Source—sink characteristics of carbon transport in Chara hispida

Abstract Rates of uptake of ¹⁴C-labelled inorganic carbon were measured for whole Chara hispida plants, detached parts of the shoot and isolated (split-chamber technique) apices, lateral branchlets and rhizoid—node complexes. The rates of inorganic carbon uptake by the rhizoid—node complex expressed per gram fresh weight whole plant were three to four orders of magnitude less than the uptake for the whole plant. Up to 70% of the carbon taken up by the rhizoid—node complex was translocated to the shoot. After 12 h exposure to ¹⁴C-labelled inorganic carbon the concentration of ¹⁴C was greater in apices than in uppermost or central internodal cells and in all lateral branchlets, regardless of whether label was supplied to the whole plant or isolated rhizoid—node complexes. Measurement of inorganic carbon uptake by detached internodal cells and detached and isolated apices and lateral branchlets showed that lateral branchlets had the greatest rates of inorganic carbon uptake. During 12 h exposure to ¹⁴C, isolated lateral branchlets translocated to the attached shoot 55% of the labelled carbon taken up; for isolated apices this value was only 13%. It is concluded that it is highly unlikely that the rhizoid of Chara hispida could acquire a significant fraction of the whole plant requirement for inorganic carbon and that apices are sink regions for photosynthate while lateral branchlets are source regions.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACT

During spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.     

Ecophysiological investigations of Chara vulgaris L. grown in a brackish water lake: ionic changes and accumulation of sucrose in the vacuolar sap during sexual reproduction

Abstract Chara vulgaris L. growing in a brackish water lake was investigated in a field study during the main growth season (May to October 1985). Sucrose content and the ionic concentrations of the cations Na⁺, K⁺, Mg²⁺ and Ca²⁺ and the anions Cl^? and SO₄^2- of the vacuolar sap were estimated. Sucrose concentration in the vacuolar sap of vegetative growing plants was negligible, but with the beginning of the sexual reproduction period (fructification) the sucrose content increased from about 2 mol m^?3 to 110 mol m^?3. This level remained constant until the end of the fructification period. In spite of the increase of the sucrose concentration the osmotic potential of the vacuolar sap was constant. This was achieved by changing the ionic concentrations accordingly; in old or vegetative growing plants the ionic content accounted for about 80% of the vacuolar osmotic potential, but was about 63% during fructification. Sucrose is considered as a major photosynthate to supply the developing antheridia and oogonia and to serve as a precursor for the starch stored in the eggs.     

Study on the phenology of Chara vulgaris in Xin’an Spring, north China

The phenology of Chara vulgaris was studied.The specimens were collected four times from April 2004 to January 2005 (one sampling per season).The water temperature,pH,dissolved oxygen,specific conductance,maximum width and maximum depth were monitored at every sampling time.Eighteen main morphological characteristics of the Chara vulgaris were also observed and measured under the microscope.The results showed that all the environmental factors had different seasonal patterns and some of the morphological characteristics had significant fluctuations,indicating differences in their seasonality.At the same time,some morphological characteristics were affected by the environmental parameters to some extent and the effect was primarily exhibited in its vegetative proportions; there was little effect on the sexual reproductive characteristics.Therefore,the relatively stable sexual reproductive characteristics can be used to identify the species.     

Source-sink Characteristic of Photoassimilate Transport in Fertile and Sterile Plants of Chara vulgaris L.*

In cuttings of Chara vulgaris consisting of 3–4 internodal cells with whorls (branchlets) transport of photoassimilates was investigated by ¹⁴C-labelling. The distribution of radioactivity in different component parts of the plant was shown as percentage and calculated in relation to fresh weight. Sucrose was identified as the main transported carbohydrate. In fertile Ch. vulgaris plants, developing oogonia and antheridia were important sinks for transported carbohydrates. Polarity of transport in the internodal cells (nutrients and assimilates) remains constant, as the main characteristic, from base to apex in fertile as well as in sterile plants. In sterile plants, the branchlets being the main source for photoassimilates and the apex the sink, transport from branchlets is mainly in the direction of the internodes. In the case of fertile plants gametangia are additional sinks redirecting transport of assimilates into the branchlets.     

Spatial Coordination of Photosynthesis and H+ Transport in Chara corallina as Studied with the Use of Local and Whole-Cell Illumination

In experiments with the alga Chara corallina Klein ex Willd., the appearance of subcellular domains with different photosynthetic activities, as well as formation of alkaline and acid zones near the cell surface were monitored with pulse-amplitude modulated microfluorometry and pH microelectrodes. After transfer of a dark-adapted cell to actinic light, the effective yield of PSII photochemistry (F/F _m) underwent different induction changes in cell regions where acid and alkaline zones were produced. The PSII effective yield decreased for 5–15 min of illumination in cell regions forming the alkaline bands but increased after the initial decline in the acid regions. The photoinduced decrease in F/F _m in the alkaline regions occurred faster than or concurrently with the change in local pH near the cell surface (pH₀). The light-induced change in pH₀ was manifested as a steep transition after a latent period of variable lengths. The kinetics of F/F _m and F _m, specific for alkaline regions, were replaced by those typical of acid regions, when the illumination area was narrowed to 2 mm. The results show that the formation of subcellular domains with different photosynthetic activities is not strictly bound to particular cell regions but is a dynamic event determined by spatial coordination of photosynthesis in a long cylindrical cell.     

Effect of GA3 Treatment on the Number of Spermatozoids and Endopolyploidy Levels of Non-Generative Cells in Antheridia of Chara vulgaris L.

The youngest nodes located under an apical bud of Chara vulgariswere isolated and cultivated in the presence or absence of 10^–5M GA₃ under laboratory conditions to form spermatozoids. GA₃increased the DNA C-value in manubria by 20% and increased thenumber of spermatozoids per antheridium over 2-fold. (Received April 27, 1998; Accepted October 8, 1998)     

Seasonal changes of ionic concentrations in the vacuolar sap of Chara vulgaris L. growing in a brackish water lake

Summary The composition of the vacuolar sap of Chara vulgaris growing in a brackish water lake was estimated weekly over 2 years (1984–1985). The ionic concentrations of the main cations Na⁺, K⁺, Ca²⁺, and Mg²⁺ and the anion Cl^- varied depending on cell age, developmental state, and season. The average of all measurements (in mM) was Na⁺: 35, K⁺: 106, Ca²⁺: 7, Mg²⁺: 23, Cl^-: 101, SO _2- ⁴ : 20, and PO _3- ⁴ : 5. At the onset of growth in May/June the ionic content was lower compared to the mean value for the year, steadily increasing until it reached its maximum above the annual mean in winter. During the period of fructification (sexual reproduction: formation of antheridia and oogonia), when up to 100 mM sucrose was accumulated in the vacuolar sap, ionic content was lowest. This resulted in a fairly constant osmotic potential throughout the year. Mg²⁺ and Ca²⁺ concentrations were correlated with the physiological age of the cells.     

The Polar Organization of the Growing Chara Rhizoid and the Transport of Statoliths are Actin-Dependent*

Horizontally positioned Chara rhizoids continue growth without gravitropic bending when the statoliths are removed from the apex by basipetal centrifugation. The transport of statoliths in centrifuged rhizoids is bidirectional: 50–60 % of the statoliths are re-transported on a straight course to the apex at velocities from 1 to 14 μm . min^?1 increasing towards the rhizoid tip. The centrifuged statoliths which are located closest to the nucleus are basipetally transported and caught up in the cytoplasmic streaming of the cell. Those statoliths which are located near the apical side of the nucleus are transported either apically or basally. A de-novo-formation of statoliths was not observed. After retransport to the apex some statoliths transiently sediment, a process which can induce a local inhibition of cell wall growth. The rhizoid bends again gravitropically only if a few statoliths finally sediment in the apex; the more statoliths that sediment in the apex the shorter the radius of bending becomes. The transport of statoliths is mediated by actin filaments which form a network of thin filaments in the apical and subapical zone of the rhizoid, and thicker parallel bundles in the basal zone where cytoplasmic streaming occurs. Both subpopulations of actin filaments overlap in the nucleus zone.     

The uptake of nitrate and ammonium nitrogen in Chara hispida L.: the contribution of the rhizoid

Abstract The uptake of ammonium and nitrate nitrogen by cultured plants of the green freshwater alga Chara hispida L. has been compared quantitatively with the contribution of its rhizoidal tissue. In the short-term, the rhizoid takes up 7–20% of the ammonium nitrogen, and about 15% of the nitrate that is taken up by whole plants under similar conditions. The uptake was studied over a range of both temperatures and external concentrations. The apparent activation energy for the uptake of NH₄⁺ and NO₃^? by the whole plant was found to be 50 kJ mol^?1 and 30 kJ mol^?1, respectively. For the rhizoid, the values were similar for both nitrogenous ions, 106 kJ mol^?1 and 70–100 kJ mol^?1. The rhizoidal uptake mechanism for ammonium nitrogen operates more efficiently compared to that in the whole plant. Nitrate is taken up by the rhizoid by a mechanism with a substrate affinity higher than in the plant taken as a whole. The possible ecological significance of the results is discussed.     

Proton extrusion in seed coats of Phaseolus vulgaris L.

Abstract. The present investigations were designed to identify proton pumps in seed coats of Phaseolus vulgaris L. Vacated seed-coat halves were exposed to bathing solutions with indicators for proton pump action and the pH changes in the media were measured. Fusicoccin increased the rate of proton extrusion from the seed coats. Orthovanadate and abscisic acid retarded the proton extrusion evoked by fusicoccin. Abolition of the proton extrusion by parachloromercuriphenylsulphonic acid was partially reversed by diethioerythritol. The extrusion was stimulated by high osmolarities (100 mol m⁻³ sorbitol), potassium ions (100 mol m⁻³ KCI) and light. Old seed coats reacted more rapidly to fusicoccin treatments than young ones. Proton pumping in seed coats and cotyledons showed differential responses to fusicoccin, K⁺ and sucrose. In contrast to seed coats, medium acidification by cotyledons was prohibited by addition of sucrose. The significance of proton pumps for photosynthate transfer in vivo is discussed.     

Discontinuities in the Temperature Function of Transmembrane Water Transport in Chara: Relation to Ion Transport

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P _d ) were observed delimiting a region of low activation energy (E _a around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high E _a (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15–30°C and lipid matrix-mediated transport beyond this region. The K⁺ channel inhibitor, tetraethylammonium as well as the Cl⁻ channel inhibitor, ethacrynic acid, diminished P _d in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K⁺ transport in Chara, only increased P _d below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K⁺ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K⁺ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K⁺ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29°C. Received: 12 August 1998/Revised: 13 November 1998     

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Natural selection and gynodioecy in Thymus vulgaris L.

Thymus vulgaris L. is a gynodioecious species with hermaphrodites (mF) and females (mS) widely distributed in southern France and in which reproduction of which is exclusively sexual. Numerous samples have been collected in the French Mediterranean region and the percentage of mS varies from 5 to 95% in different populations. By studying environmental conditions—the area covered at ground level by vegetation, stones and rocks—high percentages of mS are found to be related to a high vegetation cover and an absence or a scarcity of stones. Grasslands and old fields favour mS, while rocky sites favour mF. This relationship, which has been found both on the plains and on a mountain, is related to the most important reproductive characteristics of T. vulgaris —genetic control of male sterility, mode of pollination, percentage of selling and seed production. These relationships agree with the assumption that unstable environmental conditions such as grasslands or old fields in southern France favour maximum seed production and outcrossing. The greater stability of uncultivated areas such as rocky sites allows for the maintenance of autogamy. Consequently, it is natural selection which adjusts the percentage of male sterility in natural populations of Thymus vulgaris.     

Changes in ultrastructure of plasmodesmata during spermatogenesis in Chara vulgaris L.

Two types of plasmodesmata are found within an antheridium of Chara vulgaris: open plasmodesmata filled with electron-transparent cytoplasm, and plugged plasmodesmata, filled with an osmiophilic dense substance. Open plasmodesmata occur only between cells synchronized completely in respect of their advancement in cell-cycle progression or differentiation. Plugged plasmodesmata connect different types of cells or cells of the same type at various stages of the cell cycle. Open plasmodesmata may become plugged, and vice versa. These changes are connected with the limitation or extension of synchronization of cellular divisions and differentiation within the groups of cells in the antheridium.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

p-CMBS Modifies Extrafacial Sulfhydryl Groups at the Chara Plasma Membrane: Activation of Ca2+ Influx and Inhibition of Two Different K+ Currents

The effect of the membrane impermeant sulfhydryl group (SH) reagent, p-chloromercuribenzenesulfonic acid (p-CMBS), on electrical membrane transport properties of the giant alga, Chara corallina, was determined. In an external medium with a high K⁺ concentration (5 mM) cells typically exhibited stable membrane potentials close to the K⁺equilibrium potential. The steady-state current-voltage (I-V) relation could be dissected into two distinct components: an almost linear ohmic leak current and a voltage-dependent K⁺ current. Adding 0.5 mM p-CMBS to the external medium resulted in an immediate, short depolarization transient (resembling the time course of an action potential) and was associated with a slow down of the cytoplasmic streaming velocity. The depolarization, as well as the streaming inhibition, could be abolished by pretreating cells with the Ca²⁺ channel inhibitor, LaCl₃. This suggests that the depolarization transient reflected a p-CMBS induced Ca²⁺ influx, a scenario known to trigger membrane excitation and slow down of cytoplasmic streaming. From the I-V analysis it appeared that p-CMBS also caused a reversible inhibition of two additional transmembrane currents: (1) a reduction of a leak current and (2) a modification of the deactivation kinetics of the voltage-dependent K⁺ channels. From the I-V difference analysis, the inhibited leak current was identified as a K⁺ current, because the reversal potential was close to the estimated K⁺ equilibrium potential. Control experiments have furthermore shown that the mercapto reagent, dithiothreitol, partly reversed the effect of p-CMBS. This strengthens the view that the action of the mercurial is related to a specific and direct modification of SH groups. The p-CMBS-evoked inhibition of K⁺ currents was not abolished by the LaCl₃ pretreatment, which suggests that the effect of the SH reagent is not induced indirectly by p-CMBS-triggered Ca²⁺ influx. Therefore, it is suggested that the mercurial interacts direcly with the K⁺ transport protein.     

生物分子相互作用分析法测定甜菜组织中的钙调素

以甜菜为实验材料,经甜菜坏死黄脉病毒（beet necrotic yellow vein virus, BNYVV）侵染后,用生物分子相互作用分析（biomolecular interaction analysis,BIA）技术测定植物组织中钙调素（calmodutin,CaM）含量,分析在BNYVV侵染下植物组织中CaM的变化。该方法可以实时检测生物分子之间的相互作用,受杂质影响小,可简化样品的前处理,能够快速高通量分析大量的蛋白质样品,灵敏度高,准确性好。