Characteristics of Chlorophyll Formation in the Green Alga Golenkinia

Cells of the alga Golenkinia are bleached by growth in darknessin media containing sodium acetate. Re-greening of these cellsis light dependent; neither glucose nor intermediates of chlorophyllsynthesis can substitute. The amount of chlorophyll synthesizedis proportional to the light intensity between darkness and1,000 lux and to the duration of the exposure. Initially, onlychlorophyll a is synthesized. After 9–12 hr illumination,formation of chlorophyll b and carotenoids begins. Chlorophyllproduction apparently occurs in two stages: (1) the first 12–16hr of greening. This stage is sensitive to cyanide, azide oranaerobiosis and relatively resistant to DCMU. (2) the second16–24 hr of greening. This stage is sensitive to DCMUand relatively resistant to inhibitors of respiration. Glucosestimulates greening in both stages. The metabolic requirementsof chlorophyll synthesis are discussed. (Received December 17, 1980; Accepted June 25, 1981)     

Microfibrillar Structure, Cortical Microtubule Arrangement and the Effect of Amiprophos-methyl on Microfibril Orientation in the Thallus Cells of the Filamentous Green Alga, Chamaedoris orientalis

Microfibrillar structure, cortical microtubule orientation andthe effect of amiprophos-methyl (APM) on the arrangement ofthe most recently deposited cellulose microfibrils were investigatedin the marine filamentous green alga, Chamaedoris orientalis.The thallus cells of Chamaedoris showed typical tip growth.The orientation of microfibrils in the thick cell wall showedorderly change in longitudinal, transverse and oblique directionsin a polar dependent manner. Microtubules run parallel to thelongitudinally arranged microfibrils in the innermost layerof the wall but they are never parallel to either transverseor obliquely arranged microfibrils. The ordered change in microfibrilorientation is altered by the disruption of the microtubuleswith APM. The walls, deposited in the absence of the microtubules,showed typical helicoidal pattern. However, the original crossedpolylamellate pattern was restored by the removal of APM. Thissuggests that cortical microtubules in this alga do not controlthe direction of microfibril orientation but control the orderedchange of microfibril orientation. Amiprophos-methyl, Chamaedoris orientalis, coenocytic green alga, cortical microtubule, microfibrillar structure, tip growth     

Action Spectrum for Light-induced Chloroplast Accumulation in a Marine Coenocytic Green Alga, Bryopsis plumosa

A marine coenocytic green alga, Bryopsis plumosa exhibited multistriatetype protoplasmic streaming of a velocity less than 100 µm.min^–1.When the alga was illuminated locally, chloroplasts and othercell organelles accumulated in the illuminated zone. The actionspectrum for this reaction showed that blue light between 380and 500 nm was most effective. The velocity of chloroplast movement decreased when the cellwas totally illuminated with blue light, but no comparable changewas observed under red light illumination. Therefore, chloroplastaccumulation probably was caused by the reduced streaming ratein the illuminated zone. Electron microscopy showed cytoplasmic microtubules arrangedparallel to the cell axis in the vicinity of the chloroplasts.Chloroplast movement was inhibited heavily by treatment withantimicrotubule agents, but was little affected by cytochalasinB at a concentration of 10 µg/ml. (Received May 30, 1981; Accepted August 24, 1981)     

Effect of Metabolic Inhibitors on the Formation of Cell Walls in a Green Alga, Micrasterias crux-melitensis

The effects of cytochalasin B, N-ethylmaleimide (NEM), colchicine,vinblastine and cycloheximide on the formation of birefringentcell wall layers were studied. Birefringent layers accumulatedoutside the plasma membrane of daughter semicells when cellswere cultured in a 0.16 M mannitol solution without any inhibitors.In cells treated with 2 x 10^–5 M cytochalasin B, 3 x 10^–5M NEM, 10^–4 M vinblastine or 10^–5 M cycloheximidefor 24 hr, birefringent layers were not observed outside theplasma membrane, but were present in cells treated with 10^–2M colchicine. The possibility is discussed that substances necessaryfor wall synthesis could be transported from the cytoplasm tothe outside of the plasma membrane by a system associated withmicrofilaments, microtubules and myosin-like structures. (Received June 26, 1981; Accepted September 24, 1981)     

Arrangement of Cellulose Microfibrils in Walls of Elongating Parenchyma Cells

The arrangement of cellulose microfibrils in walls of elongating parenchyma cells of Avena coleoptiles, onion roots, and celery petioles was studied in polarizing and electron microscopes by examining whole cell walls and sections. Walls of these cells consist firstly of regions containing the primary pit fields and composed of microfibrils oriented predominantly transversely. The transverse microfibrils show a progressive disorientation from the inside to the outside of the wall which is consistent with the multinet model of wall growth. Between the pit-field regions and running the length of the cells are ribs composed of longitudinally oriented microfibrils. Two types of rib have been found at all stages of cell elongation. In some regions, the wall appears to consist entirely of longitudinal microfibrils so that the rib forms an integral part of the wall. At the edges of such ribs the microfibrils can be seen to change direction from longitudinal in the rib to transverse in the pit-field region. Often, however, the rib appears to consist of an extra separate layer of longitudinal microfibrils outside a continuous wall of transverse microfibrils. These ribs are quite distinct from secondary wall, which consists of longitudinal microfibrils deposited within the primary wall after elongation has ceased. It is evident that the arrangement of cellulose microfibrils in a primary wall can be complex and is probably an expression of specific cellular differentiation.     

Structure of Cellulose Microfibrils in Primary Cell Walls from Collenchyma

In the primary walls of growing plant cells, the glucose polymer cellulose is assembled into long microfibrils a few nanometers in diameter. The rigidity and orientation of these microfibrils control cell expansion; therefore, cellulose synthesis is a key factor in the growth and morphogenesis of plants. Celery (Apium graveolens) collenchyma is a useful model system for the study of primary wall microfibril structure because its microfibrils are oriented with unusual uniformity, facilitating spectroscopic and diffraction experiments. Using a combination of x-ray and neutron scattering methods with vibrational and nuclear magnetic resonance spectroscopy, we show that celery collenchyma microfibrils were 2.9 to 3.0 nm in mean diameter, with a most probable structure containing 24 chains in cross section, arranged in eight hydrogen-bonded sheets of three chains, with extensive disorder in lateral packing, conformation, and hydrogen bonding. A similar 18-chain structure, and 24-chain structures of different shape, fitted the data less well. Conformational disorder was largely restricted to the surface chains, but disorder in chain packing was not. That is, in position and orientation, the surface chains conformed to the disordered lattice constituting the core of each microfibril. There was evidence that adjacent microfibrils were noncovalently aggregated together over part of their length, suggesting that the need to disrupt these aggregates might be a constraining factor in growth and in the hydrolysis of cellulose for biofuel production.Growth and form in plants are controlled by the precisely oriented expansion of the walls of individual cells. The driving force for cell expansion is osmotic, but the rate and direction of expansion are controlled by the mechanical properties of the cell wall (Szymanski and Cosgrove, 2009). Expanding, primary cell walls are nanocomposite materials in which long microfibrils of cellulose, a few nanometers in diameter, run through a hydrated matrix of xyloglucans, pectins, and other polymers (Knox, 2008; Mohnen, 2008; Szymanski and Cosgrove, 2009; Scheller and Ulvskov, 2010). Native cellulose microfibrils are partially crystalline (Nishiyama, 2009; Fernandes et al., 2011). Formerly, primary wall cellulose was thought to have a unique crystal structure called cellulose IV₁ (Dinand et al., 1996), but NMR evidence suggests the presence of forms similar to the better characterized cellulose Iα and Iβ crystalline forms together with large quantities of less ordered cellulose (Wickholm et al., 1998; Sturcová et al., 2004; Wada et al., 2004). Nevertheless, cellulose is much more ordered than any other component of the primary cell wall (Bootten et al., 2004), in keeping with its key role of providing strength and controlling growth.The stiffness of the cell wall is greatest in the direction of the cellulose microfibrils, where growth is directional and the predominant microfibril orientation is usually transverse to the growth direction (Green, 1999; MacKinnon et al., 2006; Szymanski and Cosgrove, 2009). Expansion of the cell wall then requires either widening of the spacing between microfibrils (Marga et al., 2005) or slippage between them (Cosgrove, 2005), or both, and the microfibrils reorient toward the direction of growth (Anderson et al., 2010). Polymer cross bridges between microfibrils (McCann et al., 1990) are thought to resist these deformations of the cell wall nanostructure and, thus, to control the rate of growth. Until recently, most attention was focused on bridging xyloglucans, hydrogen bonded to microfibril surfaces (Scheller and Ulvskov, 2010). However, there is evidence that not all xyloglucans are appropriately positioned (Fujino et al., 2000; Park and Cosgrove, 2012a) and that other bridging polymers may be involved (Zykwinska et al., 2007). It has also been suggested that bundles of aggregated microfibrils, not single microfibrils, might be the key structural units in primary cell walls (Anderson et al., 2010), as in wood (Fahlén and Salmén, 2005; Fernandes et al., 2011). If so, single microfibrils could bridge between microfibril bundles. In summary, the growth of plant cells is not well understood, and we need more information on how cellulose orientation is controlled and on the nature of the bridging polymers, the cellulose surfaces to which these polymers bind, and the cohesion between microfibril surfaces that might mediate aggregation.Cellulose microfibrils are synthesized at the cell surface by large enzyme complexes having hexagonal symmetry, sometimes called “rosettes” (Somerville, 2006). Each complex contains multiple cellulose synthases that differ between primary cell walls and wood, although the appearance of the complexes is similar (Somerville, 2006; Atanassov et al., 2009). The simultaneous synthesis, from the same end, of all the chains in a native cellulose microfibril is why they are parallel (Nishiyama et al., 2002, 2003), in contrast to the entropically favored antiparallel structure found in man-made celluloses like rayon (Langan et al., 2001). The number of chains in a microfibril and the number of cellulose synthases in the synthetic complex are evidently related. It is commonly assumed that the number of chains is divisible by six, matching the hexagonal rosette symmetry, and 36-chain models (Himmel et al., 2007) bounded by the hydrophilic [110] and [1-10] crystal faces, as in algal celluloses (Bergenstråhle et al., 2008), have been widely adopted. The assembly and orientation of cellulose are connected, as several cellulose synthase mutants have phenotypes defective in cellulose orientation and plant form as well as depleted in cellulose content (Paredez et al., 2008). In certain other mutant lines, the crystallinity of the microfibrils appears to be affected (Fujita et al., 2011; Harris et al., 2012; Sánchez-Rodríguez et al., 2012).Therefore, a detailed understanding of the structure of primary wall cellulose microfibrils would help us to understand cellulose synthesis as well as the growth and structural mechanics of living plants (Burgert and Fratzl, 2009). Primary cell walls and their cellulose skeletons also affect food quality characteristics like the crispness of salad vegetables and apples (Malus domestica; Jarvis, 2011). When biofuels are produced from lignocellulosic biomass, lignification leads to recalcitrance (Himmel et al., 2007), but some of the cell types in Miscanthus spp., switchgrass (Panicum virgatum), and arable crop residues have only primary walls with no lignin, and recalcitrance then depends on the nature of the cellulose microfibrils (Beckham et al., 2011).A relatively detailed structure has recently been proposed for the microfibrils of spruce (Picea spp.) wood (Fernandes et al., 2011), which are 3.0 nm in diameter, allowing space for only about 24 cellulose chains. Evidence from x-ray diffraction supported a “rectangular” shape (Matthews et al., 2006) bounded by the [010] and [200] faces. There was considerable disorder increasing toward the surface, and the microfibrils were aggregated into bundles about 15 to 20 nm across, with some, but not all, of the lateral interfaces being resistant to water (Fernandes et al., 2011). Disordered domains are a feature of other strong biological materials such as spider silk (van Beek et al., 2002).Therefore, it is of interest whether any of these features of wood cellulose might also be found in the cellulose microfibrils of primary (growing) cell walls. It would be particularly useful to characterize the disorder known to be present in primary wall microfibrils, that is, to define how cellulose that is not measured as “crystalline” differs from crystalline cellulose. Many of the experiments leading toward a structure for wood cellulose were dependent on exceptionally uniform orientation of the cellulose microfibrils (Sturcová et al., 2004; Fernandes et al., 2011). However, in growing cell walls, the microfibrils are not uniformly oriented. When microfibrils are first laid down at the inner face of the primary cell wall, their orientation is normally transverse to the direction of growth, but as the cell wall expands, the microfibrils reorient so that the orientation distribution, integrated across the thickness of the expanded cell wall, becomes progressively closer to random (Cosgrove, 2005; MacKinnon et al., 2006).This technical problem does not apply to the cell walls of celery (Apium graveolens) collenchyma, which are similar in composition to other primary cell walls but have their microfibrils oriented relatively uniformly along the cell axis (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b). Some structural information on celery collenchyma cellulose has already been derived from spectroscopic and scattering experiments (Sturcová et al., 2004; Kennedy et al., 2007a, 2007b), confirming the disorder expected in a primary wall cellulose. Some of these experiments were analogous to what has been done on spruce cellulose (Fernandes et al., 2011), but insufficient data are available to specify the number of chains in each primary wall microfibril, the nature and location of the disorder, and the presence or absence of direct contact between microfibrils. Here, we report x-ray and neutron scattering and spectroscopic experiments addressing these questions and leading to a proposed structure for primary wall cellulose microfibrils. Characterizing a structure containing so much disorder presented unusual challenges, but disorder appears to be central to the enigmatic capacity of primary wall cellulose to provide high strength and yet to permit and control growth.     

Experimental Studies on the Stability of the Cortical Microtubule Cytoskeleton in Relation to Polarity and Cell Elongation in the Coenocytic Green Alga, Chaetomorpha moniligera

The stability and ordered assembly of cytoskeletal microtubules(MTs) and the relationship between cell growth and MT cytoskeletonin the coenocytic green alga, Chaetomorpha moniligera Kjellmanwere examined. The cytoplasm of cylindrical growing cells ofChaetomorpha is covered with dense arrays of longitudinallyarranged cortical MTs which constitute the MT cytoskeleton.Seventy-five percent of MTs of the cytoskeleton disappearedwithin 4 h, with 25% remaining after 20 h following cold treatment.On terminating MT assembly with amiprophos-methyl (APM), thenumber of MTs decreased by 75% within 4 h. The remaining MTsdisappeared gradually within 24 h. The MT cytoskeleton of Chaetomorphawould thus appear to be composed of at least two kinds of MTsdiffering in stability. The MT cytoskeleton returned to normalafter treatment with APM for less than 48 h. However, this didnot occur after treatment with APM for more than 48 h, and theMT arrays became random. Cell elongation ceased completely within24 h after treatment with APM for less than 48 h but was restoredwithin 24 h after removing APM. The restoration of cell elongationwas no longer evident after removaI of APM for more than 48h. The results indicate that assembly of MTs into ordered arraysdepends on cell polarity and that in turn cell elongation isdependent on the polar-dependent arrays of MTs.Copyright 1994,1999 Academic Press Cell polarity, Chaetomorpha moniligera, coenocytic green alga, cold treatment, immunofluorescence, microtubule     

Stt7-dependent Phosphorylation during State Transitions in the Green Alga Chlamydomonas reinhardtii

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b₆f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser⁵³³ in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.The primary photochemical reactions of photosynthesis are catalyzed by the pigment-protein complexes photosystem II (PSII)¹ and PSI (PSI), which are linked in series through the plastoquinone pool, the cytochrome b₆f complex, and plastocyanin in the thylakoid membranes. Upon light absorption by the antenna systems of PSII and PSI, charge separations occur across the membrane that lead to the oxidation of water by PSII and electron flow to PSI and ultimately to the reduction of NADP⁺. Because the antenna systems of PSII and PSI have different pigment composition, they are differentially sensitized upon changes in light quality and quantity. However, photosynthetic organisms have the ability to adapt to changes in light. They balance energy input and consumption in the short term through dissipation of excess absorbed light energy into heat through non-photochemical quenching and regulate absorption of excitation energy between PSII and PSI through state transitions (supplemental Fig. 1). This reversible redistribution leads to an overall increase in photosynthetic quantum yield. State transitions occur when preferential excitation of PSII reduces the plastoquinone pool. This leads to the activation of a thylakoid protein kinase as a result of the docking of plastoquinol to the Qo site of the cytochrome b₆f complex (1, 2) and to the phosphorylation of the polypeptides of the light-harvesting complex II (LHCII), a part of which migrates to PSI (state 2) (3–5). The process is reversible as preferential excitation of PSI inactivates the kinase and allows for dephosphorylation of LHCII and its return to PSII (state 1) (3, 6). In the green alga Chlamydomonas reinhardtii, the LHCII protein set consists of Type I (Lhcbm3, Lhcbm4, Lhcbm6, Lhcbm8, and Lhcbm9), Type II (Lhcbm5), Type III (Lhcbm2 and Lhcbm7), and Type IV (Lhcbm1 and Lhcbm10) proteins and of Lhcb7, CP26, and CP29 (7). Because of their nearly identical sequences and sizes, several of these Lhcbm proteins cannot be distinguished by SDS-PAGE. Most of them fractionate into four bands called P11 and P13 (Type I), P16 (Type IV), and P17 (Type III). Whereas P16 is not phosphorylated, phosphorylation events occur on P11, P13, and P17 (7, 8).The association of the mobile part of LHCII to PSI during a transition from state 1 to state 2 requires the PsaH subunit (9) and CP29, which also moves to PSI and is essential for docking LHCII to PSI (10–12). The lateral displacement of LHCII from the PSII-rich grana to the PSI-rich lamellar thylakoid regions results in transfer to PSI of about 80% of the excitation energy absorbed by LHCII in C. reinhardtii (13), a considerably higher amount than in land plants in which only 15–20% of LHCII is mobile (3). In C. reinhardtii, state transitions are associated with a reorganization of the photosynthetic electron transfer chain with a switch from linear to cyclic electron flow during a transition from state 1 to state 2 (14, 15). Thus, cells produce ATP and NADPH in state 1 but only ATP in state 2. It appears that the major function of state transitions in this alga is to adjust the level of ATP and the ATP/NADPH ratio to cellular demands (5).Thylakoid membranes contain appressed grana and nonappressed stromal domains in which PSII and PSI are enriched, respectively. Because LHCII is a major stabilizer of the grana structure (16), the movement of LHCII from PSII to PSI is expected to lead to major rearrangements of these membranes during state transitions. Indeed, based on extensive electron microscope studies, it was proposed that fusion and fission events occur at the interface between the grana and stroma lamellar domains that lead to a remodeling of the membranes (17).Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of Chlamydomonas revealed a total of 19 sites corresponding to 15 genes (18). It was shown that the major changes are clustered at the interface between the PSII core and the associated LHCII proteins during state transitions. Phosphorylation of the PSII core subunits D2 and PsbR and multiple phosphorylations of the minor LHCII antenna subunit CP29 were detected as well as phosphorylation of Lhcbm1, which belongs to the major LHCII complex (18).Although the phosphorylation of LHCII was observed many years ago (6), it is only recently that kinases involved in this process were uncovered. Fleischmann et al. (19) and Kruse et al. (20) used a genetic approach in C. reinhardtii with the aim of dissecting the signal transduction chain of state transitions. Two allelic mutants blocked in state 1 were identified that are affected in the Stt7 gene encoding a thylakoid Ser-Thr protein kinase that is required for LHCII phosphorylation during a transition from state 1 to state 2 (21). This Stt7 kinase is conserved in land plants and has an ortholog, STN7, in Arabidopsis (22).The 754-amino acid Stt7 kinase has a catalytic domain characteristic of Ser-Thr kinases (21). It contains a putative 41-amino acid transit peptide at its N-terminal end, and the protein is localized on the thylakoid membrane. Stt7 is associated with photosynthetic complexes including LHCII, PSI, and the cytochrome b₆f complex (23). Stt7 also contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys residues that are critical for its activity and state transitions (23). Moreover, the level of Stt7 decreases considerably under state 1 conditions, and the kinase acts in catalytic amounts (23). However, it is not yet known whether this kinase directly phosphorylates LHCII or whether it is part of a kinase cascade involved in the signaling pathway of state transitions.In this work, we used a mass spectrometry-based approach (24) to map the in vivo Stt7-dependent protein phosphorylation sites within thylakoid membranes isolated from the green alga C. reinhardtii subjected to state 1 and state 2 conditions. In contrast with the earlier studies via direct MS/MS sequencing of the IMAC-enriched phosphorylated peptides from thylakoid proteins (18, 25), we performed additional LC-MS/MS-based analyses using alternating collision-induced dissociation and electron transfer dissociation of peptide ions. This approach revealed novel phosphorylation sites in LHCII polypeptides, in several other membrane and membrane-associated proteins, and in the thylakoid protein kinases Stt7 and Stl1, suggesting the existence of a thylakoid protein kinase cascade. Relative quantification of phosphorylated peptides labeled with stable isotopes determined the specific Stt7-dependent phosphorylation site in CP29 linker protein under state 2. Moreover, we also identified phosphorylation sites that are redox-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent. This mapping provides new insights into the regulatory network of protein phosphorylation in algal photosynthetic membranes during state transitions.     

Oxygen Isotope Fractionation during Photosynthesis in a Blue-Green and a Green Alga

Oxygen isotope fractionation (¹⁸O/¹⁶O) at the natural abundance level has been measured during photosynthesis of a blue-green and a green alga. When sufficient attention is paid to removal of contaminating air O₂ before and during the experiments, then the photosynthetic O₂ evolved, as compared to the water O₂, had an average difference of −0.36% for a blue-green alga and −0.80% for a green alga. These experiments suggest that there is no reason to invoke an inverse isotope effect in photosynthesis as part of the explanation for the ¹⁸O enrichment in atmospheric O₂ relative to O₂ in oceanic waters. In addition, in an indirect way, the experiments also support the argument that the bulk of O₂ evolved during photosynthesis comes from water. A 10% contribution of O₂ arising from CO₂ would have been detectable in the present work.     

The Net Orientation of Wall Microfibrils in the Outer Periclinal Epidermal Walls of Seedling Leaves of Wheat

Use of the dichroic stain chlor-zinc-iodine revealed that thenet orientation of cellulose wall microfibrils in the outerparadermal wall of the epidermis of seedling wheat leaves isprincipally transverse in the extension zone. The net orientationof microfibrils changes abruptly to principally longitudinalat the end of cell elongation. The net angle of orientationof microfibrils in the extension zone was not a function ofRht-dosage (number of dwarfing alleles), and neither leaf extensionrate nor estimated maximum relative elemental rate of elongationwere functions of microfibril orientation. The results indicatethat elongation rates are not regulated by the net angle oforientation of microfibrils and support the concept that leafextension rate is regulated by the length of the extension zone.Copyright1995, 1999 Academic Press Cellulose wall microfibrils, extension zone, elongation, Rht, wheat, Triticum aestivum L     

Calcium-Sensitivity of Cortical Microtubules in the Green Alga Mougeotia

Membrane ghosts were prepared from protoplasts of the greenalga Mougeotia, and the Ca²⁺-sensitivity of microtubules onthe ghosts was examined. Microtubules on the protoplast ghosts were not depolymerizedby 3 min treatment with 1 mM Ca²⁺. As the treatment was prolonged,some depolymerization of microtubules became evident, but evenafter 10 min about 50% of the ghosts showed no depolymerization.Ca²⁺ introduced into intact protoplasts seemed to be ineffectivein depolymerizing microtubules; abundant microtubules were presenton membrane ghosts prepared from protoplasts which had beentreated with 2x10^–5M Ca²⁺-ionophore A23187 [GenBank] plus 1 mM Ca²⁺for 20 or 30 min. Neither 3 min treatment with 0.2% Triton X-100 nor with 1 mMCa²⁺ solution containing 5 min MgSO₄ and 100 mM KCl caused depolymerisationof microtubules on protoplast ghosts. However, when given successively,these treatments caused complete depolymerization of microtubules. These results suggest that Mougeotia microtubules are stableto Ca²⁺ and that the stability is conferred by a microtubule-associatedfactor which can easily be removed by Triton X-100 treatment. (Received July 19, 1985; Accepted October 25, 1985)     

Colchicine-Induced Effects in the Scaly Green Alga, Mesostigma viride: Loss of Microtubules and Paracrystal Formation

The effects of colchicine treatment upon the scale-covered,disc-shaped prasinophyte, Mesostigma viride, are profound. 2–4mg ml^–1 of the alkaloid induces the loss of pit and othercytoskeletal microtubules within 6 h of treatment. Subsequently,the organism loses its distinctive shape and becomes ellipsoidal.During treatment, prominent, intracellular crystalline bandsconsisting of 25 nm hexagonal subunits form in close proximityto the former pit region. The Golgi apparatus remains intactduring colchicine application but scale ontogenesis is distinctlyaltered. All effects are reversible upon removal of colchicine.A discussion of the cytoskeletal role of parallel series ofmicrotubules in unicellular algae is presented. Key words: Mesostigma, Colchicine, Cytoskeletal     

Interrelation between Lignin Deposition and Polysaccharide Matrices during the Assembly of Plant Cell Walls

Abstract: The modifications caused by genetic down-regulation of the enzyme cinnamoyl CoA reductase (CCR) from monolignol biosynthetic pathways on tobacco and Arabidopsis thaliana were investigated at the ultrastructural level. A typical result was that the same transformation led to similar abnormality in secondary wall formation of fibres in both plants. The cell wall alterations mainly consisted in an important disorganization and loosening of cellulose microfibrils in the inner part of the S₂ layer. This inability of the transformants to form a coherent cell wall coincided with a lack of synthesis of non-condensed forms of lignin in this disorganized region of the wall, as demonstrated by immunolabelling of lignin subunits. A similar disorganization was observed during fibre wall formation in the differentiating tissues of young Populus and A. thaliana plants. The transitory lack of organization of cellulose microfibrils, also coincided with a depletion in non-condensed forms of lignins. These results suggest that such lignin substructures may be involved in the cohesion of secondary walls during cell wall biogenesis. The mutual influence of the cellulose-hemicellulose environment and monolignol local polymerization is discussed.     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis1

Cells of the green microalga Haematococcus pluvialis were inducedto accumulate the ketocarotenoid pigment, astaxanthin. Thisinduction was achieved by the application of the following environmentalconditions: light intensity (170 µmol m~^–2s^–1),phosphate starvation and salt stress (NaCl 0.8%). These conditionsretarded cell growth as reflected by a decrease in cell divisionrate, but led to an increase in astaxanthin content per cell.Accumulation of astaxanthin required nitrogen and was associatedwith a change in the cell stage from biflagellate vegetativegreen cells to non-motile and large resting cells. It is suggestedthat environmental or nutritional stresses, which interferewith cell division, trigger the accumulation of astaxanthin.Indeed, when a specific inhibitor of cell division was applied,a massive accumulation of astaxanthin occurred. ¹ Contribution No. 55 of The Microalgal Biotechnology Laboratory (Received April 22, 1991; Accepted August 6, 1991)     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Light-Regulated Translocation of Cytoplasm in Green Alga Dichotomosiphon

Chloroplasts and other cytoplasmic granules in the freshwatercoenocytic green alga Dichotomosiphon tuberosus streamed bidirectionallyalong the longitudinal axis of its tubular body. In responseto light stimuli, the organelles migrated toward the apicalregions and accumulated there. In the dark, they migrated towardthe basal regions and stayed there. The cytoplasmic streamingand the light-regulated movement were inhibited by the presenceof 5 x 10^–4 M colchicine, but not by 100µg/ml cytochalasinB. Local illumination with blue light caused reversible accumulationof the organelles in the illuminated zone. Single arrays ofmicrotubules were found in the ectoplasmic layer of the alga,and both single and bundle arrays in the endoplasm. The endoplasmicmicrotubules disintegrated when the alga was treated for 24h with colchicine. The involvement of the microtubules in themotive force generation for the bidirectional streaming andtranslocational movement of the organelles is discussed. (Received December 27, 1985; Accepted April 16, 1986)