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The subcellular localization of a basic peroxidase (EC 1.11.1.7) isoenzyme in crisphead lettuce ( Lactuca sativa L.) leaves was studied through subcellular fractionation and protoplast and vacuole isolation. This isoenzyme is mainly located in soluble fractions. Studies using protoplast isolation and vacuole purification indicated that the soluble basic peroxidase isoenzyme is found in the vacuolar sap, probably in equilibrium with the same isoenzyme attached to tonoplast membranes. dialyzed overnight against 50 mM tris-HCl buffer, pH 7.5. Mesophyll protoplast isolation. The lower epidermis of 1.0 g of leaf tissue was removed with tweezers and immersed in 20 m M tris- mes buffer, pH 5.5, containing 1.0 m M CaCl 2 1.0% (w/v) bovine serum albumin (BSA), and 0.65 M sorbitol to prevent tissue dehydration. The leaves were incubated in a maceration medium (25 ml) containing 1% (w/v) caylase, 0.02% (w/v) pectolyase, 0.2% (w/v) pectinase, 1.0% (w/v) BSA, 1.0 mM CaCl 2 , and 0.65 M sorbitol in 20 mM tris-mes, pH 5.5, at room temperature for 45 min in darkness with occasional stirring. The released protoplasts were filtered through nylon gauze (40-μM mesh), centrifuged at 100× g for 6 min, and washed three times by resuspension in a washing buffer containing 1.0 mM CaCl 2 and 0.65 M sorbitol in 20 m M tris- mes, pH 7.3. The green precipitate was recovered by suction and viewed under a light microscope (400×) to check protoplast integrity and purity. To determine enzyme activities, mesophyll protoplasts were broken by freezing and thawing, then incubated in 1 M KCl for 30 min and centrifuged at 15,000× g for 15 min. The supernatant was desalted by dialysis. Vacuole isolation. Vacuoles were prepared from mesophyll protoplasts according to the method of Renaudin et al. (1986) but with minor modifications: 0.5 ml of protoplast suspension was added to 0.88 ml of 10 mM tris-mes, pH 7.3, containing 7.9 m M Na 2 EDTA. After 10 min at 4C and gentle shaking, the suspension was quickly mixed with 5.0 ml of 5 mM Na 2 EDTA, 0.36 M sorbitol, and 8.5% (w/v) nycodenz (5-(N-2,3-dihydroxypropylacetamido)-2,4,6- triiodo- N,N'-bis(2,3-dihydroxypropyl)isophtalamide) in 10 m M tris-mes, pH 7.3. Mesophyll vacuoles were purified by flotation and recovered in the uppermost layer (0.7 ml) after centrifugation at 160× g for 6 min at 4C. Vacuole integrity was checked by staining with 1% (w/v) neutral red in 0.65 M sorbitol and 10 mM tris- mes, pH 7.3. To determine enzyme activities, mesophyll vacuoles Russet spotting is a postharvest disorder that can develop during transport and storage of crisphead lettuce (Ke and Saltveit, 1989). This disorder is characterized by the appearance of many small brown spots along both sides of the mibrid, spots that can spread over the leaf blade (Link and Gardner, 1919). Peroxidase (EC 1.11.1.7) is an enzyme that may be crucial in this postharvest disorder due to its known participation in oxidative metabolism during plant storage (Frenkel, 1979) and its role in indole-3-acetic acid (IAA) homeostasis through catabolism (Ros Barcelo and Munoz, 1992). Ke and Saltveit (1988) reported that applying IAA greatly reduced russet spotting and IAA oxidase activities and that lignifying russet spotting-affected cells resulted in cell-wall thick- ening. IAA oxidase activity resides mainly in peroxidase isoenzymes of the most basic isoelectric point (pI) (Ros Barcelo and Munoz, 1992), and the expression of these isoenzymes is largely regulated by IAA concentrations in plant tissues (Ferrer et al.,1991). These basic peroxidase isoenzymes also play a role in flavonol turnover (Morales et al., 1993b) and lignin biosynthesis (Morales et al., 1993a). We studied the subcellular localization of a basic peroxi- dase isoenzymes in 1-month-old crisphead lettuce leaves, in which peroxidase polymorphism is mainly restricted to a sole gene product.

Subcellular Localization of a Basic Peroxidase Isoenzyme in Crisphead Lettuce