Regulation of the plasma membrane during exposure to low temperatures in suspension-cultured cells from a cryophyte (Chorispora bungeana)

Summary. As the outermost boundary of the cell, the plasma membrane plays an important role in determining the stress resistance of organisms. To test this concept in a cryophyte, we analyzed alterations of several components in plasma membranes isolated from suspension-cultured cells of Chorispora bungeana Fisch. & C.A. Mey in response to treatment at 0 and −4 °C for 192 h. When compared with the controls growing at 25 °C, both the membrane permeability and fluidity showed recovery after the initial impairment. Linolenic acid and membrane lipid unsaturation increased by about 0.8-fold following cold treatments, although the kinetics of the increase varied with the temperatures examined. During the treatments, the plasma membrane H⁺-ATPase (EC 3.6.1.3) activity increased by 78.06% at 0 °C and 100.47% at −4 °C. However, the plasma membrane NADH oxidase (EC 1.6.99.3) activity only decreased when exposed to a lower temperature (−4 °C), and remained at 63.93% after being treated for 192 h. After the treatments, the physical properties of the plasma membranes of suspension-cultured cells, especially the −4 °C treated cells, were similar to those in the wild plants. These findings indicate that the specific mechanism of cold resistance of C. bungeana is tightly linked with the rapid and flexible regulation of membrane lipids and membrane-associated enzymes, which ensure the structural and functional integrity of the plasma membrane that is essential for withstanding low temperature. Correspondence: Lizhe An, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

Abscisic acid is involved in brassinosteroids-induced chilling tolerance in the suspension cultured cells from Chorispora bungeana

The objective of this study was to investigate whether abscisic acid (ABA), a second messenger in chilling stress responses, is involved in brassinosteroids (BRs)-induced chilling tolerance in suspension cultured cells from Chorispora bungeana. The suspension cells were treated with 24-epibrassinolide (EBR), ABA, ABA biosynthesis inhibitor fluridone (Flu) and EBR in combination with Flu. Their effects on chilling tolerance, reactive oxygen species (ROS) levels and antioxidant defense system were analyzed. The results showed that EBR treatment markedly alleviated the decrease of cell viability and the increases of ion leakage and lipid peroxidation induced by chilling stress, suggesting that application of EBR could improve the chilling tolerance of C. bungeana suspension cultures. In addition, similar results were observed when exogenous ABA was applied. Treatment with Flu alone and in combination with EBR significantly suppressed cell viability and increased ion leakage and lipid peroxidation under low temperature conditions, indicating that the inhibition of ABA biosynthesis could decrease the chilling tolerance of C. bungeana suspension cultures and the EBR-enhanced chilling tolerance. Further analyses showed that EBR and ABA enhanced antioxidant defense and slowed down the accumulation of ROS caused by chilling. However, Flu application differentially blocked these protective effects of EBR. Moreover, EBR was able to mimic the effect of ABA by markedly increasing ABA content in the suspension cells under chilling conditions, whereas the EBR-induced ABA accumulation was inhibited by the addition of Flu. Taken together, these results demonstrate that EBR may confer chilling tolerance to C. bungeana suspension cultured cells by enhancing the antioxidant defense system, which is partially mediated by ABA, resulting in preventing the overproduction of ROS to alleviate oxidative injury induced by chilling.     

Low temperature acclimation to chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling temperature tolerance of rice roots was improved with increasing duration of LT-PT, but LT-PT longer than 12 h gave no additional improvement. Alcohol dehydrogenase activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h. Chilling tolerance was also improved by exogenously applied ethanol. These results suggest that LT-PT may increase chilling tolerance in rice roots owing to ethanol accumulation in the roots and LT-PT acclimation to chilling temperature may occur within 12 h.     

The role of glutathione in yeast dehydration tolerance

Among the factors that affect cell resistance against dehydration, oxidation is considered to be of great importance. In this work, we verified that both control and glutathione deficient mutant strains were much more oxidized after dehydration. Moreover, cells lacking glutathione showed a twofold higher increase in oxidation and lipid peroxidation than the control strain. While glucose 6-phosphate dehydrogenase and glutathione reductase activities did not change in response to dehydration in the control strain, the mutant strain gsh1 (glutathione deficient) showed a reduction of 50% in both activities, which could explain the high levels of oxidation shown by gsh1 cells. In conformity with these results, the mutant lacking GSH1 showed a high sensitivity to dehydration. Furthermore, the addition of glutathione to gsh1 cells restored survival rates to the levels of the control strain. We conclude that glutathione plays a significant role in the maintenance of intracellular redox balance during dehydration.     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

茄子种质资源的耐冷性鉴定

采用低温胁迫下幼苗叶片冷害指数、种子活力指数、叶片电导百分率为指标,对34份不同类型的茄子种质资源进行耐冷性鉴定.结果表明,活力指数与冷害指数、电导百分率均呈极显著负相关,电导百分率与冷害指数呈极显著正相关.通过耐冷隶属函数值和聚类分析,初步筛选出11份耐冷性较强的材料.     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

干旱胁迫对高山离子芥试管苗抗氧化系统及其活性氧代谢的影响

该试验以高山离子芥试管苗(Chorispora bungeana)为试材,采用固液培养法,设置对照(不添加PEG-6000,CK),轻度干旱胁迫(5%PEG-6000)、中度干旱胁迫(20%PEG-6000)、重度干旱胁迫(40%PEG-6000)4个干旱处理水平,分析干旱胁迫对高山离子芥幼苗抗氧化系统、活性氧代谢等部分生理特征的影响,以揭示高山离子芥在干旱胁迫下的生理响应特征,为进一步探讨其对干旱环境的适应机制奠定基础。结果显示:(1)随着干旱胁迫程度的增加以及在各时间胁迫处理下,抗氧化酶SOD活性及可溶性糖含量显著升高,POD活性、丙二醛含量、CAT活性和APX活性均经历了先升后降的过程。(2)超氧阴离子(O-·2)的产生速率和过氧化氢(H2O2)的含量均显著升高;高山离子芥试管苗叶片相对电导率呈现出升-降-升的变化趋势。(3)相关分析结果显示,MDA与相对电导率、可溶性糖、SOD、APX、O-·2及H2O2呈极显著正相关关系,可溶性糖与SOD、POD、O-·2及H2O2呈极显著正相关关系;相对电导率以及保护酶系均与O-·2、H2O2呈极显著正相关关系。研究表明,高山离子芥具有较强的耐旱性,高山离子芥试管苗在响应干旱胁迫过程中,抗氧化酶系、活性氧代谢、脂质过氧化及渗透调节物等共同参与了高山离子芥试管苗对干旱胁迫的综合抗逆性形成,从而积极启动应对外界干旱环境的耐旱响应机制。     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Coordination of light,circadian clock with temperature: The potential mechanisms regulating chilling tolerance in rice

Rice (Oryza sativa L.) is a major staple food crop for over half of the world's population. As a crop species originated from the subtropics, rice production is hampered by chilling stress. The genetic mechanisms of rice responses to chilling stress have attracted much attention, focusing on chilling‐related gene mining and functional analyses. Plants have evolved sophisticated regulatory systems to respond to chilling stress in coordination with light signaling pathway and internal circadian clock. However, in rice, information about light‐signaling pathways and circadian clock regulation and their roles in chilling tolerance remains elusive. Further investigation into the regulatory network of chilling tolerance in rice is needed, as knowledge of the interaction between temperature, light, and circadian clock dynamics is limited. Here, based on phenotypic analysis of transgenic and mutant rice lines, we delineate the relevant genes with important regulatory roles in chilling tolerance. In addition, we discuss the potential coordination mechanism among temperature, light, and circadian clock in regulating chilling response and tolerance of rice, and provide perspectives for the ongoing chilling signaling network research in rice.     

Relationship between ABA and chilling injury in mangosteen fruit treated with spermine

Theeffects of spermine on abscisic acid (ABA), hardening, and browning in storedmangosteen fruit were investigated. The hardening and browning, which areassociated with chilling injury (CI), were observed in only the skin of fruitstored at 7 °C. However, the hardening of skin was notaccompanied by moisture loss. The spermine treatment decreased the browning andhardening of the skin and extended storage time. Carbon dioxide(CO₂)production from stored fruit gradually increased with d in storage(DIS). The increase of CO₂ may be associated with the moisture lossbecause these levels coincided. ABA concentrations in the skin were highest infruit stored at 7 °C, followed by spermine treatment at 7°C, and the lowest at 13 °C. That is, thespermine treatment inhibited the increase of ABA in the skin of stored fruit.ABA concentrations in the skin may be associated with the degree of CI becausetheir fluctuations coincided. ABA metabolism in fruit stored at 7°C or 13 °C was also examined. The PA-DPApathway may not be the primary pathway of ABA metabolism because theconcentrations of PA and DPA were very low compared with those of ABA. ABAconcentrations in the aril were not significantly different between 7°C and 13 °C. This may be related to the lackof CI observed in the aril. ABA metabolism was different at each temperature.The decrease of ABA and the increase of DPA correlated at 13°C, however this correlation was not observed at 7°C. ABA metabolism may be influenced by temperature.     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.     

The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism

Both glutathione and an NADPH-dependent glutathione reductase are present in spinach (Spinacia oleracea L.) chloroplasts. It is proposed that glutathione functions to stabilise enzymes of the Calvin cycle, and it may also act to keep ascorbic acid in chloroplasts in the reduced form.Abbreviations GSH tripeptide glutathione - GSH reduced form of glutathione - GSSG oxidised form of glutathione     

The inheritance of chilling tolerance in tomato (Lycopersicon spp.)

During the past 25 years, chilling tolerance of the cultivated (chilling-sensitive) tomato Lycopersicon esculentum and its wild, chilling-tolerant relatives L. peruvianum and L. hirsutum (and, less intensively studied, L. chilense) has been the object of several investigations. The final aim of these studies can be seen in the increase in chilling tolerance of the cultivated genotypes. In this review, we will focus on low-temperature effects on photosynthesis and the inheritance of these traits to the offspring of various breeding attempts. While crossing L. peruvianum (male symbol) to L. esculentum (female symbol) so far has brought the most detailed insight with respect to physiological questions, for practical purposes, e.g., the readily cross ability, crossing programmes with L. hirsutum as pollen donor at present seem to be a promising way to achieve higher chilling-tolerant genotypes of the cultivated tomato. This perspective is due to the progress that has been made with respect to the genetic basis of chilling tolerance of Lycopersicon spp. over the past five years.     

Plasma-Membrane lipid composition and ethanol tolerance inSaccharomyces cerevisiae

Populations of cells suspended anaerobically in buffered (pH 4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration.     

The sibling species Drosophila melanogaster and Drosophila simulans differ in the expression profile of glutathione S-transferases

Two major forms of glutathione S-transferase are known in Drosophila melanogaster: GST D and GST 2. In the present paper we report the existence of a third major form of glutathione S-transferase in Drosophila simulans. Induction with phenobarbital revealed a different regulation of GST between these species. Despite the fact that these two species are closely related, there was a difference in the expression profile of the enzyme implicated in the detoxification system, suggesting variations in capacity to suit their environment.