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.     

In vivo plant impedance measurements and characterization of membrane electrical properties: The influence of cold acclimation

A procedure to measure both the real (resistance) and complex (reactance) part of plant tissue impedance is described. Bare metal electrodes were used and the impedance of electrodes was determined by obtaining measurements at four interelectrode distances. The frequency (f) dependence of the impedance of birdsfoot trefoil (Lotus corniculatus L.) stems could be modeled, approximately, by a resistor representing an extracellular resistance, a resistor representing an intracellular resistance, and a resistor and capacitor representing cell membranes. However, the membrane parameters appeared to be frequency dependent. Therefore, they were characterized by calculating a time constant at each frequency. For example, for one plant stem the time constant (τ) decreased from 1.19 × 10⁻³ sec at 101 hz to 1.36 × 10⁻⁶ sec at 100 KHz. This decrease with frequency could be described by an equation of the form: ln(τ) = a + bln(f) + c(ln[f])². Cold acclimation increased (P ⩽ 0.05) the intracellular resistance. But cold acclimation did not have a significant effect on estimates of the extracellular resistance, the membrane resistance, or the membrane time constant. Depending on the cultivar, cold acclimation either decreased or increased the estimate of membrane capacitance.     

Amylase Activity in Taproots of Medicago sativa L. and Lotus corniculatus L. Following Defoliation

Although the patterns of starch metabolism in taproots of alfalfa(Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatusL.) have been characterized, little is known regarding the activitiesof starch-degrading enzymes in taproots of these species. Ourobjective was to determine how defoliation influences starchdegradation and activities of amylases in taproots of alfalfaand birdsfoot trefoil. In Exp. 1, amylolytic activities andstarch concentrations in taproots of defoliated and undefoliatedplants were compared on days 0, 3, 7, 10, and 14 after defoliation.Taproot starch concentrations declined in defoliated plants,while increasing in taproots of undefoliated plants. Exoamylaseactivities in taproots of defoliated plants did not change withdefoliation, while endoamylase activities increased 2-fold indefoliated alfalfa and 50% in defoliated birdsfoot trefoil plantswhen compared to undefoliated plants. In Exp. 2, activity andisoform complement of amylases were monitored during seedlingdevelopment. High endoamylase activity was found in taprootsof both species at all samplings. In contrast, exoamylase accumulatedin taproots of alfalfa, but not birdsfoot trefoil, in a patternsimilar to starch accumulation. As in Exp. 1, defoliation increasedendoamylase, but not exoamylase activity in taproots of bothspecies. Taproots of both species contained one major and twominor endoamylase isoforms, but the electrophoretic mobilityof these isoforms differed between species. Activities of allisoforms, as indicated on starch-gel blots, increased in responseto defoliation. These results indicate that defoliation increasesactivity of taproot endoamylases, whose activity is associatedwith taproot starch degradation. Key words: Starch degradation, alfalfa, birdsfoot trefoil, enzymes     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

Zein accumulation in forage species (<Emphasis Type="Italic">Lotus corniculatus</Emphasis> and <Emphasis Type="Italic">Medicago sativa</Emphasis>) and co-expression of the γ-zein:KDEL and β-zein:KDEL polypeptides in tobacco leaf

Plants of birdsfoot trefoil (Lotus corniculatus), alfalfa (Medicago sativa) and tobacco (Nicotiana tabacum) were transformed by Agrobacterium with binary vectors harbouring genes that code either for wild-type and %-zein:KDEL, or for #-zein:KDEL. The maize seed storage proteins %-zein and #-zein are rich in sulphur amino acids, and our long-term goal is to improve alfalfa and birdsfoot trefoil forage quality. Significant levels of zeins were detected in leaves of birdsfoot trefoil transformants, ranging up to 0.055% and 0.30% of total extractable protein for %-zein and #-zein:KDEL, respectively. In leaves of alfalfa, the %-zein:KDEL expression level was up to 0.026% of the total extractable protein. From a F₁ population of transgenic tobacco, a plant was selected in which the amount of zeins (%-zein:KDEL plus #-zein:KDEL) accounted for 1.1% of the total extractable protein.     

Ethanol synthesis and loss from flooded roots of Medicago sativa L. and Lotus corniculatus L.

Abstract Field flooding of established alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) for up to 12 d resulted in a significant increase in alcohol dehydrogenase activity (ADH) and an increase in the K_m of ADH in both species. Root concentration of ethanol increased throughout the flooding regime in alfalfa roots. No ethanol was detected in any trefoil root samples. Alfalfa plants which had shoots removed 5 d prior to flooding accumulated significantly higher levels of root ethanol and showed flooding injury sooner, indicating a significant effect of shoots on development of flooding injury. Alfalfa and trefoil plants grown in the greenhouse were flooded and ethanol in the transpiration effluent was trapped and measured. Alfalfa transpired measurable quantities of ethanol which peaked just prior to development of shoot injury symptoms. No ethanol was detected in the transpiration effluent from trefoil shoots. Flooded roots of both alfalfa and trefoil excreted ethanol but alfalfa roots synthesized more total ethanol and retained a larger proportion in the roots than did trefoil. While the ethanol accumulation response in alfalfa and trefoil are consistent with the ethanol ‘self-poisoning’ hypothesis of flooding injury, the very small quantities of ethanol found in these roots still raises questions as to its absolute effect in the plant.     

Nitrate Effects on Nodule Oxygen Permeability and Leghemoglobin (Nodule Oximetry and Computer Modeling)

Two current hypotheses to explain nitrate inhibition of nodule function both involve decreased O2 supply for respiration in support of N2 fixation. This decrease could result from either (a) decreased O2 permeability (PO) of the nodule cortex, or (b) conversion of leghemoglobin (Lb) to an inactive, nitrosyl form. These hypotheses were tested using alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus) plants grown in growth pouches under controlled conditions. Nodulated roots were exposed to 10 mM KNO3 or KCI. Fractional oxygenation of Lb under air (FOLair), relative concentration of functional Lb, apparent PO, and O2-saturated central zone respiration rate were all monitored by nodule oximetry. Apparent PO and FOLair in nitrate-treated nodules decreased to <50% of values for KCI controls within 24 h, but there was no decrease in functional Lb concentration during the first 72 h. In nitrate-treated alfalfa, but not in birdsfoot trefoil, FOLair, apparent PO, and O2-saturated central zone respiration rate decreased during each light period and recovered somewhat during the subsequent dark period. This species difference could be explained by greater reliance on photoreduction of nitrate in alfalfa than in birdsfoot trefoil. Computer simulations extended the experimental results, showing that previously reported decreases in apparent PO of Glycine max nodules with nitrate exposure cannot be explained by hypothetical decreases in the concentration or O2 affinity of Lb.     

Metabolic response of Medicago sativa L. and Lotus corniculatus L roots to anoxia

Abstract Differential rates of fermentation and energy production have been implicated in the response of plant species to extended root anoxia. This study describes the metabolic response to anaerobiosis of waterlogging-tolerant birdsfoot trefoil (Lotus corniculatus L.) and waterlogging-sensitive alfalfa (Medicago sativa L.). Studies were carried out on glasshouse-grown plants subjected to root anaerobiosis in nutrient solution. Rate of fermentation, as estimated by CO₂ evolution, declined significantly upon anaerobiosis in both species but was proportionally less, relative to the aerobic control, in trefoil. Another indicator of carbon flux through glycolysis, the concentration of glucose-6-phosphate, was also significantly lower in trefoil roots relative to aerobic controls. Both species showed significantly increased root exudation of K⁺, sugars and andno-N, especially during the first 2 d of root anaerobiosis, indicating changes in membrane selective permeability. The energy status of roots subjected to anaerobiosis declined sharply in both species but trefoil roots maintained higher ATP/ADP ratios for up to 4 d of anaerobiosis. The results are consistent with the hypothesis that increased fermentation activity maintains a more favourable root energy status. This higher energy status may facilitate survival by maintaining crucial root activities, such as maintenance of membrane stability.     

Evaluation of the population dynamics of the forage legume (Lotus corniculatus), using matrix population models

The population dynamics of perennial crop plants are influenced by numerous factors, including management practices. Conditions in the field vary from year to year, and matrix population models are useful for evaluating population behaviour in relation to environmental variability. In Missouri, the stand persistence of birdsfoot trefoil ( Lotus corniculatus ), a perennial legume, is often limited by disease and poor seed production. A stage-based, matrix population model was developed to evaluate the population dynamics of birdsfoot trefoil in relation to clipping treatment. The plant growth stages represented in the model were seeds, seedlings, mature vegetative and reproductive plants. Two phases of population growth were evaluated in clipped and unclipped stands. Establishment-phase populations were characterized by relatively high mortality and low reproduction. Elasticity analysis indicated that growth of these populations was most sensitive to the survival of vegetative plants. Mature vegetative plants and seeds comprised the majority of surviving individuals in clipped and unclipped populations, respectively; however, establishment-phase populations under both management treatments tended toward extinction. Populations in the post-establishment phase of growth were characterized by relatively low mortality and high reproduction. Population growth in this phase of growth was most sensitive to seed production, and most individuals in these populations were at the seed stage.     

Species variation in the fixation and transfer of nitrogen from legumes to associated grasses

Summary Three legume species (alfalfa, red clover, and birdsfoot trefoil) in combination with five grass species (timothy, bromegrass, red fescue, tall fescue, and orchardgrass) were used to study N transfer in mixtures, using the 15_N dilution technique. The advantage of grass-legume mixtures was apparent. Total herbage and protein yields of grasses in mixtures were higher than those alone, especially at the later cuts. This benefit of mixed cropping is mainly due to N transfer from legumes to associated grasses. N₂-fixation and N transfer by alfalfa rated highest, red clover intermediate, and birdsfoot trefoil lowest. The importance of each pathway of N transfer from legumes appeared to differ between species. Alfalfa and red clover excreted more N than trefoil, while the latter contributed more N from decomposition of dead nodule and root tissue. The greatest advantage from a grass-legume mixture, with respect to the utilization of N released from the legume, varied with early maturing tall fescue (Kentucky 31), orchardgrass (Juno), and bromegrass (Tempo), to intermediate timothy (Climax), and least with late maturing red fescue (Carlawn). Contribution no. 817 of the Ottawa Research Station.     

Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation

Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.     

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.     

The electrical impedance spectroscopy of Scots pine needles during cold acclimation

The electrical impedance spectroscopy (EIS) was applied to current-year needles of Scots pine ( Pinus sylvestris L.) in an 8-year provenance field trial in central Finland during frost hardening. The EIS analysis of the needles using a Model-A equivalent circuit indicated a sequence of events in the needles during their cold acclimation. Some of the EIS-parameters referred to maturation phenomena occurring during the pre-hardening phase at the end of the growing season, and some parameters displayed a clear coincidence with the frost hardening itself. Significant differences between provenances were found in several of the Model-A parameters. Extracellular resistance (r_e) and β -coefficient decreased in all provenances in the pre-hardening phase in August and until mid-September. In the same phase, both the intracellular resistance (r_i) and the cell membrane time constant (τ_m) first increased and then decreased. According to τ_m, r_e and β there was a clear gradation between provenances in the pre-hardening phase. From the end of September significant differences were found in the intracellular resistance between provenances, corresponding with the differences in their hardening pattern. The dry weight (DW) content of needles increased during the study period but no clear differences were found between the provenances.     

Cold acclimation in Pinus sylvestris: Phospholipids in purified plasma membranes from needles of pine

Effects of cold acclimation on needles of Scots pine ( Pinus sylvestris L., Provenance Södra Ydre) were studied at the membrane level. Before and after a period of cold acclimation the plasma membranes were isolated from the needles by a aqueous polymer two-phase partition technique. Fatty acid composition of total lipids or of individual phospholipids from the plasma membrane showed that the plasma membrane fraction was different from the other microsomal fractions analyzed, especially the 18:2 levels of the individual phospholipids. Furthermore, the cold acclimation period did not result in a decreased saturation level in the plasma membranes. Different steps in cold acclimation reactions at the membrane level are discussed.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

Changes in leaf ultrastructure and carbohydrates inArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation

Summary We studied cell ultrastructure and carbohydrate levels in the leaf tissue ofArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Freezing tolerance of the leaves from 26 day old plants was determined after 48 h and 10 days at 4°C. Acclimation treatment of 48 h decreased the lethal freezing temperature from –5.7°C to –9.4°C. Freezing tolerance was not altered further by acclimation at 4 °C for 10 days. Ultrastructural changes in the parenchyma cells were evident after 6 to 24 h of cold acclimation. The plasma membrane showed signs of extensive turnover. Evidence of membrane invaginations and sequestering of membrane material was observed. In addition, numerous microvesicles, paramural bodies, and fragments of endoplasmic reticulum were noticed in the vicinity of plasma membrane. Modifications in the structure of cell membranes were evident after 5 days of exposure to low temperature. Small, darkly stained globules were seen on the plasma membrane, tonoplast, chloroplast envelope membrane, mitochondrion outer membrane, dictyosome cisternae membrane, and microvesicle membrane. As far as we are aware, this type of membrane modification has not been described previously in plant cells exposed to low temperature. We propose to call these structures membraglobuli. Acclimation treatment also increased the concentrations of soluble sugars and starch. These observations suggest that cold acclimation inA. thaliana induces changes in both plasma membrane properties and carbohydrate composition.     

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.     

Nitrogen fixation and vegetative regrowth of alfalfa and birdsfoot trefoil after successive harvests or floral debudding

Nitrogenase-dependent acetylene reduction, leaf, herbage, and root growth, and total nonstructural carbohydrate accumulation of alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) were compared to learn how nitrogen fixation capacity and vegetative growth respond to partial (75-85%) or total shoot and leaf removal, and floral debudding. Treatments were imposed on greenhouse-grown plants during two successive harvest cycles.