupt Tomato Fruit Ripening

Summary The treatment of green tomato fruit tissue with silver ions has been shown to prevent ripening. This paper extends this observation and demonstrates that treatment with silver ions can also arrest tomato ripening once initiated. Thus silver ions applied to discs cut from ripening fruits interrupts the normal increases in invertase and polygalacturonase activites as well as the colour change. This effect appears to be specific and not due to any toxicity of the silver ions.

Purification and properties of the arginase from Jerusalem artichoke tubers

Abstract Arginase [ l -arginine amidinohydrolase] in Jerusalem artichoke tubers occurs in a particulate fraction from which it was released in active form by detergent treatment. The particulate enzyme was purified 450-fold with ca 3% yield. The enzyme has a MW of ca 140 000 and pI of 5.3. The enzyme required Mn 2+ for activity and was unstable when Mn 2+ was removed. In tissue extracts the K m for arginine was ca 1OmM, but when purified the K m (arginine) was 145 mM. The artichoke arginase was shown to be more substrate specific than other plant and animal arginases which have been described, and to be very sensitive to competitive inhibition by indospicine, ornithine and citrulline.

Endopolygalacturonase: Messenger RNA, Enzyme and Softening in the Ripening Fruit of a Range of Tomato Genotypes

Summary Fruit from seven tomato cultivars were harvested when mature but green and allowed to ripen under conditions of regulated temperature and air flow. Fruit were analysed when pink colour was first visible at the blossom end («breaker») and 3, 6 and 9 days after breaker. Compressibility of the fruit was measured and the accumulation of the enzyme endopolygalacturonase (PG) and its messenger RNA were determined. In all genotypes, the PG mRNA abundance peaked 3 or 6 days after «breaker» and then declined. However, despite the decline in message abundance, for 4 of the 7 genotypes, the increase in PG activity between 6 and 9 days was as rapid as earlier. The two softest cultivars, Rouge de Marmande and Rutgers, accumulated most PG enzyme over 9 days and had the highest steady state levels of PG mRNA early in ripening. In Flora-Dade, a firm cultivar that accumulated over 9 days much less PG enzyme than any other cultivar, the steady state levels of PG mRNA were relatively high. Comparing rates of accumulation within cultivars or peak levels between cultivars, there was little correlation between changes in PG enzyme activity and the steady state levels of its mRNA. The correlation between enzyme activity and peak abundance of PG mRNA was much stronger when the comparison was made between populations of the genotype de Ruiters 83G38 containing the ripening-impairing genes rin or nor . In the heterozygous condition, the levels of both message and enzyme were reduced compared to the normal line; the reduction was least in the rin, intermediate in the nor and greatest in rin × nor populations. Significant levels of PG enzyme or its mRNA were not detected in populations homozygous for the mutant genes. The results indicate that the accumulation of the PG message varies in different genotypes but that post-transcriptional events other than those regulating mRNA abundance, play an important part in regulating accumulation of the PG enzyme.

Purification of three pectin esterases from ripe peach fruit

Three isoforms of pectin esterase (PE1-PE3) (pectin pectyl-hydrolase, EC 3.1.1.11.) were purified to homogeneity from ripe peach fruit (Prunus persica cv. Coronet). The three enzymes were basic proteins of M(r) 34,000 as determined by denaturing polyacrylamide gel electrophoresis but were separated by FPLC cation-exchange chromatography. The proteins were N-terminally blocked but amino acid sequences were obtained for peptides released from two of the three isoforms. The sequences revealed a threonine/lysine substitution in a comparison between isoform PE2/isoform PE3, and there were regions of sequence similarity with other plant pectin esterases. The proteins did not bind to concanavalin A and were not stained by the periodate-Schiff reagent suggesting a low or zero level of glycosylation. Polyclonal antisera to isoform PE3 also bound to isoforms PE1 and PE2. The study provides the enzyme protein sequence and immunological basis for an evaluation of the role of pectin esterases in normal and abnormal ripening of peach fruit.

Messenger RNA Changes in Tomato Fruit Pericarp in Response to Propylene, Wounding or Ripening

Summary Immature green tomato ( Lycopersicon esculentum Mill. Line 83G38) fruits were treated with propylene for 2, 4 or 6 days. The respiration rates of the propylene-treated fruit were higher than those of untreated fruit but fell when the propylene was withdrawn. Fruit exposed to propylene for 4 or more days had a higher percentage of polysomes than had untreated fruit or fruit treated for only 2 days. RNA was recovered from individual fruit, translated in a wheat germ translation system in the presence of 35 S-methionine and the products separated by gel electrophoresis and examined by fluorography. Two polypeptide products that were prominent in translations of ripening but not green fruit were seen in all normal fruits examined immediately after exposure to propylene for 2, 4 or 6 days. In fruits exposed to propylene for 2 days but returned to air for 4 days before the RNA was extracted, the ripening-related products were either not seen or were not prominent. Shifts in messenger RNA populations in response to propylene treatments were the same in the ripening-inhibited mutant rin as in the normal line but were reduced in the nonripening nor mutant line. It is concluded that the induction of the messenger RNAs for these ripening-related polypeptides is not of itself sufficient to induce ripening. Wounding either mature-green or ripe pericarp tissue induced a characteristic set of translatable messenger RNAs within 2 hours. One of the set was translated in vitro to a product of Mr 37,500 daltons. This product was also prominent in translations of the RNA of unwounded ripe fruit but was much less prominent with unwounded green fruit. It was not induced when immature fruit was given propylene treatments which did not induce ripening. It is concluded that, apart from the RNA for the 37,500 dalton product, wounding and ripening provoke distinct populations of messenger RNAs.

Changes in plastid proteins during ripening of tomato fruits

Summary Plastids and plastid fragments were recovered from homogenates of the pericarp tissue of mature-green and ripening tomato fruits by differential centrifugation followed by sucrose density gradient centrifugation. The fractions were defined by their positions in the gradient, by their chlorophyll, carotenoid and protein contents, and by the spectra of membrane proteins revealed by polyacrylamide gel electrophoresis. The light harvesting complex and photosystem proteins and the ±- and ²-subunits of coupling factor (CFI) were identified by immunological methods. The proteins of the light harvesting complex and the photosystems were the major membrane proteins in the chloroplasts recovered from mature-green fruit but were less prominent in the plastids from pink fruit and absent in the chromoplasts or ripe fruit. By contrast, the coupling factor subunits were prominent in the membrane of chloroplasts and chromoplasts. The proteins most prominent in chromoplast membranes were also detected in chloroplast fragments, which were less dense than the whole chloroplasts, and which lacked the photosystem proteins. Chloroplast membrane proteins represented about 12 per cent of the protein in mature-green tissue; about 8 per cent of the protein of ripe fruit was recovered as chromoplast membrane protein. Ribulose bisphosphate carboxylase decreased through ripening but even in the maturegreen tissue it represented only 0.3 per cent of total protein indicating that fruit chloroplasts have a very high ratio of photosystem protein to ribulose bisphosphate carboxylase relative to leaf chloroplasts.

Content of RNA and protein of the ripening banana

Abstract A possible net movement of nitrogen between peel and pulp tissues of ripening bananas was evaluated. No evidence of a movement of nitrogen from the peel to the pulp was found, nor with either tissue did the proportion of the total nitrogen extracted by 5% (w/v) trichloracetic acid change significantly during the climacteric period. Neither the content of RNA in the pulp tissue, nor the base composition of the RNA changed during the climacteric. It is concluded that no substantial increase in the amount of ribosomal material accompanies the increase in the rate of protein synthesis early in the climacteric.