Reverse Changes in Plasma Membrane Properties upon Deacclimation of Mulberry Trees (Morus bombysis Koidz.)

To gain more insight into the relation between plasma membranechanges and cold hardiness in mulberry trees (Morus bombysisKoidz. cv Goroji), biochemical and biophysical changes in theplasma membrane were studied during cold deacclimation in spring.The majority of the changes in the plasma membranes that occurredduring the cold acclimation process in the fall/winter werereversed following deacclimation in the spring. Significantdecreases in phospholipid content, degree of unsaturation inphospholipid fatty acids, and membrane fluidity were observedin the plasma membranes during cold deacclimation. The sterolto phospholipid ratio increased with decreasing cold hardiness.Reverse changes were also detected in the majority of proteinand glycoprotein components. These reversible changes in theplasma membranes are considered to be involved in the mechanismof cold hardiness of plants. ¹Contribution No. 2766 from the Institute of Low TemperatureScience. (Received July 10, 1985; Accepted October 25, 1985)     

The effect of low temperature upon the polyribosome, nucleic acid and protein content of potato leaves

Short periods of low temperature exposure, or longer periodsof cool temperature growth did not cause a decline or "run-off"of potato leaf polyribosomes. In fact, polyribosome levels werehigher in the leaves of plants grown in the cool temperatureregime. The ribosomal RNA levels were higher in cool grown leavesafter day 12 of treatment, while the protein and amino acidlevels did not exhibit a dramatic change. The results are discussedin respect to efficient plant protein synthesis in cooler climates. ¹This research was supported in part by a grant from the RockefellerFoundation. ²Scientific Journal Series Article 8745 of the Minnesota AgriculturalExperiment Station. (Received June 20, 1974; )     

Induction of Cold Hardiness by Salt Stress Involves Synthesis of Cold- and Abscisic Acid-Responsive Proteins in Potato (Solanum commersonii Dun)

Salt stress has been found to increase frost tolerance in someherbaceous species. In an attempt to understand the molecularbasis of the frost tolerance induced by salt stress, the effectof salt (100 mM NaCl) on total proteins in stem-cultured potatoplantlets (Solarium commersonn Dun) was analyzed on two-dimensionalgels. Nine salt-induced proteins were identified after 24 hsalt treatment, at which time cold hardiness increased by threedegrees. Direct comparisons of the proteins with those inducedby cold- and abscisic acid(ABA)-treatments revealed that fiveof the salt-induced proteins were also induced by cold(4°/2°C)-treatmentand seven were also induced by ABA(40µM)-treatment. Threeproteins (M_rr/pls 13/7.0, 27/6.6 and 48/6.9) were induciblein both cold- and ABA-treatments in association with frost hardening.After 6 h salt treatment, endogenous ABA levels in plantletleaves showed a transient six-fold increase before cold hardinessdeveloped. The results suggest that salt-induction of cold hardinessinvolves the synthesis of cold and ABA-responsive proteins andthe alteration of protein synthesis is mediated by ABA elevatedupon salt stress. This study also suggests that a subset ofproteins induced by cold and ABA-treatments are related to saltstress. ¹Scientific Journal Series Paper No. 20787 of the MinnesotaAgricultural Experimental Station, St. Paul, MN 55108, U.S.A. ²Present address: Department of Biochemistry Willard Hall, KansasState University, Manhattan, KS 66506, U.S.A. ³Present address: Via G.B. Marino 13, 80125-Napoli, Italy ⁴Present address: Institute of Biological Chemistry, WashingtonState Univ., Pullman, WA 99164, U.S.A.     

Ultrastructural Changes Related to Frost Hardiness in the Cortical Parenchyma Cells from Mulberry Twigs

Ultrastructural changes in the cortical parenchyma cells ofmulberry (Morus bombyciz Koidz. cv. ‘Gorogi’) twigswere related closely to the seasonal cycle of frost hardiness.Changes in hardiness from summer to winter and vice versa tookplace particularly in the plasmamembrane. During stages that result in a rapid increase in hardiness,the sequence of cytological changes for the replacement of theplasmamembrane was marked invaginations of the plasmamembrane;pinching off into vesicles in the peripheral cytoplasm; engulfmentof these vesicles into vacuoles; generation of numerous microvesiclesby the Golgi apparatus; the appearance of these microvesicleswith the plasmamembrane. Similar cytological changes that areassociated with increased hardiness occur during artificialhardening at 0°C for 5 to 20 days in both the autumn andspring. In the midwinter cells, however, the plasmamembranehas a fairly smooth structure. Thus, the highly folded structureof the plasmamembrane is not always characteristic of extremelyhardy cells. Ultrastructural changes associated with hardinessare discussed in terms of membrane renewal. ¹Contribution No. 2267 from the Institute of Low TemperatureScience. (Received October 11, 1980; Accepted December 5, 1980)     

The site of synthesis of pea chloroplast coupling factor 1

Inhibitors of cytoplasmic polyribosome function and chloroplastpolyribosome function were used to study the site of synthesisof the five subunits of coupling factor I (CF₁) in Pisum sativum.The results of these in vivo experiments are presented as evidencefor the cytoplasmic synthesis of two subunits, C and D, andthe synthesis in the chloroplast of the other three, A, B andE. ¹ Supported in part by grant PCM-74-13534 from the NationalScience Foundation. (Received April 17, 1978; )     

Dynamics of Fructan and Sucrose Biosynthesis in Crested Wheatgrass

Fructan metabolism occurs in many temperate grasses when ambienttemperatures are cool. Fructan is believed to influence thecold hardiness of grasses and to have osmoregulatory and carbohydratestorage functions. In the cold deserts of the western UnitedStates, crested wheatgrass (Agropyron spp.) usually maintainsmetabolically active leaves throughout the winter. The investigationreported here concerns nonstructural carbohydrate content ofcrested wheatgrass during a growing season (September-June).Fructan accumulated in plant parts during the fall and springwhen temperatures were near 15?C. However, fructan content waslowest when temperatures were coldest. Surose content peakedwhen fructan levels were low and when maximum daily temperaturesremained below 0?C. We propose that fructan biosynthesis inleaf vacuoles provides a sink for photosynthates when temperaturesare too cold for translocation, and growth. Stored fructan thenprovide a readily available substrate for sucrose synthesisand plant growth. Because crested wheatgrass maintains greenleaves during the winter and is capable of photosynthesis attemperatures near 0?C, less stored carbohydrates are requiredfor respiratory maintenance and spring growth than for warm-seasongrasses. ¹Cooperative investigations of the USDA-Agricultural ResearchService and the Utah Agricultural Experiment Station, Logan,Utah 84322, U.S.A. Approved as Journal Paper No. 3755. ²Plant Physiologists, USDA-ARS-Forage and Range Research, Logan,UT 84322-6300, U.S.A. (Received March 25, 1988; Accepted July 11, 1988)     

PEP Carboxylases from Two C4 Species of Panicum with Markedly Different Susceptibilities to Cold Inactivation

Phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31 [EC] ) assayedin extracts of Panicum maximum Jacq. loses up to 50% of itsactivity after incubation for 60 minutes at 0C while the enzymefrom P. miliaceum L. is completely stable under these conditions.Following dilution at room temperature the enzyme from P. maximumis labile, while that from P. miliaceum is stable. The P. maximumenzyme can be largely stabilized against dilution and againstcold-inactivation by D₂O which stabilizes hydrophobic bondsand the compatible solutes proline, betaine and trimethylamine-N-oxide.Mineral ions, previously demonstrated to be protective againstcold inactivation of pyruvate, P_i dikinase from maize, provideno protection of P. maximum PEPC against either cold or dilution.The chaotropic ion SCN^- causes partial inactivation of the enzymefrom P. miliaceum in the cold. The possible interrelationshipbetween inactivation by dilution and inactivation by cold isdiscussed. The enzyme from both species, when assayed withoutpreincubation at low temperature, exhibits similar, slightlycurvilinear Arrhenius plots; and no differences were found betweenthe two species in the temperature dependence of photosynthesis. ¹Present address: Botany Dept., University of California, DavisCA 95616 U.S.A.     

Metabolic Alterations Associated With Abscisic Acid-Induced Frost Hardiness in Bromegrass Suspension Culture Cells

Metabolic alterations associated with the induction of freezingtolerance by abscisic acid (ABA) were characterized by chemicalanalysis and by [U-^l4C]sucrose partitioning into cellular constituentsin bromegrass (Bromus inermis Leyss cv. Manchar) cell suspensioncultures. ABA caused a significant elevation in dry matter,particularly in the fraction insoluble in 85% ethanol, thatwas highly correlated with enhanced frost tolerance. Cell walls,the largest component of the insoluble fraction, increased significantlyas frost tolerance increased throughout the ABA treatment period.ABA stimulated total [¹⁴C]sucrose uptake by cells from 7% onday 1 to 97% on day 7 compared to control cells. Partitioningstudies detected a significant increase in ¹⁴CO₂ evolution at3, 5 and 7 days after ABA treatment and a significantly higherincorporation of [¹⁴C]sucrose into the ethanol insoluble fractionafter 5 and 7 days of treatment. Organic acid depletion in ABA-treatedcells was also highly correlated with the increase in hardiness.The concentration of total sugars was higher in ABA-treatedcells. The results indicate that most of the metabolic changesduring ABA-induced acclimation were similar to changes reportedfor cells acclimated in response to low temperature. ¹Oregon Agricultural Experiment Station Technical Paper No.9052 ²Present address: Department of Horticulture, University ofSaskatchewan, Saskatoon, Sask. Canada S7N 0W0 (Received November 1, 1989; Accepted March 13, 1990)     

Induction of nitrate and nitrite reductases in Anabaena cylindrica

Induction of nitrate and nitrite reductases in Anabaena cylindrica(FOGG strain) was investigated. At various stages of algal growthin the presence of nitrate or nitrite, the levels of these enzymeswere determined using cell-free preparations. Nitrate and nitritereductases were induced by the respective substrates. Nitratedid not act either as an inducer or as a repressor of nitritereductase. ¹This work was supported by grant No. 8814 from the Ministryof Education ²Department of Biology, Faculty of Science, Tokyo MetropolitanUniversitySetagaya-ku, Tokyo, Japan (Received June 18, 1970; )     

Vacuolar Membrane Lesions Induced by a Freeze-Thaw Cycle in Protoplasts Isolated from Deacclimated Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The processes of freezing injury in Jerusalem artichoke (Helianthustuberosus L.) tubers were studied using protoplasts isolatedfrom cold-acclimated and deacclimated tubers. Prior to freezing,protoplasts were preloaded with 10 µM fluorescein diacetate(FDA) in an isotonic sorbitol solution. After freeze-thawingat various temperatures, cell viability was evaluated undera fluorescence microscope. In cold-acclimated tubers, more than80% of protoplasts survived freezing to – 20°C. Bycontrast, in deacclimated tubers, the cell survival abruptlydeclined after freezing to temperatures below – 5°C.Thus, freezing tolerance differed significantly between protoplastsisolated from cold-acclimated and deacclimated tubers. Two distincttypes of cell injury, which were caused by either damage toplasma membrane (cell-lysis type) or by damage to the vacuolarmembrane (abnormal-staining type), were observed, dependingon the cold hardiness and freezing temperature. In the cellsof the abnormal-staining type, shrinkage of the central vacuolarspace and simultaneous acidification of the cytoplasmic spacewere characteristically observed immediately before completecell-rehydra-tion during thawing. The decrease in freezing toleranceof protoplasts after deacclimation was suggested to be due mainlyto destabilization of the vacuolar membrane by freeze-induceddehydration stress. ¹Contribution no. 3945 from The Institute of Low TemperatureScience, Hokkaido University. This research was supported inpart by the grant from Japan Society for the Promotion of Science(JSPS-RFTF 96L00602) ²Present address: Tohoku National Agricultural Experiment Station, Morioka, Iwate, 020-01 Japan     

Elicitor Activity of a Fungal Product Assessed at the Single-Cell Level by a Novel Gel-Bead Method

Elicitor from Erysiphe pisi was incorporated into gel beads.Individual beads were placed on single cells from barley coleoptiles.The elicitor induced unusual cytoplasmic responses and temporaryresistance to infection in coleoptile cells. The technique isapplicable to assessment of elicitor activity at the single-celllevel. ¹Contribution no. 118 from the Laboratory of Plant Pathology,Mie University. ²Present address: Laboratory of Plant Pathology & GeneticEngineering, College of Agriculture, Okayama University, Okayama,700 Japan     

Seasonal variations in freezing curves of stem sections of Cornus stolonifera MICHX.I

Comparisons of freezing curves have been used to determine theviability of plant parts exposed to stress. To gain understandingof the natural seasonal variations in freezing curves, uniformtwig sections of red-osier dogwood (Cornus stolonifera MICHX.)were collected throughout the year from a single clone and subjectedto controlled freezing while the tissue temperature was recorded.The supercooling of samples ranges from –2 to –7,but the variation was random and unpredictable. There was noapparent relationship between supercooling and the season ofthe year or the hardiness of the tissue. The freezing pointdepression, as estimated by the temperature of the first freezingplateau, was always between –0.25 and –1.0 andbore no relationship to hardiness or season. The freezing curveswere basically of three types: Summer and winter curves withtwo distinct freezing points; Early autumn curves with 3distinctfreezing points and spring curves with one prominent first freezingpoint which tended to mask the second freezing point. ¹Scientific Journal Series paper No. 6628, Minnesota AgriculturalExperiment Station. This research was supported in part by agrant from the Louis W. and MAUD HILL Family Foundation. ²Present Address: Horticulture Department, University of Wisconsin,Madison, Wisconsin, U.S.A.     

Seasonal Changes in Plasma Membranes and Mitochondria Isolated From Jerusalem Artichoke Tubers. Possible Relationship to Cold Hardiness

Plasma membranes and mitochondria were isolated from Jerusalemartichoke tubers during cold acclimation from September to December.The protein and lipid contents of the membranes were analyzedwith reference to physiological properties of the tubers, especiallycold hardiness. As cold hardiness increased from autumn to winter,the content of phospholipids and sterols on a mg protein basisincreased by 20–30% in plasma membranes, but little changewas observed in mitochondria. Minor changes were observed inthe fatty acid composition of phospholipids either in plasmamembranes or mitochondria. Membrane fluidity, assessed by fluorescentpolarization of 1,6-diphenyl-1,3,5-hexatriene, was found tobe relatively constant in both membranes during the season.One dimensional SDS-polyacrylamide gel electrophoresis revealedseasonal changes in proteins and glycoproteins in plasma membranes,but not in mitochondrial membranes. Plasma membrane ATPase increasedin specific activity from September to December, which was morenoticeable at higher assay temperatures. However, irrespectiveof the season, the plasma membrane ATPase had an inflectionon the slope of the Arrhenius plot around 15C. These resultssuggest that plasma membranes, in contrast to mitochondria,undergo several molecular changes from autumn to winter, whichmay be related to cold acclimation of the tubers. ¹ Contribution No. 2668 from the Institute of Low TemperatureScience. ² Present address: Crop Development Centre, University of Saskatchewan,Saskatoon, Canada S7N 0W0.     

Survival of poplar callus at super-low temperatures after cold acclimation

Callus derived from the cambial area of poplar twigs survivedfreezing down to –120?C or even the temperature of liquidnitrogen (–196?C), after cold acclimation. Exposure tocool preconditioning temperatures for a length of time beforebeing acclimated at 0?C is essential for enhancing fully thecallus hardiness. ¹ Contribution No. 1248 from the Institute of Low TemperatureScience. (Received May 30, 1973; )     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VII. Oxidation of sulfite by a cell-free extract of Thiobacillus thiooxidans

Properties of the cell-free extract, prepared from a strainof Thiobacillus thiooxidans by sonic disruption followed byfractionation with centrifugatiori, were investigated with referenceto its sulfite-oxidizing activity. Without the addition of cofactors the particulate fraction(F-P)catalyzed oxidation of sulfite with oxygen or bacterial cytochromec-552 obtained from Pseudomonas stutzeri as electron acceptor.TMPD reduced by ascorbic acid was also oxidized by F-P. Thesoluble fraction(F-S) showed no activity in oxidizing sulfiteand TMPD, but stimulated TMPD oxidation by F-P. Oxygen uptake with either sulfite or TMPD as substrate was inhibitedby KCN, NaN₃, CO and c-phenanthroline. CO-Inhibition was reversedby light. Reduction of cytochrome c-552 by sulfite was insensitiveto these agents. Antimycin A markedly inhibited sulfite oxidation with eitheroxygen or cytochrome c-552 as electron acceptor, but was withouteffect on TMPD oxidation. DDC and SAO, both strong inhibitors of sulfur oxidation, didnot affect sulfite and TMPD oxidations. Cytochromes of the a, b and c types were contained in F-P. Thesecytochromes were rapidly reduced when F-P was incubated withsulfite. Cytochrome(s) of the c type was present in F-S, too. ¹VI.=References (3) ²Partly supported by a grant from the Ministry of Education ³Present address: Sanyo Women's College, Hatsukaichi, Hiroshima738, Japan ⁴Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima 734, Japan (Received May 15, 1970; )     

Further studies on ribulose-l,5-diphosphate carboxylase from Rhodopseudomonas spheroides and Rhodospirillum rubrum

Some enzymic properties of RuDP carboxylase isolated from Rhodopseudomonasspheroides and Rhodospirillum rubrum, grown under autotrophic,semi-autotrophic and heterotrophic conditions were studied.Regardless of the growing condition, the nature of the enzymefrom each respective bacterium remained unchanged. The molecularweight of R. spheroides and R. rubrum RuDP carboxylase was estimatedto be approximately 2.4x10⁵ and 8.3x10⁴, respectively. Bothenzymes require Mg⁺⁺. Their kinetic properties were also examined. ¹Part XII. Structure and Function of Chloroplast Proteins ²Supported in part by research grants from the Ministry of Educationof Japan (No. 8719), USPHS (AM. 10792-03) and the Asahi Press(Tokyo) (Received March 18, 1970; )     

Glycine-serine transformation in the photosynthetic bacterium Chromatium vinosum

The metabolic transformation of glycine into serine in the photosyntheticbacterium Chromatium vinosum was accompanied by the evolutionof CO₂ due to decarboxylation of glycine. Isonicotinylhydrazideinhibited both ¹⁴CO₂ evolution and the formation of ¹⁴C-serinefrom ¹⁴C-glycine. The results indicate that a glycine-serinetransformation reaction takes place which is analogous to thatoccurring in green leaf tissues. Glycine may be metabolisedthrough serine by this reaction. The light stimulation of ¹⁴CO₂evolution and ¹⁴C-serine formation from 14C-glycine by the Chromatiumcells are judged to be results of the light-induced enhancementof ¹⁴C-glycine uptake by the bacterial cells. ¹This is paper 53 in the series "Structure and Function of ChloroplastProteins" and paper 7 of the series "Biosynthetic Mechanismof Glycolate in Chromatium". Paper 6 of the latter series isRef. 3 by Asami and Akazawa (1978). ²This study was aided by research grants from the Ministry ofEducation, Science and Culture of Japan and the Nissan ScienceFoundation (Tokyo). ³Postdoctoral Fellow (1980) of the Japan Society for the Promotionof Science. (Received May 20, 1980; )     

Influence of leaf age, light, dark, and iron deficiency on polyribosome levels in maize leaves

The relations between leaf age and polyribosome levels werestudied with dark-and light-grown maize (Zea mays L.) seedlings.In general, polyribosome levels decline with the period of growthin darkness. Light induces an increase in the polyribosome levelin dark-grown seedlings. The response can be detected after30 min exposure to light. Seven or eight-day-old dark-growncorn seedlings, used in the present study, have high levelsof polyribosomes when greened in the light. This is indicativeof healthy seedlings, competent in protein synthesis. The polyribosomelevels in iron deficient maize plants were significantly differentfrom plants grown under complete nutrient solution, while thereis no significant difference among plants suffering differentdegrees of iron deficiency. (Received November 18, 1977; )