Donald J. Huber

Polyuronides in Avocado (Persea americana) and Tomato (Lycopersicon esculentum) Fruits Exhibit Markedly Different Patterns of Molecular Weight Downshifts during Ripening

Avocado (Persea americana) fruit experience a rapid and extensive loss of firmness during ripening. In this study, we examined whether the chelator solubility and molecular weight of avocado polyuronides paralleled the accumulation of polygalacturonase (PG) activity and loss in fruit firmness. Polyuronides were derived from ethanolic precipitates of avocado mesocarp prepared using a procedure to rapidly inactivate endogenous enzymes. During ripening, chelator (cyclohexane-trans-1,2-diamine tetraacetic acid [CDTA])-soluble polyuronides increased from approximately 30 to 40 [mu]g of galacturonic acid equivalents (mg alcohol-insoluble solids)-1 in preripe fruit to 150 to 170 [mu]g mg-1 in postclimacteric fruit. In preripe fruit, chelator-extractable polyuronides were of high molecular weight and were partially excluded from Sepharose CL- 2B-300 gel filtration media. Avocado polyuronides exhibited marked downshifts in molecular weight during ripening. At the postclimacteric stage, nearly all chelator-extractable polyuronides, which constituted from 75 to 90% of total cell wall uronic acid content, eluted near the total volume of the filtration media. Rechromatography of low molecular weight polyuronides on Bio-Gel P-4 disclosed that oligomeric uronic acids are produced in vivo during avocado ripening. The gel filtration behavior and pattern of depolymerization of avocado polyuronides were not influenced by the polyuronide extraction protocol (imidazole versus CDTA) or by chromatographic conditions designed to minimize interpolymeric aggregation. Polyuronides from ripening tomato (Lycopersicon esculentum) fruit extracted and chromatographed under conditions identical with those used for avocado polyuronides exhibited markedly less rapid and less extensive downshifts in molecular weight during the transition from mature-green to fully ripe. Even during a 9-d period beyond the fully ripe stage, tomato fruit polyuronides exhibited limited additional depolymerization and did not include oligomeric species. A comparison of the data for the avocado and tomato fruit indicates that downshifts in polyuronide molecular weight are a prominent feature of avocado ripening and may also explain why molecular down-regulation of PG (EC 3.2.1.15) in tomato fruit has resulted in minimal effects on fruit performance until the terminal stages of ripening.

Methyl de-esterification as a major factor regulating the extent of pectin depolymerization during fruit ripening: a comparison of the action of avocado (Persea americana) and tomato (Lycopersicon esculentum) polygalacturonases.

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.

Acidified phenol alters tomato cell wall pectin solubility and calcium content

Abstract The inactivation of cell wall enzymes is often accomplished by exposing the wall to phenol-acetic acid-water (PAW; 2:1:1, w/v/v) during isolation. We investigated the influence of this solvent on the solubility characteristics of pectin and calcium in cell wall derived from tomato pericarp tissue. When placed in 50 mM NaOAc at pH 6.5, cell wall from ripe fruit which had not received a PAW treatment released from 25 to 30 μg galacturonic acid equivalents (mg cell wall) −1 . The inclusion of the chelator cyclohexane- trans - 1,2-diamine tetraacetic acid (CDTA) increased recoveries from non-PAW treated wall to nearly 160 μg mg −1 . In contrast, PAW-treated wall from ripe fruit when placed in buffer without chelator released 170 μ mg −1 ; recoveries were not enhanced in the presence of chelator. Pectin solubility was also enhanced in PAW-treated wall from mature-green pericarp. PAW-treated wall exhibited a 35–50% reduction in calcium and showed greatly enhanced susceptibility to hydrolysis by polygalacturonase. Buffered phenol was examined as a possible alternative to PAW. Cell wall exposed to Tris-buffered phenol (pH 7.0) were enzymically inactive, showed no reduction in calcium and exhibited chelator-dependent pectin solubilization.

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (Mr), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the Mr of unbranched cell-wall polymers in the Mr range of 106–107 Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.

Pectin degradation in ripening and wounded fruits

Pectin depolymerization during fruit ripening has been shown to be largely due to pectinolytic enzymes, including polygalacturonases (E.C. 3.2.1.15) and pectinmethylesterases (E.C. 3.2.1.11). Studies have shown that these enzymes are not the primary determinants of softening, although participation in texture changes during the late stages of ripening seems evident. Pectin depolymerization differs significantly between various fruit types, notably avocado and tomato, even though levels of extractable PG activity in these fruits are similar. Collective evidence indicates that the activities of some cell wall enzymes are restricted in vivo, with maximum hydrolytic potential expressed only in response to tissue disruption or wounding. In contrast, other enzymes reported to participate in pectin degradation, notably b-galactosidases/exo-galactanases, exhibit in vitro activity far below that anticipated to be required for the loss of cell wall galactosyl residues during ripening. Factors controlling in vivo hydrolysis have not been fully explored but might include apoplastic pH, cell wall inorganic ion levels, non-enzymic proteins including the noncatalytic b-subunit and expansins, wall porosity, and steric hindrances. Recent studies of cell wall metabolism during ripening have demonstrated an orderly process involving, in the early stages, cell wall relaxation and hemicellulose degradation followed, in the later stages, by pectin depolymerization. A limited number of studies have indicated that radical oxygen species generated either enzymically or non-enzymically might participate in scission of pectins and other polysaccharides during ripening and other developmental processes. Similar mechanisms might also occur in response to wounding, an event typically followed by an oxidative burst. Cell wall degradation as influenced by physical wounding could be of particular relevance to the deterioration of lightly processed fruits.

Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit

Perception of the plant hormone ethylene is essential to initiate and advance ripening of climacteric fruits. Since ethylene receptors negatively regulate signaling, the suppression is canceled upon ethylene binding, permitting responses including fruit ripening. Although receptors have autophosphorylation activity, the mechanism whereby signal transduction occurs has not been fully determined. Here we demonstrate that LeETR4, a critical receptor for tomato (Solanum lycopersicum) fruit ripening, is multiply phosphorylated in vivo and the phosphorylation level is dependent on ripening stage and ethylene action. Treatment of preclimacteric fruits with ethylene resulted in accumulation of LeETR4 with reduced phosphorylation whereas treatments of ripening fruits with ethylene antagonists, 1-methylcyclopropene and 2,5-norbornadiene, induced accumulation of the phosphorylated isotypes. A similar phosphorylation pattern was also observed for Never ripe, another ripening-related receptor. Alteration in the phosphorylation state of receptors is likely to be an initial response upon ethylene binding since treatments with ethylene and 1-methylcyclopropene rapidly influenced the LeETR4 phosphorylation state rather than protein abundance. The LeETR4 phosphorylation state closely paralleled ripening progress, suggesting that the phosphorylation state of receptors is implicated in ethylene signal output in tomato fruits. We provide insights into the nature of receptor on and off states.

Alterations in Structural Polysaccharides during Liquefaction of Tomato Locule Tissue

The locule tissue of tomato (Lycopersicon esculentum, Mill.) undergoes extensive liquefaction during ripening. In this study, the solubility, molecular mass, and glycosyl composition of locule pectic and alkali-soluble polysaccharides were examined with the aim of identifying features contributing to the unique properties of this tissue. Ethanol-insoluble solids were prepared from de-seeded locule tissue from tomato fruit at the immature green (IMG), mature green, and breaker stages of development. Ethanol-insoluble pectins were extracted sequentially in H2O, 50 mM trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, 50 mM Na2CO3, and 4 M KOH. At the IMG stage, nearly 85% of the locule pectins were solubilized by water, trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid, and Na2CO3 solutions. Solubility increased only slightly with further locule development. The noncovalently associated polymers were of high molecular mass throughout liquefaction. Polymers extracted in mild alkali were of considerably lower molecular mass. Locule pectins in IMG fruit were heavily glycosylated with galactose, arabinose, and xylose. All pectin classes exhibited similar deglycosylation trends during liquefaction. Locule hemicelluloses were rich in glucose, xylose, and arabinose. These polymers collectively showed molecular mass downshifts with minimal compositional changes during liquefaction. The KOH-soluble material also included xylose-rich acidic polymers not matching the neutral sugar profile of the noncovalently associated pectic polymers.

Ethylene feedback mechanisms in tomato and strawberry fruit tissues in relation to fruit ripening and climacteric patterns

Exposing pericarp tissue excised from immature tomato fruit to 4.5 mmol l 1 C2H4 revealed a negative C2H4 feedback mechanism in relation to its biosynthesis since ACC concentration and C2H4 production by the tissue were reduced. An opposite trend (positive C2H4 feedback mechanism) was observed in pericarp tissue excised from fruit at the pink stage. At the mature-green stage however, tissue showed a transition from negative to positive C2H4 feedback mechanism with the onset of tissue ripening. In strawberry tissues excised from green, white and half-coloured fruits however, C2H4 application caused a short-term increase in C2H4 production followed by a sharp reduction to the control level along with a marked reduction in ACC levels. In both tomato and strawberry fruit tissues, C2H4 application significantly induced ACC oxidase (ACO) activity at all ripening stages, as measured by in vivo ACC conversion to C2H4. This strongly suggests that ACC synthesis is the limiting step in C2H4 autocatalysis and the only limiting step in C2H4 autoinhibition. In tomato pericarp tissues, C2H4 autoinhibition and autocatalysis caused by C2H4 application in immature and pink fruits, respectively, were eliminated when tissues were transferred to air and re-occurred when tissues were returned back to C2H4. These responses did not occur in all strawberry tissues due to the sharp reduction in C2H4 production with the time course of C2H4 application. Inhibiting C2H4 action with STS pretreatment inhibited both negative and positive C2H4 feedback mechanisms in both tomato and strawberry tissues indicating that C2H4 feedback mechanism is one sort of C2H4 action. In addition, only tomato fruit tissue showed significant increases in CO2 production with C2H4 application. In contrast to the nonclimacteric behaviour of strawberry fruit which exhibits only a negative C2H4 feedback mechanism, these data strongly suggest that the transition of the C2H4 feedback mechanism from negative to positive, which occurs in tomato fruit only with ripening initiation and progress, may be the reason behind the climacteric behaviour of tomato fruit.

Exoglucanases fromZea mays L. seedlings: their role inβ-D-glucan hydrolysis and their potential role in extension growth

Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall β-D-glucan and their possible role in extension growth. An exo-β-1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both β1–4 and β1–3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-β-D-glucanase. Similarly, the capacity of the purified exo-β-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-β-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a β-glucosidase and did not appear to participate in the hydrolysis of wall β-D-glucan.

Ethylene evolution and endo-beta-mannanase activity during lettuce seed germination at high temperature

Altas temperaturas durante a embebicao das sementes de alface podem atrasar ou inibir a germinacao e o endosperma parece ser o responsavel na restricao da protrusao da radicula. O envolvimento da enzima endo-b-mananase durante a germinacao de sementes de alface a 35°C e a influencia do etileno na regulagem desta enzima foram estudados. Sementes das cultivares Dark Green Boston (DGB) e Everglades (EVE) foram germinadas em agua ou em solucoes de 10 mmol L-1 de 1-aminociclopropano-1-acido carboxilico (ACC), 10 mmol L-1 de amino-etoxi-vinil-glicina (AVG), ou 20 mmol L-1 de tiossulfato de prata (STS). Sementes foram ainda osmoticamente condicionadas em solucoes de polietilenoglicol (PEG), ou PEG + ACC, PEG + AVG, ou PEG + STS. Sementes nao tratadas germinaram 100% a 20°C. A 35°C, EVE germinou 100%, enquanto DGB germinou somente 33%. A germinacao a 35°C aumentou em sementes osmoticamente condicionadas ou sementes que receberam ACC durante a incubacao. Maior evolucao de etileno foi detectada em EVE do que em DGB durante a germinacao a 35°C. AVG nao inibiu a germinacao de DGB a 35°C, enquanto que STS inibiu. Maior atividade de endo-b-mananase nas sementes foi observada em EVE quando comparado com DGB. Fornecimento de ACC tanto durante o condicionamento osmotico como durante a germinacao, aumentou a atividade de endo-b-mananase, enquanto que AVG e STS proporcionaram um decrescimo ou ausencia da atividade enzimatica. O etileno pode minimizar o efeito inibitorio de altas temperaturas na germinacao de sementes de genotipos sensiveis de alface devido ao aumento da enzima endo-b-mananase, possivelmente levando ao enfraquecimento do endosperma.