Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.     

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene)

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.     

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.     

Developmental regulation of phospholipase D in tomato fruits

The catabolism of phospholipids initiated by phospholipase D (PLD, EC 3.1.4.4) is an inherent feature of developmental processes that include fruit growth and ripening. In cherry tomatoes (Lycopersicon esculentum Mill.), soluble and membrane-associated PLD activities increased during fruit development, which peaked at the mature green and orange stages. The increase in PLD activity was associated with a similar increase in the intensity of a 92 kDa band as demonstrated by western blot analysis. A full-length cDNA having 2430 bp and encoding a putative polypeptide with 809 amino acids, was isolated using tomato RNA, RT-PCR and 5' and 3' rapid amplification of cloned ends (RACE). Analysis of the primary and secondary structures showed the presence of the C2 domain, the PLD domain and several other features characteristic of PLD alpha. Microtom tomato plants transformed with antisense PLD alpha cDNA, were similar to untransformed plants and showed normal fruit set and development. The ethylene climacteric was delayed by over 7 d in the antisense PLD fruits, indicative of a slower ripening process. The leaves and unripened fruits of antisense PLD microtom plants possessed lowered PLD activity and PLD protein, as demonstrated by western blotting. However, during ripening, PLD activity in the transgenic fruits was maintained at a higher level than that in the untransformed control. Immunolocalization of PLD in microtom tomato fruits revealed the cytosol-membrane translocation of PLD during fruit development. The ripe fruits of antisense PLD celebrity plants possessed lowered PLD expression and activity and showed increased firmness and red colour. These results suggest that the expression of antisense PLD cDNA could be variable in different tomato varieties. The potential role of PLD in ethylene signal transduction events is discussed.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

PG与番茄果实成熟的关系

系统比较了转多聚半乳糖醛酸酶（PG）反义基因和对照番茄果实成熟过程中绿熟、转色、粉顶、粉红、全红5个时期的PG活性和与其相关的生理、生化组分的动态变化。实验表明,转基因果与对照果相比,PG活性始终处于较低水平,PG活性强烈被抑制是在全红期;果实的硬度、贮藏寿命指数都高于对照果;番茄红素合成积累进程被延缓;可溶性果胶含量、电解质外渗百分率均低于对照果。外源乙烯刺激引起对照果PG活性剧增,而转基因果表现钝化。讨论了PG活性与果实成熟、耐贮性及软化的关系,及对外源乙烯刺激的敏感性。首次明确提出PG活性在对照果中极大地表达,在转基因果中强烈被抑制是在全红期 ,而不是在整个成熟期;PG活性在果实软化中起直接和重要作用;PG活性的高低与番茄红素的合成与积累有关。     

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.     

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a λgtll expression library constructed from MG pericarp poly(A)⁺ RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.     

转反义PG基因番茄果实细胞结构变化的研究

经细胞学观察发现,转反义PG基因番茄果实在不同成熟期及存放前后,其果皮外面几层细胞的厚度都比未转基因的厚1～5 μm,细胞结构、细胞质和细胞核等的状态都有明显区别。尤以贮存后更为明显,未转基因果实的果皮细胞结构解体、细胞质凝聚、细胞核变的模糊程度都比转基因的严重。经外源乙烯处理后,转基因和未转基因果实的细胞结构也有相似的变化。结果表明：反义PG基因的转入降低了PG活性,并且减弱了外源乙烯的作用,延缓了果实的衰老,提高了耐贮性能,从而起到果实保鲜作用。     

Analysis of Transgenic Tomatoes with Expression of Antisense Polygalacturonase Gene

Authors have been transfered antisense polygalacturonase (PG, E, C, 3, 2, 1, 15) gene into two tomato cultivars (Lycopersicon esculentum cv. “Lichun” and “Qingfeng”) by Agrobacterium-mediated transformation and obtained homozygotes with expression of antisense PG gene. Analysis of Northern blot and PG activity showed that antisense PG gene had stably expressed in T0～T2 generations of transgenic tomatoes, and this expression could be inherited and enhanced in the progeny. For example, PG activity of To, T1 and T2 generation fruits in line T2-19 of"Lichun” was dropped to 27%, 20% and 4% respectively. But the remarkable decrement of PG activity did not have obvious inhibitory effect on normal fruit softening. The transgenic plants of T0～T2 generations grew and developed normally. Average yield per plant and average weight per fruit in these transgenic plants were a little more than those in nontransgenic control. The content determination of vitamin C, soluble sugar and soluble solid substances indicated that transgenic fruits kept the original texture and taste. The contents of vitamin C and soluble solid substances of “Lichun” T2-19 and soluble sugar of “Qingfeng” T2-46 were higher than those of the control. Stored in 28～32℃ for 30 days, all of nontransgenic fruits and about 50% of the transgenic ones were infected by pathologcal bacteria, a few of remainders could be perserved for 50 days. These evidences suggested that the resistance of the transgenic fruits to pathological bacteria during preservation has been strengthened. The approach which the authors used to study regulation of tomato fruit ripening will be desirable to improve the storability of certain famous and precious fruits.     

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.     

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.