Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Improvement of Oil Rape Hardening and Frost Tolerance

We studied the chemical composition of autumnal rosette leaves of winter rape hybrid Kasimir in stands of different density and its influence on plant wintering. The average duration of winter wheat growth in autumn was 95.7 days. For this period, rape accumulated 24.7% of dry weight in rosette leaves and 29.7% of dry weight in a terminal bud, whereas the sum of active temperatures was 857.3 °C . The content of dry matter, protein, and sulfur increased with increased crop density. In contrast, the total content of sugars, lipids, potassium, and phosphorus decreased. Tight correlations were observed between crop density and total sugar content (r = −0.877) and between crop density and the N/K ratio (r = 0.920) in the terminal bud. Rape overwintering depended directly on the total level of sugars in the leaves of autumnal rosette (r = 0.684) and in the terminal bud (r = 0.720) and on the lipid content in leaves (r = 0.623) and terminal bud (r = 0.832). The winter rape hybrid overwintered best in stands of low density (21–40 plants/m²) (r = –0.900). Changes in the protein composition in tissues of developing inflorescence and root collar (the appearance of novel proteins, dehydrins in particular, and disappearance of some other proteins) under the influence of auxin-like compounds TA-12 and TA-14 are one of the principal biochemical characteristics of oilseed rape cold hardening.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 532–539.Original Russian Text Copyright © 2005 by Velicka, Rimkeviciene, Novickiene, Anisimoviene, Brazauskiene.     

Cold-induced physiological and biochemical responses of three grapevine cultivars differing in cold tolerance

In this study the cold tolerance potential of three Vitis vinifera cultivars including ‘Red Sultana’, ‘White Sultana,’ and ‘Flame Seedless’ was evaluated under greenhouse condition. After 15 leaves stage in average, the grapevine plants were subjected to cold stress regimes (4, 0 and ? 4 °C) and compared with control plants (24 °C). A clear increase in leaf electrolyte leakage (EL), thiobarbituric acid reactive substances (TBARS), and H₂O₂ concentrations was observed with decreasing temperature from 4 to ? 4 °C in all grapevine cultivars. Chilled plants showed marked increases in their abscisic acid (ABA), soluble sugars, and proline contents in compared to control vines. Upon exposure to cold stress, the EL, TBARS, H₂O₂, and relative water content of ‘Red Sultana’ were found to be lower compared to ‘White Sultana’ and ‘Flame Seedless’. Under 0 °C condition, ‘Red Sultana’ had the highest superoxide dismutase, guaiacol peroxidase and catalase activities, which was approximately twofold higher than those of all other cultivars. Soluble sugars such as glucose, fructose, and sucrose increased from 4 to ? 4 °C. These increments were higher in ‘Red Sultana’ compared to other cultivars which was concomitant with higher accumulation of endogenous ABA concentration in this cultivar. Higher accumulation of ABA and soluble sugars in ‘Red Sultana’ confirmed the key roles of these compounds in cold tolerance which could be applied as a cold tolerance marker for early selection of grapevine cultivars with the aim to establish vineyards in cold winter regions.     

自交对白菜型冬油菜生理生化特性及抗寒性的影响

为了解自交对白菜型冬油菜后代生理生化特性及抗寒性的影响,以6份白菜型冬油菜为材料,研究了在越冬降温前与降温后,自交和开放授粉后代植株叶片生理生化指标及越冬率的变化。结果表明:(1)除MDA含量外,无论是降温前还是降温后,白菜型冬油菜自交后代植株叶片中的SOD、CAT、POD的活性以及可溶性蛋白和游离脯氨酸含量都较开放授粉表现出不同程度的降低,其幼苗越冬率也较开放授粉植株显著降低。(2)与降温前相比,降温后所有测定指标在两种授粉方式后代中都表现为上升的趋势,其中以CAT活性变化最为显著。研究认为,自交会使白菜型冬油菜后代植株叶片中保护酶活性及可溶性蛋白和游离脯氨酸含量明显降低,MDA含量升高,导致幼苗的越冬率显著下降,进而引起其植株抗寒性减弱。     

Changes in the carbohydrate and hormonal status in <Emphasis Type="Italic">Tulipa bifloriformis</Emphasis> bulbs forced into bloom in a greenhouse and in the open ground

Time-course of ABA, cytokinins, monosaccharides (MS), and water-soluble polysaccharides (WSP) contents were followed during cold dormancy period in the bulbs of tulip (Tulipa bifloriformis Vved.) that stayed over winter in the open ground or were cold-forced into bloom in a greenhouse. In both cases, the level of monosaccharides and water-soluble polysaccharides in the storage scale tissues increased, whereas the MS/WSP ratio in the bulbs planted in the open remained essentially the same and in case of forcing treatment decreased almost fivefold. In the apical bud tissues, the level of monosaccharides also rose in both cases, but the MS/WSP ratio in the open was greater throughout the whole experiment. The level of cytokinins in the apical bud tissues in the open was higher than in the forcing case, although the changes in their total content were identical following both treatments. Following the forcing treatment, the contents of free and bound ABA in the apical bud tissues increased reaching their peaks by the end of cold period. In the open, there were two peaks of free ABA: in October (when early frosts occurred) and in March (at the end of the wintering period). The winter forcing treatment resulted in rapid depletion of energy and plastic resources in T. bifloriformis and the death of 20% of embryonic flower buds (in the open, all flower buds survived). Nevertheless, plant adaptation potential ensured the development of generative shoots with 4–5 normal flowers, which makes it possible to use this species of multiflorous tulip for winter forcing in a greenhouse.     

北方寒旱区白菜型冬油菜的越冬安全性及经济效益和生态效益

为了进一步明确白菜型冬油菜在北方寒旱区的越冬安全性、扩大冬油菜的种植区域,本研究通过2008—2013年57点18个白菜型冬油菜品种的多点试验,统计分析不同生态区不同品种越冬率的变化情况.结果表明： 研究期间,北方旱寒区白菜型冬油菜的越冬率在70%～90%,且年份间、品种间变化不明显,稳定性高,经济效益显著.以甘肃天水为起点,含甘肃全部,西藏拉萨、林芝以南,青海民和以东,至新疆乌鲁木齐、拜城,阿勒泰、塔城以南,喀什以东;向东沿黄河一线到宁夏、内蒙古临河以南、陕北、山西祁县周边、河北大名、天津、北京、山东潍坊以北、辽宁葫芦岛南部、吉林延边南部等地为白菜型冬油菜安全越冬地区.陇油6号、陇油7号、陇油8号、陇油9号为安全越冬白菜型冬油菜品种.     

Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Cold acclimation of wheat (Triticum aestivum): Properties of enzymes involved in proline metabolism

Two cultivars of wheat ( Triticum aestivum L.), a winter wheat, Kharkov, and a spring wheat, Glenlea, were acclimated under controlled conditions at 2 temperatures, 5°C and 25°C with a 12-h photoperiod. Water content, protein and proline concentrations were determined. Enzymatic properties (activity and apparent energy of activation) were investigated for enzymatic systems involved in 2 pathways of proline metabolism, the glutamic acid and ornithine pathways. Four enzymes were studied, proline dehydrogenase (PDH, EC 1.5.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), glutamine synthetase (GS, EC 6.3.1.2) and ornithine transaminase (OT, EC 2.6.1.13). Cold acclimation led to an accumulation of proline, a decrease in water content and an increase in soluble protein, especially in winter wheat. For both cultivars, cold acclimation modulated enzyme properties of PDH and GDH. Increased activities of GS and OT were observed as a result of cold acclimation in both cultivars, with the greatest increase in Kharkov. The apparent energy of activation of these 2 enzymes decreased, particularly for Kharkov, which accumulated proline in cold conditions.     

Analogues of auxin modifying growth and development of some monocot and dicot plants

The dependence of morphogenetic processes in the formation of vegetative and generative organs in spring oilseed rape and barley on exogenously applied physiological analogues of auxin: 2,4-D (2,4-dichlorphenoxyacetic acid), NAA (naphthalene-1-acetic acid), TA-12 (1-[2-chloroethoxycarbonyl-methyl]-4-naphthalenesulfonic acid calcium salt) and TA-14 (1-[2-dimethylaminoethoxicarbonylmethyl]naphtalene chlormethylate) were investigated. The experiments were performed with hypocotyl tissue cultures of oilseed rape and barley microspores in vitro. The auxin analogues applied revealed differences of morphogenetic competence in dedifferentiation-redifferentiation processes that occurred in oilseed rape cultures. TA-12 and TA-14 applied together with NAA and BA (6-benzylaminopurine) caused more intensive callus growth in comparison with 2,4-D. Rhizogenesis was induced when 2,4-D was substituted by TA-12. Compound TA-14, unlike TA-12, facilitated the appearance and development of cotyledons in callus tissues. Hower the compounds TA-12 and TA-14 have no positive effect in monocot plant — barly anther culture for callogenesis and regeneration in comparison to indole-3-acetic acid (IAA). TA-14 and TA-12 showed similar but not identical auxin properties and demonstrated high efficiency as modifiers of rape-dicot plant growth and morphogenesis.     

Profiling the Changes of Molecular Species in Membrane Lipids during Cold acclimation in Winter and Spring Cultivars of Rapeseed (Brassica napus)

The changes of molecular species in membrane lipids during cold acclimation (CA) in winter and spring cultivars of rapeseed (Brassica napus) were profiled with ESI MS/MS based lipidomics. The membrane fluidity, the critical properties for plants to tolerate freeing injuring, was examined with double bond index (DBI) and carbon number of the fatty acid of the glycrolipid. The results indicated that the molecular composition of membrane lipids in the two cultivars under both normal growth condition and CA were similar to that of Arabidopsis reported previously and that DBI in spring cultivar accumulation subtly more than that in winter cultivar. The results suggested that CA of rapeseeds needs more than 3 days and that spring cultivar were more sensitive to CA, which was confirmed by the CA induced proline accumulation.     

莼菜冬芽越冬生理研究

按季节测定了莼菜生长周期中可溶性蛋白质、可溶性糖、脯氨酸以及水分的含量 ,测定结果表明冬芽越冬阶段其体内可溶性蛋白质、可溶性糖以及水分的含量较高 ,而脯氨酸却是一年中的最低。这说明冬芽越冬期间其抗寒性的维持可能与体内高浓度的可溶性蛋白质、可溶性糖以及水分有关 ,而与脯氨酸无直接的关系。     

Identification and analysis of cold stress-inducible genes in Korean rapeseed varieties

The rapeseed plant (Brassica napus) is one of the top five oil-producing crops in the world. However, its potential for global cultivation is limited due to its sensitivity to cold. We selected five varieties of rapeseed popular in Korea and analyzed their sensitivity to cold stress. Three — ‘Naehan’, ‘Sandongchae’, and ‘Yudal’ — proved more tolerant than the other two — ‘Youngsan’ and ‘Tammi’. Because rapeseed is in the same family (Brassicacae) as Arabidopsis, one would expect that both share similar genes, with protein coding sequences that show >86% homology. We used a cDNA microarray enriched with Arabidopsis cold stress-related genes to examine the molecular background for differences in sensitivity among rapeseed varieties. Their hierarchical clustering patterns for expression were highly correlated with their cold-sensitive phenotypes. For example, expression patterns were similar between ‘Naehan’ and ‘Sandongchae’ as well as between ‘Yudal’ and our Arabidopsis samples. Using co-expression gene sets developed in this laboratory, we further analyzed expression patterns for three cultivars. Three distinct gene sets were commonly enriched in ‘Naehan’ against ‘Youngsan’ and ‘Tammi’, indicating that they could be the core gene sets that confer a high degree of cold tolerance in ‘Naehan’. Knockout plants with down-regulated expression of GSTF2, TRXh5, LTI30, and LACS4 were highly cold-sensitive whereas overexpression of those four genes led to a reversal of this sensitivity. In fact, overexpression of GSTF2 and LTI30 enhanced tolerance, with plants that over-expressed GSTF2 having extremely high tolerance that enabled them to acclimate almost completely to freezing conditions. By comparison, only about 40% of the wild type demonstrated a similar acclimation. Therefore, the genes described here could be useful in furthering our knowledge about other stress responses in rapeseed, thereby facilitating the production of cold-tolerant plants.     

气候变化背景下中国南方地区季节性干旱特征与适应Ⅱ.基于作物水分亏缺指数的越冬粮油作物干旱时空特征

近年来中国南方地区的季节性干旱频繁发生,对当地越冬作物造成严重影响.本文基于中国南方地区268个气象站点1959-2009年的地面气象观测资料,以作物水分亏缺指数为指标,分析了中国南方地区越冬作物冬小麦和油菜的季节性干旱时空特征.结果表明:对于中国南方地区而言,西南、淮北和华南部分地区的越冬作物在生育阶段内易发生干旱.在长江中下游地区,冬小麦在生育中后期的干旱发生范围和强度增加,且随着生育期后移,其干旱越加严重;油菜在冬前生育阶段和生育后期的干旱发生范围和强度呈增加趋势.在西南地区,冬小麦和油菜在冬前生育期的干旱发生范围和强度均显著增加.20世纪90年代初以来,研究区域冬小麦和油菜在生育阶段中后期发生干旱的范围和强度都增加.     

Exogenous dormancy-breaking substances positively change endogenous phytohormones and amino acids during dormancy release in ‘Anna’ apple trees

A 2-season trial was conducted to verify the effects of foliar applications of some dormancy-breaking substances (DBS) on dormancy release in buds of ‘Anna’ apple (Malus sylvestris, Mill) trees, as well as on metabolic changes in the contents of phytohormones, proline and arginine in buds during their release from dormancy. The efficiency of early bud break induced by Dormex?, potassium nitrate, mineral oil, calcium nitrate and thiourea was noticed in varying degrees. Although Dormex? was distinguished, all DBS hastened bud break, shortened flowering duration, improved bud break% and fruit-set%, increased the contents of gibberellic acid, indole-3-acetic acid, proline and arginine, but reduced abscisic acid content in buds as compared to the control. These results were positively reflected in the final tree yield. Accordingly, it is concluded that the use of Dormex?, at a rate of 4 %, could be recommended for reaching bud break as early as possible and improving ‘Anna’ apple tree yield under the short winters in Egypt and similar regions by regulating the contents of proline, arginine and phytohormones in buds.     

两种冠温型冬小麦籽粒蛋白质和淀粉组分积累动态

 于2001～2003年对河南省主要推广的3个冬小麦 (Triticum aestivum)品种冠温特征、籽粒蛋白质和淀粉组分积累动态进行了研究。结果表明：‘豫麦50’在灌浆后期冠层温度明显降低,表现为冷尾型,而‘豫麦34’、‘豫麦70’冠层温度有上升的趋势,表现为暖尾型 ,在灌浆末期冷尾型与暖尾型小麦冠层温度相差超过2.5 ℃。不同冠温小麦籽粒蛋白质和淀粉组分积累动态表现出差异：暖尾型小麦清蛋白、球蛋白积累量在灌浆后期显著高于冷尾型品种,最终醇溶蛋白积累量品种间差异不显著,麦谷蛋白积累量在花后15 d以前差异不显著,但开花 20 d后暖尾型小麦麦谷蛋白积累量增大,与冷尾型小麦差异达到1%显著水平,最终麦谷蛋白与总蛋白质含量比例亦表现出同样的趋势,麦谷蛋白含量与灌浆中、后期冠层温度和整个灌浆期平均冠层温度均达到显著正相关,相关系数分别为0.7781、0.865、0.968 7;不同冠温型 小麦淀粉组分积累量与直/支差异不大,但淀粉糊化特性表现出明显的差异,除稀懈值外, 暖尾型小麦与冷尾型小麦峰值粘度、低谷粘度、最终粘度、糊化时间、糊化温度和反弹值差异分别达到1%显著水平。     

Metabolic changes associated with cold-acclimation in contrasting cultivars of barley

Cereal plants become more resistant to freezing when first exposed to a period of cold-acclimation. Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance is still poorly understood. Two cultivars of barley ( Hordeum vulgare L.), the winter barley Onice and the spring barley Gitane, were acclimated under controlled conditions under an 8-h photoperiod at 4°C (light) and 2°C (dark) for 21 days. Changes in free proline, ABA, water-soluble carbohydrates and free fatty acids were measured to assess their involvement in cold-acclimation and to explain the different frost-resistant capacities of the two cultivars. Exposure of barley plants to low temperature resulted in an equal increase in proline in both cultivars. During the first days of cold acclimation, ABA levels showed a peak in the frost-resistant cultivar, lasting about 24 h, followed by a decrease. The water soluble carbohydrates reached their highest content after 3 days of hardening, although after 14 to 21 days of acclimation the carbohydrate content was similar to that of unhardened plants. The frost-resistant Onice had a much higher free fatty acid content than the frost-sensitive Gitane. Furthermore in Onice 86% of free farty acids was represented by unsaturated molecular species. Inolenic acid alone being 71%. In contrast, in the frost-sensitive cultivar only 31% of free fatty acids was unsaturated and a large amount of 9-oxo-nonanoic acid, a product present in the linolenic acid cascade, was also detected.
The ABA content after 2 days of hardening and the free fatty acid composition were clearly different between the two cultivars and may explain, at least in part, the different frost-resistant capacities of Onice and Gitane.     

Assessment of proline function in higher plants under extreme temperatures

Climate change and abiotic stress factors are key players in crop losses worldwide. Among which, extreme temperatures (heat and cold) disturb plant growth and development, reduce productivity and, in severe cases, lead to plant death. Plants have developed numerous strategies to mitigate the detrimental impact of temperature stress. Exposure to stress leads to the accumulation of various metabolites, e.g. sugars, sugar alcohols, organic acids and amino acids. Plants accumulate the amino acid ‘proline’ in response to several abiotic stresses, including temperature stress. Proline abundance may result from de novo synthesis, hydrolysis of proteins, reduced utilization or degradation. Proline also leads to stress tolerance by maintaining the osmotic balance (still controversial), cell turgidity and indirectly modulating metabolism of reactive oxygen species. Furthermore, the crosstalk of proline with other osmoprotectants and signalling molecules, e.g. glycine betaine, abscisic acid, nitric oxide, hydrogen sulfide, soluble sugars, helps to strengthen protective mechanisms in stressful environments. Development of less temperature-responsive cultivars can be achieved by manipulating the biosynthesis of proline through genetic engineering. This review presents an overview of plant responses to extreme temperatures and an outline of proline metabolism under such temperatures. The exogenous application of proline as a protective molecule under extreme temperatures is also presented. Proline crosstalk and interaction with other molecules is also discussed. Finally, the potential of genetic engineering of proline-related genes is explained to develop ‘temperature-smart’ plants. In short, exogenous application of proline and genetic engineering of proline genes promise ways forward for developing ‘temperature-smart’ future crop plants.     

葡萄早熟芽变与其亲本果实发育特征比较分析

‘峰早’和‘洛浦早生’葡萄分别是‘巨峰’和‘京亚’的早熟芽变,本研究对比分析了早熟芽变与其亲本间在果实发育过程中与成熟有关的生理指标动态变化的差异。结果表明,与亲本相比,两个早熟芽变品种在果实横径、纵径、单粒重的增长速率,叶绿素、类黄酮和总酚含量变化趋势,以及β-半乳糖苷酶活性等方面的变化趋势差别不大。可溶性固形物和可溶性糖的变化趋势、类胡萝卜素的含量及脂氧合酶（LOX）活性的变化趋势在两对芽变与亲本间无明显一致的趋势,表现出的情形比较复杂。但两芽变品种花色素苷的增长速率在花后50 d均显著快于亲本,且在成熟时的绝对含量均大于对应时期亲本,这可能是早熟芽变品种果实先着色的原因之一。两芽变与亲本间果胶甲酯酶（PE）活性和多聚半乳糖醛酸酶（PG）活性变化模式相一致,且两芽变的PG酶活性增长速率均明显大于其亲本,这种变化幅度也与早熟芽变性状变化的幅度相关,所以PG酶活性增加加快了芽变品种的成熟软化,这可能是芽变比亲本早熟的原因之一。     

仙客来组织培养中再生器官类型及增殖稳定性比较

对‘胜利女神’(Cyclamen persicum cv. Victoria’)和‘大红’(C. persicum cv. ‘Dahong’)两个仙客来品种成苗叶片诱导的愈伤组织,在6-BA与NAA配合的12个分化培养基上继代培养,第一次继代主要产生大量的不定芽芽点,从第二次继代后会得到5种类型的再生器官,分别为不定芽芽点、多芽球茎、多芽新梢、单芽球茎和叶片状芽丛。通过对不同再生器官增殖稳定性的试验表明,‘大红’品种的多芽球茎和多芽新梢在继代培养中再生相同器官的稳定性比较强,由于多芽球茎的增殖率大于多芽新梢,在继代中选择多芽球茎为再生体系较理想;不定芽芽点在以后的继代中会分散产生各种再生器官,不宜作为增殖器官;单芽球茎和叶片状芽丛继代分散性强,为不正常再生器官。‘胜利女神’品种多芽球茎和多芽新梢继代的稳定性比‘大红’品种差,易于产生多种不正常器官。