(Genitourin Med 1995;71:402-404)

ITwo different forms of invertase are found in pollen of lily (Lilium anratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partilly purifed and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K, of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are ,@-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germtion. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.Invertase (EC 3.2.1.26) or ,B-fructofuranoside fructohydrolase hydrolyzes sucrose and related oligosaccharides. Sucrose represents the major soluble sugar present in the majority of angiosperm pollen grains and is also the most commonly detected sugar in natural substrates for pollen germination (25). Germinating pollen grains show an extremely high rate of growth, i.e. 400 Am/h (13) and thus have a high demand for carbon skeletons required for pollen tube wall synthesis as well as a substrate for respiration. In vivo, the pollen tubes of lily grow on the surface of stigmatic and stylar canal secretion products. By means of labeling experiments, it has been shown that carbohydrate material of the secretion product of styles is used by growing pollen tubes for tube wall synthesis (17). Although invertase was among the first enzyme activities recognized in pollen, no detailed characterization of this pollen enzyme has been attempted. In the present study, we report the purification and properties of invertase from the pollen of Lilium auratum and discuss its functional significance. 12,000g for 10 min, and the supernatant was dialyzed for 20 h at 2C against extraction buffer and the dialyzed extracts were then used as a crude extract for enzyme assays. The wall pellets were again washed with extraction buffer three times and the washings were discarded. The resulting pellet was then suspended in 50 mM citrate-phosphate buffer (pH 6.0), and the suspension was used as a source of wall-bound invertase. Aliquots of wall fraction were assayed for hexokinase activity (11) to ascertain the absence of contaminant cytoplasmic proteins. The enzymic activity of soluble invertase was assayed as follows: a 0.05-ml sample of invertase preparation was added to 0.45 ml of 0.1 M acetate buffer (pH 4.8) which contained sucrose at a final concentration of 100 mm. The mixture was incubated for 20 min at 35C. The reactions were terminated by placing the reaction tubes in a boiling water bath for 5 min. After cooling the tubes to 35C, glucose released in the reaction mixture was estimated by a colorimetric assay using glucose oxidase and peroxidase. The assay was carried out by adding 0.5 ml ofchromogen reagent (4) to 0.5 ml ofthe invertase reaction mixture. In some instances, the reducing sugars relea...

The synthesis of jB-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-j%-Dgalactoside is used as substrate giving a bluegreen final reaction product. We have recently detected oilseed rape plants which are heterozygous for the fi-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The (1,3) and the presence or absence of alcohol dehydrogenase (ADH) in maize pollen (4), and segregation for f,-galactosidase activity in oilseed rape pollen (9). These markers all appear to be products of postmeiotic transcription of the haploid genome. However, the exact stage of pollen development at which synthesis of gametophytic proteins is initiated is not known. In the present report, we have used a recently developed quantitative cytochemical method (10) in combination with cytological stains to study the pattern of synthesis of f,-galactosidase.Plants of oilseed rape, Brassica campestris, which are heterozygous for the Gal locus, produce pollen grains where 50% carry the mutant gal allele and are therefore deficient for f-galactosidase activity (9). Anthers from such plants are particularly useful when studying the initiation of postmeiotic genetic activity because the enzyme-deficient pollen grain serve as an internal control for the visual observation and quantitation of the enzyme-specific reaction product. The presence of enzyme-deficient pollen enables ready identification of the first developmental stage where Gal grains show enzyme activity. MATERIALS AND METHODSBrassica campestris, oilseed rape, plants of Gal/gal genotype (9, 10) were raised to flowering under constant growth conditions with a 250/180 C day/night temperature regime and a 14-h photoperiod.Buds at various stages of development were removed from an inflorescence of one of these plants and the six anthers were dissected from each bud. Bud and anther lengths were recorded. For the assessment of pollen quality, two of the anthers from each bud were kept in Petri dishes lined with moist filter paper, prior to being used for the FCR' test (5). This same method was employed for identification of microspore and pollen developmental periods (12). The FCR method enables precise definition of the vacuolate period of microspore development. To identify the occurrence of pollen grain and generative cell mitosis, a modification of the method of Coleman and Goff (2) was employed. Two anthers were fixed in 3:1 ethanol:acetic acid for 2 h, washed in 70% ethanol, rinsed in water, and stained in DAPI, a nucleic acid fluorochrome (0.5 ,g/ml) and the grains viewed by incident light fluorescence microscopy.The remaining two anthers from each bud were employed for enzyme cytochemistry. The anthers were pla...

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