Rhythmic Leaflet Movement in Albizzia julibrissin: Effect of Electrolytes and Temperature Alteration

The rhythmic movement of darkened Albizzia leaflets is accompanied by K⁺ flux in pulvinule motor cells whose turgor changes control opening and closing. The azide-sensitive open phase is promoted by an increase in temperature from 16 to 33C (Q₁₀ = 3), implying active transport of K⁺ ions during this period. The azide-insensitive closed phase is less temperature-sensitive and has a Q₁₀ less than 1, implying diffusion or some other physical process as the predominant pathway of K⁺ flux at this time. Thus rhythmic leaflet movement is probably due to oscillation in active K⁺ transport or membrane permeability or both. External electrolytes (0. 1 n) alter leaflet angle during the open, but not the closed, phase of the rhythm. All chlorides except NH₄⁺ promote opening, with divalent more effective than monovalent ions. Some anions promote and others inhibit opening; activity is not correlated with charge. It is likely that electrolytes alter leaflet movement by altering K⁺ flux, accomplishing this by interacting with key macromolecules in motor cell membranes.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.     

Rhythmic potassium flux in albizzia: effect of aminophylline, cations, and inhibitors of respiration and protein synthesis

Rhythmic leaflet movement in Albizzia is controlled by rhythmic K⁺ flux in pulvinal motor cells. The angle assumed by darkened leaflets during the open phase of the rhythm can be altered by various compounds and changes in temperature; such treatments are ineffective during the closed phase. In all cases, effects on leaflet angle are correlated with and probably a consequence of K⁺ flux in pulvinal motor cells. Incubation at low temperature (6C) or on sodium azide (1.0 mm) reduces K⁺ in the ventral motor region and increases K⁺ in the dorsal motor region, thereby decreasing leaflet angle. Incubation on cycloheximide (0.1 mm) or sodium acetate (0.05 m) inhibits protein synthesis; if the incubation period immediately precedes the opening phase, these compounds prevent both K⁺ flux into the ventral motor cells and leaflet opening. Magnesium nitrate (0.05 m), supplied after leaflets have started to open, promotes K⁺ secretion from the dorsal motor cells and increases the angle of opening.     

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

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

Histological Compartmentation of Phosphate in Vicia faba L. Leaflet : Possible Significance to Stomatal Functioning

Guard cells, epidermal cells, palisade parenchyma cells, and spongy parenchyma cells of Vicia faba L. leaflet were analyzed for inorganic phosphate and phosphorus. On a molar basis, cells in the epidermal layer contained about 15-fold more inorganic phosphate than mesophyll cells did. Although a metabolic role for this asymmetric distribution cannot be defined unequivocally, we note that high epidermal inorganic phosphate would buffer against pH changes in the epidermis during stomatal movements.     

Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase

Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg²⁺. Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K_m(PEP) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K_m(PEP) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K_m(PEP) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed.     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Coupled Diffusion of Peripherally Bound Peptides along the Outer and Inner Membrane Leaflets

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D_P, of PLL differed by a factor of √2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D_M, were even more pronounced. Although D_M remained larger than D_P on PLL binding to the first membrane leaflet, D_M dropped to D_P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of ∼20 nm² membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PLL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.     

湖南安息香属植物的叶片比较解剖学研究

利用光学显微镜和扫描电镜对湖南产安息香属10种植物叶表皮微形态和解剖结构进行了观察和测量。结果表明:(1)表皮均由单层细胞构成,细胞形状为多边形和无规则形,垂周壁式样为平直、弓形、波状;(2)气孔器大小及密度在种间的差异明显;(3)气孔外拱盖内缘主要为波状和浅波状,气孔器外围角质纹饰以越南安息香的鸟巢状和栓叶安息香的碗状为显著区别特征;(4)老鸹铃、垂珠花和白花龙的气孔器保卫细胞两极有"T"形加厚;(5)栓叶安息香和大果安息香叶上表皮由大型细胞组成;(6)叶肉分化为栅栏组织和海绵组织,二者的厚度及比值在种间有明显的差异,其中越南安息香栅栏组织分化为2层;(7)大果安息香和芬芳安息香的主脉维管组织构成一圈封闭的环状结构,而其他种则为开放的半圆形结构;(8)叶片及主脉的厚度在种间差异明显。安息香属植物的表皮微形态及解剖结构特征比较稳定,可作为种间鉴定和分类的重要依据。     

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.     

SUMMATION OF DISSIMILAR STIMULI APPLIED TO LEAFLETS OF SENSITIVE BRIER (SCHRANKIA)

1. The pinnæ of sensitive brier or Schrankia are favorable for demonstrating summation of dissimilar stimuli. 2. The demonstration was made as follows: A time of day was chosen when the closure of a single proximal leaflet did not provoke closure of the next distal leaflet. An irritating gas was applied to the pinna. A few seconds later, a single leaflet was touched; it closed, induced closure of the next distal leaflet, and inaugurated a wave of closure which proceeded until all leaflets on the same side of the mid-rib were closed.     

Models of voltage-dependent conformational changes in NaChBac channels

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 3₁₀ helix, whereas in the closed model, the first part of S4 forms a 3₁₀ helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 3₁₀ portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K_v channels are discussed.     

Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats

Vertebrate telomeres consist of tandem repeats of T₂AG₃ and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.     

Measurement of biological activity in materially closed microbial ecosystems

Gas phase oxygen concentrations of materially closed, energetically open miniature microbial ecosystems were measured periodically. Our results indicate: (i) closed systems remain biologically active for at least 9 years, (ii) Po₂ values might serve as an indicator of stability, (iii) each closed ecosystem seems to seek its own unique final Po₂ state, and (iv) ecosystem response to experimentally depleted Po₂ suggests the presence of positive feedback control.     

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNA_i^Met ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which P_i is released from eIF2·GDP·P_i. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and P_i release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of P_i release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui⁻ mutations in numerous factors. We conclude that both of eIF5''s functions, regulating P_i release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.     

Redifferentiation of leaflet tissues during midrib gall development in Copaifera langsdorffii (Fabaceae)

The tree Copaifera langsdorffii is a superhost for galling herbivores. This plant species has great morphogenetic potential, and responds differently to the stimuli of more than 20 gall-inducing insects. Among these, an undescribed species of Cecidomyiidae induces a midrib gall in which a radial cecidogenetic field is generated and the leaflet tissues redifferentiate. Our objectives were to assess the amplitude of this cecidogenetic field, in which the leaflet tissues were influenced by the feeding action of the cecidomyiid; how the final gall shape was generated; and if tissue redifferentiation conferred any adaptive value on the galling herbivore. Leaflet morphogenesis followed the pattern described in the literature for simple leaves, resulting in a mesophytic arrangement. Tissue redifferentiation due to gall formation revealed that in a midrib gall, abaxial epidermal cells divided to enlarge the gall; spongy parenchyma cells originated the storage tissue, secretory structures, and vascular bundles; palisade parenchyma cells became homogeneous; and adaxial epidermis originated the nutritive tissue. Cell elongation, a necessary step towards cell redifferentiation, is triggered by an increase in water transport to the gall site and vacuole pressure due to neoformed xylem bundles. The generation of the final shape of the midrib gall involved repetitive histological steps in response to the amplitude of the cecidogenetic field. The largest impact of the cecidomyiid feeding action occurred in gall tissues redifferentiated from protoderm and adaxial ground meristem, which provided advantages to the gall maker of the C. langsdorffii midrib gall in terms of nutritional value, microenvironment, and protection against natural enemies.     

Transbilayer Colocalization of Lipid Domains Explained via Measurement of Strong Coupling Parameters

When micron-scale compositional heterogeneity develops in membranes, the distribution of lipids on one face of the membrane strongly affects the distribution on the other. Specifically, when lipid membranes phase separate into coexisting liquid phases, domains in each monolayer leaflet of the membrane are colocalized with domains in the opposite leaflet. Colocalized domains have never been observed to spontaneously move out of registry. This result indicates that the lipid compositions in one leaflet are strongly coupled to compositions in the opposing leaflet. Predictions of the interleaflet coupling parameter, Λ, vary by a factor of 50. We measure the value of Λ by applying high shear forces to supported lipid bilayers. This causes the upper leaflet to slide over the lower leaflet, moving domains out of registry. We find that the threshold shear stress required to deregister domains in the upper and lower leaflets increases with the inverse length of domains. We derive a simple, closed-form expression relating the threshold shear to Λ, and find Λ = 0.016 ± 0.004 k_BT/nm².     

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO₂ fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH¹⁴CO₃, and the kinetics of ¹⁴C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO₂ fixation is similar in the different mesophyll tissues from which these cells were derived.