Galactolipid Synthesis in Vicia faba Leaves: IV. Site(s) of Fatty Acid Incorporation into the Major Glycerolipids

The fatty acids of the major glycerolipids of Vicia faba leaves were analyzed immediately following ¹⁴CO₂ feeding. The leaves were fractionated into chloroplast and cytoplasmic fractions and the location of radioactivity in the fatty acids determined. The results indicate that the major site of incorporation of fatty acids is in the phospholipids. Phosphatidylcholine contained the highest level of radioactivity in the cytoplasmic fraction, whereas phosphatidylglycerol contained radioactivity in both the chloroplast and cytoplasmic fractions. The galactolipids contained very little radioactivity in comparison, this radioactivity being confined to high speed centrifugal fractions believed to contain the envelopes of the chloroplast. Our results suggest that phosphatidylcholine is a major site of incorporation of fatty acids (mainly in oleic acid) in the cytoplasm, whereas phosphatidylglycerol is also a site of incorporation involving both oleic and palmitic acids, inside and outside the chloroplast.     

Galactolipid Synthesis in Vicia faba Leaves: I. Galactose, Glycerol, and Fatty Acid Labeling after CO(2) Feeding

The galactose, glycerol, and fatty acids of mono- and digalactosyl diglycerides (MGDG and DGDG) have been separated and analyzed for ¹⁴C activity after ¹⁴CO₂ feeding of Vicia faba leaf discs. Fully expanded and developing leaves were analyzed at time intervals following feeding during continuous illumination. In addition, fully expanded leaves were analyzed after similar times in complete darkness. In all cases, ¹⁴C was incorporated very rapidly into galactose, whereas glycerol and fatty acids were labeled much more slowly and over a longer period of time. The data are consistent with the galactosylation of a diglyceride to MGDG which is in turn galactosylated to DGDG. The data suggest that the formation of diglycerides suitable for galactosylation to MGDG is slow in comparison to the galactosylation process. It is also suggested that DGDG may be formed from more than one pool of MGDG. The complete analysis of the ¹⁴C incorporation into galactose appears to represent the only satisfactory method of comparing galactolipid synthesis by ¹⁴C incorporation. Estimates of comparative rates of synthesis of MGDG and DGDG have been made on this basis.     

Galactolipid Synthesis in Vicia faba Leaves: II. Formation and Desaturation of Long Chain Fatty Acids in Phosphatidylcholine, Phosphatidylglycerol, and the Galactolipids

The labeling kinetics of the fatty acids of phosphatidylcholine (PC), phosphatidylglycerol (PG), monogalactosyldiglyceride (MGDG), and digalactosyldiglyceride (DGDG) were examined after ¹⁴CO₂ feeding and incubation of leaf discs of Vicia faba over 72 hours in continuous light. The results indicate a rapid accumulation and turnover of radioactivity into PC and PG fatty acids (oleic acid in PC and oleic and palmitic acids in PG). Radioactivity accumulates in MGDG and DGDG fatty acids much more slowly and continuously over 72 hours. Most of this activity is found in linoleic and linolenic acids; very little activity is found in the more saturated fatty acids. Little or no desaturation occurs in situ in conjunction with the galactolipids. The results suggest that PC and PG may act as “carriers” for MGDG and DGDG fatty acid synthesis. Analyses of the labeling patterns of the molecular species of MGDG after ¹⁴CO₂ and ¹⁴C-acetate feeding confirm that MGDG is formed by galactosylation of a preformed diglyceride containing predominantly unsaturated fatty acids.     

Wilting of Leaves in Vicia faba L.

Wilting of leaves of Vicia faba L., which occurs when the pressurepotential (_p) is zero, and the leaf-water potential () at wiltingboth depend entirely upon the solute potential at incipientplasmolysis (_so) and not on soil-water status. Wilting in V.faba is acropetal; this is consistent with the hypothesis thatthere is a gradient of decreasing _so up the plant and that wateris transferred from the lower to the upper leaves, hasteningthe overall water loss from the lower leaves to the point when_p is zero. The gradient in _so up the plant is of the order of3–8 bar. It is proposed that wilting when _p>0 (i.e. > _so) shouldbe ‘apparent wilting’ and that when _p0 (i.e. _so),‘true wilting’.     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Some Observations on Invertase Activity in Roots of Vicia faba L.

Invertase activity has been determined at intervals along primaryroots of Vicia faba as they elongated from 0·5 to 8 cm.Little activity was evident in 0·5–1·0 cmlong primaries but in those 2–8 cm in length the mainpeak of enzyme activity was associated with the region of cellelongation. Changes took place in the pattern of invertase activityalong the primary roots as they lengthened and these changeshave been correlated with fluctuations in both the rate of rootelongation and the supply of sucrose to the root from the cotyledons.The presence of a root cap did not increase the activity ofthis enzyme in the apical 1 mm of these roots. Invertase activity was higher in lateral root primordia thanin most parts of the primary root basal to the meristem, presumablybecause of the presence of sucrose in the adjacent cavity inthe cortex of the primary root. The peaks of invertase activityfound basal to the region of cell elongation in 3–8 cmlong primary roots probably resulted from the development ofroot pnmordia in these parts of the root.     

Starch Degradation and Distribution of the Starch-Degrading Enzymes in Vicia faba Leaves (Diurnal Oscillation of Amylolytic Activity and Starch Content in Chloroplasts)

Subcellular localization of the starch-degrading enzymes in Vicia faba leaves was achieved by an electrophoretic transfer method through a starch-containing gel (SCG) and enzyme activity measurements. Total amylolytic and phosphorolytic activities were found predominantly in the extrachloroplastic fraction, whereas the debranching enzymes showed homogenous distribution between stromal and extrachloroplastic fractions. Staining of end products in the SCG revealed two isoforms of [alpha]-amylase (EC 3.2.1.1) and very low [beta]-amylase activity (EC 3.2.1.2) in the chloroplast preparation, whereas [alpha]- and [beta]-amylase exhibited higher activities in the crude extract. However, it is unclear whether the low [alpha]- and [beta]-amylase activities associated with the chloroplast are contamination or activities that are integrally associated with the chloroplast. Study of the diurnal fluctuation of the starch content and of the amylase activities under a 9-h/15-h photoperiod showed a 2-fold increase of the total amylolytic activity in the chloroplasts concurrent with the starch degradation in the dark. No fluctuation was detectable for the extrachloroplastic enzymes. The possible roles and function of the chloroplastic and extrachloroplastic hydrolytic enzymes are discussed.     

The Galactolipid, Phospholipid, and Fatty Acid Composition of the Chloroplast Envelope Membranes of Vicia faba. L

The galactolipid, phospholipid, and fatty acid composition of chloroplast envelope membrane fractions isolated from leaves of Vicia faba L. has been determined. The major lipids in this fraction are: monogalactosyldiglyceride, 29%; digalactosyldiglyceride, 32%; phosphatidylcholine, 30%; and phosphatidylglycerol 9%. The lipid composition of the chloroplast envelope membranes is qualitatively similar to that of the lamellar membranes isolated from the same plastids, but the proportion of each lipid present is very different. The total galactolipid to total phospholipid ratio was 1.6: 1 in the envelope and 11.1: 1 in the lamellae. The monogalactosyldiglyceride-digalactosyl-diglyceride ratio was 0.9: 1 in the envelope and 2.4: 1 in the lamellae. Both membranes lack phosphatidylethanolamine.     

Compartmentation in Vicia faba Leaves: I. Kinetics of C in the Tissues following Pulse Labeling

Leaflets of Vicia faba were pulse-labeled with ¹⁴CO₂ to follow the subsequent movement of photosynthate between leaf tissues. Samples were taken during a ¹²CO₂ chase, quick frozen, freeze-substituted, and embedded in methacrylate. Paradermal sections provided tissue samples consisting only of upper epidermis, palisade parenchyma, spongy parenchyma and veins, spongy parenchyma, or lower epidermis. Most CO₂ fixation occurred in the palisade parenchyma, but its ¹⁴C content declined rapidly after labeling. Concomitant with the decline of activity in the palisade parenchyma, there was an increase in activity in the spongy parenchyma and upper epidermis and a slow increase in the lower epidermis. Activity in the palisade parenchyma and spongy parenchyma eventually reached similar levels and remained constant. Tissue samples containing veins were consistently the most radioactive, and activity in those samples showed a decline. Very little change occurred in the insoluble fraction from any tissue. The results support previous assumptions regarding the pathway of assimilate transport to the veins, and demonstrate the rapidity of such transport. Sucrose is apparently the principal mobile compound.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

Altered features in the secondary structure of Vicia faba 5.8s rRNA.

We have re-examined the nucleotide sequence of Vicia faba (broad bean) 5.8S rRNA using partial chemical degradation and a new approach to high temperature (65-80 degrees C) sequencing gels. The results indicate that the secondary structure was not completely disrupted in previous studies (Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nucleic Acid Res. 8, 1259-1272) and explain ambiguities between the nucleotide sequence and T1 ribonuclease digests. Despite this revision, estimates in the secondary structure suggest that this 5.8S rRNA differs from previously examined examples in two respects, more open conformations in both the "GC-rich" and "AU-rich" stems. The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. These studies and theoretical considerations generally supported the "burp gun" model previously proposed for all 5.8S rRNAs and were inconsistent with the recently suggested "cloverleaf" configuration. More importantly, they were also consistent with more open stem structures in this higher plant.     

High Respiratory Activity of Guard Cell Protoplasts from Vicia faba L.

The rate of O₂ uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O₂ uptake was inhibited by respiratory inhibitors,but stimulated by respiratory uncouplers. On a Chi basis, theactivities of Cyt c oxidase and NADH-Cyt c reductase, mitochondrialenzymes, were about 27 and 35 times higher in GCPs than in MCPs.On a Chi basis, the ATP content was about 9 times higher inGCPs. The amount of ATP in GCPs was decreased by respiratoryinhibitors, an energy transfer inhibitor, and uncouplers ofoxidative phosphorylation. On a volume basis, GCPs had 8- to10-fold higher respiratory activities than MCPs, but had a lowChi content and lacked the activity of NADP-glyceraldehyde-3-phosphatedehydrogenase (NADP-GAPD), the Calvin cycle enzyme. From theseresults, we concluded that oxidative phosphorylation plays amain role in ATP production in guard cells and that guard cellshave a heterotrophic feature. Salicylhydroxamic acid (SHAM)in combination with KCN or NaN₃ strongly inhibited O₂ uptake,indicating the presence of cyanide-resistant respiration inguard cells. Phenylmercuric acetate (PMA), a potent inhibitorof stomatal opening, reduced the ATP content of GCPs by about90%, whereas it had a relatively small effect on the ATP levelof MCPs. The specific effect of PMA on GCPs is discussed. (Received March 24, 1983; Accepted June 8, 1983)     

Thymidine Kinase Activity and its Relationship to DNA Synthesis in the Primary Root of Vicia faba, L.

The enzyme thymidine kinase can be induced, or activated, inthe apical 6 cm of the primary root of Vicia faba. The activityof this enzyme is correlated with the amount of thymidine triphosphatepresent in successive 6-mm segments of the root. Thymidine isproduced by cell death. Some of this thymidine may reach themeristem and be used by the apical cells in DNA synthesis, onceit is phosphorylated by thymidine kinase. Most remains in thebasal region of the root, however, where it is used by differentiatingcells. Non-radioactive thymidine, at the concentrations used,has no effect on root growth by elongation.     

蚕豆叶片尿囊素酶性质初探

酰尿输出型蚕豆有一定程度合成和同化酰脲的能力(刘承宪和黄维南1987a),蚕豆叶片尿囊素酶(B-ALNase)和酰脲输出型大豆叶片尿囊素酶(S-ALNase)(Thomas等1983)不同,是热敏感的(刘承宪和黄维南 1987b)。我们进一步分离和纯化B-ALNase,并作了初步的鉴     

Compartmentation in Vicia faba Leaves: II. Kinetics of C-Sucrose Redistribution among Individual Tissues following Pulse Labeling

Leaflets of Vicia faba L. were pulse labeled with ¹⁴CO₂ and the kinetics of ¹⁴C-sucrose redistribution among individual tissues was followed. Sucrose specific activity in the whole leaf peaked about 15 minutes after labeling and declined with a half-time of about 80 minutes. In one experiment, leaflet discs taken at various times during the ¹²CO₂ chase were quick frozen, freeze-substituted, and embedded in plastic. The tissue was sectioned paradermally and sections of palisade parenchyma, of spongy parenchyma, and of spongy parenchyma that contained veins were collected. Water extracts from these sections were assayed for sucrose specific activity. Sucrose specific activity in the palisade parenchyma was higher than that of the spongy parenchyma and reached a maximum in both tissues 9 to 15 minutes after labeling. Sucrose specific activity initially declined rapidly in the palisade parenchyma followed by a period during which little or no loss occurred. Sucrose specific activity in sections containing veins peaked at 15 minutes with a maximum value substantially higher than either mesophyll tissue, indicating that recently synthesized sucrose was preferentially exported from the mesophyll. Decline of activity in these sections containing veins continued for the remainder of the experiment. Sucrose specific activity in lower epidermal peels peaked several minutes after that of the whole leaflet and remained lower. Sucrose specific activity in upper epidermal peels was variable (probably due to contamination), but the limited data suggest that the sucrose specific activity there reached somewhat higher values than those of the lower epidermis. The experiments indicate that each leaf tissue contains a kinetically identifiable sucrose pool (which we refer to as “histological compartmentation”), and that further compartmentation may occur at the intracellular level. A simulation of leaf sucrose compartmentation is presented.     

Hydrogen Peroxide-dependent Synthesis of Flavonols in Mesophyll Cells of Vicia faba L.

Mesophyll cells of Vicia faba contain kaempferol and quercetinglycosides. When isolated mesophyll cells were treated with0.1 mM H₂O₂ for 2 h, the levels of these flavonols increasedby 10–70% of the control values (mean values, 19.6% and34.4% for kaempferol and quercetin glycosides, respectively).Such increases in levels of flavonols were also observed inisolated vacuoles of mesophyll cells. However, when mesophyllcells and vacuoles were treated with 10 mM H₂O₂₎degradationof flavonols was observed. These data suggest that H₂O₂ hastwo effects on the metabolism of flavonols: induction of theirsynthesis and stimulation of their oxidation. (Received March 6, 1989; Accepted July 10, 1989)     

The Respiration of Mature Field Bean (Vicia faba L.) Leaves During Prolonged Darkness

Dark respiration in attached and detached mature leaves of thefield bean (Vicia faba L.) was studied whilst leaves experiencedup to 60 h of darkness. The results showed: (1) the initialrespiration rate to vary according to the irradiance duringthe previous photoperiod; (2) the dark respiration rate (perunit area) of attached leaves to be essentially constant duringa normal 12 h night although there was a rapid loss in leafd. wt during this time; (3) after 12 h, the respiration rateof attached leaves decayed to an asymptotic value at about 36h; (4) the respiration rate of leaves detached at the end ofthe photoperiod and maintained in the dark on deionised water,decayed only after 36 h of darkness; (5) there was no differencebetween the respiration rate of attached and detached leavesduring the normal 12 h night. It is concluded that the dark respiration of attached fieldbean leaves is intially related to the synthesis and translocationof sucrose in addition to maintenance. After about 36 h, whenthe rate of CO₂ efflux is more or less steady, the CO₂ effluxreflects the intensity of maintenance processes only. The maintenancerespiration rate (determined after 60 h in the dark) rangedfrom 062 to 151 mg CO₂ (g d. wt)^–1 h^–1 but wasrelatively unaffected by several applied treatments. Vicia faba L., field bean, respiration, maintenance, nitrate, non-structural carbohydrate, export     

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 CaCl₂, 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.