Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca²⁺-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.Actin filaments, one of the major components of the cytoskeleton, are organized into a highly ordered architecture and are involved in various kinds of cell motility. Their architecture is regulated by several kinds of actin-binding proteins, including cross-linking proteins, severing proteins, end-capping proteins, and monomer-sequestering proteins in animal, protozoan, and yeast cells (Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove and Vancompernolle, 1992). In plant cells the organization of the actin cytoskeleton also changes remarkably during the cell cycle or during developmental processes, and it is suggested that actin-binding proteins are involved in their dynamic change. However, little is known about actin-binding proteins in plant cells.Only a low-M_r actin-binding and -depolymerizing protein, profilin, in white birch (Betula verrucosa; Valenta et al., 1991), maize (Zea mays; Staiger et al., 1993; Ruhlandt et al., 1994), bean (Phaseolus vulgaris; Vidali et al., 1995), tobacco (Nicotiana tabacum; Mittermann et al., 1995), tomato (Lycopersicon esculentum; Darnowski et al., 1996), Arabidopsis (Arabidopsis thaliana; Huang et al., 1996), and lily (Lilium longiflorum; Vidali and Hepler, 1997), and an ADF in lily (Kim et al., 1993), rapeseed (Brassica napus; Kim et al., 1993), and maize (Rozycka et al., 1995; Lopez et al., 1996), have been identified by biochemical or molecular biological means.The native and recombinant forms of these proteins are capable of binding to animal or plant actin (Valenta et al., 1993; Giehl et al., 1994; Ruhlandt et al., 1994; Lopez et al., 1996; Perelroizen et al., 1996; Carlier et al., 1997). Plant profilin expressed in mammalian BHK-21 cells (Rothkegel et al., 1996) or profilin-deficient Dictyostelium discoideum cells (Karakesisoglou et al., 1996) was able to functionally substitute for endogenous profilin in these cells. The introduction of plant profilin into living stamen hair cells by microinjection caused the rapid reduction of the number of actin filaments (Staiger et al., 1994; Karakesisoglou et al., 1996; Ren et al., 1997). These results indicate that plant profilin and ADF share many functional similarities with other eukaryote profilins and ADFs.It is well known that the actin cytoskeleton undergoes dynamic changes in organization during hydration and activation of the vegetative cells of pollen grains (Pierson and Cresti, 1992). Before hydration actin filaments exist as fusiform or spiculate structures (a storage form), but they are rearranged to form a network upon hydration (Heslop-Harrison et al., 1986; Tiwari and Polito, 1988). In the growing pollen tube there are strands or bundles of actin filaments parallel to the long axis (Perdue et al., 1985; Pierson et al., 1986; Miller et al., 1996) that are involved in cytoplasmic streaming (Franke et al., 1972; Mascarenhas and Lafountain, 1972) and transport of vegetative nuclei and generative cells to the growing tip (Heslop-Harrison et al., 1988; Heslop-Harrison and Heslop-Harrison, 1989). Characterization of the function of actin-binding proteins is essential to understanding the regulation of actin organization during the developmental process of pollen. Since only a small number of vacuoles containing proteases develop in pollen grains and pollen tubes at a younger stage, pollen tubes are suitable materials for isolating and biochemically studying actin-binding proteins responsible for organizing actin filaments into various forms.In a previous paper we reported that several components in a crude extract prepared from lily pollen tubes, including a 170-kD myosin heavy chain and 175-, 135-, and 110-kD polypeptides, could be coprecipitated with exogenously added F-actin (Yokota and Shimmen, 1994). We also found that rhodamine-labeled F-actin was tightly bound to the glass surface treated with the fraction containing the 135- and 110-kD polypeptides (Yokota and Shimmen, 1994). These results suggested that either one or both of the 135- and 110-kD polypeptides possesses an F-actin-binding activity. In the present study, we purified the 135-kD polypeptide from lily pollen tubes by biochemical procedures and then characterized its F-actin-binding properties and distribution in the pollen tubes. This protein was able to bundle F-actin isolated from chicken breast muscle and colocalized with actin-filament bundles in pollen tubes. We refer to this protein as P-135-ABP (Plant 135-kD Actin-Bundling Protein).     

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The K_m values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg²⁺ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary plant metabolism (Herrmann, 1995). It provides chorismate for the synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used in protein biosynthesis, but also serves as a precursor for a wide variety of aromatic substances (Herrmann, 1995; Weaver and Hermann, 1997; Fig. ​Fig.1a).1a). Chorismate is also the starting point of a biosynthetic pathway leading to phylloquinones (vitamin K₁) and anthraquinones (Poulsen and Verpoorte, 1991). The first committed step in this pathway is the conversion of chorismate into isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991; Fig. ​Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the synthesis of shikimate-pathway-derived compounds, and its distribution over the various pathways is expected to be tightly regulated. Elicited cell cultures of Catharanthus roseus provide an example of the partitioning of chorismate. Concurrently, these cultures produce both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In bacteria DHBA is synthesized from isochorismate (Young et al., 1969). Elicitation of C. roseus cell cultures with a fungal extract induces not only several enzymes of the indole alkaloid biosynthetic pathway (Pasquali et al., 1992) but also ICS (Moreno et al., 1994). Information concerning the expression and biochemical characteristics of the enzymes that compete for available chorismate (ICS, CM, and AS) may help us to understand the regulation of the distribution of this precursor over the various pathways. Such information is already available for CM (Eberhard et al., 1996) and AS (Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS. Figure 1a, Position of ICS in the plant metabolism. SA, Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism as a precursor of o-succinylbenzoic acid, an intermediate in the biosynthesis of menaquinones (vitamin K₂) (Weische and Leistner, 1985) and phylloquinones (vitamin K₁; Poulsen and Verpoorte, 1991). In bacteria isochorismate is also a precursor of siderophores such as DHBA (Young et al., 1969), enterobactin (Walsh et al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid (Serino et al., 1995). Although evidence from tobacco would indicate that salicylic acid in plants is derived from Phe via benzoic acid (Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it cannot be excluded that it is also synthesized from isochorismate. In the secondary metabolism of higher plants, isochorismate is a precursor for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and Leistner, 1992), naphthoquinones (Müller and Leistner, 1978), catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids (Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS activity was detected in protein extracts of cell cultures from plants of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes encoding ICS have been cloned from bacteria such as Escherichia coli (Ozenberger et al., 1989), Pseudomonas aeruginosa (Serino et al., 1995), Aeromonas hydrophila (Barghouthi et al., 1991), Flavobacterium K_3–15 (Schaaf et al., 1993), Hemophilus influenzae (Fleischmann et al., 1995), and Bacillus subtilis (Rowland and Taber, 1996). Both E. coli and B. subtilis have two distinct ICS genes; one is involved in siderophore biosynthesis and the other is involved in menaquinone production (Daruwala et al., 1996, 1997; Müller et al., 1996; Rowland and Taber, 1996). The biochemical properties of the two ICS enzymes from E. coli are different (Daruwala et al., 1997; Liu et al., 1990). Sequence analysis has revealed that the bacterial ICS enzymes share homology with the chorismate-utilizing enzymes AS and p-aminobenzoate synthase, suggesting that they share a common evolutionary origin (Ozenberger et al., 1989).Much biochemical and molecular data concerning the shikimate pathway in plants have accumulated in recent years (Schmid and Amrhein, 1995; Weaver and Hermann, 1997), but relatively little work has been done on ICS from higher plants. The enzyme has been partially purified from Galium mollugo cell cultures (Ledüc et al., 1991, 1997), but purification of the ICS protein to homogeneity has remained elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate partitioning over the various pathways. Furthermore, we studied ICS in C. roseus to gain insight into the biosynthesis of DHBA in higher plants (Moreno et al., 1994). In this paper we report the first purification, to our knowledge, of ICS to homogeneity from a plant source and the cloning of the corresponding cDNA.     

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.The ultrastructure and physical properties of plant cell walls are known to be affected by boron deficiency (Kouchi and Kumazawa, 1976; Hirsch and Torrey, 1980; Fischer and Hecht-Buchholz, 1985; Matoh et al., 1992; Hu and Brown, 1994; Findeklee and Goldbach, 1996). Moreover, boron is predominantly localized in the cell wall when plants are grown with suboptimal boron (Loomis and Durst, 1991; Matoh et al., 1992; Hu and Brown, 1994; Hu et al., 1996). In radish, >80% of the cell wall boron is present in the pectic polysaccharide RG-II (Matoh et al., 1993; Kobayashi et al., 1996), which is now known to exist as a dimer that is cross-linked by a borate ester between two apiosyl residues (Kobayashi et al., 1996; O''Neill et al., 1996). Dimeric RG-II is unusually stable at low pH and is present in a large number of plant species (Ishii and Matsunaga, 1996; Kobayashi et al., 1996, 1997; Matoh et al., 1996; O''Neill et al., 1996; Pellerin et al., 1996; Kaneko et al., 1997). The widespread occurrence and conserved structure of RG-II (Darvill et al., 1978; O''Neill et al., 1990) have led to the suggestion that borate ester cross-linked RG-II is required for the development of a normal cell wall (O''Neill et al., 1996; Matoh, 1997).One approach for determining the function of boron in plant cell walls is to compare the responses to boron deficiency of growing plant cells that are dividing and synthesizing primary cell walls with those of growth-limited plant cells in which the synthesis of primary cell walls is negligible. Suspension-cultured cells are well suited for this purpose because they may be reversibly transferred from a growth phase to a stationary phase. Continuous cell growth phase is maintained by frequent transfer of the cells into new growth medium (King, 1981; Kandarakov et al., 1994), whereas a stationary cell population is obtained by feeding the cells with Suc and by not subculturing them. Cells in the stationary phase are characterized by mechanically stabilized primary walls and reduced biosynthetic activity. Here we describe the responses of suspension-cultured Chenopodium album L. cells in the growth and stationary phases to boron deficiency. These cells have a high specific-growth rate, no significant lag phase, and reproducible changes in their wall pore size during the transition from the growth phase to the stationary phase (Titel et al., 1997).     

Arabidopsis Roots and Shoots Have Different Mechanisms for Hypoxic Stress Tolerance

Arabidopsis has inducible responses for tolerance of O₂ deficiency. Plants previously exposed to 5% O₂ were more tolerant than the controls to hypoxic stress (0.1% O₂ for 48 h) in both roots and shoots, but hypoxic acclimation did not improve tolerance to anoxia (0% O₂). The acclimation of shoots was not dependent on the roots: increased shoot tolerance was observed when the roots of the plants were removed. An adh (alcohol dehydrogenase) null mutant did not show acclimation of the roots but retained the shoot survival response. Abscisic acid treatment also differentiated the root and shoot responses; pretreatment induced root survival in hypoxic stress conditions (0.1% O₂) but did not induce any increase in the survival of shoots. Cycloheximide blocked both root and shoot acclimation, indicating that both acclimation mechanisms are dependent on protein synthesis.The supply of O₂ to plant tissues may be restricted under certain environmental conditions (Hook and Crawford, 1978). When air spaces normally present in the soil become saturated with water, the root environment becomes hypoxic or anoxic as a result of O₂ consumption by respiring roots and microorganisms and the insufficient diffusion of O₂ through water (Armstrong, 1979). O₂ deficiency is thought to be a major determinant in the adverse effects of waterlogging on crops and other plant species (Jackson et al., 1991). Plants have evolved inducible metabolic mechanisms to cope with these ephemeral, low-O₂-stress conditions. When exposed to low-O₂ conditions, plants switch to the expression of “anaerobic” polypeptides (Sachs et al., 1980, 1996). The induction of these proteins may be responsible for the tolerance to O₂ deficiency that would otherwise be lethal. A number of anaerobic polypeptides have been identified as enzymes involved in glycolysis and ethanol fermentation (for a recent review, see Vartapetian and Jackson, 1997), and this supports the view that when O₂ is limiting, oxidative catabolism of sugars is hindered and ethanolic fermentation acts as an alternative energy-producing pathway.Ethanol is the main end product of anaerobic metabolism in plants (Smith and ap Rees, 1979; Good and Muench, 1993). Unlike lactate, which is also generated under O₂ deficiency, ethanol is a relatively nontoxic end product (Jackson et al., 1982) and does not lead to the acidification of the cytoplasm, a major determinant in intolerance to O₂ deficiency (Roberts et al., 1984, 1985). The induction of glycolytic enzymes probably reflects the need for increased glycolysis to compensate for the lower ATP yield of ethanol fermentation.The importance of ethanol fermentation is supported by studies of adh (alcohol dehydrogenase) null mutants in a number of species (Schwartz, 1966; Harberd and Edwards, 1982; Jacobs et al., 1988; Matsumura et al., 1995), which report reduced tolerance to O₂ deficiency in these plants.Some plant tissues exposed to a period of mild hypoxia show more tolerance to subsequent hypoxic or anoxic stress than plants kept in fully aerated conditions before the stress (for review, see Drew, 1997; see also more recent work on tomato [Germain et al., 1997] and rice [Ellis and Setter, 1999]).In this study we examined the survival of Arabidopsis plants after exposure to anoxic or hypoxic stress. Our results demonstrate that hypoxic pretreatment protects against hypoxic stress and that different mechanisms of acclimation to hypoxic stress are operative in root and shoot tissues.     

Expression of a Soybean Gene Encoding the Tetrapyrrole-Synthesis Enzyme Glutamyl-tRNA Reductase in Symbiotic Root Nodules

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor δ-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3′ portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.Soybean (Glycine max) and numerous other legumes can establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review, see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to ammonia within nodules, which is assimilated by the plant host to fulfill its nutritional nitrogen requirement. The high energy requirement for nitrogen fixation necessitates efficient respiration by the prokaryote within the microaerobic milieu of the nodule. The plant host synthesizes a nodule-specific hemoglobin (leghemoglobin) that serves to facilitate oxygen diffusion to the bacterial endosymbiont and to buffer the free oxygen concentration at a low tension (for review, see Appleby, 1992). Both of these functions require that the hemoglobin concentration be high, and, indeed, it exceeds 1 mm in soybean nodules (Appleby, 1984) and is the predominant plant protein in that organ. Once thought to be confined to legume nodules, hemoglobins are found throughout the plant kingdom, and leghemoglobin likely represents a specialization of a general plant phenomenon (for review, see Hardison, 1996). A gene encoding a nonsymbiotic hemoglobin has been identified in soybean and other legumes (Andersson et al., 1996); therefore, expression in nodules involves the specific activation of a subset of genes within a gene family. Leghemoglobin genes may have arisen from gene duplication, followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized from common precursors; chlorophyll is quantitatively the major tetrapyrrole in plants, with heme and other tetrapyrroles being present in minor amounts. Legume root nodules represent an exception, in which heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in nonphotosynthetic tissues. Heme is synthesized from the universal tetrapyrrole precursor ALA by seven successive enzymatic steps; chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O''Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O''Brian, 1991, 1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al., 1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells (Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called the C₅ pathway (Fig. ​(Fig.1);1); the latter two steps are committed to ALA synthesis and are catalyzed by GTR and GSAT, respectively (for review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA or genes encoding GTR (Gtr, also called HemA) and GSAT (Gsa) have been identified in several plant species (Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to a nuclear factor found in both tissues. (Frustaci et al., 1995). In this study we isolated soybean Gtr1 and characterized the genetic basis of GTR expression in root nodules. Figure 1C₅ pathway for ALA synthesis. The committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed. Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA^Glu also participate in protein synthesis. The gene designations in plants are shown in parentheses ...