Koch J L, Nevins D J
Department of Vegetable Crops, University of California, Davis, California 95616.
Plant Physiol. 1989 Nov;91(3):816-22. doi: 10.1104/pp.91.3.816.
Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces beta-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development.
对细胞壁分离程序进行了评估，以确定其对番茄（Lycopersicon esculentum L.）果实细胞壁总果胶含量和甲酯化程度的影响。水匀浆可释放大量缓冲液可溶性糖醛酸，即5.2毫克糖醛酸/100毫克细胞壁。溶解似乎是由多聚半乳糖醛酸酶II的同工酶A和B介导的自水解作用的结果，因为在50%乙醇存在下匀浆并加热后，细胞壁残渣中的糖醛酸释放可被抑制。热灭活细胞壁中的甲酯化程度为94摩尔%，显著高于水匀浆中的甲酯化程度（56摩尔%）。结果表明，细胞壁相关酶介导的自水解作用是番茄果实果胶体外溶解的原因。内源性酶也导致细胞壁制备过程中甲酯化程度降低。热灭活细胞壁制备方法优于所研究的其他方法，因为它减少了加热过程中的β-消除作用，并使可能改变果胶结构的组成酶失活。这种热灭活细胞壁制备物用于随后的果胶结构酶分析。纯化的番茄果实多聚半乳糖醛酸酶和部分纯化的果胶甲酯酶用于评估番茄果实成熟过程中组成底物的变化。用多聚半乳糖醛酸酶处理来自绿熟期和转色期的热灭活细胞壁，可释放14%的糖醛酸。多聚半乳糖醛酸酶释放多聚糖醛酸的程度取决于果实发育阶段。在转色期，释放了21%的果胶部分，在红熟期该值增加到糖醛酸的最大值28%。用纯化的番茄果胶酯酶预处理细胞壁，使所有成熟阶段的细胞壁对多聚半乳糖醛酸酶的敏感性相同。从数量上看，多聚半乳糖醛酸酶从所有经果胶酯酶处理的细胞壁中释放糖醛酸的量相当于对成熟阶段细胞壁进行多聚半乳糖醛酸酶处理的量。多聚半乳糖醛酸酶释放的糖醛酸聚合物含有半乳糖醛酸、鼠李糖、半乳糖、阿拉伯糖、木糖和葡萄糖。随着发育，观察到半乳糖醛酸和鼠李糖的释放增加（在绿熟期这些聚合物中分别为40%和6%，在红熟期分别为54%和15%）。在发育过程中，外源性多聚半乳糖醛酸酶释放的半乳糖和阿拉伯糖的量减少（从绿熟果实细胞壁中的41%和11%分别降至红熟期的11%和6%）。外源性多聚半乳糖醛酸酶从细胞壁中释放的少量葡萄糖和木糖（4 - 7%）在整个果实发育过程中保持不变。
