Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid.

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl(2), and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H(+)-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H(+)-translocating ATPase located at the plasma membrane of the mesophyll cells.

Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid.

The effect of gibberellic acid (GA(3)) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA(3) (10 micromolar) applied to the source leaf, enhanced export of [(14)C]sucrose (generated by (14)CO(2) fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA(3) was applied 24 hours prior to the (14)CO(2) pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA(3) increased the proportion of sucrose exported. After a 10-min exposure to [(14)C]GA(3), radioactivity was found only in the source leaf. Following a 2 hour exposure to [(14)C]GA(3), radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [(14)C]GA(3) in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA(3) effect in the lower stem with time after treatment. We conclude that in the short term, GA(3) enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA(3) is outside the source leaf. GA(3) accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA(3) is rapidly metabolized in the lower stem thus attenuating any GA effect.

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation.

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with (14)CO(2), stem sections were cut and the efflux characteristics of (14)C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of (14)C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [(14)C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [(14)C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.

Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells.

A recombinant plasmid containing 2/3 of the rat skeletal muscle actin structural gene plus 730 bp of its 5' flanking region, spliced to the 3' end of the human epsilon-globin gene, was introduced into cells of the rat myogenic line L8. Myogenic clones carrying the actin/globin chimeric gene were isolated. In many of these clones, the expression of the gene greatly increased during differentiation (up to greater than 50-fold) and, in some clones, the amount of the chimeric gene transcripts in the differentiated cultures exceeded that of the native muscle actin gene transcripts. Furthermore, the temporal relation between differentiation of the cultures and the accumulation of the transcripts from the transferred genes was very similar to that of the native skeletal muscle actin gene, suggesting a similar mechanism of regulation. Endonuclease S1 analysis indicated a correct initiation and termination of the mRNA but suggested that a fraction of the chimeric actin/globin transcripts was not properly processed. To test whether the increased expression of the transferred gene which occurred during differentiation was determined by DNA sequences in the 5' region of the muscle actin gene, a plasmid (p alpha-CAT) containing 730 bp of the 5' flanking region of the rat skeletal muscle actin gene (plus the exon of the 5' untranslated region, and 25 bp of the first intron), spliced to the bacterial structural gene coding for chloramphenicol acetyl transferase (CAT), was constructed and introduced into L8 cells. In the majority of the isolated clones containing this plasmid, CAT activity increased many-fold during differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pithiness in plants: I. The effect of mechanical perturbation and the involvement of ethylene in petiole pithiness in celery.

Mechanical perturbation (MP) applied to celery (Appium graveolens L. cv. Florida 683) leaf petioles or ethephon application to the plant did not induce thigmomorphogenesis (inhibition of elongation and increase in thickness of the petiole). However, the two treatments did cause the parenchyma breakdown which leads to pithiness or increased natural pithiness, mainly at the base of the petiole. Nevertheless, MP (but not ethephon) decreased the severity of drought-stress or GA3-induced pithiness. Although MP stimulates ethylene production, mainly at the middle part of the petiole, it seems that the protection by MP of the petiole may not be directly mediated by ethylene production. The exposure of the plant to drought stress brought about an increase in ethylene evolution. Upon reirrigating the plants, the first steps of pithiness were accompanied by a sharp decline in ethylene production. This decrease might be due to membrane disruption. The increase in ethylene production during drought stress may be one of the events which stimulate pithiness of the celery leaf petiole.

The erythrocyte membrane site for the effect of temperature on osmotic fragility.

The osmotic fragility of human erythrocytes is well known to decrease as the temperature is elevated. The cellular site for the temperature effect was studied by assessing possible roles of hemoglobin and of membrane lipids and by taking advantage of the unique response of camel erythrocytes to temperature. It is concluded that the erythrocyte membrane is the site for the temperature effect on osmotic fragility. The human erythrocyte is likely to rupture in protein--lipid boundary regions in the membrane, from which cholesterol is apparently excluded.

Unique properties of the camel erythrocyte membrane, II. Organization of membrane proteins.

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein:lipid ratio of 3:1). Membrane proteins of camel erythrocytes were compared with those of human eruthrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an "integral skeleton", is a dominant structural feature stabilizing the camel erythrocyte membrane.

Elevated microviscosity in membranes of erythrocytes affected by hereditary spherocytosis.

Erythrocytes affected by hereditary spherocytosis (HS), obtained from several splenectomized patients, showed a varying degree of elevated osmotic fragility. In order to evaluate a possible role of the erythrocyte membrane lipids in HS, microviscosity of the membrane lipid core was measured by a fluorescence-polarization technique. Intact HS-affected red cells, as well as their ghost membranes and liposomes prepared from their lipid extract, all showed a distinctly higher micro-viscosity than the respective normal control. The increased microviscosity correlated with the severity of HS. The data support the proposition that the defect in HS-affected red cells is associated, at least in part, with alterations in the membrane lipids.

Effect of Water Stress on Ethylene Production by Detached Leaves of Valencia Orange (Citrus sinensis Osbeck).

Detached leaves of Valencia oranges, Citrus sinensis Osbeck, emanated ethylene at markedly higher rates and contained more endogenous ethylene when placed under water stress at 55% relative humidity than when placed in water-saturated air. Water stress induced defoliation from detached branches. Relieving the water stress of such leaves by transfer to a mist chamber resulted in lowering of the rates of ethylene emanation to the level occurring in leaves maintained continuously in a mist chamber. This ability to recover from the water stress was evident for only up to 10 to 20 hours of stress, when the relative turgidity of the leaves was 50 to 60%. Beyond that time the level of ethylene emanation of stressed leaves was not lowered by rehydration in a mist chamber; these overstressed leaves could not reabsorb their original water content. Ethylene emanation was in high correlation with the relative turgidity of detached leaves of oranges.