Accumulation of 19-kDa Plasma Membrane Polypeptide during Induction of Freezing Tolerance in Wheat Suspension-Cultured Cells by Abscisic Acid

Suspension-cultured cells derived from immature embryos of winterwheat (Triticum aestivum L. cv. Chihoku) were used in experimentsdesigned to obtain clues to the mechanism of the ABA-induceddevelopment of freezing tolerance. Cultured cells treated with50 µM ABA for 5 d at 23°C acquired the maximum levelof freezing tolerance (LT50; -21.6°C). The increased freezingtolerance of ABA-treated cells was closely associated with theremarkable accumulation of 19-kDa polypeptides in the plasmamembrane. The 19-kDa polypeptide components were isolated bypreparative gel electrophoresis and were further separated intoone major (AWPM-19) and other minor polypeptide components byTricine-SDS-PAGE. N-terminal ami no acid sequence of AWPM-19was determined, and a cDNA clone encoding AWPM-19 was isolatedby PCR from the library prepared from the ABA-treated culturedcells. The cDNA clone (WPM-J) encoded a 18.9 kDa hydrophobicpolypeptide with four putative membrane spanning domains andwith a high pi value (10.2). Expression of WPM-1 mRNA was dramaticallyinduced by 50 µM ABA within a few hours. These resultssuggest that the AWPM-19 might be closely associated with theABA-induced increase in freezing tolerance in wheat culturedcells. (Received January 20, 1997; Accepted March 31, 1997)     

Relationship between Proline and Abscisic Acid in the Induction of Chilling Tolerance in Maize Suspension-Cultured Cells

Both proline and abscisic acid (ABA) induce chilling tolerance in chilling-sensitive plants. However, the relationship between proline and ABA in the induction of chilling tolerance is unclear. We compared the time course of the increase in chilling tolerance induced by proline and ABA, and the time course of the uptake of both into the cultured cells of maize (Zea mays L. cv Black Mexican Sweet) at 28[deg]C. The plateau of proline-induced chilling tolerance preceded by 12 h the plateau of ABA-induced chilling tolerance. The uptake of exogenous ABA into the cells reached a plateau in 1 h, whereas the uptake of exogenous proline gradually increased throughout the 24-h culture period. Although the proline content in ABA-treated cells was 2-fold higher than in untreated cells at the end of the 24-h ABA treatment at 28[deg]C, the correlation between the endogenous free proline content and the chilling tolerance in the ABA-treated cells was insignificant. Isobutyric acid treatment, which resulted in a larger accumulation of proline in the cells than ABA treatment, did not increase chilling tolerance. The induction of chilling tolerance by proline and ABA appeared to be additive. Cycloheximide inhibited ABA-induced chilling tolerance, but it did not inhibit proline-induced chilling tolerance. Newly synthesized proteins accumulate in ABA-treated cells at 28[deg]C while the chilling tolerance is developing (Z. Xin and P.H. Li [1993] Plant Physiol 101: 277-284), but none of these proteins were observed in the proline-treated cells. Results suggest that proline and ABA induce chilling tolerance in maize cultured cells by different mechanisms.     

Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 × 10⁻⁵ molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23°C) for 7 days acclimated to −28°C (LT₅₀) when treated with ABA, or to −5°C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H₂O) or as grams water per gram dry weight (gram H₂O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H₂O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H₂O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H₂O/10⁶ cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10⁶ cells increased to 3.3 milligram/10⁶ cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10⁶ cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Carrier-Mediated Uptake of Abscisic Acid by Suspension-Cultured Amaranthus tricolor Cells

Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K_m of 3.74 ± 0.43 micromolar and an apparent V_max of 1.5 ± 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0°C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by ΔpH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the ΔpH by the proton ionophore carbonylcyanide-m-chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711.     

Alteration of Gene Expression Associated with Abscisic Acid-Induced Chilling Tolerance in Maize Suspension-Cultured Cells

ABA induces chilling tolerance in maize (Zea mays L., cv Black Mexican Sweet) suspension-cultured cells at 28[deg] C when ABA was added to the culture medium at least 6 h prior to chilling (4[deg] C), and this induction can be inhibited by blocking protein synthesis with cycloheximide treatment (Z. Xin, P.H. Li [1992] Plant Physiol 99: 707-711). De novo synthesis of proteins and changes in poly(A+) RNAs were investigated during the ABA induction of chilling tolerance at 28[deg] C as well as during chilling exposure. At 28[deg] C, ABA increased the net synthesis of 11 proteins. Five of these proteins, whose net synthesis was also increased by chilling (4[deg] C), were called group I ABA-induced proteins; the remaining six proteins, whose net synthesis was not altered by chilling, were called group II ABA-induced proteins. Chilling suppressed the net synthesis of three proteins. ABA treatment prior to chilling did not alleviate this suppression. ABA applied at the inception of chilling induced neither chilling tolerance nor accumulation of any of the group II proteins; however, once the group II proteins appeared, they were continually synthesized even in a chilling regimen. ABA induced seven in vitro translation products at 28[deg] C. Three of these products could also be induced by chilling; the remaining four were induced by ABA only at 28[deg] C. These results suggest that ABA-induced alteration of protein synthesis at 28[deg] C is associated with an increased chilling tolerance in maize suspension-cultured cells.     

Role of Abscisic Acid in the Induction of Desiccation Tolerance in Developing Seeds of Arabidopsis thaliana

In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation-specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance.     

Role of the Ring Methyl Groups in Abscisic Acid Activity in Erucic Acid Accumulation in Oilseed Rape (Brassica napus L.)

Modification of the structure of abscisic acid (ABA) has been reported to result in modification of its physiologic activity. In this study we tested the effect of removing methyl groups from the ring and of chirality of ABA on activity in microspore-derived embryos of oilseed rape (Brassica napus L.). The natural (+)-ABA molecule induced growth inhibition and an increase in the amount of erucic acid accumulated in the oil at medium concentrations less than 1 μm. (−)-ABA showed similar effects. Removing the 7′-methyl group resulted in a dramatic decrease in activity: (+)-7′-demethyl-ABA retained some activity as a growth inhibitor; a 10–100 μm concentration of this compound was needed for a response, and (−)-7′-demethyl-ABA was almost completely inactive. Similar effects were observed with regard to elongase activity, which catalyzes erucic acid biosynthesis from oleic acid. Removal of the 8′- and 9′-methyl groups resulted in a more complex response. These compounds all showed intermediate activity; for growth inhibition, the presence of the 9′-methyl was the more important determinant, whereas chirality dominated the response on erucic acid accumulation, with the (+)-enantiomers being more active. Received July 25, 1997; accepted October 31, 1997     

Exogenous Abscisic Acid Mimics Cold Acclimation for Cacti Differing in Freezing Tolerance

The responses to low temperature were determined for two species of cacti sensitive to freezing, Ferocactus viridescens and Opuntia ficus-indica, and a cold hardy species, Opuntia fragilis. Fourteen days after shifting the plants from day/night air temperatures of 30/20[deg]C to 10/0[deg]C, the chlorenchyma water content decreased only for O. fragilis. This temperature shift caused the freezing tolerance (measured by vital stain uptake) of chlorenchyma cells to be enhanced only by about 2.0[deg]C for F. viridescens and O. ficus-indica but by 14.6[deg]C for O. fragilis. Also, maintenance of high water content by injection of water into plants at 10/0[deg]C reversed the acclimation. The endogenous abscisic acid (ABA) concentration was below 0.4 pmol g-1 fresh weight at 30/20[deg]C, but after 14 d at 10/0[deg]C it increased to 84 pmol g-1 fresh weight for O. ficus-indica and to 49 pmol g-1 fresh weight for O. fragilis. Four days after plants were sprayed with 7.5 x 10-5 M ABA at 30/20[deg]C, freezing tolerance was enhanced by 0.5[deg]C for F. viridescens, 4.1[deg]C for O. ficus-indica, and 23.4[deg]C for O. fragilis. Moreover, the time course for the change in freezing tolerance over 14 d was similar for plants shifted to low temperatures as for plants treated with exogenous ABA at moderate temperatures. Decreases in plant water content and increases in ABA concentration may be important for low-temperature acclimation by cacti, especially O. fragilis, which is widely distributed in Canada and the United States.     

Detection of Two Membrane Polypeptides Induced by Abscisic Acid and Cold Acclimation: Possible Role in Freezing Tolerance

Membrane proteins labeled in vivo from cold-acclimated and ABA-treatedalfalfa seedlings of two cultivars differing in cold-tolerancehave been compared by SDS polyacrylamide gel electrophoresisand fluorography. Results thus obtained indicate that severalqualitative changes occur in the membrane protein-profile specificallyin response to cold acclimation or ABA treatment. While somepolypeptides disappear from the non-acclimated protein patterns,others specifically appear in response to acclimation. Separationby two-dimensional gel electrophoresis and fluorography hasconfirmed the above and has enabled us to detect two proteinsof Mr 42 kDa and 120 kDa that are induced by both acclimationand ABA treatment in the freezing tolerant cultivar. (Received November 30, 1987; Accepted February 22, 1988)     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

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)     

Uptake of Abscisic Acid by Suspension-Cultured Phaseolus coccineus L. Cells: Evidence for Carrier Participation

The uptake of abscisic acid (ABA) by suspension-cultured Phaseoluscoccineus L. cv. ‘Prizewinner’ (runner bean) cellshas been studied. In addition to non-mediated diffusive uptakeof ABAH, a saturable component of uptake occurs which is demonstrableas inhibition of uptake of submicromolar concentrations of [2-¹⁴C]ABAby increasing concentrations of nonradioactive ABA to a constantplateau level. Saturation is not due to competition for extracellularbinding sites or cytoplasmic acidification and can only be partiallyaccounted for by saturation of metabolic enzymes. It is consideredlargely to represent carrier-mediated ABA uptake. The maximumvelocity of the carrier is greater at pH 4.0 than at pH 5.0,although the apparent Michaelis constants are similar (about2.0 mmol m^–3). No carrier activity was detectable abovepH 6.0. The carrier is unaffected by indol-3-yl acetic acid(IAA), gibberellin A₃, benzyladenine or 2, 3, 5-triiodobenzoicacid (TIBA). Use of protein-modifying reagents suggested a roleof histidine residues in carrier activity. Reagents expectedto modify the transmembrane pH ( pH) and electrical ( E) gradientswere used to study the driving forces for ABA uptake. Therewas no apparent effect of E, indirectly monitored by effectson an electrogenic TIBA-sensitive component of IAA transport,on ABA uptake. Both diffusive and saturable components weremodified in proportion by changes in pH, suggesting a commonsensitivity to this driving force. The carrier is suggestedto act as an ABA^–/H⁺ symport. Key words: Abscisic acid, Phaseolus coccineus L., Transport, Suspension culture     

The Effect of Abscisic Acid on the Freezing Tolerance of Callus Cultures of Lotus corniculatus L

The effects of growth temperature (2°C and 24°C), abscisic acid (ABA) concentration, duration of exposure to ABA, and light were assessed for their ability to induce acclimation to freezing temperatures in callus cultures of Lotus corniculatus L. cv Leo, a perennial forage legume. The maximal expression of freezing tolerance was achieved on B₅ media containing 10⁻⁵ molar ABA, at 24°C for 7 or 14 days. Under these culture conditions, the freezing tolerance of the callus approximated that observed in field grown plants. In contrast, low temperatures (2°C) induced only a limited degree of freezing tolerance in these cultures. Viability was assessed by tetrazolium reduction and by regrowth of the callus. The two assays often differed in their estimates of absolute freezing tolerance. Regression analysis of the temperature profile suggested that there may be two or more distinct populations of cells differing in freezing tolerance, which may have contributed to the variability between viability assays.     

转vgb基因油菜的耐涝性

油菜是我国重要的油料作物和蛋白质饲料作物,涝害严重影响了我国油菜产业的发展,提高油菜的耐涝能力对于我国油菜可持续发展具有非常重大的意义。本研究采用PCR、Southern杂交等方法对转vgb基因油菜植株进行鉴定,15 d淹涝实验结果显示,转基因油菜的超氧化物歧化酶、丙二醛和脯氨酸含量在淹涝前后的变幅显著低于对照。对淹水后的农艺性状进行调查,转vgb基因的油菜相比较对照抗涝性明显得到增强,证明vgb基因在油菜中的表达对油菜的抗涝性具有显著作用。     

Abscisic Acid and Desiccation Tolerance in Mosses

In higher plants the phytohormone ABA is involved in processes that are connected to water deficit, like stomatal closure or desiccation tolerance. In bryophytes, also containing ABA in their tissues, physiological functions remained uncertain for a long time. Quite recently, several papers have shown different effects of exogenously applied ABA: stomatal closure in Anthoceros, drought hardening in Funaria and production of the landform in Riccia. In all these cases the relevant conditions (water deficit) enhance the endogenous ABA level significantly. For induced desiccation tolerance, ABA serves as a mediator to induce specific proteins (dehydrins) strongly connected with this tolerance. Therefore, it can be concluded that in bryophytes ABA has the same function as in higher plants. It acts as a mediator in stress conditions.