Solute Accumulation in Plant Cells: III. Treatments which Arrest and Restore the Course of Absorption and Growth in Cultured Carrot Explants1

Prior papers have dealt with the range of the growth responsesof carrot explants to the composition of the ambient media andhow these affected the solute concentration and compositionof the tissue. They have also dealt with the sequential eventsalong the time course of growth of the tissue explant. Thispaper presents results and conclusions derived from experimentswhich exploit changes in the growing explants when their normalcourse of growth and solute uptake is interrupted by exposingthem sequentially to different ambient media. After explants were induced to grow during an initial 6 days,they were placed in a minimal nutrient medium which lacked growthstimuli and salts, notably potassium ions. Thus the internalsalt concentrations of the cells declined as they continuedto divide and enlarge at a reduced rate and their osmotic valuewas maintained by storage of organic solutes (sugar). The subsequentresponses of these ‘low salt’ cells to differentnutrient regimes were studied. When salts were resupplied, growth was stimulated somewhat andthe osmotic value of the cells increased as salts were accumulated;with a renewed full nutrient medium and a full complement ofgrowth substances, the cultures re-embark upon their growthand attain the average cell size and composition as if theyhad not been reversibly arrested. Thus reversible trends insolute composition of cells may be superimposed upon their normaldevelopmental course by alternately withholding and restoringthe stimuli to their growth and by changing the balance betweenorganic and inorganic solutes supplied in the medium. The emphasis is on the control of osmotic value in the cellsas they enlarge and mature and this over-rides the changes inthe solutes they receive (salts or sugars) via the ambient medium.The effects here observed were induced by sequential changesin the culture medium, but these obviously relate to similarresponses of cells in the intact plant body as their growthand solute supply is modified through interactions between organsas the plant grows.     

Solute Accumulation in Plant Cells: II. The Progressive Uptake of Non-electrolytes and Ions in Carrot Explants as They Grow

An earlier paper established the range of solute compositionthat may obtain in aseptically cultured carrot explants growingin different media which regulate either cell enlargement orcell division in the explants. This paper concentrates uponthe most rapidly growing cultures, containing cells which individuallypass through their cycle of division and enlargement and collectivelytrace a sigmoidal curve of growth for the explant as a whole.The time course of growth is interpreted in terms of the numberand average size of the cells and, in different phases, in termsof the changes in solute uptake and content of the cells. Thesedata are correlated with the concomitant metabolic characteristicsof the tissue, notably its protein synthesis. In the early exponential growth phase of the explant, when theemphasis is on cells in division, the organic solutes whichare absorbed are used to create form and complex substance;concomitantly, the cells develop a specific requirement forpotassium, selectively preferred to sodium, and balanced byorganic anions rather than halide. These relationships changeas cells develop; the emphasis is then upon the maintenanceof osmotic value in cells with enlarging vacuoles. The developingvacuoles preferentially store organic solutes but, later, thesesolutes may be replaced by natural salts (KC1, NaCl) when organicsupplies are depleted. This latter accumulation of salts doesnot display as markedly the disparity between potassium, sodium,and chloride which was so evident in the cell multiplicationphase of growth. Superimposed upon these relationships are certain clonal differenceswhich are interpreted compatibly with the above concepts. Thedata obtained on many clones reinforce the view that the changesin ion relations of cells with growth, as noted above, are compensatedby accumulation of organic solutes as cells build osmoticallyactive concentrations of solutes in a system that primarilycontrols the internal activity of their water.     

Solute Accumulation In Plant Cells IV. Effects of Ammonium lons on Growth and Solute Content

Procedures previously described were used to study growth andsolute content of aseptically cultured carrot explants as affectedby supplementary salts in the medium. The salts chosen (KC1,KNO₃, NH₄,Cl, and NH⁴,NO₃) contrasted, with appropriate controls,the effects due to nitrate and ammonium. Growth was measuredin terms of fresh weight, the number and average size of cells:solute concentrations were recorded for total solutes, sugars,soluble nitrogen compounds, and the electrolytes K⁺, Na⁺, C1^–,NO^–₃, and organic acids. The time-response curves of thecultures were traced at a fixed concentration of the added saltsand the effects due to the concentration of the supplementarysalts were tested after a fixed time period, For the same nitrogensource the concentrations of metabolites and solutes in cellswere very similar despite some clonal differences in their growth.When cells in a nitrate medium were small and dividing, thecultures had a low osmotic value, contained K⁺ as the principalcation balanced by organic acid, had relatively low sugar content,and their enriched total nitrogen content emphasized proteinrather than soluble nitrogen compounds. Later, as the cellsbecame older and larger, salts (K⁺, organic anions, Cl^–)contributed substantially to their increased osmotic value butthey accumulated sugar as their main, osmotically active solute,and the ratio of soluble to protein nitrogen declined as proteinsynthesis progressed. The extra nitrogen supplied by the additionalpotassium nitrate contributed more to protein and caused potassium,organic acids, and sugars to accumulate to higher levela. Supplementaryammonium salts required that more sugar be metabolized to organicnitrogen compounds (e.g. glutamine), contributed more to solublethan to protein nitrogen, and sharply reduced. both the osmoticvalue of the cells and the potassium linked to organic anions.The selectivity of the growing cells for K⁺ over Na⁺ and theirdiscrimination. between alkali cations (Ka⁺+Na⁺) and halides(C1^–) were relaxed in the presence of ammonia. Attentionis drawn to the implications of these results for the accumulationof solutes, organic and inorganic, by dividing and enlargingcells.     

Multiphasic osmotic adjustment in a euryhaline cyanobacterium

Abstract Transfer of Synechocystis PCC6714 from a freshwater medium to a saline medium caused the cells to shrink; rapid entry of NaCl resulted in a partial recovery of cellular volume within 2 min. Active extrusion of internal Na⁺ in exchange for extracellular K⁺ then occurred (within 20 min). Finally, the low- M _r carbohydrates sucrose and glucosylglycerol were accumulated and internal KC1 levels declined. In long-term growth experiments, the relative importance of sucrose as a component of the low- M _r organic solute fraction decreased and glucosylglycerol became the single most important intracellular solute. These observations demonstrate that several inorganic and organic solutes are involved in osmotic adjustment in this cyanobacterium, with sequential changes in the relative importance of each solute following transfer to a saline medium.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.     

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.     

Investigations on the growth and metabolism of cultured explants of Daucus carota

Summary Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K⁺, Na⁺, Cl^–, total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K⁺ over Na⁺ strongly toward Na⁺, a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K⁺ vs. Na⁺; organic anions vs. Cl^–) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.     

Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.     

Storage of Osmotically Active Compounds in the Taproot of Daucus carota L.

The osmotic potential of cell sap from the storage root, lateralroots and shoots of carrot (Daucus carota L., cv. AmsterdamseBak) was calculated from the concentration of osmotically activecompounds in these tissues. The osmotic potential of the taprootdid not change with age prior to and during the storage of osmoticallyactive sugars, as sucrose and reducing sugars. The increased contribution of soluble sugars in the osmoticpotential was accompanied by a proportionally decreased contributionof potassium and organic acids. Before storage of soluble sugarsin the taproot occurred, potassium and organic acids contributed80% to the total osmotic value, in contrast with lateral roottissue, where potassium and nitrate were the main osmotic solutes.The concentration of osmotically active solutes was lower inlateral root tissue than in storage root tissue. Shoot tissueresembled taproot tissue before storage, in having potassiumand organic acids as the main osmotic solutes. The concentrationof osmotically active solutes was highest in shoot tissue andit increased towards the end of the experimental period. The calculated osmotic potentials were in good agreement withthe experimental values, as determined from the molecular depressionof the freezing point of cell sap. Storage of reducing sugarsand sucrose is discussed in relation to acid and neutral invertaseactivities. Key words: Daucus carota, Osmotic solutes, Sugar storage, Invertase activity     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K⁺/Na⁺ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.     

Osmoregulation in the Avena coleoptile in relation to auxin and growth

A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e. to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin-induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2-deoxy-d-glucose or 3-O-methyl-d-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. The rate of solute uptake is even greater in the presence of auxin or fusicoccin, but the osmotic concentration rises only slightly because of the water taken up during growth. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells.     

Solute accumulation in the deep-sea bacterium Photobacterium profundum

The identity and amounts of intracellular solutes in the deep-sea bacterium Photobacterium profundum strain SS9 were studied using nuclear magnetic resonance techniques. P. profundum strain SS9, a moderate piezophile which grows optimally at 20-30 MPa primarily accumulated glutamate and betaine, with lesser amounts of alanine, beta-hydroxybutyrate (beta-HB) and oligomers composed of the beta-HB units when grown at 0.1 MPa to early stationary phase. When grown at the optimal pressure, the cells preferentially increased intracellular concentrations of beta-HB and beta-HB oligomers, while the amino acid pools remained relatively constant. Since the organic solutes increased with increasing external NaCl in the medium, they are functioning as osmolytes. The beta-HB molecules represent a novel class of osmolytes, termed 'piezolytes,' whose cellular levels responded to hydrostatic pressure as well as osmotic pressure. Factors such as cell growth stage and temperature were also examined for their effect on the solute distribution in these cells.     

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m⁻³ NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na⁺) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K⁺ and Ca²⁺ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.     

Spatial and temporal distribution of solutes in the developing carrot taproot measured at single-cell resolution

The time-course and spatial distribution of sugars and ions in carrot (Daucus carota L.) was studied at fine resolution using single cell (SiCSA) and tissue analysis. Four phases of osmolyte accumulation in the taproot were identified: an amino acid (germination) phase, when internal sources of amino acids provide seedlings with osmotica; an ion phase, when inorganic and organic ions were the main solutes; a hexose phase, when concentrations of glucose and fructose sharply increased and reached their maximum; and a sucrose phase, when sucrose became the major solute. Spatial distribution of sugar in taproot cells showed a general trend of highest concentration on both sides of the vascular cambium (some 200 mM sucrose, 150 mM glucose) and a minimum in the pith (some 100 mM sucrose, 60 mM glucose) and in periderm. Electrolytes (e.g. potassium) followed a distribution generally reciprocal to that of sugars; minimum in the tissue adjacent to the cambium (some 10 mM) and maximum in the pith and periderm (some 60-100 mM). The cambial cells contained unexpectedly low concentrations of sugars and potassium. These spatial and temporal patterns indicate that amino acids, other electrolytes and sugars are interchangeable in the tissue osmotic balance. The nature of the solute is developmentally determined both temporally and spatially. During the accumulation of electrolytes following the initial amino acid phase, osmotic pressure to 420 mosmol kg-1 rises and then remains constant despite large changes in the concentration of individual solutes. This indicates that osmotic pressure is regulated independently of the individual concentrations of solutes.     

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

OSMOTIC ADJUSTMENT AND DYNAMIC CHANGES IN DISTRIBUTION OF LOW MOLECULAR WEIGHT SOLUTES BETWEEN CELLULAR COMPARTMENTS OF CARROT AND BEET ROOT CELLS EXPOSED TO SALINITY

Carrot cells (Daucus carota L.) in suspension culture exposed to medium containing 150 mM NaCl plasmolyzed immediately and deplasmolyzed within 35 to 40 hr. Three days after exposure to NaCl the cells resumed proliferation. Accommodation to salinity and renewal of growth was accompanied by absorption of Na⁺ from the external medium. On completion of deplasmolysis, K⁺ concentration in the cytosol doubled and Na⁺ concentration approximated that of K⁺. The vacuolar K⁺ concentration was practically unchanged while Na⁺ accumulated to a concentration double that of K⁺. Cl^−- accumulation started later and eventually exceeded that of Na⁺ plus K⁺. Malate was redistributed during accommodation to salinity and eventually returned to its initial level. Amino acid content in the cytosol increased fivefold, while in the vacuole it remained unchanged. These results show that: 1) recovery from osmotic shock requires absorption of easily penetrating solute, mainly Na⁺; 2) distribution of solutes, absorbed or synthesized in cells exposed to salinity, is a dynamic process; 3) cells could grow and proliferate in high NaCl content in the cytosol; 4) red beet root cells grown in the presence of NaCl contain higher cytoplasmic Na⁺ than K⁺; and 5) during adjustment to salinity small spherical carrot cells survive the osmotic shock and do not show any detectable damage.