Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion.

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.

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : II. Regulation of ALA-Synthesizing Capacity by Phytochrome.

When dark-grown cucumber (Cucumis sativus L.) seedlings previously exposed to white light for 20 hours were returned to darkness, the ability of isolated chloroplasts to synthesize 5-aminolevulinic acid dropped by approximately 70% within 1 hour. The seedlings were then exposed to light, and the synthetic ability of the isolated chloroplasts was determined. Restoration of the synthetic capacity was promoted by continuous white or red light of moderate intensity. Intermittent red light was also effective. Blue and far-red light did not restore the synthetic capability. Blue light given after a red pulse did not enhance the effect of the red light. Far-red light given immediately after each red pulse prevented the stimulation due to intermittent red light. Restoration of the biosynthetic activity by in vivo light treatments was inhibited by cycloheximide indicating the requirement for translation on 80 S ribosomes for the in vivo light response. These findings suggest that the majority of the plastidic 5-aminolevulinic acid synthesis is under phytochrome regulation.

Labeling of porphobilinogen deaminase by radioactive 5-aminolevulinic acid in isolated developing pea chloroplasts.

A protein had been previously described, which was labeled by radioactive 5-aminolevulinic acid in isolated developing chloroplasts. In the present study we have shown that this protein (Mr approximately equal to 43,000) probably exists as a monomer in the chloroplast stroma. The labeling is blocked if known inhibitors of 5-aminolevulinic acid dehydratase are added to the incubation mixture, and is markedly decreased in intensity if nonradioactive 5-aminolevulinate or porphobilinogen are added to the incubation mixture; other intermediates in the porphyrin biosynthetic pathway, uroporphyrinogen III, uroporphyrin III, and protoporphyrin IX, do not decrease the labeling of the 43-kDa protein appreciably. Nondenaturing gels of the proteins isolated from the incubation with radioactive 5-aminolevulinic acid were stained for porphobilinogen deaminase activity. A series of red fluorescent bands was obtained which coincided with the radioactive bands visualized by autoradiography. It is concluded that the soluble chloroplast protein that is labeled in organello by radioactive 5-aminolevulinic acid is porphobilinogen deaminase.

Phytochrome Regulation of the Response to Exogenous Gibberellins by Epicotyls of Vigna sinensis.

The elongation rate of cowpea epicotyls from whole cowpea (Vigna sinensis) seedlings and derooted and debladed plants (explants) increased after the main light period (8-hour duration) was extended with either continuous low intensity tungsten light or brief (5 minutes) far-red (FR) irradiation. This end-of-day FR effect was reversed by red (R) irradiation suggesting the involvement of phytochrome. These results confirm and extend those obtained previously with other species. Localization studies indicate the epicotyl to be the site of the photoreceptor. Treatment of cowpea seedlings with paclobutrazol, a gibberellin (GA) biosynthetic inhibitor, abolished the FR promoted epicotyl elongation, indicating a role for GAs in this process. There was no significant difference in epicotyl elongation rates of R plus FR irradiated explants treated with GA(1) or GA(20) and R irradiated explants treated with GA(1). However, R irradiation inhibited subsequent epicotyl elongation of GA(20) treated explants. Moreover, the observation, using GC-MS, that GA(1) and GA(20) are native GAs in cowpea lends support to the concept that phytochrome may control the conversion of endogenous GA(20) to GA(1) in cowpea.

Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L.

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 (GA(20)), the product (GA(1)), 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 GA(1) under red irradiation and in darkness. The lele plants grew in response to GA(20) and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA(20) 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 3beta-hydroxylation of GA(20) to GA(1) in genotype lele is due to a Pfr-induced blockage in the expression of that activity.

An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth.

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.

The effect of abscisic acid on the uptake of potassium and chloride into Avena coleoptile sections.

The effect of abscisic acid (ABA) on uptake of potassium ((86)Bb(+) or (42)K(+)) by Avena sativa L. coleoptile sections was investigated. ABA lowered the potassium uptake rate within 30 min after its application and inhibition reached a maximum (ca. 75%) after 2 h. The inhibition of K(+) uptake increased with ABA concentration over a range of 0.03 to 10 µg/ml ABA. At a higher K(+) concentration (20 mM) the percentage inhibition decreased. The percentage inhibition of K(+) uptake by ABA remained constant with external K(+) varied from 0.04 to 1.0 mM. After a loading period in 20 mM K(+) ((86)Rb(+)), apparent efflux of potassium was only slightly increased by ABA. Experiments in which growth was greatly reduced by mannitol or by omission of indole-3-acetic acid from the medium indicated there was no simple quantitative correspondence between ABA inhibition of coleoptile elongation and ABA inhibition of K(+) uptake. Chloride uptake was also inhibited by ABA but to a smaller degree than was K(+) uptake. No specificity for counterions was observed for K(+) uptake. Uptake of 3,0-methylglucose and proline were inhibited by ABA to a much smaller extent (14 and 11%) than that of K(+), a result which suggests that ABA acts on specific ion uptake mechanisms.

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684.

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2 degrees the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2 degrees was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2 degrees varied 3-fold among the plants treated. The reaction was not first order. At 25 degrees the reaction followed at 690 nm was complete in 20 to 60 sec. Q(10)'s varied from 2.8 to 3.7 between 2 degrees and 25 degrees . Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.

An effect of light on the production of ethylene and the growth of the plumular portion of etiolated pea seedlings.

The production of ethylene by etiolated pea epicotyls (Pisum sativum L., cv. Alaska) is confined to the plumule and plumular hook portion of the epicotyl, and occurs at a rate of about 6 mul.kg(-1).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 ethylene production and a corresponding increase in plumular expansion. Far-red irradiation following the red light treatment decreased the red effect to the level achieved by the far-red alone, suggesting that the ethylene production mechanism is controlled by phytochrome and thus that the ethylene intervenes as a regulator in the phytochrome control of plumular expansion.

Isolation of phytochrome from the alga mesotaenium and liverwort sphaerocarpos.

Phytochrome has been isolated from the green alga Mesotaenium and the liverwort Sphaerocarpos. The Mesotaenium pigment had absorption peaks at 649 and 710 nm for the P(R) and P(FR) forms, respectively. Corresponding difference spectrum maxima for the Sphaerocarpos 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 Mesotaenium pigment are very similar to that reported for phytochrome isolated from etiolated seedlings of higher plants. Methods are described which allow efficient separation of phytochrome from highly pigmented light-grown material.