MATERIALS AND METHODSA technique of centrifuging pea epicotyl sections which extracts watersoluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests Reactions occurring in the cell wall are often difficult to study because of the problems in separating the solution that bathes the cell wall from the complex solution within the cell. Any process which ruptures the plasmalemma leads to a mixing of intracellular and extracellular components, making it difficult to determine whether compounds such as enzymes and water-soluble polysaccharides were localized in the cell wall or the cytoplasm.Abeles et al.(1) devised a method of centrifuging pea stem sections to remove the extracellular solution. They found that pea stem sections could be centrifuged at 3,000g, and a solution was removed which contained cellulase, an enzyme believed to be present in the cell wall. Using a similar technique, Stafford and Bravinder-Bree (13) localized a peroxidase isozyme within the cell wall of sorghum, but some cytoplasmic isozymes were also detected. Ferrari and Amison (6) refined this technique further and found that centrifuging pea stem sections at 500g released cellulase. However, some of the cells must have been broken because a cytoplasmic enzyme, malate dehydrogenase, was also found in the solution spun from the sections.The present study describes a method of centrifuging soluble substances from the cell walls of plant stem sections and evaluates the damage to the cells and contamination of the cell wall solution by the cytosol. ' Present address: Department of Botany, University of California, Berkeley, Calif. 94720.General Procedure. Seeds of Pisum sativum var. Alaska were grown in Vermiculite for 7 days in darkness. Under fluorescent room lights and using a specially designed harvesting rack, 1.2-cm sections were excised approximately 1 cm below the apical hook. All subsequent operations were performed in the light. These sections were packed vertically in the barrel of a 20-ml plastic syringe (20 mm i.d.) cut off at the 6-ml mark to form a small tube (Fig. 1). The apical end of the sections rested against a 0.32-cm porous polyethylene disk in the bottom of the tube (Bel-Art Products, Pequanock, N.J.). Once packed, the sections were maintained in a vertical position, retaining the original apical end above the basal end, except during centrifugation. Each tube contained 95 to 105 sections weighing approximately 3 g. Among tubes packed for each experiment, the number of sections varied by less than four per tube and the weight varied by less than 0.1 g/tube. The packed tubes were placed in distilled H20 covering the bottom I to 3 mm of tissue. One h after excising the sections, the tubes were connected to a circulating pump and 20 mm Kphosphate adjusted to pH 6.0 with HCI was passed through the tubes at 200 ml/min -tube for 30 min at 26 C to remove cytoplasmic contamination from cut surfaces. The total volume of...

Summiarv. The produiction of ethylene by etiolated pea epicotyls (Pisuim sativum11 L., cv. Alaska) is confined to the plumule and plumular hook portion of the epicotyl, and occurs at a rate of about 6 pLlkg-l.hr-1. Such a rate is sufficient to give physiologically active concentrations of ethylene within the tissue. Exposure of etiolated seedlings to a single dose of red light caused a transient decrease in ethylenc produiction and a corresponding increase in plumuilar expansion. Far-red irradiation following the red light treatment decreased the red effect to the level achieved by the far-red alone, suiggesting that the ethylene produiction mechanism is controlled by phytochrome and thuis that the ethylene intervenes as a regulator in the phytochrome control of pltimuilar expansion.A relationship between the production of ethylene an(l the inhibition of plumular expansion in etiolatedI pea epicotyls can be 'deduced from several recent observations. First, the rate of ethylene productioin in the growing epicotyl does not increase in proportion to the increasing mass or ntumber of cells (5)

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA20), the product (GA1), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA1 under red irradiation and in darkness. The lele plants grew in response to GA20 and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA20 and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3j@-hydroxylation of GA20 to GA1 in genotype lele is due to a Pfr-induced blockage in the expression of that activity.Light grown dwarf peas lack the ability to convert GA202 to GA1 (6, 7), yet GA1 and GA8 were recently identified in extracts ofetiolated dwarf pea shoots (3). We will present evidence which suggests that etiolated dwarf pea plants lose the ability to convert GA20 to GA, upon formation of Pfr as a consequence ofexposure to R. Attempts by earlier workers to measure light-induced changes in GA metabolism or GA levels in tall and dwarf peas led to inconsistent results (reviewed in Smith [20]). Some experiments indicated phytochrome-mediated changes in GA metabolism, while other reports suggested an effect of light on "sensitivity" to endogenous GAs.Some genes which help determine plant stature in Pisum have recently been characterized (6,7,13,14,17). At least five loci have been shown to influence stem elongation, including the Na and Le loci, which affect GA metabolism. The recessive na allele apparently blocks an early step in GA biosynthesis, since nana plants do not contain detectable levels of GAs (13). The early C-13 hydroxylation pathway of GA biosynthesis predominates in peas (3,8

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.

Sumuwiiii(iry. Phytcchrc'me has been isolated from the green alga Alesotaenimn and the liverwort Spiwerocarpos. The Mlesotaenhiuin p gment had absorption peaks at 649 and 710 n-m for the P1 and P1. form's, respotcively. Corre-pon-ding difference spectrum maxima for the Sphw(erocorpos pigment were at 655 and 720 nm. While the absorption maxima differ, the reversibility and efficiency with which red and far-red light transform the MAesotaenitnm pigment are verv similar to that reported for phvtochrome isolated from etiolated seedlings of higher plants. Mcthods are described wxhich allow efficient separation of phytochrome from highly pigmente(d light-grown mater al.