Electrophoretic studies of several dehydrogenases in relation to the cold tolerance of alfalfa

The influence of ionic composition and pH of extractant on the relationship between the extracted proteins and the cold tolerance of Vernal and Arizona Common alfalfa (Medicago sadiva L.) was examined. Five environments were used to induce different tolerance levels.The quantity of protein extracted from plants was influenced by the hardening environment, cultivar, and ionic composition and pH of 29 extractants. Extractants with a pH below 6 generally extracted less protein.The measured cold tolerance of the plants was correlated with the quantity of protein detected in many of the 14 regions of the electrophoresis gel columns regardless of extractant but was most dosely associated with the protein in either region 7 or 8 with nine of ten extractants. The purpose of this research was to examine the influence of ionic nature and pH of extractants on the relationship between extractable proteins and cold tolerance. Sources of variation in cold tolerance were cultivars of alfalfa and hardening environments. MATERIALS AND METHODSPlant Material. Cans with perforated bottoms and a volume of 3.78 liters were filled with a sandy loam soil and seeded with approximately 50 seeds of cold-tolerant Vernal or cold-sensitive Arizona Common alfalfa (Medicago sativa L.). The seedlings were allowed to grow in the greenhouse for 7 months and then were moved outside in April. The plants were allowed to reach a flowering stage before clipping to maintain high food reserve levels. Insects were controlled by spraying with malathion and Sevin.In late September, the containers were randomly assigned to one of five environments: (a) day and night temperatures of 7 and 2 C, respectively, and a photoperiod of 8 hr; (b) day and night temperatures of 16 and 10 C, respectively, and a photoperiod of 12 hr; (c) natural environment in the field at Morgantown, W. Va.; (d) greenhouse conditions maintained for vigorous growth; and (e) day and night temperatures of 27 and 21 C, respectively, with a photoperiod of 16 hr. Light in the growth chambers was supplied by six cool-white, 40-w fluorescent lamps and four 60-w incandescent bulbs/chamber. This lighting system provided a light intensity of approximately 12,900 lux at the plant tops. Relative humidity was regulated at approximately 80% in the chambers. A randomized block design was used with three replications for each environmental regime.After the plants had been subjected to the environments for 44 to 46 days, they were sampled for cold tolerance determinations and protein analyses. Plant roots were trimmed to a length of approximately 2.5 cm and crowns to 5 cm. All dead material and plant leaves were removed from the samples. A 10-g portion of prepared crown and root sample was used to measure cold tolerance using the technique of Dexter et al. (2). The remainder of each sample was immediately frozen in liquid N2, lyophilized, ground in a Wiley mill to pass through a 40-mesh screen, and stored at -20 C in air-tight vials that were enclosed in plastic bags.

Section: Discussionmentioning

confidence: 75%

Extractant Influence on the Relationship between Extractable Proteins and Cold Tolerance of Alfalfa

Faw

Shih²,

Jung³

1976

“…However, after 7 d of deacclimation, total protein content reached a concentration almost twice that of preacclimation levels. Incorporation of [35Sjmethionine into protein was reduced during the first 4 d of low temperature exposure, but approached NA levels by the 7th d (Table II) Changes in protein electrophoretic patterns characteristic of CA tissues have been demonstrated for a number of species (3,7,16,27). When spinach leaf proteins from NA and CA tissue were separated on SDS gels and stained with Coomassie blue, banding patterns were very similar (Fig.…”

mentioning

confidence: 81%

“…This concept is supported by much indirect evidence (5,10,16,20,21), but only recently has direct evidence to support an altered gene expression hypothesis been reported. In spinach, a qualitative alteration in the translatable mRNA content of leaf tissue was observed during cold acclimation (11).…”

mentioning

confidence: 99%

Induction of Freezing Tolerance in Spinach Is Associated with the Synthesis of Cold Acclimation Induced Proteins

Guy

Haskell²

1987

136

Spinach (Spinacia okracca L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold accfimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrientagar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deaccfimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with 3ISimethionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visnalized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonaccimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M, of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M, 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deaccimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M, 79,000, pl 4.8) similar to that of a HSP (M, 83,000, pl 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.

“…Differences in the lipid constituents of membranes rather than enzymes in these two groups of plants are usually considered to produce the observed variations in Arrhenius plots (26,27). However, changes in isoenzymes occur with cold acclimation (development of resistance to freezing) and deacclimation (19) along with changes in the amounts of membrane-bound and water-soluble enzymes (12,19). This suggests that, at least in part, the enzyme complement of tissues could influence responses to low temperature in some species.…”

mentioning

confidence: 99%

“…Dehydrogenases are of particular importance in cold acclimation due to their production of reduced nucleotides necessary for energy transduction (1,19 All dehydrogenases were assayed spectrophotometrically utilizing a Gilford 2000 spectrophotometer equipped with a H20-jacketed cuvette chamber for temperature control. GDH and MDH were assayed as described before (9) with pellet and supernatant activites added to give total activity.…”

mentioning

confidence: 99%

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination

Duke

Schrader²,

Miller³

1977

The influence of low temperature on soybean (Glycine max [L.] Meff. cv. Wells) energy transduction via mitochondrial respiration and dehydrogenases was investigated in this study during imbibition and germination. Mitochondria were isolated from embryonic axes of seeds treated at 10 and 23 C (control) by submergence in H20 for 6 hours and maintenance for an additional 42 hours in a moist environment.Arrhenius plots of initial respiration rates revealed that those from cold-treated axes had respiratory control (RC) ratios of near 1.0 above an inflection in the plot at 8 C. Arrhenius plots of control axes mitochondrial respiration showed RC rtios of 2.8 above and 5.0 below an inflection temperature of 12.5 C. Energies of activation for mitochondrial respiration between 20 and 30 C for the cold and control treatments were 7.8 and 15.6 kcal/mole, respectively. These data indicate possible differences in mitochondria membranes, degree of mitochondrial integrity, and mitochondrial enzyme complement between the two treatments.Gluhtmate dehydrogenase (GDH), malate dehydrogenase (MDH), adcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6P-DH), and NADP-isocitrate dehydrogenase (NADP-ICDH) were sayed from whole seeds and axes (after germination) during the 48 bours of temperature treatments. Activity of these dehydrogenases decased during the first 6 hours with the exception of MDH. After germination at 23 C (48 hours) all five debydrogenases increased in activity. Arrhenius plots of cotyledon debydrogenase activities indicated that one inflection temperature between 6 and 18 C was present for each enzyme assayed. Differences were seen in Arrhenius plots of axes dehydrogenase activities with the two temperature treatments in the cases of GDH and MDH from mitochondrial pellets and with differences in enzyme extraction media. These data suggest that the temperature treatments yield differences in mitochondrial enzyme complement. There were no detectable inflection temperatures for the activities of G6P-DH and ADH extracted from axes. Arrhenius plots of NADP-ICDH activity indicated extreme cold sensitivity. The dopes of the plots for axes NADP-ICDH were very similar to those for mitochondrial respiration (23 C trtment) suggesting that this enzyme may limit mitochondrial respiration at low temperature in soybean tissues grown at moderate temperatures.