Changes in fatty acid composition in wheat cultivars of contrasting hardiness

142

There is mounting evidence to indicate that membranes, primarily the plasma membrane, is intimately involved in cold acclimation and freezing injury (11,18,19,(24)(25)(26). Thus, it follows that plant plasma membranes must undergo certain chemical and biophysical changes during cold acclimation in order to withstand freezing stresses.There are several reports on changes in cellular membranes during cold acclimation (6,7,27). However, these are based on analytical data obtained from crude membrane fractions or total membrane extracts. Therefore, it is uncertain whether the reported changes are related to the changes in the plasma membranes per se during cold acclimation. Based on these facts, it is important to know what changes in the plasma membranes are responsible for the development offreezing tolerance during cold acclimation. A major problem encountered has been the difficulty ofisolating the plasma membrane in a high degree ofpurity needed for detailed studies. Recently, we have succeeded in preparing plasma membranes from light-grown winter rye seedlings with a high degree of purity using an aqueous two-polymer phase partition system (29).In the present study, Evaluation of Freezing Resistance. Freezing tolerance of the crown was evaluated by the ion-leakage test. One g fresh weight of tissues were cut into 5-mm-long pieces and were frozen in test tubes at -3°C for 2 h with small pieces of ice. Thereafter, the tissues were cooled in steps of 5°C at 2-h intervals and held at the desired temperatures for 2 h. After thawing at OC, the tissues were immersed in 5 ml of distilled H20 and incubated at room temperature for more than 5 h by gently shaking before measuring the conductivity. The tissues frozen directly in the liquid nitrogen and unfrozen were taken as 100% and 0% in the relative conductivity, respectively.Isolation of Plasma Membranes. Crown tissues were homogenized with a Polytron PT 20 at the medium speed for 90 s in a medium consisting of 0.5 M sorbitol, 50 mm MOPS2-KOH (pH 7.6), 10 mm EGTA neutralized with KOH, 2.5 mm potassium metabisulfite, 4 mM SHAM, 1 mm PMSF, 5% (w/v) soluble PVP (mol wt 24,500), and 1% (w/v) defatted BSA. The homogenate was passed through two layers of gauze and then two layers of Miracloth, and then centrifuged at 14,000g for 15 min and 156,000g for 20 min. The 156,000g pellet (14,000-156,000g fraction) was used for plasma membrane isolation using the aqueous two-polymer phase partition technique as reported previously (29). The purity of the isolated plasma membrane was determined by phosphotungstate-chromate staining and by specific aggregation in the presence ofZnCl2 or at pH 4.5 as reported previously (29). The isolated plasma membranes were suspended in 0.5 M sorbitol-3 mm Tris-maleate at pH 7.3 and kept at -70°C until use.Lipid Extraction. Total lipids were extracted from membrane samples according to the method of Bligh and Dyer (1) with the 2 Abbreviations: MOPS, 34N-morpholino)propanesulfonic acid;

Section: Resultsmentioning

confidence: 60%

mentioning

confidence: 99%

Involvement of Plasma Membrane Alterations in Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

Uemura¹,

Yoshida²

1984

142

“…Increased asaturation of fatty acids is therefore probably an important part of the mechanism of cold adaptation in winter wheat.BASF 13-338 also prevents the increase in per cent dry weight in roots and shoots during hardening and causes a decrease in root lipid phosphorus and total fatty adds.The concurrent incea in linoleic acid and decream in linolenic acid in the treated plats, while the level of the otber fatty adds is but ittle affected, suggest that BASF 13-338 specifically inhibits linoleic acid desuae.Although linolenic acid content greatly increases in the roots of winter wheat during frost hardening, it accumulates to the same extent in cultivars showing a large difference in frost resistance (3,7,12). Increased unsaturation of membranes does not seem directly involved in the cold-hardening process in winter wheat.…”

mentioning

confidence: 89%

“…Although linolenic acid content greatly increases in the roots of winter wheat during frost hardening, it accumulates to the same extent in cultivars showing a large difference in frost resistance (3,7,12). Increased unsaturation of membranes does not seem directly involved in the cold-hardening process in winter wheat.…”

mentioning

confidence: 89%

Simultaneous Inhibition of Linolenic Acid Synthesis in Winter Wheat Roots and Frost Hardening by BASF 13-338, a Derivative of Pyridazinone

Willemot¹

1977

Treatment of 12-day-old winter wheat (Triticum aestivum) plants with BASF 13-338 {4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone} 36 hous before frost hardening simultaneousy and completely inhibit accumulation of linolenic acid in the roots during the hardening period and the acquiition of frost resistance. Increased asaturation of fatty acids is therefore probably an important part of the mechanism of cold adaptation in winter wheat.BASF 13-338 also prevents the increase in per cent dry weight in roots and shoots during hardening and causes a decrease in root lipid phosphorus and total fatty adds.The concurrent incea in linoleic acid and decream in linolenic acid in the treated plats, while the level of the otber fatty adds is but ittle affected, suggest that BASF 13-338 specifically inhibits linoleic acid desuae.Although linolenic acid content greatly increases in the roots of winter wheat during frost hardening, it accumulates to the same extent in cultivars showing a large difference in frost resistance (3,7,12). Increased unsaturation of membranes does not seem directly involved in the cold-hardening process in winter wheat. However, the close correlation between percentage of linolenic acid and degree of frost resistance during both hardening and dehardening of the hardy winter wheat cultivar Kharkov (unpublished data) casts doubt on this conclusion. It is possible that linolenic acid accumulation is a prerequisite to hardening, but that all cultivars have acquired this characteristic and that less hardy cultivars have their frost resistance limited by other factors.To test this hypothesis an attempt was made to prevent frost hardening of winter wheat by specifically blocking linolenic acid synthesis during the hardening period by treating the plants with 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone (Sandoz 9785,. St. John and Christiansen (9) showed that treatment of germinating cotton seeds with this compound reduced both the tolerance of the seedlings to chilling and the low temperature-induced increase in linolenic acid content of the polar lipids in the root tips. MATERIALS AND METHODSWinter wheat (Triticum aestivum, cv. Kharkov) was grown and hardened as described previously (11 contained 15 seedlings and 400 g of sand-vermiculite (1:1, v/v). During hardening the humidifier was stopped. Before and during hardening the frost resistance of the plants was tested by controlled freezing. The plants were cut above the crown and the pots were transferred to a programmed freezer (12). The temperature was lowered at a rate of 2 C/hr, equilibrated for 12 hr at -4 C, and kept for 2 hr at each test temperature. At the end of each pause pots were withdrawn and allowed to thaw slowly for 36 hr at 4 C. Survival counts were made after 3 weeks of recovery under initial growth conditions. Frost hardiness was expressed as the temperature at which 50% of the plants died (killing temperature, LT5o).Twelve days after sowing and 36 hr before being moved to the hardening chamber, the plants were tre...

“…A recent study (16) into the effect of temperature on respiration of mitochondria from the same four cultivars grown at 2 and 24 C revealed no correlation between cold hardiness of the cultivars and the transition temperatures for mitochondrial and tissue segment respiration. The observed increase in fatty acid unsaturation of both mitochondrial membranes (11) and total membranes (1) (1,11,15,17), has failed to establish a correlation between cold hardiness and changes in the structural and functional properties of mitochondria and other cell membranes. It has generally been shown that numerous changes occur in the cell which are associated with growth at low temperature but not with the development of cold hardiness.…”

Section: Methodsmentioning

confidence: 99%

Swelling and Contraction of Mitochondria from Cold-hardened and Nonhardened Wheat and Rye Seedlings

Pomeroy

1976

A comparison of mitochondria isolated from 2 and 24 C grown winter wheat (Triticum aestivum L.) and winter rye (Secak cereak L.) seedlings revealed no correlation between changes in swelling and contraction characteristics and extent of cold hardiness. The swelling response changed markedly due to growth at low temperature, but the change was similar for the four cultivars examined. The swelling response was also observed to change rapidly during aging of isolated mitochondria, either at 2 or 24 C. Spontaneously swollen mitochondria, isolated from 24 C grown seedlings, contracted abruptly upon addition of certain oxidizable substrates, but this response was lost when seedlings were transferred from 24 to 2 C. Studies on the effect of various substrates and respiratory inhibitors on the swelling and contraction responses indicate that inhibitors which reduce or stop electron flow through the electron transport chain also inhibit substrate induced mitochondrial contraction.Although the swelling and contraction characteristics of isolated plant mitochondria have been thoroughly investigated, the mechanisms involved in these processes are not completely understood. Numerous studies (3,13,18,22) have demonstrated a passive, energy-independent swelling of plant mitochondria in the presence of isomolar salt solutions, or isomolar solutions of monosaccharide sugars. The existence of an energy-dependent (active) swelling component in plant mitochondria has also been demonstrated (2,5,19,21). Addition of certain oxidizable substrates to swollen mitochondria results in an immediate rapid contraction of the mitochondria while the addition of ATP initiates contraction of swollen corn mitochondria (18), but not swollen castor bean endosperm mitochondria (20). Earlier studies on the effect of specific inhibitors of electron transport and oxidative phosphorylation on swelling and contraction (18,20) postulated that high energy intermediates are associated with the active swelling and contraction observed in plant mitochondria. More recent investigations of energized swelling and contraction have led to interpretation of these phenomena in chemiosmotic terms, suggesting that the energy required is derived from changes in the electrochemical proton gradient (2).Recent studies (11,15) in our laboratories on the mechanism of cold hardening in plants have been directed to investigations of structural and functional properties of wheat and rye mitochondria in relation to growth at low temperature. These studies have revealed a marked increase in the level of fatty acid unsaturation during growth at low temperature, but no differences were observed among cultivars of contrasting cold hardiness. The functional properties of mitochondria from cold hardy cultivars were similar to those from less hardy cultivars. Electron spin resonance studies identified three possible temperature-dependent structural transitions in the mitochondrial membranes, and the shift in one of these appeared to be quantitatively greater in the cold hardy ...