Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.     

Biochemical changes during embryogeny in Hordeum distichum

Changes in fr. and dry wt, soluble reducing sugars, protein. total carbohydrate, DNA, RNA, sucrose synthetase activity and invertase activity were recorded for the developing embryo of Hordeum distichum var Julia over the period 18–60 days after anthesis. Fresh wt increased until 45 days whereupon rapid dehydration commenced. Reducing sugar concentration remained low throughout development but total carbohydrate and protein accumulated rapidly over the initial period to reach maximum values at around 50 days. DNA concentration remained relatively constant throughout the middle and later stages of development, but RNA, on the other hand, increased rapidly to reach a maximum value at maturity. Sucrose synthetase (assayed in the direction of sucrose cleavage) was considerably more active with UDP than ADP and reached a maximum value around 35 days after anthesis. When assayed in the direction of sucrose synthesis the peak of activity was slightly later in development and doubled in value. Invertase activity was appreciable and was still present at maturity.     

PROTEINS AND GLYCOPROTEINS IN MYELIN PURIFIED FROM THE DEVELOPING BOVINE AND HUMAN CENTRAL NERVOUS SYSTEMS

Myclin was purified from bovine and human midbrain at various stages of prenatal and postnatal development. Basic protein and proteolipid proteins were the major individual proteins at all stages. The specific activity of 2′3′-cyclic nucleotide 3′-phosphohydrolase remained constant in the bovine myclin during development but decreased slightly in human myelin. A high molecular weight glycoprotein with electrophoretic mobility similar to that previously reported in rodent myelin (QUARLES et al., 1973) was present in both bovine and human myelin at all stages of development. The intensity of staining of this glycoprotein with periodic acid-Schiff reagents per mg total myelin protein was less in mature bovine and human myelin than in rat myelin.     

Cellular compartmentalization of salt ions and protective agents with respect to freezing tolerance of leaves

Summary Evidence was given that the freezing tolerance of Halimione portulacoides, a wintergreen halophyte, can be explained by protection of sensitive cellular membranes in vivo. Experiments were done with cloned cuttings of a plant from the German North Sea coast. One series received no NaCl (O-plants) the other 3% NaCl (NaCl-plants) in the nutrient solution. During the annual course Na⁺ and Cl^- of the O-plant leaves remained on a nearly constant low level. In the leaves of the NaCl-plants Na⁺ and Cl^- concentrations changed strongly during year and reached a maximum in winter. Potassium was always on a low level. The freezing tolerance curves showed a minimum in summer and a maximum in winter. The small difference between the freezing tolerance peaks of the NaCl- and O-plants indicated that the increased salt stress did not affect freezing tolerance very much. Freezing stress in cellular membranes, like thylacoids, acts in the same way as increasing salt concentration; consequently both together must amplify the stress. For the analysis of their ion contents chloroplasts of H. portulacoides were non-aqueously isolated from leaves with different freezing tolerance during the year. In midwinter, when freezing tolerance was highest, the chloroplasts of the NaCl-plants contained about 250 mM chloride (O-plants c. 150 mM), while the non-plastidic fraction of the cell contained about 1 M (O-plants c. 400 mM) chloride. On the other hand citrate reached high concentrations in the chloroplasts in winter. Non-volatile organic acids like citrate are known to compensate colligatively the injurious action of the inorganic salt ions on thylacoids in vitro (Heber and Santarius, 1976). The molar proportion between chloride and citrate in H. portulacoides chloroplasts decreased with increasing freezing tolerance and reached values which were protective on chloroplast membranes in vitro. This relationship in vivo with H. portulacoides provides evidence supplying the concept of colligative protection of cellular membranes. Besides citrate also malate may act as a colligatively protecting agent against the amplified salt stress by freezing.     

Derepressive Formation of l-Leucine-Pyruvate Transaminase under Nitrogen-Free Conditions in Gluconobacter suboxydans

l-Leucine-pyruvate transaminase activity increased 6- to 20-fold in 3 hr when Gluconobacter suboxydans cells grown on yeast extract-medium were transferred to and incubated in a nitrogen-free medium. The increase in enzyme activity was influenced remarkably by the age and concentration of cells used. The phenomenon depended upon de novo synthesis of enzyme protein.

The enzyme activity in cell-free extracts of cells incubated under a nitrogen-free condition decreased remarkably after heat treatment at 50°C (pH 6.0) or after freezing and thawing. The level of such enzyme inactivation was high in extracts of cells in the early stages of induction and low in later stages.     

Temporal cell wall changes during cold acclimation and deacclimation and their potential involvement in freezing tolerance and growth

Plants adapt to freezing stress through cold acclimation, which is induced by nonfreezing low temperatures and accompanied by growth arrest. A later increase in temperature after cold acclimation leads to rapid loss of freezing tolerance and growth resumption, a process called deacclimation. Appropriate regulation of the trade-off between freezing tolerance and growth is necessary for efficient plant development in a changing environment. The cell wall, which mainly consists of polysaccharide polymers, is involved in both freezing tolerance and growth. Still, it is unclear how the balance between freezing tolerance and growth is affected during cold acclimation and deacclimation by the changes in cell wall structure and what role is played by its monosaccharide composition. Therefore, to elucidate the regulatory mechanisms controlling freezing tolerance and growth during cold acclimation and deacclimation, we investigated cell wall changes in detail by sequential fractionation and monosaccharide composition analysis in the model plant Arabidopsis thaliana, for which a plethora of information and mutant lines are available. We found that arabinogalactan proteins and pectic galactan changed in close coordination with changes in freezing tolerance and growth during cold acclimation and deacclimation. On the other hand, arabinan and xyloglucan did not return to nonacclimation levels after deacclimation but stabilized at cold acclimation levels. This indicates that deacclimation does not completely restore cell wall composition to the nonacclimated state but rather changes it to a specific novel composition that is probably a consequence of the loss of freezing tolerance and provides conditions for growth resumption.     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

Free Amino Acid, Protein and Water Content Changes Associated with Seed Development in Araucaria angustifolia

The free amino acid, protein, water and dry matter contents were determined during the seed development of Araucaria angustifolia. Soluble and insoluble proteins in the mature seed represent 4.2 % of the fresh matter. The embryonic axis stored the greatest amount of soluble proteins, while cotyledons both with the embryonic axis showed the largest quantities of insoluble proteins in the mature seed. The greatest concentration of free amino acids was detected during the stage when cotyledons start to develop. Glutamic acid, aspartic acid, alanine and serine were predominant in the whole seed while arginine, lysine and γ-aminobutyric acid were present in great amounts only in cotyledons and embryonic axis. Although megagametophyte was important as a source of free amino acids, it was not the major protein storage organ in the mature seed. In the embryogenetic process, the rise of cotyledons is closely related to physiological and biochemical changes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in the Electrophoretic Patterns of the Soluble Proteins of Winter Wheat and Rye following Cold Acclimation and Desiccation Stress

The degrees of freezing tolerance acquired by winter wheat (Triticum aestivium L.) and rye (Secale cereale L. cv Puma) were similar following a 4-week cold conditioning and a 24-hour desiccation stress. Soluble proteins were extracted from shoots of cold-conditioned or desiccation-stressed seedlings and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Quantitative changes in the electrophoretic patterns of the soluble proteins of the different cultivars grown in different environments were detected, but the changes were not equivalent following cold conditioning and desiccation stress. The abundance of two polypeptide bands showed a significant increase correlated to the degree of freezing tolerance and, hence, the polypeptides in these bands may play a role in the development of freezing tolerance.     

Nutrient allocation in the gonads of the sea urchin Arbacia dufresnii in different stages of gonadal development

This study reports nutrient allocation in different stages of gonadal development for two populations of the sea urchin Arbacia dufresnii off the Patagonian coast of Argentina (Nuevo Gulf and San Jorge Gulf). The biochemical composition of gonads was used to assess nutrient allocation by measuring ash, soluble protein, lipid and trichloroacetic acid-soluble carbohydrate concentrations, and absolute contents over a 24-month period. Reproductive output in terms of energy was calculated for females. Results were correlated with histological stage of the gonads. Soluble proteins were the main component for the Nuevo Gulf population while unmeasured organic material (i.e. insoluble proteins and nucleic acids, especially in testes) was prevalent in gonads from San Jorge Gulf. Soluble protein and lipid concentrations followed the gonadal cycle, while carbohydrate concentration was almost negligible, especially in the Nuevo Gulf population. The different patterns in the gonadal cycle in the two populations were reflected in the biochemical composition of gonads. Concentrations and contents of the biochemical components and reproductive output were higher in the population from San Jorge Gulf owing to the larger size of gonads and gametes. These findings contribute to the better understanding of the plasticity of the reproductive biology of A. dufresnii in different environments.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

Protein kinase C (calcium/phospholipid-dependent protein kinase) in developing chick heart: selective localization to atrium versus ventricle and changes in activity levels during cardiogenesis

Activity levels of calcium/phospholipid-dependent protein kinase were examined in preparations of atria and ventricles from embryonic chick hearts at various stages of development. Activity of protein kinase C was much higher in atria than ventricles. Protein kinase C activity underwent a progressive increase in atria during cardiogenesis, being highest just prior to hatching, followed by a profound decrease in activity after hatching. In contrast, activity of cyclic AMP-dependent protein kinase (protein kinase A), while also higher in atria than ventricles, remained relatively constant at the developmental stages examined, likewise decreasing following hatching. These progressive changes in atrial protein kinase C activity suggest a potential regulatory role for this enzyme in cardiogenesis.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

ABA and Low Temperature Induce Freezing Tolerance via Distinct Regulatory Pathways in Wheat

The role of ABA in the induction of freezing tolerance was investigatedin two wheat (T. aestivum L.) cultivars, Glenlea (spring var)and Fredrick (winter var). Exogenous application of ABA (5x10^–5M for 5 days at 24°C) increased the freezing tolerance ofintact plants by only 3°C (LT₅₀) in both cultivars. Maximalfreezing tolerance (LT₅₀ of –9°C for Glenlea and –17°Cfor Fredrick) could only be obtained with a low temperaturetreatment (6/2°C; day/night) for 40 days. These resultsshow that exogenously applied ABA cannot substitute for lowtemperature requirementto induce freezing tolerance in intactwheat plants. Furthermore, there was no increase in the endogenousABA level of wheat plants during low temperature acclimation,suggesting the absence of an essential role for ABA in the developmentof freezing tolerance in intact plants. On the other hand, ABAapplication (5x10^–5 M for 5 days at 24°C) to embryogenicwheat calli resulted in an increase of freezing tolerance similarto that achieved by low temperature. However, as in intact plants,there was no increase in the endogenous ABA level during lowtemperature acclimation of calli. These results indicate thatthe induction of freezing tolerance by low temperature is notassociated with an increase in ABA content. Using an antibodyspecific to a protein family associated with the developmentof freezing tolerance, we demonstrated that the induction offreezing tolerance by ABA in embryogenic wheat calli was correlatedwith the accumulation of a new 32 kDa protein. This proteinis specifically induced by ABA but shares a common antigenicitywith those induced by low temperature. These results suggestthat ABA induces freezing tolerance in wheat calli via a regulatorymechanism different from that of low temperature. (Received June 15, 1993; Accepted September 16, 1993)     

IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis

The aim of this work was to study morphological and biochemical aspects during zygotic embryogenesis in O. catharinensis, by measuring changes in the endogenous concentrations of proteins, amino acids, polyamines (PAs), indole-3-acetic acid (IAA) and abscisic acid (ABA). Buffer-soluble and insoluble protein contents were determined by spectrometry, and amino acids, PAs, IAA and ABA concentrations were determined by high performance liquid chromatography. Total amino acid accumulation, predominantly asparagine, occurred when the embryo showed completely developed cotyledons, with posterior reduction in the mature embryo. This decrease in total amino acid concentration in the mature embryo may result from their use in storage␣as well as for LEA protein synthesis. Free putrescine (Put) concentration decreased, while free spermine (Spm) increased during embryo development. This suggest a role for Put in the initial phases of embryogenesis when high rates of cell division occur, while elevated concentration of Spm are essential from the middle to the end of embryo development, when growth is mainly due to cell elongation. An IAA peak in zygotic embryos occurred during initial development, suggesting a link between growth and cellular division as well as with the establishment of bilateral symmetry. ABA concentration declined during initial stages of development then increased at the mature embryo stage, suggesting a possible relationship with dormancy and recalcitrance characteristics. Our results show that changes in the phytohormones (IAA, ABA and PAs) concentrations in combination with amino acids are likely important factors determining the developmental stages of O.␣catharinensis zygotic embryos.     

Growth and ion accumulation of two rapid-cycling Brassica species differing in salt tolerance

The response of two rapid-cycling Brassica species differing in tolerance to seawater salinity was studied over a period of 24 days. In response to 8 dS m⁻¹ salinity, the two Brassica species showed clear differences in the changes in relative growth rate (RGR), net assimilation rate (NAR) and leaf area ratio (LAR). The RGR of B. napus was slightly reduced by salinity, wheareas the RGR of B. carinata was largely reduced in the early stages of salinization. LAR of B. napus was affected by salinity in the later stages of growth and significantly correlated with the reduction in RGR. On the other hand, the NAR of B. carinata was decreased by salinity, corresponding to the decrease of the RGR of B. carinata. The NAR of B. napus was not significantly affected by salinity according to analysis of covariance. The shoot concentrations of Na, Mg and Cl increased while the concentrations of K and Ca decreased sharply during the first 5 days of salinization; subsequently, all ion concentrations remained relatively constant. The concentrations of Na, K, Ca, Mg and Cl in the root were similarly affected by salinity. There were no significant differences of ion concentrations between species that could be related to the differences in salt tolerance. Thus, the differences in salt tolerance between species can not be related to differences in specific-ion effects, but may be related to some factor that reduces the NAR of B. carinata during the early stages of growth.     

THE EXTENT OF AXOPLASMIC TRANSPORT DURING DEVELOPMENT, DETERMINED BY MIGRATION OF VARIOUS RADIOACTIVELY-LABELLED MATERIALS

Abstract— Several isotopic precursors have been monocularly injected into chick embryos and into day-old or 15-day-old chicks. After various intervals, the incorporation of various isotopes into acid insoluble material within the retina of the injected eye and within the optic lobes, was determined. Radioactive proline and fucose were used as precursors of protein and glycoprotein respectively while uridine was used as an RNA precursor. The proportion of rapidly migrating proteins and glycoproteins was reduced during maturation. The extent of RNA migrating also appeared to decline during development. The proportion of synthesized protein that was transported was relatively constant and independent of the amino acid used. Around 30 per cent of retinally synthesized glycoprotein migrated distally and this migrating material appeared to contain very few sialic acid residues. A considerable amount of retinally synthesized gangliosides also appeared rapidly in the distal regions of the optic nerve.     

Soluble HLA class I molecules: biological significance and clinical implications

Soluble class I human leukocyte antigens (sHLAs) have been detected in serum, sweat, lymphatic fluid, urine and cerebrospinal fluid. Their biological function has, however, remained a puzzle. The physiological concentration of sHLA varies more than tenfold depending on the phenotype of the individual, and is significantly upregulated in various diseases and during inflammation. This suggests that sHLAs might serve as a marker of pathological changes. Recent experiments have shown that, in vitro, sHLAs can modulate T-cell reactivity and induce cell-activated apoptosis, implicating sHLAs in the induction and maintenance of peripheral tolerance. Therefore, sHLAs have the therapeutic potential to induce tolerance to transplants.