Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures

Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7[prime]hydroxyABA (7[prime]OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures. A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated. Racemic ABA, (+)-ABA, ([plus or minus])-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ([plus or minus])-7[prime]OHBA, (-)-DHABA, ([plus or minus])-ABA ald, and ([plus or minus])-ABA alc had a moderate effect, and PA was inactive. If the relative cellular water content decreased below 82%, dehydrin gene expression increased. Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance. PA had no effect on the induction of RAB proteins; however, ([plus or minus])- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance. Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance. The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.     

Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses

The stress inducibility of dehydrin protein production in seedlingsof castor bean was analysed by subjecting them to ABA and variouswater-deficit-related treatments including desiccation, waterstress, high salt, high osmolarity, and low temperature. A furthergoal was to determine whether the immature seed (at stages priorto major dehydrin synthesis) would respond in a similar mannerto these stresses. A number of dehydrin-like proteins increasedin seedlings subjected to the various stress treatments. Inthe endosperm, these appear to be different from the dehydrin-relatedpolypeptides that are induced during late seed development andwhich persist following germination/growth of mature seeds.In the endosperm of seedlings, ABA, water stress and desiccationinduced the same dehydrin polypeptides, while high osmolarity,high salt and low temperature induced a different set. Stress-specificdifferences in dehydrin synthesis were also found in the cotyledonsand radicle of castor bean seedlings; however, dehydrins indu-cibleby exogenous ABA were consistently produced. Immature seedstreated with ABA or subjected to stress responded by producingdehydrin-like proteins associated with late development; however,the same proteins were induced following detachment of immatureseeds from the parent plant and maintenance on water. When seedlingswere exposed simultaneously to GA and either ABA, high salt,or low temperature, dehydrin production was suppressed. It isconcluded that dehydrin production in castor bean is tissue-specificand is dependent upon the physiological stage of the seed. Inthe endosperm, the response to different stresses may rely uponmore than one signal trans-duction pathway. Key words: Dehydrin, castor bean, ABA, desiccation     

Modulation of Dehydration Tolerance in Soybean Seedlings (Dehydrin Mat1 Is Induced by Dehydration but Not by Abscisic Acid)

Germinated soybean (Glycine max L. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. Increased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin Mat1 mRNA levels. The accumulation of Mat1 mRNA is dehydration dependent but insensitive to ABA. Induction of Mat1 mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.     

Abscisic acid-induced heat tolerance in Bromus inermis Leyss cell-suspension cultures. Heat-stable, abscisic acid-responsive polypeptides in combination with sucrose confer enhanced thermostability.

Increased heat tolerance is most often associated with the synthesis of heat-shock proteins following pre-exposure to a nonlethal heat treatment. In this study, a bromegrass (Bromus inermis Leyss cv Manchar) cell suspension cultured in a medium containing 75 microM abscisic acid (ABA) without prior heat treatment had a 87% survival rate, as determined by regrowth analysis, following exposure to 42.5 degrees C for 120 min. In contrast, less than 1% of the control cells survived this heat treatment. The heat tolerance provided by treatment with 75 microM ABA was first evidenced after 4 d of culture and reached a maximum tolerance after 11 d of culture. Preincubation with sucrose partially increased the heat tolerance of control cells and rendered ABA-treated cells tolerant to 45 degrees C for 120 min (a completely lethal heat treatment for control cells). Comparative two-dimensional polyacrylamide gel electrophoresis of cellular protein isolated from heat-tolerant cells identified 43 ABA-responsive proteins of which 26 were heat stable (did not coagulate and remained soluble after 30 min at 90 degrees C). Eight heat-stable, ABA-responsive proteins ranging from 23 to 45 kD had similar N-terminal sequences. The ABA-responsive (43-20 kD), but none of the control heat-stable, proteins cross-reacted to varying degrees with a polyclonal antibody directed against a conserved, lysine-rich dehydrin sequence. A group of 20- to 30-kD heat-stable, ABA-responsive proteins cross-reacted with both the anti-dehydrin antibody and an antibody directed against a cold-responsive winter wheat protein (Wcs 120). In ABA-treated cells, there was a positive correlation between heat- and pH-induced coagulation of a cell-free homogenate and the heat tolerance of these cells. At 50 degrees C, control homogenates coagulated after 8 min, whereas cellular fractions from ABA-treated cells showed only marginal coagulation after 15 min. In protection assays, addition of heat-stable, ABA-responsive polypeptides to control fractions reduced the heat-induced coagulation of cell-free homogenates. Sucrose (8%) alone and control, heat-stable fractions enhanced the thermostability of control fractions, but the most protection was conferred by ABA-responsive, heat-stable proteins in combination with sucrose. These data suggest that stress-tolerance mechanisms may develop as a result of cooperative interactions between stress proteins and cell osmolytes, e.g. sucrose. Hypotheses are discussed implicating the role of these proteins and osmolytes in preventing coagulation and denaturation of cellular proteins and membranes.     

Effect of Temperature, Light, Nutrients and Dehardening on Abscisic Acid Induced Cold Hardiness in Bromus inermis Leyss Suspension Cultured Cells

The effects of culture conditions on abscisic acid (ABA)-inducedfreezing tolerance were determined in smooth bromegrass Bromusinermis Leyss cv. Manchar) cell suspension cultures. Bromegrasscultures initiated with 2 g fr wt of cells achieved maximumfreezing tolerances (greater than –32?C) at 25 to 30?Cin the presence of 75 to 100 µM ABA. High levels of freezingtolerance induced by ABA were correlated with high growth ratesat 25 and 30?C. In control cells, incubation at 10?C inducedoptimum levels of hardiness with minimal growth. Prolonged exposure(6 weeks) of cells to 3?C, with or without ABA, increased freezingtolerance only by several degrees. Exogenous ABA concentrationsgreater than 100 µM were not inhibitory to growth. Repeatedexposure to ABA, however, retarded growth and made the cellstolerant to temperatures below –40?C. Removal of ABA fromthe medium resulted in dehardening of the cells both at 25 and3?C. Nitrogen had a marginal effect on ABA-induced hardeningat 25?C, but inhibited age-dependent hardening of control cellcultures. Light had no effect on the freezing tolerance of culturedcells. Addition of 10% sucrose, 30 min prior to freezing, tobromegrass cells treated with ABA for 4 days increased freezingtolerance more than 15?C. These observations are discussed inrelation to the contrasting behaviour of the low temperatureand photoperiod dependent cold acclimation of plants (Received July 14, 1989; Accepted October 23, 1989)     

Differential mechanisms to induce dehydration tolerance by abscisic acid and sucrose in Spathoglottis plicata (Orchidaceae) protocorms

Abscisic acid (ABA) and sucrose are known to induce dehydration tolerance of in vitro plant cells and tissues. The present study reports the presence of different mechanisms by which sucrose and ABA improve dehydration tolerance of Spathoglottis plicata (orchid) protocorms. Orchid protocorms were generated aseptically from seeds on Murashig and Skoog medium, and then treated for 7 d in medium containing 10 mg L^?1 ABA and/or 10% (w/v) sucrose. Dehydration tolerance of protocorms was determined at ～25 °C under various drying conditions at relative humidity from 7 to 93%. The actual rate of water loss (i.e. drying rate) was determined using the rate constant of tissue water loss during drying according to the first‐order kinetics. Drying rate affected dehydration tolerance. ABA treatment reduced drying rate and increased dehydration tolerance of protocorms at all relative humidity values tested. However, when compared on the basis of actual drying rates, there was no difference in dehydration tolerance between control and ABA‐treated protocorms, suggesting that ABA‐induced tolerance was correlated with the drying rate reduction. Sucrose treatment was more effective than ABA treatment for the induction of dehydration tolerance. Interestingly, sucrose only slightly affected drying rate. ABA treatment significantly enhanced the synthesis of dehydrin, whereas sucrose treatment primarily resulted in sucrose accumulation. Sucrose treatment also affected protein turnover during drying, causing a significant decrease in protein content in protocorms. Slow drying promoted the degradation of high molecular weight proteins and enhanced the synthesis of low molecular weight dehydrin. The data suggest that different physiological mechanisms are probably involved in the induction of dehydration tolerance by ABA and sucrose treatment.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS) and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass cells.     

Changes in the Translatable RNA Population during Abscisic Acid Induce Freezing Tolerance in Bromegrass Suspension Culture

Abscisic acid (ABA) has been shown to increase freezing toleranceof bromegrass (Bromus in-ermis Leyss cv. Manchar) cell suspensioncultures from a LT₅₀ (the temperature at which 50% cells werekilled) of –7 to – 30?C in 5 days at 23?C. Our objectivewas to study the qualitative changes in the translatable RNApopulation during ABA induced frost tolernace. In vitro translationproducts of poly(A)⁺ RNA isolated from bromegrass cells withor without 75 µM ABA treatment for various periods oftime were separated by 2D-PAGE and visualized by fluorography.SDS soluble proteins from the same treatments were also separatedby 20-PAGE. After 5 days treatment, at least 22 new or increasedabundance SDS soluble polypeptides were observed. From fluorographs,29 novel or increased abundance in vitro translation productscould be detected. The pattern of changes between ABA inducedSDS-soluble proteins and translation products from the 2D gelswere similar. A time course study (0–7 days) showed that17 of the 29 translation products were detected after 1 dayABA treatment, and at least 14 were present after 1 h. Coldtreatment (+4?C) induced fewer changes in the pool of translatableRNA than with ABA treatment. Three translation products inducedby cold appear to be similar to 3 of the ABA induced translationproducts. The majority of the ABA inducible translatable RNAsappeared at 10 µM or higher which coincides with the inductionof freezing tolerance. Many of these ABA inducible RNAs persisted7 days after ABA was removed from the media and correspondinglythe LT₅₀ (–17?C) was still well above the control level(–17?C). The results suggest that ABA alters the poolof translatable RNAs during induction of freezing tolerancein bromegrass suspension culture cells. ¹Oregon Agricultural Experiment Station Technical Paper No.9256. (Received August 3, 1990; Accepted October 18, 1990)     

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.     

脱落酸对水稻抽穗开花期高温胁迫的诱抗效应

为探究脱落酸(ABA)对水稻(Oryzasativa)抽穗开花期高温胁迫的诱抗效应,以江西省主推水稻品种黄华占为材料,于孕穗期用蒸馏水、ABA溶液(10、50和100μmol·L^-1)、氟啶酮(FLU)和原花青素(PC) 6种溶液进行叶面喷施,然后移入对照(CK)和高温胁迫(HS)环境处理8天,考查籽粒活性氧(ROS)积累、抗氧化防御能力、产量构成及相关基因的表达。结果表明,高温胁迫下,水稻的穗长、穗重、结实率、千粒重和产量与超氧阴离子和过氧化氢含量呈显著负相关。高温胁迫下,喷施ABA显著上调了ABA应答和抗氧化防御基因的表达,籽粒中活性氧含量下降了8.24%–31.35%;喷施ABA显著增加了水稻的穗长、穗重、结实率和千粒重,显著上调了产量形成基因的表达,增产12.73%–20.77%。高温胁迫下,喷施FLU可抑制ABA的生物合成,导致ROS过量积累和水稻减产;喷施抗氧化剂PC则抑制ROS过量积累,使产量增加。以上结果表明,高温胁迫下,孕穗期喷施ABA不仅能够激发ABA信号通路,而且上调抗氧化防御能力和产量形成基因的表达,进而提高水稻在抽穗开花期的耐热性,达到增...     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.