Freezing Tolerance and Alteration of Translatable mRNAs in Alfalfa (Medicago sativa L.) Hardened at Subzero Temperatures

We analyzed changes in populations of translatable mRNAs occurringin crowns of the cold-tolerant alfalfa (Medicago sativa L.)cv. Apica (CT) and the cold-sensitive cv. CUF-101 (CS) aftertheir acclimation at low nonfreezing temperatures and at subzerotemperatures. Both cultivars showed very similar translationprofiles under all treatments. Low temperatures induced significantchanges in the populations of translatable mRNAs. We observeda relationship between the accumulation of cold-regulated (COR)translation products and freezing tolerance within cultivars.Moreover, at least three COR translation products were specificto the CT and might be related to hardiness potential in alfalfa.Whereas extension of the cold acclimation period at 2C reducedcold tolerance, incubation at subzero temperatures increasedor maintained freezing tolerance. This increased hardiness wasassociated with enhanced translation of COR polypeptides andalso with the appearance of new translatable mRNAs. This is,to our knowledge, the first report of altered gene expressionin plants incubated at subzero temperatures. Marked changesin populations of translatable mRNAs at temperatures below freezingmight be related to previous reports that alfalfa achieves maximumhardiness under snow cover when the soil has frozen. Translationin the presence of [³H]glycine showed that a large proportionof the COR genes encode for glycine-rich proteins (GRPs) andthat some of the GRPs are specific to the CT. (Received May 29, 1992; Accepted October 13, 1992)     

Early Developments in RNA, Protein, and Sugar Levels during Cold Stress in Winter Rye (Secale cereale) Leaves

Changes in freezing tolerance of winter rye (Secale cerealeL. cv. Voima) were determined for leaf tissues during a 1-weekcold stress, which was performed by transferring the 7-d-oldseedlings from a greenhouse (25°C, long day) to 3°Cand short day conditions. The development of cold hardeningwas shown by using an ion leakage test and by determining theamounts of carbohydrates, soluble proteins and RNA. The firstevidence of the development of freezing resistance was foundafter 1 d at low temperature, i.e. an LT₅₀ value increased from-5 to -7°C. Plants cold treated for 7 d reached an LT₅₀value of -9°C. This increase in freezing tolerance was foundto be associated with the increased levels of soluble carbohydrates,total RNA and soluble proteins. These metabolic changes indicatethe association with adjustment of growth and cell metabolismto low temperatures at the beginning of cold acclimation ofwinter rye.Copyright 1994, 1999 Academic Press Secale cereale L., winter rye, cold stress, proteins, RNA, sugars     

Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: proteins associated with the cytoplasmic polysomal and non-polysomal mRNAs (messenger ribonucleoprotein complex)

Cytoplasmic polysomal and non-polysomal mRNA-associated proteincomplexes were isolated from, and characterized in, developingsomatic and zygotic embryos of alfalfa (Medicago sativa L.).³⁵S-methionine-labelled intact embryos were irradiated withultraviolet light (UV) in situ to cross-link mRNA and proteinsoccurring within one bond length, and the polysomal and non-polysomalfractions were extracted. Then the mRNA-protein complexes wereisolated from the fractions and separated using two cycles ofaffinity chromatography on an oligo(dT)-cellulose column. Followingdigestion with RNase A and T1 and micrococcal nuclease, mRNA-associatedproteins were separated by SDS-PAGE. Several polypeptides of 15–150 kDa were associated withthe polysomal and non-polysomal (ribonucleoprotein, mRNP) fractionsof alfalfa embryos after UV-cross-linking. Many of the polypeptidesassociated with the polysomal and non-polysomal mRNAs were qualitativelysimilar, although their concentration in the two fractions wasdifferent. However, some developmentally stage-specific polypeptideswere found to be associated with the non-polysomal mRNA fractionduring the early stages of embryogenesis (precotyledonary) ofsomatic embryos. Thus the presence of mRNPs during embryogenesishas been demonstrated, and proteins intimately associated withthe mRNAs identified. Key words: Embryogenesis, translational control, protein synthesis, messenger ribonucleoproteins, alfalfa (Medicago sativa L.)     

Cold-Induced Alterations in Plasma Membrane Proteins That are Specifically Related to the Development of Freezing Tolerance in Cold-Hardy Winter Wheat

The objective of this study was to identify plasma membraneproteins that are specifically induced by cold acclimation inwheat (Triticum aestivum L.). Two cultivars with a marked differencein the genetic ability to cold-acclimate, namely, spring wheat(cv. Chinese Spring) and winter wheat (cv. Norstar), were usedas the experimental material. After four weeks of growth ina cold chamber, the freezing tolerance in the shoots of winterwheat increased to –18°C, whereas it increased onlyto –8°C in the shoots of spring wheat. In the caseof roots from both cultivars, freezing tolerance increased onlyslightly after the growth in the cold environment. Cold acclimationinduced remarkable changes in the electrophoretic patterns ofplasma membrane proteins which depended on both the cultivarand the tissue examined. Levels of polypeptides with molecularmasses from 22 to 31 kDa decreased in both the root and shootplasma membranes from both cultivars. Among these polypeptides,levels of those of 28 and 26 kDa decreased abruptly after oneweek of cold acclimation. By contrast, levels of polypeptidesof 89, 83, 52, 23, 18 and 17 kDa increased specifically in theshoots of winter wheat. The increases in the levels of the 23-,18- and 17-kDa polypeptides were proportional to the developmentof freezing tolerance. Freeze-fracture electron microscopy ofplasma membranes from shoot cells revealed that the number ofintramembrane particles on the fracture faces decreased markedlyin winter wheat after cold acclimation, but to a lesser extentin spring wheat. These results suggest that the plasma membranesmight undergo molecular reorganization during cold acclimation. ¹Contribution no. 3709 from the Institute of Low TemperatureScience, Hokkaido University.     

Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [³⁵S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.     

Temporal and nutritional factors modulate responses to abscisic acid and osmoticum in their regulation of storage protein synthesis in developing seeds of alfalfa (Medicago sativa L.)

Storage protein synthesis during alfalfa (Medicago sativa L.)seed development displays a temporal pattern and is regulatedboth by ABA and low osmotic potentials. Deposition of the 2S,7S and 11S storage proteins occurs maximally at the mid–to late stages of development. Osmoticum, but not ABA alonecould effectively induce storage protein synthesis at earlystages of development in isolated alfalfa embryos placed onwater for several days. Neither ABA nor osmoticum alone couldmaintain storage protein synthesis in isolated late-stage embryosplaced on water. On the other hand, isolated developing embryoscultured on a nutrient (Murashige and Skoog) medium could maintainstorage protein synthesis in the presence of ABA and osmoticum,alone or in combination, for up to 14 d after being dissectedfrom the pod. Of the components of this medium, the inorganicsalts appeared to be the most important. The response to ABAand osmoticum varied with the time of isolation during developmentwith the greatest enhancement of storage protein synthesis inisolated embryos coinciding with the time of maximum synthesiswithin the seed when in planta. Addition of ABA and osmoticumafter the time of maximum storage protein synthesis did notelevate the amount of synthesis, but rather prolonged the timeover which it could take place. Thus, while the amplitude ofstorage protein synthesis could be modified by ABA or osmoticum,the inherent temporal pattern, with maximum synthesis occurringonly at mid– to late stages of embryo development, couldnot. Key words: Embryogenesis, Medicago sativa L., alfalfa, abscisic acid, osmotic potential, storage protein synthesis, nutrient supply     

Effect of Cold Acclimation on Bulk Tissue Electrical Impedance: II. Measurements with Alfalfa and Birdsfoot Trefoil at Nonfreezing Temperatures

Stem and electrode electric impedance at 14 frequencies were monitored during cold acclimation of alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.). Cold acclimation significantly increased high frequency (e.g. 1.11 megahertz) resistance and reactance but not low frequency (49 hertz) resistance and reactance of both species. High frequency resistance of living stems was equivalent to the average resistance at all frequencies of dead stems and the resistance of dead stems (y) was related to stem water content (x):y = 9.28 − 4.11x + 0.47x², R = 0.92. The low-high frequency (49 hertz/1.11 megahertz) resistance ratio decreased during cold acclimation. A time constant believed to be a function of membrane resistance and capacitance was not affected by cold acclimation.     

Low Temperature Affects Pattern of Leaf Growth and Structure of Cell Walls in Winter Oilseed Rape (Brassica napus L., var. oleifera L.)

Three-week acclimation of winter oilseed rape (Brassica napusL. var. oleifera L.) plants in the cold (2 °C) resultedin a modified pattern of leaf cell enlargement, indicated bythe increased thickness of young leaf blades and modified dimensionsof mesophyll cells, as compared with non-acclimated tissuesgrown at 20/15 °C (day/night). The thickness of leaf cellwalls also increased markedly during cold acclimation but itdecreased in response to a transient freezing event (5 °Cfor 18 h followed by 6 or 24 h at 2 °C, in the dark). Cellwalls of the upper (adaxial) epidermis were most affected. Theirultrastructure was modified by cold and freezing treatmentsin different ways, as revealed by electron microscopy. Possiblereasons for the cold- and freezing-induced modifications inthe leaf and cell wall morphology and their role in plant acclimationto low temperature conditions are discussed. Copyright 1999Annals of Botany Company Acclimation, Brassica napus var. oleifera, cell wall ultrastructure, cold, freezing, leaf structure, winter oilseed rape.     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Characterization of a Small Sulphur-Rich Storage Albumin in Seeds of Alfalfa (Medicago sativa L.)

A 2S albumin fraction was characterized in seeds of alfalfa{^{Medicago sativa} L.). This low molecular weight (LMW) familyof disulphide-bonded proteins represents a major nitrogen andsulphur storage reserve for the alfalfa seed Characteristicof seed storage proteins, the 2S albumins are abundant in nitrogen-richglutarrune/glutamate/asparagine/aspartate (32%) In addition,this LMW fraction is high in cysteine (9%) and methionine (4%),amino acids which are under-represented in legume seed globulins.These 2S proteins start to accumulate during the early cotyledonstage of development, and are mobilized following germinationPulse-chase labelling experiments show that the 2S proteinsare synthesized as 'preproproteins', similar to 2S proteinsin other seeds. However, alfalfa 2S albumins are immunologicallyunrelated to these proteins. Key words: Seed development, sulphur-containing 2S storage protein, alfalfa (Medicago sativa)     

Alfalfa Nuclei Contain Cold-Responsive Phosphoproteins and Accumulate Heat-Stable Proteins during Cold Treatment of Seedlings

We have examined whether low temperature, as a pervasive thermodynamicstimulus, is sensed independently in different parts of thecell by studying low temperature responses of phosphoproteinsin isolated nuclei. The isolated alfalfa (Medicago saliva) nucleirespond to cold by rapid and reversible changes in phosphorylationlevel of their proteins. The population of such cold-regulatedphosphoproteins and the cold-stimulation of their phosphorylationare greater in a freezing-tolerant cultivar Apica than in asensitive cultivar Trek. With a 4-day cold treatment of theseedlings, additional proteins showing cold-stimulated phosphorylationappear in the nucleus of Apica while there is little changein the case of Trek. Furthermore, nuclei from cold-treated seedlingsof Apica, but not of Trek, show a large accumulation of heat-stableproteins. These results support the view that the low temperaturesensing and acclimation occur in all vital parts of the celland that accumulation of heat-stable nuclear proteins may berelated to freezing tolerance. (Received October 14, 1996; Accepted November 11, 1996)     

Use of the Pressure Bomb for Hydraulic Conductance Studies

Stem sections of alfalfa (Medicago sativa L.) were subjectedto forced flows of both water and high molecular weight dextransolutions. In either case, stems responded in accordance withthe Ohm's law analogy for flow, i.e., flow was a linear functionof pressure, at pressures ranging from 7.0–500 kPa andvacuum from 25.69 kPa. Since conductance values were the samefor both pressure and vacuum powered flows, it is possible toextrapolate conductance values determined with a pressure bombto negative pressure gradients experienced by plants. Key words: Pressure bomb, Conductance, Medicago sativa     

The response of tomato roots (Lycopersicon esculentum Mill.) to iron deficiency stress: alterations in the pattern of protein synthesis

Dicotyledon plants adapt to iron (Fe) deficiency through a seriesof reactions that increase the ability of the plant to assimilateFe and to increase the efficiency of Fe utilization. In an attemptto gain an insight into these adaptive processes, the specificchanges in protein synthesis associated with the onset of theFe deficiency response in tomato roots (Lycopersicon esculentumMill cv. Rutgers) have been investigated. Roots were grown underFe—sufficient and —deficient conditions, and thepattern of protein synthesis was analysed by in vitro translationof root mRNA and by in vivo labelling of root proteins. Polypeptideswere resolved by two—dimensional polyacrylamide gel elec—trophoresis.Seven polypeptides were identified by in vitro translation,whose synthesis was significantly increased during Fe deficiency.The increase was probably specific to Fe deficiency in thatthe polypep—tide synthesis was not increased during phosphatedeficiency stress, was less prominent following prolonged Fedeficiency and was decreased following re—supply of Feto the hydroponic medium. The pattern of in vitro translation of mRNA isolated from Fe—deficientroots was compared to the results obtainedin vivo followingradiolabelling of proteins. In these analyses, eight polypeptideswere identified, tentatively including the seven polypeptidespreviously identified by in vitro translation. All polypeptideswere characterized with regard to molecular mass and pl andtheir localization in the cell, whether being membrane boundor soluble. It is suggested that members of this group of polypeptidesare involved in the response of the root to Fe deficiency: althoughtheir functions remain to be identified. Key words: In vitro protein synthesis, iron, iron deficiency, root, 2-dimensional PAGE     

Developmental control of xylem hydraulic resistances and vulnerability to embolism in Fraxinus excelsior L.: impacts on water relations

The freezing tolerance of many plants, such as pea (Pisum sativum),is increased by exposure to low temperature or abscisic acidtreatment, although the physiological basis of this phenomenonis poorly understood. The freezing tolerance of pea shoot tips,root tips, and epicotyl tissue was tested after cold acclimationat 2C, dehydration/rehydration, applications of 10^–4M abscisic acid (ABA), and deacclimation at 25C. Tests wereconducted using the cultivar ‘Alaska’, an ABA-deficientmutant ‘wil’, and its ‘wildtype’. Freezinginjury was determined graphically as the temperature that caused50% injury (T₅₀) from electrical conductivity. Endogenous ABAwas measured using an indirect enzyme-linked immunosorbant assay,and novel proteins were detected using 2-dimensional polyacrylamidegel electrophoresis. The maximum decrease in T₅₀ for root tissuewas 1C for all genotypes, regardless of treatment. For ‘Alaska’shoot tips and epicotyl tissue, exogenous ABA increased thefreezing tolerance by –1.5 to –4.0C, while coldtreatment increased the freezing tolerance by –7.5 to–14.8C. Cold treatment increased the freezing toleranceof shoot tips by –9 and –15C for ‘wil’and ‘wild-type’, respectively. Cold acclimationincreased endogenous ABA concentrations in ‘Alaska’shoot tips and epicotyls 3- to 4-fold. Immunogold labeling increasednoticeably in the nucleus and cytoplasm of the epicotyl after7 d at 2C and was greatest after 30 d at the time of maximumfreezing tolerance and soluble ABA concentration. Cold treatmentinduced the production of seven, three, and two proteins inshoot, epicotyl, and root tissue of ‘Alaska’, respectively.In ‘Alaska’ shoot tissue, five out of seven novelproteins accumulated in response to both ABA and cold treatment.However, only a 24 kDa protein was produced in ‘wil’and ‘wild-type’ shoot and epicotyl tissues aftercold treatment. Abscisic acid and cold treatment additivelyincreased the freezing tolerance of pea epicotyl and shoot tissuesthrough apparently independent mechanisms that both resultedin the production of a 24 kDa protein. Key words: Pisum sativum, cold acclimation, immuno-localization     

Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature