Variations of phosphorylation of protein membrane components in pea root meristems during germination

A number of endogenous substrates have been found in the 27,000g meristematic root membrane fraction of pea seedlings. The identities of such proteins remain unknown, but their phosphorylation patterns change during germination. The presence of different protein kinase activities, or different protein phosphatase activities besides the occurrence of metabolic variations in membranes, have been indicated as key factors that could act simultaneously to give variations in protein phosphorylation pattern during germination. The absence, previously reported, of such protein kinase activity in membranes extracted from differentiated root tissue has been related to a change in protein composition. This fact leads to the hypothesis that protein phosphorylation of such membranes could have a physiological role at least in meristematic tissues. However, no clear-cut indication of which physiological role such protein phosphorylations fulfill emerges from present data. Nevertheless, the coincidence of onset of DNA synthesis and variation in protein phosphorylation pattern of these membranes suggests that these two events could have some physiological dependence.     

Cyclin-dependent kinase-like proteins in pea nuclei: their presence and role in cell proliferation

Summary Although many putative cdk (cyclin-dependent kinase) homologue genes have been identified in higher plants, their function and involvement in cell proliferation are still unclear. In this work we investigated the presence and distribution of cdk-like proteins in root tip meristem nuclei at different germination times (before, during, and after the onset of cell proliferation) and in nuclei of differentiated leaves. Nuclear cdk-like proteins were found in the root meristem throughout seed germination with a higher amount in actively proliferating cells, but were not detected in differentiated leaf. Characterization of the detected pea cdk-like proteins by immunoblotting led to the identification of two specific principal proteins of 33.2 and 34 kDa with the cdk conserved motif PSTAIRE. The p33.2 protein was also recognized by the anti-human p33^cdk2 antibody, suggesting that the p33.2 and p34 proteins could be pea homologues of human p33^cdk2 and p34^cdk1, involved in the G₁-S and G₂-M transitions, respectively. Additional analysis of pea cdk protein localization has shown partial localization of these proteins at DNA replication sites during the G₁ to S transition. These microscopical and biochemical data support the hypothesis that, in pea nuclei as in mammals, many PSTAIRE-cdks are present with different functions related to cell proliferation, one of which is probably involved in the control of the G₁-S transition.Abbreviations Cdk cyclin-dependent kinase - HU hydroxyurea - BrdU bromodeoxyuridine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101 - PI propidium iodide     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts

Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743–745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L₆ myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system.We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f₂b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10^?5 M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase.Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems.The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane missing data     

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high K_m and a low K_m protein kinase activity while only the high K_m activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.     

Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation     

Catabolism of glucose in pea stem section during root formation and its inhibition by kinetin and ethionine

The catabolism of specifically¹⁴C-labelled glucose during the root formation and its inhibition by kinetin and ethionine in the etiolated pea stem sections were studied. The formation of root meristematic foci in the pericycle region of sections was accompanied by the decrease of the C₆/C₁ ratio. Such a result and activation of pentose phosphate cycle, which was also checked by another method, suggested increased participation of pentose phosphate cycle in glucose oxidation. The above mentioned changes were also found after the prevention of root formation by ethionine and, therefore, do not seem to be specific for the meristematic foci formation. The growth of newly formed roots was closely connected with the rise of C₆/C₁ values. The increase of CO₂ release from the first carbon atom of glucose molecule was recorded after the inhibition of root formation by ethionine. The rise of C₆/C₁ values and decrease of pentose phosphate cycle activity was observed after the treatment of pea stem sections by kinetin in the first 64 hours after sectioning. In this case root formation was prevented and the growth of lateral buds was stimulated. The secondary xylem formation which took place later was accompanied by the activation of the pentose phosphate cycle. These phenomena are discussed in relation to cell division and biosynthesis of lignin-precursors.     

Effect of water stress on root meristems in woody and herbaceous plants during the first stage of development

We investigated the effect of water stress on the root system architecture of pine saplings and pea seedlings during the first stage of development. Attention was focused on meristematic tissue situated at the root tip because of the leading role played by the tissue in the planning of root system architecture. The data showed that both species are extremely sensitive and that plants arrest their growth immediately during water stress treatment. When stress treatment was not intense, both species recovered growth but presented modifications in the root system architecture. In pine saplings, the modification in root system architecture was the consequence of fine root meristems not recovering from water stress. The saplings survived by producing new lateral meristems from the cortical tannin zone above the fine root tip. In the case of pea seedlings, the meristematic tissues in the primary root arrested proliferation during water stress although they recovered when the event occurred during the first hours of germination. The response was different when water stress was enforced on older seedlings. In this case, root meristems never completely recovered their proliferation despite the increase in proline content observed in the cells. The modification of root system architecture in pea seedlings depended on the arrest of primary root elongation and the formation of new root laterals. As regards the primary roots, water stress treatment induced along the axis the formation of irregular ‘swellings’ in the cortical zone above the meristematic zone. Anatomical investigations suggested that such swellings may have derived from the changes in elongation direction of derivatives. The formation of new laterals was observed in hydroponic cultures when water stress treatment was enforced slowly and prolonged for a long time. The production of new lateral meristems may have been a similar response of woody and herbaceous plants to water stress conditions. It is not known whether these new meristems present characteristics of resistance to water stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Redox modulation of osmotic water permeability in plasma membranes isolated from roots and shoots of pea seedlings

Partitioning in a biphasic polymer system was used to isolate plasmalemma (PM) from roots and shoots of etiolated pea seedlings. The membrane preparations were used to assess the osmotic water permeability (P _os) with the stopped-flow method. The Western-blot technique was employed to determine the membrane content of the PIP-family of aquaporins, and their activity was estimated by measuring the rate of osmotic vesicle shrinking in the presence of inhibitors, HgCl₂ and AgNO₃. Monobromobimane fluorescent dye was used to determine the quantity of sulfhydryl groups in cell membranes and follow the effect of SH-oxidizing (diamide) and SH-reducing (dithiothreitol and tributylphosphine) agents on P _os of the root PM and oligomerization of aquaporins. The shoot PM was shown to combine high P _os with low aquaporin content. In the root PM, P _os was lower and the aquaporin content greatly exceeded that in the shoots. HgCl₂ and AgNO₃ did not decrease the rate of osmotic shrinking in root membrane vesicles, whereas considerably (by 40–50%) inhibited this index in the shoot membranes. Root and shoot PM preparations dramatically differed in their SH-group contents: the former exceeded the latter sixfold. When added to the homogenization medium, diamide and tributylphosphine affected the content of SH-groups and P _os in the root PM. In the roots, diamide decreased the quantity of SH-groups almost twofold and increased P _os fourfold, and the introduction of tributylphosphine produced a twofold increase in the quantity of SH-groups with only slight decrease in the P _os. Immunological analysis of membranes isolated in the presence of diamide showed that the ratio between the monomer and dimer forms of aquaporins in two membrane preparations depended on the presence of dithiothreitol in the denaturing buffer apparently because dithiothreitol exposed and reduced disulfide bonds essential for monomer interactions and inaccessible for interaction with redox modifiers of SH-groups in the membrane. Because of their inaccessibility, these modifiers could not cause the changes of P _os in the root PM produced by oxidation and reduction of SH-groups. This phenomenon is probably related to the change in the status of SH-groups in two cysteine residues at the N-end of the aquaporin loop C oriented outward into the apoplast.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

The presence of a p53-like protein during pea seed maturation and germination

ABSTRACT

The mechanisms that allow monitoring of DNA damage and the activation of repair systems in plants are poorly known. In mammalian cells the tumor suppressor protein p53 plays an important role in the checkpoint pathway induced by DNA damage. In this work, we investigated the presence and distribution of the p53-like protein in pea root tip nuclei and its role during early germination in relation to DNA damage. In pea seed, PFGE and TdT assays show that DNA fragmentation occurs during maturation and dry seed storage, and that this DNA fragmentation is repaired at the beginning of germination before the onset of proliferation. In the same seeds, the p53-like protein was found during maturation and germination. Immunoblotting characterization of this protein led to the identification of a single specific protein of about 94 kDa, more abundant at the beginning of the hydration process than in actively cycling cells. Furthermore, the p53-like protein revealed different nuclear distribution patterns, probably in relation to the formation of DNA fragments in dry seeds, and to the reactivation of repair mechanisms during early germination. These data suggest that the presence of a p53-like protein in quiescent or proliferating pea embryo cells is related to DNA damage, and serves for the maintenance of genetic information and the development of normal seedlings.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Aluminum inhibits root growth and induces hydrogen peroxide accumulation in Plantago algarbiensis and P. almogravensis seedlings

We have evaluated the impact of aluminum (Al) on germination, relative root growth, Al accumulation in roots tips, H₂O₂ levels, plasma membrane integrity, pigment levels, protein content, and the activities of superoxide dismutase (SOD) and catalase (CAT) in seedlings of the endangered Portuguese species Plantago algarbiensis and Plantago almogravensis. We found that up to 400 μM Al had no impact on the germination percentage in either species but inhibited root growth in a concentration-dependent manner (more severely in P. algarbiensis). Al accumulation in the root tips of both species was concentration dependent up to 200 μM but declined thereafter despite the absence of membrane damage. We observed a concentration-dependent induction of SOD activity but no change in CAT activity resulting in the accumulation of H₂O₂ (a known growth inhibitor), although its impact in P. almogravensis may be partially ameliorated by the accumulation of carotenoid pigments. Our data suggest an association between Al uptake, H₂O₂ production, and the inhibition of root growth during early seedling development in P. algarbiensis and P. almogravensis, although the latter is more tolerant towards higher concentrations of the metal.     

Limited proteolysis of the catalytic subunit of cAMP-dependent protein kinase — A membranal regulatory device?

Brush border membranes isolated from rat small intestine were found to possess a cAMP-dependent protein kinase activity. Upon addition of cAMP, a rapid, time-dependent inactivation of this enzyme occurs, which was found to be due to a proteolytic activity identified in the membranes. This activity could not be assigned to previously known brush border proteases. The inactivation and the proteolytic degradation of the kinase could be reproduced also with the pure catalytic subunit of cAMP-dependent protein kinase (C) from rabbit skeletal muscle (M.W. 40000) which was cleaved by the membranal proteolytic activity with concomitant quantitative appearance of a degradation product (M.W. 30000) devoid of kinase activity. The membranal proteolytic activity appears to be specific for C since: (1) it does not degrade the other endogenous proteins in the membrane preparation; (2) it does not degrade any of six arbitrarily chosen proteins from other sources; (3) it catalyzes a limited proteolysis of C which could not be simulated by other proteolytic enzymes such as trypsin, clostripain, chymotrypsin and papain. The attack of C by the membranal protease is blocked by the presence of the nucleotide substrate of the kinase (MgATP). In addition, the undissociated and inactive form of the enzyme (R₂C₂) does not lose its potential enzymatic activity, and neither its catalytic nor its regulatory subunits are digested by the protease. The specific, restricted and limited action of the protease, together with the prevention of its action by the substrate and the regulatory protein (R) of the kinase raise the possibility that the membranal protease may have a distinct physiological (possibly regulatory) assignment.     

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca²⁺-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca²⁺ (K_0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca²⁺-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with ³²P-phosphate. Overall, the results show the existence of a Ca²⁺-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca²⁺-dependent phosphorylation.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. I. ANATOMICAL CHANGES IN THE EMBRYOS

Anatomical changes in the radicle and shoot meristems of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides were studied. When germination of seeds was stimulated by cowpea (Vigna unguiculata) root exudate, growth occurred mainly in the radicular pole of embryos and minimally in the plumular pole, resulting in seedlings with elongated radicles. Maximum radicle elongations of about 3 mm in A. vogelii and 2 mm in S. gesnerioides were recorded during a period of 8 and 11 days, respectively. Analysis of the radicular tip during the course of seed germination revealed that the activity of the meristematic tissue progressively decreased until it completely disappeared. When germinated seeds were cultured on nutrient agar media, the radicle meristem of A. vogelii continued to grow producing a normal root with a root cap. On the other hand, the radicles of cultured S. gesnerioides seeds elongated only slightly before meristematic activity ceased. During continued growth of seedlings of both species on agar media, lateral roots whose tips had typical angiosperm root topography, were initiated from the radicle.