Levels of ethylene, ACC, MACC, ABA and proline as indicators of cold hardening and frost resistance in winter wheat

Changes in ethylene production and in the contents of 1-aminocydopropane-1-carboxylic acid (ACC), 1-(malonylamin6)-cyclopropane-1-carboxylic acid (MACC), abscisic acid (ABA) and L-proline were determined after 40 days of cold hardening at 4°C in three wheat cultivars differing in frost resistance. Proline and especially ABA accumulated with hardening in all varieties in parallel with the degree of frost resistance, e.g. proline and ABA increases in the non-resistant cv. Slávia were 2x and 5x, whilst in the resistant cv. Mironovská 808 increases were 4X and 20X. Ethylene production and MACC level showed no significant changes with hardening in any of the cultivars after 40 d, but ACC levels did increase with hardening. The production of ethylene, ACC and MACC was studied during hardening. Ethylene production decreased sharply at low temperature and rose rapidly (within 1 day) on return to normal temperature, while ACC production reacted in the opposite direction. MACC levels rose rapidly during the first 4 days of cold, then more slowly for about 2 weeks, thereafter decreasing again steadily. The only varietal differences occurring at maximum levels were correlated with the degree of frost resistance.     

Changes m Ethylene Production in Relation to l-Aminocyclopropane-l-Carboxylic Acid and Its Malonyl Conjugate in Waterlogged Wheat Plants

When wheat seedlings were subjected to waterlogging, 1-aminocyelopropane-l-carboxylic acid (ACC), an ethylene precursor, accumulated in large quantity in roots. In shoots, ACC and ethylene production also increased, but declined with the prolonged periods of waterlogging. However, ACC content in roots maintained in high level during the whole period of waterlogging. Drainage caused a drastic drop in both ACC content and ethylene production in waterlogged plants to control level. 1-(malonylamino) cyclopropane-l-carboxylic acid (MACC) level in roots subjected to waterlogging showed little changes. However, MACC content in shoots kept increasing during the 9-days period of waterlogging. At later period of waterlogging (longer than 5 days) when ACC and ethylene production bad dropped, the. level of MACC continued to increase. Draining stopped this increasing, but did not reduced its level. When exogenous ACC was introduced into the leaves via transpiration stream, the ability of leaves of waterlogged plant to convert ACC to MACC was much higher than control. The data presented showed that at the later stage of waterlogging, the conversien of a great quantity of ACC to MACC in waterlogged wheat plants is the cause of the reduction of ethylene production and ACC content. It was suggested that the formation of MACC is another way of regulation in ethylene biosynthesis. Among leaves of different ages, the enhancement of ethylene, ACC and MACC content was more pronounced in older leaves than in younger laves during the waterlogging period. The physiological significance of adaptation to waterlogging stress was discussed.     

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Cold acclimation of Ilex aquifolium under natural conditions with special regard to the photosynthetic apparatus

Frost resistance of leaves of holly ( Ilex aquifolium L.) increased from about −9°C in late summer to −24°C in mid-winter. The gradual rise in cold hardiness occurred when the minimum air temperature dropped to 0°C or below and was closely related to increase in the cellular sap concentration. Predominantly, the decrease in the osmotic potential of the cellular sap was caused by sugar accumulation, mainly of sucrose. The capacity of net photosynthesis of the leaves, as well as the total lipid and protein content and the proportion of individual lipids of the thylakoid membranes, did not significantly change during cold acclimation. The gradual shift towards desaturation in the fatty acids of the thylakoid lipids during the hardening period was neither correlated with alterations in the frost resistance nor did it affect the potential efficiency for various light-induced chloroplast membrane reactions such as linear photosynthetic electron transport, photophosphorylation and the proton gradient (ΔpH). It is suggested that in holly leaves reduction in cell volume changes during freeze-thawing and cryoprotection by sugars could play a dominant role for the increase in frost resistance. Seasonal changes in the degree of unsaturation of polar lipids of the thylakoids could contribute to maintain optimal functional efficiency of the membranes at low temperatures rather than to avoid freezing damage.     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

Effect of Low Temperature upon Lipid and Fatty Acid Composition of Roots and Leaves of Winter Rape Plants

When winter rape plants were transferred from favourable temperature conditions (25/20°C day/night temperature) to 5°C, the frost resistance of the leaves was increased whereas the frost tolerance of the roots remained unaffected. This permitted an analysis of the changes in lipid and fatty acid composition both as related to functioning of the plant at low temperature alone (roots) and as related to adaptation to freezing and functioning at low temperature (leaves). — Transfer of the plants to 5°C lead to an increase in the level of linolenic acid in roots and leaves. This increase was most evident in the phosphatidyl choline and ethanolamine fractions of the leaves, and in the neutral lipids and in an unidentified phospholipid from the roots. It was concluded that upon transfer of the plants to 5°C a general and non-specific increase in linolenic acid level contributed to functioning of the rape plants at low temperature; and that parallel but minor increases in linolenic acid level of digalactosyl diglyceride, phosphatidyl inositol and the unknown phospholipid in roots and leaves could only contribute to low-temperature functioning in specific membrane enzyme locations. Combined adaptation of the leaves to freezing tolerance and low-temperature functioning was correlated with a higher level of phosphatidyl choline and ethanolamine, predominantly esterified with linolenic acid.     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

Development of Frost Tolerance in Winter Wheat as Modulated by Differential Root and Shoot Temperature

Abstract: Winter wheat plants ( Triticum aestivum L. cv. Urban), grown in nutrient solution, were exposed to differential shoot/root temperatures (i.e., 4/4, 4/20, 20/4 and 20/20 C) for six weeks. Leaves grown at 4C showed an increase in frost tolerance from - 4C down to - 11 C, irrespective of root temperature. In 4/20 C plants, high root temperature decreased the rate of hardening of the leaves, but did not influence the final level of frost tolerance. In roots grown at 4C frost tolerance increased from - 3 C down to - 4 C, independently of shoot temperature. An accumulation of soluble sugars in the leaves was only observed when both shoot and root were grown at 4C and was not correlated with final frost tolerance achieved. However, the rate of hardening was correlated with the soluble sugar concentration. An increase in root soluble sugar concentration was exclusively observed in roots exposed to a temperature of 4C, irrespective of shoot temperature. Proline concentration only increased in plant parts exposed to a temperature of 4C. The present results indicate that the importance of root temperature in low-temperature hardening of winter wheat is limited, even though exposure to differential root and shoot temperatures brought about pronounced changes in growth, soluble sugar concentration, insoluble sugar concentration and proline concentration in roots and leaves.     

Conversion of Phosphatidyl Choline to Phosphatidic Acid in Freeze Injured Rye and Wheat Cultivars

The effect of exposure to freezing temperature (?15°C) on leaf phospholipid composition of hardened rye (Secale cereale L.) and hardened wheat cultivars (‘Miranovskaja 808’, ‘Bezostaja 1’, ‘Short Mexican’ and ‘Penjamo 62’), which differ in their resistance to frost, was investigated. Hardening took place under natural conditions. All the seedlings attained an equal level of linolenic acid in their leaves during hardening. Exposure to freezing temperatures resulted in a loss of phosphatidyl choline and accumulation of phosphatidic acid in the leaves. The ratio of phosphatidic acid to phosphatidyl choline, but not the level of poly-unsaturated fatty acids in the leaves, was related to their ability to survive at low temperatures. As freezing injury is caused by the formation of ice crystals in both extra- and intracellular space, it is probable that the plasma membranes of the investigated cultivars differed with respect to their water permeability. It is concluded that the plants, depending on the degree of their resistance to cold, produce an unknown substance of lipidic nature upon exposure to cold, with the aid of which they adjust the transitional state of their membranes to the prevailing temperature and, at the same time, facilitate the efflux of water from the cells.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Does availability of potassium affect cold hardening of Scots pine through polyamine metabolism?

The effect of different potassium availability on the polyamines and frost resistance of Scots pine seedlings ( Pinus sylvestris L.) during cold hardening was studied. Scots pine seedlings were grown applying different rates of potassium by using the relative addition rate technique followed by a 2- or 9-week hardening period with decreased light intensity, day length and temperature. After 2 weeks of treatment the seedlings were not hardened (LT₅₀, =−11°C) and showed no differences in frost resistance, although differences in the polyamine levels between the K levels were observed. After 9 weeks of hardening the seedlings at the low, medium and high K levels showed a mean frost resistance (LTs.₅₀) of −81, −63, and −47°C, respectively. A negative effect of K on the frost resistance of the needles was also found in adult trees in September. The results indicate that at the early stage of cold hardening, potassium or free polyamine levels do not affect the frost resistance of Scots pine needles. However, in hardened seedlings and adult trees potassium displays a negative and putrescine a positive correlation with frost resistance, whereas spermidine and spermine show no correlation.     

Porphyrinogenesis in maize as affected by bidentate chelators of iron

Intracellular compartmentation of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) in wheat ( Triticum aestivum L. cv. Kanzler) and barley ( Hordeum vulgare L. cv. Gerbel) leaves was studied using different methods: first, the isolation of intact vacuoles from protoplasts and, second, a non-aqueous fractionation procedure. The two methods gave similar results. ACC concentrations were similar in the extravacuolar space and in the vacuole, whereas MACC was accumulated in the vacuolar space. Transport studies revealed that no specific carrier for ACC exists at the tonoplast. MACC transfer across the tonoplast was enhanced by 120% in the presence of ATP. MACC competitively inhibited malate transport into the vacuole indicating that the same transfer system catalyzes the transfer of the two dicarboxylates.
It is concluded that malonylation of ACC is not a prerequisite for the transport of ACC through the tonoplast.     

春小麦水分胁迫响应中的ACC、MACC合成及乙烯的释放

水分胁迫使两个抗旱性不同的春小麦 (TriticumaestivumL .)品种“8139”(抗旱性较弱 )和“5 0 4”(抗旱性较强 )叶片ACC和MACC含量于胁迫初期下降后期升高 ,ACC合酶活性持续升高 ,乙烯释放量在 8139中下降而在5 0 4中先大幅升高而后下降。两种作用效果相反的抑制剂MGBG (抑制SAMDC活性 )和AOA (抑制ACC合酶活性 )均明显影响了两品种春小麦叶片以上各指标的变化。结果表明 ,水分胁迫下作物乙烯的释放量并不与其合成直接前体ACC的量成正相关 ;胁迫乙烯在抗性品种中于胁迫初期的升高可能是植物胁迫信号传导的响应之一 ,是一种干旱适应现象 ,可能与作物的干旱忍耐形成有关 ,而MACC具有调节胁迫乙烯释放的特殊生理作用。     

Regulation of frost resistance during cold de-acclimation and re-acclimation in oilseed rape. A possible role of PSII redox state

A possible role of photosynthetic apparatus during cold de-acclimation was studied in oilseed rape ( Brassica napus var. oleifera ). Plants of spring (Star) and winter (Górczañski) cultivars were cold acclimated at + 5°C, and de-acclimated during 4 weeks at combinations of + 12 and + 20°C operating in the light or/and dark, with a 12-h photoperiod. Evidence is presented that the photosynthetic apparatus may be involved in temperature perception during de-acclimation. De-acclimation was faster under a 20/12°C (day/night) treatment than under the reverse 12/20°C (day/night). De-acclimation rate was constant when the day temperature was constant, irrespective of the night temperature both under cold day temperature regimes (12/20, 12/12°C (day/night) and warm-day treatments (20/12, 20/20°C (day/night). The fast decrease in frost resistance observed under warm-day de-acclimation was always accompanied by an acceleration of elongation growth. In the spring cultivar, elongation growth increased starting from the second week of de-acclimation, regardless of temperature conditions. Once elongation growth had commenced during de-acclimation, it continued throughout the period necessary for re-acclimation to low temperature. Re-acclimation to the initial freezing tolerance level was only possible when plant elongation was reduced. In addition re-acclimation of the photosynthetic apparatus to low temperature was impossible in fast growing plants. A possible relationship between PSII, growth rate and frost resistance during cold acclimation and de-acclimation is discussed.     

Frost tolerance in cell cultures of potato

Different methods of freezing and of estimating frost damage in cell cultures of Solanum tuberosum L. and a number of wild Solanum species were compared. Frost-killing temperatures (FKT, i.e. the temperature resulting in 50% of the maximum possible frost damage) in leaves of these species were -6°C ( S. acaule ), 5°C ( S. me-gistacrotobum ), -4.5°C ( S. commersonii ) and -3°C ( S. polytrichon and S. tubero-sum ) No appreciable species differences were found in FKT when cells were submerged in either buffer or medium and frozen. However, differences did exist when cells were frozen in a non-submerged condition: S. acaule and S. commersonii callus were more sensitive to frost than suspensions, whereas suspensions of the other species were the most sensitive. Measurement of freezing damage by either electrolyte leakage or by 2,3,5-triphenyltetrazolium chloride viability assays revealed similar FKT values. Cell cultures of S. acaule showed better frost tolerance than S. tuberosum (FKT values were -4.5 to -6°C and -2.5°C. respectively), however, frost tolerance of S. megistacrolobum and S. commersonii was only poorly expressed at the cell level (FKT values were between -2 and -3°C). Variant cell lines previously selected for resistance to the amino acid analogues hydroxyprotine, aminoethylcysteine and 5-methyhryptophan appeared to be more tolerant to frost than the wild type S. tuberosum clone.     

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

Treatment of 12-day-old winter wheat (Triticum aestivum) plants with BASF 13-338 {4-chloro-5 (dimethylamino)-2-phenyl-3(2H)-pyridazinone} 36 hours before frost hardening simultaneously and completely inhibits accumulation of linolenic acid in the roots during the hardening period and the acquisition of frost resistance. Increased unsaturation of fatty acids is therefore probably an important part of the mechanism of cold adaptation in winter wheat.