Conversion of the polygalacturonase isoenzymes from ripening tomato fruits

From pericarp tissue of ripening tomato ( Lycopersicom esculentum Mill. cv. Sonato), two isoenzymes of polygalacturonase, PGl and PG2, can be extracted. With water hardly any polygalaeturonase activity is extracted; with 0.5 M NaCl predominantly PG2 is found and subsequent extraction with 1.25 M NaCI delivers mainly PGl. A partly purified PGl solution gradually decomposes into PG2. Conversion of PG2 into an isoenzyme that resembles PGl, but differs from it, can be brought about by a faetor (eonvertor) that occurs at low levels in free form in unripe and early-ripe fruits as well as in leaves. Convertor (CV) ean be set free from PGl by a short treatment at 100°C, at which temperature the convertor activity itself also decreases.
The in vitro activities and several characteristics of the isoenzymes and CV as found by us differ from that found by others, probably because of carefully optimized methods. It is suggested that the CV anchors PG2 onto the cell wall, forming PGl. Thus PGl would constitute the form that depolymerizes the peetins in the middle lamellae.     

Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation

Ultrastructural changes in the pericarp of tomato (Lycopersicon esculentum Mill) fruit were followed during ripening. Ethylene production was monitored by gas chromatography and samples analyzed at successive stages of the ripening process.

Changes in the cytoplasmic ultrastructure were not consistent with the suggestion that ripening is a `senescence' phenomenon. A large degree of ultrastructural organization, especially of the mitochondria, chromoplasts, and rough endoplasmic reticulum, was retained by ripe fruit.

Striking changes in the structure of the cell wall were noted, beginning with dissolution of the middle lamella and eventual disruption of the primary cell wall. These changes were correlated with appearance of polygalacturonase (EC 3.2.1.15) isoenzymes. Application of purified tomato polygalacturonase isoenzymes to mature green fruit tissue duplicated the changes in the cell wall noted during normal ripening. Possible roles of the polygalacturonase isoenzymes in cell wall disorganization are discussed.

     

Molecular characterization of tomato fruit polygalacturonase

Summary Using the expression vector gt11 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.     

Purification and characterization of tomato polygalacturonase converter

Extracts of ripe tomatoes contain two forms of polygalacturonase (PG I and PG II). A heat-stable component that binds PG II to produce PG I has been isolated from tomato fruit. This component has been named polygalacturonase converter (PG converter). The PG converter has been purified by gel filtration, ion-exchange chromatography and chromatofocusing. It appears to be a protein with a relative molecular mass of 102000. It was readily inactivated by papain and pronase. The converter was labile at alkaline conditions, and treatment of PG I at pH 11 released free PG II. A similar factor with a lower molecular mass was extracted from tomato foliage.     

Carbohydrate composition and physical properties of tobacco phosphodiesterase

Some physical and chemical properties of phosphodiesterase from cultured tobacco cells were studied. The enzyme contained ca 50% carbohydrate consisting of residues of arabinose, glucose, glucosamine, galactose, mannose and xylose. Analyses showed that the enzyme had a sedimentation coefficient, s_20,w of 16 S, a Stokes'radius of 5.7 nm and a calculated specific volume of 0.66 ml/g. The MW of the enzyme was calculated to be 300 000 from these values.     

Insertional inactivation of the tomato polygalacturonase gene

The site-selected insertion (SSI) procedure was used to generate insertional knockout mutations in the gene for tomato polygalacturonase (PG), a critical enzyme in fruit ripening. Previously, it had been shown that the Dissociation (Ds) elements in a select group of tomato plants frequently inserted into PG, at least in somatic tissues. DNA isolated from pollen produced by progeny of these plants was screened by SSI to identify plants likely to transmit the insertions in PG to progeny. These results identified one family as likely candidate for yielding germinally transmitted insertions. Four thousand progeny were screened and five were found containing germinally transmitted Ds insertions in PG, one of which contained two Ds insertions in PG. The Ds elements were stabilized by genetically removing the transposase and four of the five insertions were recovered as homozygous in the next generation. Enzymatic analysis of fruit from these individuals demonstrated that there was at least a 1000-fold reduction in polygalacturonase levels in those plants bearing Ds insertions in PG exons. Individuals with modified PG sequences due to the sequence footprint, resulting from excision of the element, were identified using the single-strand conformational polymorphism (SSCP) method. Enzymatic analysis of fruit from a plant homozygous for one such excision allele showed a significant reduction in polygalacturonase activity. Since there is no transgenic material left in PG, this demonstrates the ability to modify a gene of commercial value in planta and subsequently removing all transgenic material.     

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in Never ripe mutants and the lack of activity in ripening inhibitor mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.Abbreviations PG polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

Immunocytolocalization of polygalacturonase in ripening tomato fruit

Using tissue blotting and immunocytolocalization, we have investigated the appearance and accumulation of polygalacturonase (PG) during tomato (Lycopersicon esculentum Mill.) fruit ripening. Results show that PG first appears in the collumella region followed by sequential appearance in the exopericarp and endopericarp, respectively. Detectable levels of PG were not present in the locular material containing seeds. This result indicates that PG synthesis initiates at the central collumella region of tomato fruit during ripening.     

Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase

Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.     

In vitro synthesis and processing of tomato fruit polygalacturonase

The in vitro processing of tomato fruit polygalacturonase (PG) (poly[1,4-α-d-galacturonide]glucanohydrolase, EC 3.2.1.15) was studied. Complete chemical deglycosylation of a mixture of mature, purified PG 2A and PG 2B isozymes (45 and 46 kilodaltons; respectively) with trifluoromethane sulfonic acid yielded a single polypeptide of 42 kilodaltons. Similarly, N-terminal amino acid sequencing of the PG 2A/2B isozyme mixture yielded a single 21 amino acid N-terminal sequence, suggesting that the two isozymes result from differential post-translational processing of a single polypeptide. Translation of PG mRNA in vitro results in the synthesis of a single polypeptide with an apparent molecular weight of 54 kilodaltons. Nucleotide sequence analysis of a full-length PG cDNA clone indicates that the large size difference between the PG in vitro translation product and the mature isozymes is due to the presence of a 71 amino acid (8.2 kilodaltons) domain at the N-terminus of in vitro translated PG, consisting of a hydrophobic signal sequence followed by a highly charged prosequence. To determine the precise cleavage site of the signal sequence, PG mRNA was translated in vitro in the presence of canine pancreas microsomal membranes. This resulted in the production of two glycosylated PG processing intermediates with apparent molecular weights of 58 and 61 kilodaltons. The PG processing intermediates were shown to be sequestered within the lumen of the microsomal membranes by protease protection and centrifugational analysis. Deglycosylation of the PG processing intermediates with endoglycosidase H yielded a single polypeptide with an apparent molecular weight of 54 kilodaltons. The production of two distinct, glycosylated processing intermediates from the single in vitro translated PG polypeptide suggests a mechanism by which the differential glycosylation observed for the mature PG 2A and PG 2B isozymes may occur. Edman degradation of ³H-labeled 58 and 61 kilodalton PG processing intermediates indicates that the site of signal sequence cleavage is after amino acid 24 (serine). These results suggest that the proteolytic processing of PG occurs in at least two steps, the first being the co-translational removal of the 24 amino acid signal sequence and the second being the presumed post-translational removal of the remaining highly charged 47 amino acid prosequence.     

加工番茄多聚半乳糖醛酸酶(PG)基因的cDNA克隆和全序列测定

以加工番茄"87-5”为材料,采用反转录PCR(RT-PCR)技术将加工番茄PG基因的cDNA序列克隆到pGEM-T载体上,并进行了全序列测定分析.结果表明,加工番茄的PG基因的cDNA与国内外报道的番茄PG基因的cDNA,在碱基序列及氨基酸序列上均有差异.说明番茄的PG基因具有多态性.     

Purified tomato polygalacturonase activity during thermal and high-pressure treatment

Extracted tomato polygalacturonase was purified by cation-exchange chromatography (and gel filtration) and characterized for molar mass, isoelectric point, as well as optimal pH for polygalacturonase activity. The enzymatic reaction of purified tomato polygalacturonase on polygalacturonic acid as substrate was investigated during a combined high-pressure/temperature treatment in a temperature range of 25 degrees to 80 degrees C and in a pressure range of 0.1 to 500 MPa at pH 4.4 (the pH of tomato-based products). The optimal temperature for initial tomato polygalacturonase activity in the presence of polygalacturonic acid at atmospheric pressure is about 55 degrees to 60 degrees C. The optimal temperature for initial tomato polygalacturonase activity during processing shifted to lower values at elevated pressure as compared with atmospheric pressure, and the catalytic activity of pure tomato polygalacturonase decreased with increasing pressure, which was mostly pronounced at higher temperatures. The elution profiles of the degradation products on high-performance anion-exchange chromatography indicated that for both thermal and high-pressure treatment all oligomers were present in very small amounts in the initial stage of polygalacturonase activity. The amounts of monomer and small oligomers increased with increasing incubation times, whereas the amount of larger oligomers decreased due to further degradation.     

Function of the polygalacturonase convertor in ripening tomato fruit

In extracts from pericarp tissue of ripening tomato ( Lycopersicon esculentum Mill. cv, Sonato) fruits, two isoenzymes of polygalacturonase (E.C. 3.2.1.15), PG1 and PG2, are usually found. Also in such extracts, or as part of PG1, a convertor (CV) occurs. Incubation of PG2 with this CV gives rise to PG1 or a different isoenzyme, PGx, that is also stable at 65°C but differs in _pH optimum and size from PG1. It appears that CV has two affinity sites that can bind with PG2 or with a polydextran. PG1 is an extraction artifact, consisting of one molecule of CV and two molecules of PG2. PGx is made up of one molecule of CV and one molecule of PG2. It is the CV part of PGx that binds to polydextrans such as Blue Dextran 2000, Sephadex G-100, and cell wall preparations. In this last form PGx is the physiologically active form of the enzyme, solubilizing demethylated pectin.
On Sephacryl S-300, CV appears to have a molecular weight of 81 kDa, but because of its heat stability and partial leakage through a 10 kDa cut-off membrane, it might be a much smaller, rod-like molecule. The polygalacturonase convertor might be a lectin without intrinsic enzyme activity, with a function to immobilize, stabilize and activate enzymic proteins in the cell wall.     

Analysis of tomato polygalacturonase expression in transgenic tobacco.

Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening. Polygalacturonase is synthesized as a glycoprotein precursor that undergoes numerous cotranslational and post-translational processing steps during its maturation, yielding three isozymes in tomato fruit, PG1, PG2A, and PG2B. To investigate the physiological roles of the three isozymes and the functional significance of the polygalacturonase processing domains in its intracellular transport and activity, we have examined polygalacturonase expression in transgenic tobacco plants. A full-length polygalacturonase cDNA was placed under control of the cauliflower mosaic virus 35S promoter and introduced into tobacco by way of Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants indicated that (1) immunologically detectable polygalacturonase can be extracted from leaves, roots, and stems of transgenic tobacco plants; (2) only PG2A and PG2B were detectable in transgenic tobacco; (3) the polygalacturonase isozymes present in transgenic tobacco were electrophoretically indistinguishable from the tomato isozymes; (4) the N-terminal sequence, degree of N-linked glycosylation, and extent of oligosaccharide processing were similar in polygalacturonase from transgenic tobacco and tomato; (5) polygalacturonase was properly localized in cell walls of transgenic tissue; (6) the protein was enzymatically active in vitro; however, (7) accumulation of PG2A and PG2B in cell walls of transgenic tobacco did not result in pectin degradation in vivo. These results indicated that tomato polygalacturonase was properly processed and transported to the cell wall of tobacco. However, accumulation of the two polygalacturonase isozymes expressed in this heterologous host was insufficient to promote polyuronide degradation in tobacco leaf tissue.