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.     

Partial Turgor Pressure Regulation in Chara canescens and its Implications for a Generalized Hypothesis of Salinity Response in Charophytes

Concentrations of ions and sucrose in the vacuolar sap of Chara canescens growing in an oligohaline lake (1.5 ‰) were estimated over the main growth period of the plants. During fructification vacuolar sap contained a mean of 41 mol m^?3 (range 10.2–61.8) sucrose. The mean turgor pressure was 239 mosmol kg^?1 (range 219–264). In long- and short-term experiments these plants were subjected to increasing salinities up to 22 ‰. When salinity was increased from 1.5 to 4.4 ‰ turgor pressure was restored to only 80 % of the initial value. This reduced level of turgor pressure was maintained up to a salinity of 22 ‰. The increase in vacuolar osmotic potential was due to the monovalent ions Na⁺, K⁺ and Cl^?. The relative amounts of Na⁺ and K⁺ participating in the regulation process were dependent on external salinity. The regulatory mechanisms observed in the brackish water species Ch. canescens are compared with those reported from freshwater and euryhaline species.     

ATP-driven proton pumping in two species of Chara differing in salt tolerance

Chara corallina is an obligate freshwater alga, while C. buckellii can be grown in salt and freshwater culture. When grown in fresh water, C. buckellii has electrophysiological properties similar to C. corallina, but when cultured in salt water, it has a less negative membrane potential and has a higher conductance. We show in internally perfused, tonoplast-free cells that the ATP-dependence of the two species cultured in fresh water is similar, although C. buckellii hyperpolarizes at lower ATP concentrations. We determined the pump parameters in perfused and intact cells. Using both techniques, C. corallina and C. buckellii cultured in fresh water show similar values of E_p, G_p and I_p. However, there is a significant difference between the two techniques: E_p is more negative (–400 to –700 mV) in perfused cells than in intact cells (–220 to –260mV); G_p is lower (0·1–0·2 versus 0·3–0·9 S m^?2); and I_p is higher (40–60 versus 10–18 mA m^?2). Salt-cultured C. buckellii was compared with freshwater C. buckellii using intact cells; G_p and E_p were similar, but I_p was much higher in salt-cultured cells (60 versus 15mA m^?2). This higher pump rate is due to the depolarization of the membrane of salt-cultured algae, which is caused by a higher passive conductance. The significance of the less negative membrane potential and the higher rate of proton pumping is discussed with respect to the banding pattern and salt stress.     

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.     

Salinity relationships in a freshwater population of eastern mosquitofish

Tolerance limit to elevated ambient salinity, and plasma osmotic regulatory capabilities were evaluated in a freshwater population of Gambusia holbrooki. The upper tolerance limit of ambient salinity was found to be 25‰. Plasma osmotic concentrations trended sharply upward with increasing salinity, but in a stepwise fashion, unlike patterns in related, but more euryhaline species. These laboratory observations are consistent with field observations that natural populations of G holbrooki are generally restricted to fresh and dilute brackish waters.     

Ecophysiology of the hypotonic response in the salt-tolerant charophyte alga Lamprothamnium papulosum

The ecophysiology of the hypotonic response was studied in the charophyte alga, Lamprothamnium papulosum, which was grown in a marine (SW; 1072 mosmol kg^–1) and a brackish (1/2 SW; 536 mosmol kg^–1) environment. The cells produced an extracellular mucilage identified by histochemical staining as a mixture of sulphated and carboxylated polysaccharides. The thickness and chemical composition of the mucilage layer was a function of environmental salinity and cell age. Mucilage progressively increased in thickness from the apex (9 SW cells: 12·6 ± 1·8 μm; 15 1/2 SW cells: 4·8 ± 0·7 μm) to the base of the plants (15 SW cells: 44·8 ± 3·3 μm; nine 1/2 SW cells: 23·8 ± 2·5 μm); with a corresponding increase in the sulphated proportion. The mucilage was significantly thicker in SW plants. Hydraulic conductivity (Lp) at the apex of SW plants, measured by transcellular osmosis, was 8·3 × 10^–13 m s^–1 Pa^–1. This was close to Lp of freshwater Chara (8·5 × 10^–13 m s^–1 Pa^–1) which lacked mucilage. Basal SW cells with thicker mucilage had a smaller apparent Lp of 3·5 × 10^–13 m s^–1 Pa^–1. The electrophysiology of the resting state and hypotonic response was compared in cells from the two environments based on current/voltage (I/V) analysis. The resting potential difference (PD) and conductance differed (11 SW cells: – 102·4 ± 10·1 mV, eight SW cells: 18·6 ± 2·4 S m^–2; 19 1/2 SW cells: –125·7 ± 5·9 mV, 8·3 ± 0·8 S m^–2). The type of cellular response to a hypotonic shock (decrease of 268 mosmol kg^–1) also differed. In 1/2 SW plants, only the apical cells with thin mucilage responded classically with depolarization, conductance increase, Ca²⁺ influx, cessation of cytoplasmic streaming, and K⁺ and Cl^– effluxes. Older cells making up the bulk of the plants responded with depolarization, but continued cytoplasmic streaming, and had only a small increase in conductance; or depolarized transiently without altering the I/V profile, conductance or streaming speed. Most cells remained depolarized and in the K⁺ state 1 h post-shock. Cells treated with the K⁺ channel blocker tetraethylammonium chloride also depolarized and remained depolarized. The SW cells depolarized but otherwise responded minimally to a 268 mosmol kg^–1 drop in osmolarity and required a further 268 mosmol kg^–1 down-step to elicit a change in the conductance. A spectrum of responses was measured in successively older and more mucilaginous cells from the same marine plant. We discuss the ecophysiological significance of the mucilage layer which modulates the cellular response to osmotic shock and which can be secreted to different degrees by plants inhabiting environments of different salinity.     

Short-term regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress in the charophyte alga Nitellopsis obtusa

Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m^?3 NaCl solution unless more than several mol m^?3 Ca²⁺ is added. Short-term effects of NaCl stress on the cytosolic concentration of Ca²⁺ ([Ca²⁺]_c), cytosolic pH (pH_c) and vacuolar pH (pH_v) were studied in relation to the external concentration of Ca²⁺ ([Ca²⁺]_e). Changes in [Ca²⁺]_c were measured with light emission from a Ca²⁺-sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pH_c and pH_v were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m^?3 NaCl (0–1 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_e), [Ca²⁺]_c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca²⁺]_c was not influenced by the level of [Ca²⁺]_c (0.1 and 10 mol m^?3). In some cases, the transient increase in [Ca²⁺]_c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m^?3 NaCl (0.1 mol m^?3 [Ca²⁺]_c), pH_c decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pH_v increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pH_c and pH_v were suppressed when [Ca²⁺]_e was raised from 0.1 to 10mol m^?3. These results show that the charophyte alga Nitellopsis can regulate [Ca²⁺]_c, pH_c and pH_v under NaCl stress in the short term and that the protective effect of Ca²⁺ on salinity stress is apparently unrelated to perturbation of Ca²⁺ and pH homeostasis.     

Salinity tolerance of Kosteletzkya virginica. II. Root growth, lipid content, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicot halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m ³ NaCl, and the effects of external salinity on root growth, ion and water levels, and lipid content were examined in successive harvests. Root growth paralleled shoot growth trends, with some enhancement observed at 85 mol m ³ NaCl and a reduction noted at the higher salinities. Root Na⁺ content increased with increasing external NaCl, but remained constant with time for each treatment. K⁺ content, although lower in salt-grown plants after 14 d salinization, subsequently increased to levels comparable to unsalinized plants. A strong K⁺ affinity was reflected in the increased K⁺/Na⁺ selectivity of salt-grown plants and by their low Na⁺/K⁺ ratios. Cl levels rose in salinized plants and values were double or more those for Na⁺, indicating the possibility of a sodium-excluding mechanism in roots. Root phospholipids and sterols, principal membrane constituents, were maintained or elevated and the free sterol/phospholipids ratio increased in salinized K. virginica plants, suggesting retention of overall membrane structure and decreased permeability. This response, considered in light of root calcium maintenance and high potassium levels, suggests that salinity-induced changes in membrane lipid composition may be important in preventing K⁺ leakage from cells.     

Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata, but not in downward bending by bowing of Chara rhizoids

The localization of cytoplasmic free calcium and a dihydropyridine (DHP) receptor, a putative calcium channel, was recorded during the opposite graviresponses of tip-growing Chara rhizoids and Chara protonemata by using the calcium indicator Calcium Crimson and a fluorescently labeled dihydropyridine (FL-DHP). In upward (negatively gravitropically) growing protonemata and downward (positively gravitropically) growing rhizoids, a steep Ca²⁺ gradient and DHP receptors were found to be symmetrically localized in the tip. However, the localization of the Ca²⁺ gradient and DHP receptors differed considerably during the gravitropic responses upon horizontal positioning of the two cell types. During the graviresponse of rhizoids, a continuous bowing downward by differential flank growth, the Ca²⁺ gradient and DHP receptors remained symmetrically localized in the tip at the centre of growth. However, after tilting protonemata into a horizontal position, there was a drastic displacement of the Ca²⁺ gradient and FL-DHP to the upper flank of the apical dome. This displacement occurred after the apical intrusion and sedimentation of the statoliths but clearly before the change in the growth direction became evident. In protonemata, the reorientation of the growth direction started with the appearence of a bulge on that site of the upper flank which was predicted by the asymmetrically displaced Ca²⁺ gradient. With the upward shift of the cell tip, which is suggested to result from a statolith-induced displacement of the growth centre, the Ca²⁺ gradient and DHP receptors became symmetrically relocalized in the apical dome. No major asymmetrical rearrangement was observed during the following phase of gravitropic curvature which is characterized by slower rates of bending. Labeling with FL-DHP was completely inhibited by a non-fluorescently labeled dihydropyridine. From these results it is suggested that FL-DHP labels calcium channels in rhizoids and protonemata. In rhizoids, positive gravitropic curvature is caused by differential growth limited to the opposite subapical flanks of the apical dome, a process which does not involve displacement of the growth centre, the calcium gradient or calcium channels. In protonemata, however, it is proposed that a statolith-induced asymmetrical relocalization of calcium channels and the Ca²⁺ gradient precedes, and might mediate, the rearrangement of the centre of growth, most likely by the displacement of the Spitzenk?rper, to the upper flank, which results in the negative gravitropic reorientation of the growth direction. Received: 13 February 1999 / Accepted: 25 June 1999     

A comparative study of the effects of NaCl salinity on respiration, photosynthesis, and leaf extension growth in Beta vulgaris L. (sugar beet)

Abstract. Three parameters influencing the capacity for carbon accumulation, i.e. photosynthesis, respiration, and leaf extension growth, were studied in Beta vulgaris L. (sugar beet) cultured in nutrient solution containing 0.5 to 500 mol m⁻³ NaCl. Leaf extension growth was the parameter most sensitive to salinity: the initial rate of leaf extension and final leaf length each declined linearly with increase in external NaCl concentration. Photosynthetic O₂ evolution of thin leaf slices did not decline until salinity levels reached 350 to 500 mol m⁻³ NaCl, while respiratory O₂ consumption was not affected by salinity throughout the range. The results suggest that the influence of salinity on the capacity for carbon accumulation in B. vulgaris occurs primarily through reduction in the area of photosynthetic surface.     

Salinity tolerance of Kosteletzkya virginica. I. Shoot growth, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicotyledonous halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m^-3 NaCl, and the effects of external salinity on shoot growth and ion content of individual leaves were studied in successive harvests. Growth was stimulated by 85 mol m^-3 NaCl and was progressively reduced at the two higher salinities. Growth suppression at high salinity resulted principally from decreased leaf production and area, not from accelerated leaf death. As is characteristic of halophytic dicots. K. virginica accumulated inorganic ions in its leaves, particularly Na⁺ and K⁺. However, the Na⁺ concentration of individual leaves did not increase with time, but remained constant or even declined, seeming to be well-coordinated with changes in water content. A striking feature of the ion composition of salinized plants was the development of a dramatic gradient in sodium content, with Na⁺ partitioned away from the most actively growing leaves. Salt-treated plants exhibited a strong potassium affinity, with foliar K⁺ levels higher in salinized plants than unsalinized plants after an initial decrease. These results suggest that selective uptake and transport, foliar compartmentation of Na⁺ and K⁺ in opposite directions along the shoot axis, and the regulation of leaf salt loads over time to prevent build-up of toxic concentrations are whole-plant features which enable K. virginica to establish favourable K⁺-Na⁺ relations under saline conditions.     

Relative importance of osmotic-stress and ion-specific effects on ABA-mediated inhibition of leaf expansion growth in Phaseolus vulgaris

The osmotic and ion-specific components of salt-induced inhibition of leaf expansion growth were investigated in beans grown from 12 h to several days in either NaCl-containing solution cultures, an isosmotic concentrated macronutrient solution, or a vermiculite–compost mixture with low Na⁺ but high Cl^– availability. Inhibition of leaf expansion and leaf ABA increase was more intense in the NaCl than in the isosmotic macronutrient treatment. Root Na⁺ was highly correlated to inhibition of leaf expansion and leaf or xylem sap ABA. When Na⁺ was sequestered in soil, salinized plants showed no reduction in leaf expansion or ABA increase, regardless of the presence of high leaf Cl^– concentrations. Stomatal conductance exhibited an exponential relationship with the reciprocal value of xylem sap ABA. Our results indicate that an ion-specific effect caused by Na⁺ in roots may account for an ABA-mediated reponse of both stomatal closure and leaf expansion inhibition.     

Osmotic stress, endogenous abscisic acid and the control of leaf morphology in Hippuris vulgaris L

Abstract. Previous reports indicate that heterophyllous aquatic plants can be induced to form aerial-type leaves on submerged shoots when they are grown in exogenous abscisic acid (ABA). This study reports on the relationship between osmotic stress (e.g. the situation encountered by a shoot tip when it grows above the water surface), endogenous ABA (as measured by gas chromatography-electron capture detector) and leaf morphology in the heterophyllous aquatic plant, Hippuris vulgaris. Free ABA could not be detected in submerged shoots of H. vulgaris but in aerial shoots ABA occurred at ca. 40ng (g fr wt)⁻¹. When submerged shoots were osmotically stressed ABA appeared at levels of 26 to 40ng (g fr wt)⁻¹. These and other data support two main conclusions: (1) Osmotically stressing a submerged shoot causes the appearance of delectable levels of ABA. (2) The rise of ABA in osmotically stressed submerged shoots in turn induces a change in leaf morphology from the submerged to the aerial form. This corroborates the hypothesis that, in the natural environment, ABA levels rise in response to the osmotic stress encountered when a submerged shoot grows up through the water/air interface and that the increased ABA leads to the production of aerial-type leaves.     

Functional analysis of TaDi19A, a salt-responsive gene in wheat

A salinity stress upregulated expressed sequence tag (EST) was selected from a suppression subtractive hybridization cDNA library, constructed from the salinity-tolerant wheat cultivar Shanrong No. 3. Sequence analysis showed that the corresponding gene (named TaDi19A ) belonged to the Di19 family. TaDi19A was constitutively expressed in both the root and leaf of wheat seedlings grown under non-stressed conditions, but was substantially up-regulated by the imposition of stress (salinity, osmotic stress and cold), or the supply of stress-related hormones [abscisic acid (ABA) and ethylene]. The heterologous over-expression of TaDi19A in Arabidopsis thaliana increased the plants' sensitivity to salinity stress, ABA and mannitol during the germination stage. Root elongation in these transgenic lines showed a reduced tolerance to salinity stress and a reduced sensitivity to ethophon. The expression of the ABA signal pathway genes ABI1 , RAB18 , ERD15 and ABF3 , and SOS2 (SOS pathway) was altered in the transgenic lines. TaDi19A plays a role in the plant's response to abiotic stress, and some possible mechanisms of its action are proposed.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Influence of Environmental Salinity on Messenger RNA Levels of Growth Hormone, Prolactin, and Somatolactin in Pituitary of the Channel Catfish (Ictalurus punctatus)

Pituitary growth hormone (GH), prolactin (PRL), and somatolactin (SL) messenger RNA levels in channel catfish (Ictalurus punctatus) were examined under various environmental and physiological conditions. Catfish were sampled following salinity challenge, during the winter (December) and spring or summer (April or July), and at different sizes (15–18 g, 620–664 g, and 956–1134 g). When catfish (956–1134 g) were transferred from freshwater to saline water containing 8 ppt NaCl, their plasma [Na⁺] increased significantly above values in the freshwater control group until they were transferred back to freshwater. Pituitary GH mRNA levels were low for the first 24 hours following transfer to saline water, but thereafter were significantly elevated above control values until the fish were transferred back to freshwater. Pituitary GH mRNA levels were highest in July and lowest in December. Growth hormone mRNA levels were also elevated in the size groups 15–18 g and 956–1134 g in July when compared with December values. Pituitary PRL mRNA levels increased for the first 24 hours following transfer to saline water (956–1134 g), but thereafter were significantly lower than control values until the fish were transferred back to freshwater. Pituitary PRL mRNA levels were highest in April and July and lowest in December, and were also elevated in the size groups 620–664 g and 956–1134 g. Pituitary SL mRNA levels were unaffected in catfish transferred to saline water; however, levels were significantly elevated in catfish of the 956–1134-g size group sampled in April when compared with December. These results suggest the involvement of GH in adaptation to brackish water and of PRL in adaptation to freshwater in the catfish, and seasonal and size-related differences in pituitary GH, PRL, and SL mRNA levels. Received May 17, 2000; accepted October 30, 2000