Studies on chilling injury in plant cells II. Ultrastructural changes in cells rewarmed at 26?C after chilling treatment

Both the restoration and deterioration of ultrastructures wereobserved during therewarming of cultured cells of Cornus stoloniferain which chilling at 0?C had caused an apparent change in themorphology of the organelles. Complete restoration of the ultrastructures,moderately altered by the 12-hr chilling, took place within12 hr of wanning at 26?C. Even in cells chilled for 24 hr, severelyaltered ultrastructures were partially or completely repairedin more than fifty percent of the treated cells. Some cellschilled for 24 hr, however, displayed further deteriorationof their ultrastructures during rewarming. Restoration of therough endoplasmic reticulum and the development of polysomesin recovering cells were characteristic of the early stage ofrewarming. Rupture of the tonoplast was sometimes observed duringrewarming of cells chilled for 24 hr. A possible role for therough endoplasmic reticulum and for the integrity of the tonoplastin cell recovery during the chill-warm sequence is discussed. ¹Contribution No. 2026 from the Institute of Low TemperatureScience, Hokkaido University. ²This work was supported in part by Grant 248004 from the Ministryof Education. (Received November 6, 1978; )     

Cell membrane permeability and respiratory activity in chilling stressed callus

To improve our understanding of the mechanism of chilling injuryin chill-sensitive callus (Cornus stolontfera), early changesin cell permeability and respiratory activity were studied.Partial leakage of amino acids and an abrupt increase in permeationand oxidation of added dopamine were characteristic of chilledcallus in the late stage of chilling at 0?C (48 hr), when mostof the callus sustained severe injury. However, little or nochange in cell permeability was observed in the early stageof chilling (within 24 hr), when calli retained their viabilityfor growth after transfer to a warm temperature. These resultssuggest that changes in the cell membranes per se are by nomeans the primary step in cell injury. Temporary depressionof respiratory activity was detected soon after chilling for12 hr, but activity appeared to return to the original levelon further chilling up to 24 hr. An irreversible dysfunction,however, occurred in the respiratory system on prolonged chillingup to 48 hr. This implies that irreversible impairment of mitochondrialfunctions may not be involved in the early stage of the cellinjury. A possible relationship between these observed changesand ultrastructural changes in chilled cells is discussed. ¹Contribution No. 2153 from the Institute of Low TemperatureScience. (Received June 6, 1979; )     

Ultrastructural features of chilling injury: injured cells and the early events during chilling of suspension-cultured mung bean cells

The ultrastructure of cells of mung bean (Vigna radiata L. var. Wilczek) in suspension culture was studied during chilling. During such treatment, three kinds of injured cells were observed: swollen cells, cells with broken vacuolar membranes, and cells with shrunken plasma membranes. Swelling was observed from the early stages of chilling, and in most cells during chilling. The other two types of cells were observed at the late stages of chilling. At the early stage of chilling, whorls of rough endoplasmic reticulum that surrounded clear regions of cytoplasm were observed. At the same time, markedly rough vacuolar membranes, plastids and mitochondria with vacuoles, enlargement of Golgi vesicles, and dilation of the ER were seen. These changes preceded the swelling of cells. These ultrastructural features of chilling injury are discussed in terms of biochemical observations. The disruption of the vacuolar membrane and the shrinking of the plasma membrane are discussed in terms of destruction of the cytoskeleton.     

Chilling injury in cotton (Gossypium hirsutum L.) Prevention by abscisic acid

Cotton (Gossypium hirsutum L.) intact seedlings and isolatedcotyledonary discs were exposed to chilling (4?C) under humidconditions which prevented dehydration. The damage resultingfrom chilling was estimated by means of electrolyte leakageand survival in whole seedlings and by the electrolyte leakageand necrotic areas in isolated cotyledonary discs. Also, theeffect of chilling on membrane phospholipids and cellular reducedglutathione was determined. Within the first two and three daysof chilling, there was a marked reduction in the reduced glutathioneand membrane phospholipid levels without electrolyte loss andnecrosis. This reduction was completely prevented by pretreatmentwith abscisic acid. Prolonging the chilling period resultedin decreased survival in whole seedlings and in progressiveincrease in electrolyte leakage and necrosis in isolated cotyledonarydiscs. Pretreatment with abscisic acid prior to chilling almostcompletely prevented this chilling injury when exposure to 4?Cwas less than 5 days. Even with longer chilling periods, theabscisic acid pretreatment greatly reduced the damage. ³Incumbent of the Seagram Chair in Plant Science. (Received July 21, 1979; )     

Mechanism of chilling injury in sweet potato VII. Changes in mitochondrial structure during chilling storage

Mitochondrial protein-N amounts per the contents of heme a,cytochrome b and cytochrome c, the tightly bound componentsin the mitochondrial inner membrane, were not changed during14 days of chilling stored sweet potatoes. However, the lipid-Pamount per amount of protein-N in mitochondria decreased byabout 20% during the 14 day chilling period. Via electron microscopy, two types of mitochondria were foundin the mitochondrial fraction prepared from healthy sweet potatoes.One, which had reticulately-developed cristae and extremely-electrondense matrix spaces, was called Form A. The other, which hadcristae which were not clearly distinguished from the matrixspaces, was called Form B. In the fraction prepared from 14day chill-stored sweet potatoes, a third type of mitochondriawas found besides Forms A and B. This one, which had an extremelyswollen form, was called Form C. Form C is thought to occur through the degradation of Form Aor B, concomitant with the release of phospholipid from boththe inner and outer membranes during chill-storage. It is quitelikely that Form C occurs during physiological deteriorationin sweet potato root tissues, which proceeds irreversibly duringchill-storage. ¹ This paper constitutes Part 95 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received March 7, 1972; )     

Subcellular localization, of glucosyl transferases involved in lectin glucosylation in Pisum sativum seedlings

Cellular membranes from 4 to 5-days old etiolated pea seedlingswere isolated by isopyenic centrifugation. Marker enzymes wereused to measure the purity of the subcellular fractions. Theformation of dolichy!-P-glucose and of glucosylated lectin wastested in all the fractions. Both activities were associatedwith the endoplasmic reticulum fractions. When the membraneswere prepared in the presence of EDTA, a shift in both activitiesto a lower density in the gradient was observed. These results are in agreement with the current assumption thatglycosylation of proteins via lipid-linked sugars is a cotranslationalprocess. ¹ Present address: Centra de Investigaciones Agron?micas, INTA,C.C. 25, Castelar, Argentina. ² Present address: Department of Biochemistry, University ofMiami, P. O. Box 016129, Miami, FL 33101, U.S.A. ³ To whom correspondence should be addressed. Present address:Instituto de Investigaciones Biologicas, Universidad Nac. deMar del Plata, C. C. 1348, 7600 Mar del Plata, Argentina. (Received April 24, 1979; )     

ULTRASTRUCTURAL CYTOLOGY OF CALLUS CULTURES OF STREPTANTHUS TORTUOSUS AS AFFECTED BY TEMPERATURE

Tissue culture cells of Streptanthus tortuosus var. orbiculatus (Cruciferae) which have acquired a spherical viruslike particle located in their nucleoli, designated cell line STV, developed supergranal chloroplasts and lost the ability to differentiate vascular tissues. The effect of temperature on the ultrastructural cytology of one line of the STV tissue, STV-I, was compared with the effect of temperature on the ultrastructural cytology of tissue culture cells lacking the viruslike particles (control cell lines). At 4 C, the cellular and ultrastructural appearance of control tissue culture cells differed from that of tissue grown at 22 C by producing increased amounts of endoplasmic reticulum and dictyosomes and by reduction of chloroplast thylakoids. STV-I cells were generally moribund as a result of 4 C treatment. Chloroplast thylakoids were also reduced in control tissue following growth at 10 C and the apparent quantities of endoplasmic reticulum and dictyosomes were similar to those observed in control cells grown at the control temperature (22 C), but less than those observed in tissue subjected to 4 C. STV-I tissue grown at 10 C demonstrated increased endoplasmic reticulum and dictyosomes and reduction of polysomal configurations. The mitochondrial morphology was variable and the cells contained supergranal chloroplasts and proplastids. At the control temperature (22 C), the fine structural appearance of control tissue culture cells was typical of parenchyma cells, but STV-I cells contained mitochondria of variable morphology and two types of chloroplasts— normal and supergranal. Control tissue grown at 30 C also contained proplastids, but these proplastids contained starch in contrast to the proplastids in control tissue grown at low temperatures. The ultrastructural cytology of STV-I cells grown at elevated temperature (30 C) was characterized by enlarged mitochondria containing massive lipid bodies and the presence of protoplastids with starch and supergranal chloroplasts.     

Antioxidant enzyme responses to chilling stress in differentially sensitive inbred maize lines

Antioxidant enzyme activities were determined at the first,third and fifth leaf stages of four inbred lines of maize (Zeamays L.) exhibiting differential sensitivity to chilling. Plantswere exposed to a photoperiod of 16:8 L:D for one of three treatments:(a) control (25C), (b) control treatment plus an exposure toa short-term chilling shock (11C 1 d prior to harvesting),and (c) long-term (11 C constant) chilling exposure. Catalase(CAT; EC 1.11.1.6 [EC] ), ascorbate peroxidase (ASPX; EC 1.11.1.11 [EC] ),superoxide dismutase (SOD; EC 1.15.1.1 [EC] ), glutathione reductase(GR; EC 1.6.4.2 [EC] ), and mono-dehydroascorbate reductase (MDHAR;EC 1.6.5.4 [EC] ) activities were assessed. Reducing and non-reducingsugars and starch concentrations were determined as generalmetabolic indicators of stress. Reduced activities of CAT, ASPX,and MDHAR may contribute to limiting chilling tolerance at theearly stages of development in maize. Changes in levels of sugarand starch indicated a more rapid disruption of carbohydrateutilization in comparison to photosynthetic rates in the chilling-sensitiveline under short-term chilling shocks and suggested a greaterdegree of acclimation in the tolerant lines over longer periodsof chilling. Key words: Antioxidant enzymes, differential chilling sensitivity, maize, soluble carbohydrates, Zea mays     

STRATIFICATION AND SUBSEQUENT BEHAVIOR OF PLANT CELL ORGANELLES

Living excised roots of pea were centrifuged at 20,000 g for 24 hours, and the behavior of organelles was followed by electron microscopy at various intervals after centrifugation. With these forces, organelles are not perceptibly or irreversibly damaged, nor is the viability of the whole root destroyed. Organelles stratify generally in the order of lipid (centripetal pole), vacuoles, smooth endoplasmic reticulum and dictyosomes, proplastids (without starch), mitochondria, rough endoplasmic reticulum, proplastids with starch. The nucleus distends from the vacuolar region to the extreme centrifugal pole of the cell, while the chromatin and nucleolus seek the centrifugal pole of the nucleus. During the redistribution of organelles the rough endoplasmic reticulum is among the first to reorient, and possible explanations for this are discussed. Mitochondria can be stretched elastically many times their original length, but proplastids seem fairly rigid. Small vacuoles, forced together during centrifugation, apparently may fuse to form a large unit. Lipid droplets, on the other hand, tend to remain separate. Dictyosomes and smooth endoplasmic reticulum layer in the same region of the centrifuged cell, indicating a density similarity between these two organelles.     

Alterations in Protein Synthesis in vivo in Chilling Sensitive Mung Bean Hypocotyls Caused by Chilling Stress

The effects of chilling on protein synthesis in vivo in etiolatedhypocotyls of Vigna radiata L. were investigated. After exposureof the tissues to 0?C for various periods of time, proteinswere labeled with [³⁵S]-methionine at 26?C. The total amountof ³⁵S incorporated into soluble and membrane proteins was reversiblyreduced by chilling for 24 h, during which time the tissuessuffered no injury. Further prolonged chilling produced an irreversibledecline both in the incorporation of radioactivity and in cellviability as assessed by the extent of leakage of electrolyte.The ³⁵S-labeled proteins in the soluble and the total membranefractions were analyzed quantitatively. Chilling of etiolatedhypocotyls for one or two days induced the syntheses of twosoluble proteins (82 and 74 kDa) and one membrane protein (80kDa). Moreover, three heat-shock proteins (HSPs) that were inducedby heat stress (41 ?C, 4h) had the same electrophoretic mobilitiesas those of the proteins induced directly or indirectly by thechilling treatment. ¹Contribution No. 3170 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 30, 1988)     

The mechanism of chilling injury in sweet potato IX. The relation of chilling to changes in mitochondrial respiratory activities

To examine the mitochondrial activity of chilling-stored sweetpotatoes a method of isolating mitochondria with a good respiratorycontrol (RC) ratio from healthy sweet potato tissue was established.Mitochondria were isolated from two varieties of sweet potatoes(Norin No. 1, moderately sensitive to chilling, and OkinawaNo. 100, very sensitive) kept at about 0°C for about 15to 40 days. Respiratory activity was measured with an oxygenelectrode apparatus. Mitochondrial activities of chilling-storedNorin No. 1, i.e. the RC ratio, respiratory rate at state 3and ADP/O ratio decreased about 2 to 3 weeks after the beginningof incubation. The decline in the RC ratio was most sensitive.Diminution of the activities when malate was used was seen earlierthan when succinate was used. When activities were measuredusing succinate at low concentration (0.2 M) of mannitol, thedecrease in activities was more conspicuous than at a high concentration(0.7 M). Similar experiments with Okinawa No. 100 also showedthe decline in these activities. However, the three kinds ofactivities simultaneously decreased, and the decline appearedfaster than in the case of Norin No. 1. ¹ This paper constitutes part 99 of the phytopathological chemistryof sweet potato with black rot and injury. ² Current address: Nomura Research Institute, Kamakura, Kanagawa247, Japan. (Received June 6, 1972; )     

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg·1⁻¹) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.     

Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 4°C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28°C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28°C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.     

Chilling injury in cotton (Gossypium hirsutum L.) : Effects of antimicrotubular drugs

When exposed to 4°C for more than three days, intact cotton(Gossypium hirsutum L.) seedlings and isolated cotyledonarydiscs suffered chilling injury as shown by the leakage of electrolytesfrom the tissue and the development of necrotic areas. Applicationof antimicrotubular drugs such as colchicine, demecolcine orpodophyllotoxin during chilling significantly accelerated andenhanced tissue damage. Lumicolchicine, the stereoisomer ofcolchicine, was ineffective. Non-chilled tissues showed hardlyany damage when treated with the same levels of antimicrotubulardrugs. Prior treatment with 10^–5 M abscisic acid (ABA)prevented the appearance of symptoms of damage caused by chillingand the antimicrotubular drugs during the first 2 to 3 daysand greatly reduced it at later stages. Our present resultssuggest that chilling damage may be due at least in part, tothe cold-induced disassembly of microtubules. Furthermore, themode of action of ABA might be related to factors which influencethe physiological stability of the microtubule network. ¹Preliminary report of this work was presented at the 10th InternationalConference on Plant Growth Substances, Madison, Wisconsin, 1979. ³Incumbent of the Seagram Chair in Plant Sciences. (Received April 15, 1980; )     

Studies on the Growth in Culture of Plant Cells: XXI FINE STRUCTURAL FEATURES OF ACER PSEUDOPLATANULS L. CELLS RESPONDING TO 2,4-DICHLOROPHENOXYACETIC ACID WITHLDRAWAL IN TURBIDOSTAT CULTURE

A culture of Acer pseudoplatanus L. grown in the presence ofan equilibrium level of 2,4-dichlorophenoxyacetic acid (2,4-D)of 1?5 ? 10^–7 M (state IV culture) showed, in comparisonwith one of a similar specific growth rate but in which theequilibrium level of 2,4-D was 2?3 ? 10^–6 M (state Iculture), an enhanced degree of cell aggregation, enhanced meancell volume, and the presence of cells giving a generalizedlignin reaction with extracellular lignin-positive material.The state IV culture showed a proportion (10–15 per cent)of cells having ultrastructural features not observed in thestate I culture. Some of the cells, located at the surface ofthe cellular aggregates, were small, rounded, highly cytoplasmic,and rich in rough endoplasmic reticulum. Further within theaggregates there occurred some cells showing abnormal or incompletecytokinesis and having irregularly thickened walls. Locatedcentrally in the aggregates were cells showing massive accumulationsof electron-dense material and with cell walls showing bandsof thickening alternating with thinner wall regions traversedby plasmodesmata. The latter cells are interpreted as cellsshowing intense polyphenol metabolism and imperfect xylogenicdifferentiation.     

Gibberellic acid and the fine structure of barley aleurone cells

Summary This paper describes the ultrastructural changes in barley aleurone cells following exposure to gibberollic acid (GA₃) for 10–12 hr and longer. These changes involve a further proliferation of the endoplasmic reticulum (ER), distention of the endoplasmic reticulum (ER) cisternae (12–16 hr of GA₃) and proliferation of vesicles from the ER and dictyosomes (14–22 hr). Accompanying these changes is a reduction in the size of the aleurone grains and a decrease in the number of spherosomes. Plastids and microbodies however appear to increase in number during this period of GA₃ treatment. The relevance of these ultrastructural changes to GA₃-stimulated synthesis of hydrolases is discussed.The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged. Supported by National Science Foundation grant GB-8332.     

Effects of two free radical scavengers and intermittent warming on chilling injury and polar lipid composition of cucumber and sweet pepper fruits

Cucumber (Cucumis sativus L.) and sweet pepper (Capsicum annuumL.) fruits were chilled at 2.5?C for different periods and thentransferred to 20?C and subsequently evaluated for chillinginjury. Sodium benzoate at 10 nw or ethoxyquin at 9.2 mM, appliedas a 5-min dip before chilling, increased the degree of unsaturationof 18-carbon fatty acids in the polar lipids and reduced theseverity of chilling injury. Intermittent warming to 20?C for24 hr at 3-day intervals also alleviated the chilling symptomsand increased fatty acid unsaturation of the polar lipids incucumber and pepper fruits. (Received August 22, 1978; )     

The nature of the dual effect of auxin on root formation in Azukia cuttings

In disbudded Azukia stem cuttings, auxin exerted a dual effecton root formation. The first phase of auxin action is identifiedwith the acceleration of cell division, especially longitudinaldivision. In cuttings treated with auxin during the first 24hr, longitudinally divided cells were observed in all 12 rootprimordia, while in water-treated cuttings, such cells wereobserved only in 8 root primordia. The second phase is the promotionof the reaction in which root primordia unable to develop furtherwithout auxin supply develop into roots. Irrespective of thetreatment during the first 24 hr, the auxin-treatment duringthe second 24 hr increased the number of roots protruding fromthe cuttings. Portulal applied during the first 24 hr increased the numberof root primordia which contained longitudinally divided cells.Gibberellin applied during the first 24 hr inhibited both transverseand longitudinal divisions in root primordia. ¹ Supported in part by Grant No. 139011 from the Ministry ofEducation, Japan. ² Present address: Junior College of Toyo University, Hakusan,Bunkyo-ku, Tokyo 112, Japan. (Received June 13, 1978; )     

Isolation of membranes containing protease from the cotyledons of germinating pea seeds

Membranes containing protease were isolated from the cotyledonsof germinating pea seeds by differential and two successivesucrose density gradient centrifugations. Membranes were recoveredin the microsomal fraction, but could be clearly separated frommembranes carrying antimycin A-insensitive NADH-cytochrome creductase EC 1.6.2.1 [EC] ), which seem to come from the endoplasmicreticulum. The density of protease-containing membranes was1.135. Membranes contained a lower amount of phospholipid ascompared with the endoplasmic reticulum. Electron microscopicobservations also showed that the membranes were different fromthe smooth-surfaced endoplasmic reticulum. ¹Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received September 12, 1973; )     

The effect of tunicamycin on Leishmania braziliensis cell growth, cell morphology and ultrastructure

The effect of tunicamycin (TM) on Leishmania braziliensis promastigotes in culture has been studied. TM at different concentrations (2, 4, 6 micrograms/ml) inhibits promastigote growth as the mean generation time of control cells, 36 hr, is changed to 41, 46 and 55 hr, respectively. Cells remain viable after long exposure to 2 micrograms/ml of TM and can be cultured in the presence of the drug for several generations. Under these conditions cells tend to round up and many "ruffle"-like structures appear at the parasite cell surface. At the ultrastructural level, cell coat disappears and the rough endoplasmic reticulum appears distended. Other structures remain unaltered by the drug treatment. The changes in cell morphology are discussed in relation to changes in cell surface morphology. The possible use of these TM-transformed cells as experimental systems for host-parasite studies is also considered.