The Effects of Water Deficits on Slices of Beetroot and Potato Tissue:3

The effects of osmotically induced water deficits on the metabolismof aged beetroot and potato discs were investigated. In thispaper the tissue-water relationships in two osmotica, mannitol,and Carbowax 1540, are described. Tissues in equilibrium with Carbowax solutions had lower freshweights than those in isotonic mannitol solutions, particularlyat water potentials below –0.7 J g^–1 with potatoand –2.0J g^– with beetroot. Potato discs killedby freezing and thawing lost water to Carbowax but not to mannitolsolutions. The extra effectiveness of Carbowax solutions inlowering fresh weight was attributed to an osmotic effect acrossthe cell wall. Carbowax was found to penetrate plasmolysed potatotissue, however, at a rate of about 3.0 mg (g fresh weight)^–1h^–. The extent to which water uptake occured on retransfer to waterwas unaffected by the nature of the soulte used, but dependon the degree of dehydration. The following phases were clearlydefined: (1) recovery to the fresh weight at full turgor, whenthe water potential of potato tissue was not reduced below –0.5J g^– and of beetroot below –1.2 J g^–1; (2)a declining degree of recovery with decreasing water potentialover the ranges –0.5 to –1.0 J g^–1 and –1.2to –3.0 J g^–1 for potato and beetroot, respectively,and (3)in potato, the absence of recovery of fresh weight followingreduction of the water potential below –1.0 J g^–1.     

用渗透胁迫鉴定小麦种子萌发期抗旱性的方法分析

本以聚乙二醇(PEG)-6000、甘露醇和蔗糖作为渗透剂模拟水分胁迫,胁迫溶液渗透势范围在-0．25MPa到-1．50MPa,分析适于进行小麦种子水分胁迫萌发试验的条件,以鉴定小麦萌发期的抗旱性。结果表明,蔗糖溶液易诱发霉茵,胚芽不能正常生长。渗透势为-0．25MPa的PEG-6000及-0．50MPa的甘露醇胁迫已经显抑制了胚芽伸长;-0．50MPa的PEG-6000及-1．00MPa的甘露醇显抑制种子萌发,随着胁迫强度增加,种子相对发芽率及胚芽长度减小,主要是因为渗透胁迫降低了种子吸水速度,胚芽的相对含水量和渗透势均低。在渗透势相同的胁迫条件下,PEG-6000对小麦种子萌发各项检测值的抑制作用均大于甘露醇。如果目的是通过鉴定小麦种子在高渗溶液中的萌发情况,评价萌发期的抗旱性。选用-0．50MPa的PEG-6000或-1．00MPa的甘露醇较为理想,若同时考虑降低试验成本,则应首选-0．50MPa的PEG-6000。     

Rapid Changes in Cell Wall Yielding of Elongating Begonia argenteo-guttata L. Leaves in Response to Changes in Plant Water Status

Elongation and epidermal cell turgor (P) of Begonia argenteoguttata L. leaves were simultaneously measured to determine the wall-yielding behavior of growing leaf cells in response to changes in plant water status. Rapid changes in plant water status were imposed by irrigating the rooting media with solutions of −0.20 and −0.30 MPa mannitol. These treatments caused decreases in P of 0.09 and 0.17 MPa, respectively. The decreases in P were complete within 10 min, and P did not change thereafter. Following treatments, leaf elongation was nil for periods of 25 to 38 min. Subsequently, elongation recovered to steady rates that were 45 or 75% lower than in the well-watered controls. Leaves of plants that were pretreated with −0.30 MPa of mannitol and rewatered showed an increase in P of 0.19 MPa, which was complete within 15 min; P did not change thereafter. Rewatering caused a several-fold increase in leaf elongation rates, which subsequently declined while P was increasing, to reach steady rates similar to that of the controls. Several estimates of elastic deformation indicated that most of the elongation responses to altered P were due to changes in irreversible deformation. The results showed that the initial effects of changes in P on leaf elongation were partially compensated for by changes in the cell wall-yielding properties. We conclude that linear relationships between P and adjusted growth rates are not necessarily indicative of constant wall-yielding properties. Instead, these relationships may reflect the effect of P on wall-loosening processes.     

Preliminary Investigation on Protein Bodies of Soybean Seeds

With the purpose of separation and isolation of protein bodies from soybean, soybean seeds were homogenized in oil and fractionated by successively adjusting densities of extracts with carbon tetrachloride. Isolated protein bodies consist of about 10% nitrogen, 0.8% phosphorus, 8.5% sugar, 7% ash and 0.5% RNA. Over 93% of protein in the bodies is found as particle-bound protein which is insoluble in 15% Carbowax 6000 solution but soluble in 10% sodium chloride solution. Protein bodies in intact cells, isolated bodies and those treated with Carbowax 6000 solution were respectively observed by electron-microscopy.     

Combined Carbowax-Paraffin Technic for Microsectioning of Fixed Tissues

A combined Carbowax-paraffin technic for microsectioning fixed tissues gave ribbon sections as do paraffin infiltrated and embedded tissues. Blocks of formalin or alcohol fixed tissues 2 mm. thick were infiltrated with H.E.M. (Polyethlene Glycol: Carbowax, Hartman-Leddon Co.) or with one of the following polyethylene glycol ester waxes (Glycol Products Co., Inc.) for 4 hours at (61°C.): Polyethylene Glycol 600(Di) Stearate; Carbowax 1000-(Mono) Stearate; Carbowax 4000 (Mono) Stearate; Carbowax 4000 (Mono) Laurate; Carbowax 6000 (Mono) Oleate. The Carbowax infiltrated tissues were placed for 10 minutes in xylene (61° C.), into paraffin (61° C.) for 30 minutes, then into molten paraffin contained in separate molds. (The xylene passage can be excluded for preparations which preclude its use). The blocks were hardened rapidly by submerging in ice water and were fastened to carriers as in the usual paraffin technic. Tissues were cut 6 µ thick. Segments of ribbon were spread on a water bath and mounted on slides. After drying, tissues were stained directly with hematoxylin-eosin or were carried through xylene and alcohols as in routine paraffin preparations prior to staining. The Sudan III fat stain and Best's carmine stain for glycogen were applied as in usual technics. Cellular detail was well preserved and structures did not show the extent of distortion and shrinkage encountered in ordinary paraffin technic preparations.     

An improved method for staining cells of the endocrine polypeptide (APUD) series by masked metachromasia: application of the principle of ''fixation by excluded volume''.

Synopsis Masked metachromasia is demonstrated by staining with a metachromatic basic dye, after acid hydrolysis of suitably fixed tissue. We report that the addition of 20% Carbowax 20M (an inert polymer, mol. wt. about 20000) to the hydrolysis mixture improved the reaction. The improved method gives increased metachromasia, greater tolerance to variations in hydrolysis conditions, and demonstrates a greater proportion of cells — presumably due to a lower threshold of sensitivity. Lower molecular weight polymers (Carbowax 1000, Carbowax 6000) are less effective.     

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat: I. EFFECTS ON SIZE, WEIGHT, TOTAL PROTEIN, DNA, AND PHOSPHORUS

Seedlings of wheat (Triticum durum, cv. Balcarceño-INTA)were water-stressed in darkness with 20% polyethylene glycol(PEG) 6000 or 0.3 M mannitol added to the root medium. At differenttimes and up to a total of 36 h of treatment the coleoptileand primary leaves were cut and analysed. The height and freshweight of shoots were lower in treated plants than in controlplants. Dry weight was not significantly different between controland water-stressed plants. Total protein concentration decreasedsignificantly (P < 0.01) after 36 h of PEG 6000 treatment.Total DNA concentration decreased in controls but not significantly(P < 0.025) in treated seedlings. This result was interpretedas indicating that cell elongation prevailed over cell divisionin controls and that cell enlargement was affected in stressedplants. Total phosphorus concentration fell in control and treatedseedlings. However, phosphorus specific radioactivity increasedby 116% in control plants, 93% in mannitol-treated plants, and22% in PEG 6000-treated seedlings. These data suggest that anearly metabolic effect of water stress may be on phosphorusturnover in shoots.     

Transient changes in length and growth of wheat coleoptile segments following treatments with osmotica and auxin

The dependence of elongation on the osmotic potential of the medium was investigated, using coleoptile segments (CS) of Triticim aestivum L. (cv. Hartri) and an optoelectronic device. The study aimed at separating the osmoelastic response from the irreversible growth response when an osmoticum (mannitol) was added, and to compare both processes in order to consider the possibility of growth-induced reduction in turgor pressure. The prompt inhibition of elongation registered just after addition of 50 mM mannitol as well as the subsequent resumption of the original elongation rate could be quantitatively explained by the extent and the kinetics of the osmoelastic relaxation. An initial reduction in the irreversible elongation component by mild osmotic stress could not be demonstrated. Above a critical value, the irrevesible growth was insensitive to a further increase in water potential. The minimum turgor pressure required to drive steady growth was not far from zero in both the presence and absence of auxin. The rate (r) of osmotically caused shortening per unit change of water potential was determined from the kinetics of CS shortening induced by addition of mannitol at nearly isotonic concentration (300 mM). This parameter relates a fractional change in length to the difference in water potential between inside and outside, and was assumed to depend largely on the hydraulic resistance of the tissue and cuticle. It was found to be independent of IAA. The relatively low value of Γ suggests significant reduction of turgor at high growth rates. In accordance with this conclusion, the extent of osmoelastic shortening after a transfer to 300 mM mannitol (dependent on wall strain) was significantly decreased in the presence of IAA. Addition of 100 μM IAA to CS growing at a constant rate induced pronounced oscillations in the rate of elongation, which may be connected with the change in elastic cell wall strain. Whereas the steady state growth rate before the addition of IAA was the same in the presence and in the absence of 50 mM mannitol, the maximum growth rate found after addition of IAA was substantially reduced in the mannitol variant.     

Carbohydrate Catabolism in Azospirillum amazonense

The nitrogen fixer Azospirillum amazonense grew on the various disaccharides, hexoses, and pentoses tested in this study but not on polyols and on some tricarboxylic acid cycle intermediates. An active transport system was detected for sucrose and glucose but not for mannitol and 2-ketoglutarate. Six A. amazonense strains were examined for 16 carbon-metabolizing enzymes, and the results indicate that these strains employ the Entner-Doudoroff pathway to catabolize sucrose, fructose, and glucose. The hexose monophosphate and Embden-Meyerhof-Parnas pathways were not detectable.     

Na, Cl, and Water Transport by Rat Colon

Segments of the colon of anesthetized rats have been perfused in vivo with isotonic NaCl solutions and isotonic mixtures of NaCl and mannitol. Unidirectional and net fluxes of Na and Cl and the net fluxes of water and mannitol have been measured. Net water transport was found to depend directly on the rate of net Na transport. There was no water absorption from these isotonic solutions in the absence of net solute transport, indicating that water transport in the colon is entirely a passive process. At all NaCl concentrations studied, the lumen was found to be electrically negative to the surface of the colon by 5 to 15 mv. Na fluxes both into and out of the lumen were linear functions of NaCl concentration in the lumen. Net Na absorption from lumen to plasma has been observed to take place against an electrochemical potential gradient indicating that Na is actively transported. This active Na transport has been interpreted in terms of a carrier model system. Cl transport has been found to be due almost entirely to passive diffusion.     

Investigation of plant water relations with divided root systems of soybean

Soybean (Glycine max) was grown with root systems divided between adjacent cartons containing nutrient solution or soil. By adding polyethylene glycol (Carbowax 6000) to reduce solute potential or withholding water to reduce soil matric potential until water absorption from that side stopped, the root xylem water potential could be ascertained. Carbowax appeared to increase root resistance. An imbalance technique is described with which soil moisture contents of adjacent containers were followed individually. The patterns of water absorption obtained following repeated additions of water or addition of CaCl₂ solutions to one side indicated soil hydraulic conductivity became limiting at a soil water potential of −2 bars. A high concentration of CaCl₂ added to one side greatly reduced transpiration and produced severe plant injury. With part of the root system developing in nutrient solution, growth of roots into and water absorption from soil were slow; however, reduction of solute potential in the solution side greatly increased water absorption from the soil side.     

Chloroplast ultrastructure of Hypericum perforatum plants regenerated in vitro after cryopreservation

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min^?1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min^?1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.     

Infiltrating and Embedding Tissues with Mixtures of Polyethylene Glycols and Polyvinylacetate Resins

The use of water-soluble polyethylene glycol polymers (Carbowax, Hydrowax) as embedding media can be extended and facilitated by incorporating a water insoluble polyvinylacetate resin, AYAF (Union Carbide Co.). A combination of 7.5% resin added by heating to a 3:1 mixture of polyethylene glycols 1540 and 4000 gives blocks which may be cut at 2-3 μ. Sections can be floated and properly expanded on an ordinary water bath in a manner which may be impossible with Carbowax alone because of section fragility. This may require judicious adjustment of surface tension by the prior addition of minute quantities of the wax. On water, polyethylene glycol dissolves out of tissues, which remain supported by the resin. After attachment to albumen-coated slides, residual resin may, at option, be removed by a 1-2 min immersion in methyl alcohol without visible impairment of fat content. Abopon is used for mounting. The method appears suitable for the study of intracellular lipids, particularly in tissues which cannot be conveniently handled after Carbowax alone.     

Growth Interactions during Bacterial Colonization of Seedling Rootlets

Rootlet elongation and bacterial growth on rootlets were determined after inoculation of cucumber and spinach seedlings with Pseudomonas strains differing in production of siderophores and HCN. Siderophore producers grew more profusely than nonproducers on both species and promoted rootlet elongation on cucumber. Coinoculation of siderophore producers and nonproducers resulted in restricted growth of the latter. The total populations of nonproducers of HCN in the presence of HCN producers were not decreased, but the tenacity of their association with the rootlet surface was altered.     

Ontogeny of hypertonic preabsorptive inhibitory control of intake in neonatal rats

The ontogenetic development of postingestive inhibitory control of ingestion by the osmotic load of a preload was examined in rats. On postnatal days 6 (P6) and 12 (P12), pups were deprived for either 6 or 24 h. Gastric preloads (5% body wt) of water, mannitol (a sugar alcohol that is not absorbed) in six concentrations [from 0.125 M (hypotonic) to 1.0 M (hypertonic)], or sham preloads were administered 5 min before a 30-min intake test. Compared with sham treatment, isotonic mannitol (0.25 M), a probe of volumetric control, significantly reduced intake on P12, but not on P6. Compared with isotonic mannitol, the three highest hypertonic concentrations (0.5, 0.66, and 1.0 M) significantly decreased intake on P12, at both levels of deprivation. On P6, 0.66 and 1.0 M mannitol reduced intake after 24 h, but not after 6 h, of deprivation. Thus, on P6, the hypertonic control was detectable only after prolonged deprivation and the volumetric control was not present. On P12, both controls were observed and the hypertonic control was more potent than on P6.     

Mannitol and Fructose Catabolic Pathways of Pseudomonas aeruginosa Carbohydrate-Negative Mutants and Pleiotropic Effects of Certain Enzyme Deficiencies

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.     

Promotion of Infection Thread Formation by Substances from Rhizobium

An extrinsic substance (ES-6000) was isolated from the periplasmic space of Rhizobium trifolii (strain 4S) cells by osmotic shock, using a high-density sucrose solution. This substance promoted infection thread formation in root hairs of white clover when inoculated together with the infectious strain (4S). However, ES-6000 obtained from another rhizobial species and from strain A1, which is a noninfectious mutant strain obtained from strain 4S, did not have this effect. The promoter in the ES-6000 from strain 4S is a relatively small molecule since it passed through a hollow-fiber membrane (molecular weight, 6,000). This substance was also recognized as an R_f 0.1 fraction by paper chromatography. Sucrose was effective in promoting nodulation and root elongation.     

Involvement of Abscisic Acid in Mesocotyl Growth in Etiolated Seedlings of a Foxtail Millet Dwarf Mutant

Etiolated seedlings of foxtail millet (Setaria italica Beauv.) dwarf mutant CH84113 were treated with various concentrations of abscisic acid (ABA), mefluidide, mannitol, or polyethylene glycol (PEG) 6000. It was found that these chemicals, at suitable concentrations, could increase mesocotyl length significantly, whereas these chemicals at higher concentrations had an inhibitory effect. Endogenous levels of ABA in mesocotyl were measured by enzyme-linked immunosorbent assay. It was found that endogenous ABA increased progressively in a chemical (ABA, mefluidide, mannitol, or PEG 6000) concentration-dependent manner, indicating that the effects of these chemicals on mesocotyl growth may be mediated by increased endogenous ABA levels. On the other hand, S-3307, an inhibitor of the oxidative reactions in gibberellin (GA) biosynthesis, inhibited the elongation of mesocotyl significantly. When ABA and GA₃ were applied simultaneously, the effect on mesocotyl growth was additive. These results imply that ABA and GA may control different processes in the regulation of mesocotyl growth. Received October 27, 1997; accepted May 11, 1998     

Mannitol metabolism in the phytopathogenic fungus Alternaria alternata

Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC-MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.     

Ion and water fluxes in the ileum of rats

Studies have been carried out on the movement of salt and water across the small intestine of the rat. Segments of the ileum of anesthetized rats have been perfused in vivo with unbuffered NaCl solutions or isotonic solutions of NaCl and mannitol. Kinetic analysis of movements of Na²⁴ and Cl³⁶ has permitted determination of the efflux and influx of Na and Cl. Net water absorption has been measured using hemoglobin as a reference substance. Water was found to move freely in response to gradients of osmotic pressure. Net water flux from isotonic solutions with varying NaCl concentration was directly dependent on net solute flux. The amount of water absorbed was equivalent to the amount required to maintain the absorbed solute at isotonic concentration. These results have been interpreted as indicating that water movement is a passive process depending on gradients of water activity and on the rate of absorption of solute. The effluxes of Na and Cl are linear functions of concentration in the lumen, but both ions are actively transported by the ileum according to the criterion of Ussing (Acta Physiol. Scand., 1949, 19, 43). The electrical potential difference between the lumen and plasma has been interpreted as a diffusion potential slightly modified by the excess of active Cl flux over active Na flux. The physical properties of the epithelial membrane indicate that it is equivalent to a membrane having negatively charged uniform right circular pores of 36 Å radius occupying 0.001 per cent of the surface area.