Ronald W. Wilen

A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis)

The objective was to investigate the expression of a lipid transfer protein gene (LTP) both in bromegrass (Bromus inermis) cells and seedlings after exposure to abiotic stresses, abscisic acid (ABA), anisomycin, and sphingosine. A full-length cDNA clone BG-14 isolated from bromegrass suspension cell culture encodes a polypeptide of 124 amino acids with typical LTP characteristics, such as a conserved arrangement of cysteine residues. During active stages of cold acclimation LTP expression was up-regulated, whereas at the final stage of cold acclimation LTP transcript level declined to pre-acclimation level. A severe drought stress induced the LTP gene; yet, LTP expression doubled 3 d after re-hydration. Both temperature and heat shock duration influence LTP induction; however temperature is the primary factor. Treatment with NaCl stimulated accumulation of LTP mRNA within 15 min and the transcripts remained at elevated levels for the duration of the salinity stress. Most interestingly, Northern blots showed LTP was rapidly induced not only by ABA, but also by anisomycin and sphingosine in suspension cell cultures. Of the three chemicals, ABA induced the most rapid and highest response in LTP expression as well as highest freezing tolerance, whereas sphingosine was the least active for both LTP expression and freezing tolerance.

Competitive Inhibition of Abscisic Acid-Regulated Gene Expression by Stereoisomeric Acetylenic Analogs of Abscisic Acid.

The properties of two enantiomeric synthetic acetylenic abscisic acid (ABA) analogs (PBI-51 and PBI-63) in relation to ABA-sensitive gene expression are reported. Using microspore-derived embryos of Brassica napus as the biological material and their responsiveness to ABA in the expression of genes encoding storage proteins as a quantitative bioassay, we measured the biological activity of PBI-51 and PBI-63. Assays to evaluate agonistic activity of either compound applied individually showed a dose-dependent increase in napin gene expression on application of PBI-63. Maximal activity of about 40 [mu]M indicated that PBI-63 was an agonist, although somewhat weaker than ABA. PBI-63 has a similar stereochemistry to natural ABA at the junction of the ring and side chain. In contrast, PBI-51 showed no agonistic effects until applied at 40 to 50 [mu]M. Even then, the response was fairly weak. PBI-51 has the opposite stereochemistry to natural ABA at the junction of the ring and side chain. When applied concurrently with ABA, PBI-63 and PBI-51 had distinctly different properties. PBI-63 (40 [mu]M) and ABA (5 [mu]M) combined gave results similar to the application of either compound separately with high levels of induction of napin expression. PBI-51 displayed a reversible antagonistic effect with ABA, shifting the typical ABA dose-response curve by a factor of 4 to 5. This antagonism was noted for the expression of two ABA-sensitive genes, napin and oleosin. To test whether this antagonism was at the level of ABA recognition or uptake, ABA uptake was monitored in the presence of PBI-51 or PBI-63. Neither compound decreased ABA uptake. Treatments with either PBI-51 or PBI-63 showed an effect on endogenous ABA pools by permitting increases of 5- to 7-fold. It is hypothesized that this increase occurs because of competition for ABA catabolic enzymes by both compounds. The fact that ABA pools did not decrease in the presence of PBI-51 suggests that PBI-51 must exert its antagonistic properties through direct competition with ABA at a hormone-recognition site.

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.

Effects of abscisic acid (ABA) and ABA analogs on freezing tolerance, low-temperature growth, and flowering in rapeseed

Brassica napus and B. campestris are grown in Western Canada in areas subject to unseasonable frosts. At the seedling stage, cultivars of Brassica are very sensitive to frosts of -2° to-5°C, which are either lethal or delay the development of the plant. Seedlings of B. napus and B. campestris, germinated and grown at 10°C (16-h photoperiod), were treated with a foliar spray of either 100 μM racemic abscisic acid (ABA), 100 μM of various ABA analogs, 0.1% acetone, or were untreated. Freeze tests indicated 2°C of frost tolerance could be gained in B. napus following an application of three ABA analogs. In B. campestris, three analogs also increased freezing tolerance approximately 1.5°C. The analogs 2′,3′ dihydro ABA and acetylenic divinyl methyl-ABA were effective in both species. Plant fresh weight and dry weight increased in treated plants relative to control or acetone-treated plants after 3 weeks at 10°C. The effect of frost and/or analog treatment on flowering was determined in both species. In B. campestris and B. napus, a mild frost advanced flowering by approximately 2 days compared with nonfrozen control plants. The promotive effect of frost on flowering decreased with increasing severity of the frost. Several of the analog treatments, particularly 2′,3′ dihydro ABA and acetylenic divinyl ABA, advanced flowering by 2–3 days in both species. The benefit of these ABA analog treatments on flowering was enhanced additionally by a mild frost. Plants treated with either ABA, 2′,3′ dihydro ABA, 2′,3′ acetylenic dihydro ABA, or acetylenic divinyl ABA flowered up to 5 days earlier than control plants.

Dehydrin gene expression and leaf water potential differs between spring and winter cereals during cold acclimation.

Summary Spring and winter cultivars of wheat (Triticum aestivum L.) and rye (Secale cereale L.) were cold acclimated using controlled environment and natural conditions. With respect to freezing tolerance, winter cereal seedlings could be distinguished from their spring counterparts by their initiation of acclimation at a warmer temperature, increasing in freezing tolerance sooner, and by achieving greater freezing tolerance at the end of the acclimation period. The timing and extent of expression of a family of dehydrin genes correlated with the increase in measured freezing tolerance in both spring and winter genotypes. The expression of these genes was detected sooner in the winter types, and dehydrin mRNA accumulated to higher levels in the winter cereals. Dehydrin transcripts could be detected throughout the acclimation period in winter cereals, but were only moderately expressed in spring cereals in response to acclimation. Similar results were obtained using western blot analysis with a dehydrin carboxy terminal antibody. Crown moisture content (CMC), crown osmotic potential (COP) and leaf water potential decreased in spring and winter cereals in response to acclimating conditions in both controlled environment and field conditions, but were lowest in fully acclimated winter cereals. However, the onset and rate of decrease in CMC and COP did not differ between the spring and winter genotypes, suggesting that neither CMC nor COP were involved in the initial regulation of dehydrin gene expression. Leaf water potential (LWP) also declined at similar rates in the spring and winter cereals in the field between September and November. However, a difference in LWP was observed between spring and winter wheat subjected to a cold shock treatment. The winter genotype LWP decreased within 10 h of exposure to 2 °C, reached significantly lower levels than prior to the cold shock, but returned to pre cold-shock level after 7 days at 2 °C. In contrast, the decline in leaf water potential in spring wheat was slower and less pronounced than in winter wheat. These results correlate well with those observed with dehydrin gene expression and suggest a relationship between water potential and cold-induced gene expression.

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, 2′3′ 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 μM (−) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 μM (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 μM. Pretreatment of cells with (−) DHA (20 or 50 μM) had no effect on freezing tolerance when 25 μM (+) ABA was added. The induction of freezing tolerance by 25 μM (−) ABA was completely inhibited by the presence of 20 μM (−) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 μM (−) DHA in cells treated with 2.5 or 7.5μM (+) ABA, and in cells treated with 25 μM (−) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 μM. 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.

A Comparison of the Cold Hardiness Potential of Spring Cereals and Vernalized and Non-Vernalized Winter Cereals

Both spring and winter cereals cold acclimate in response to low temperatures, however the level of freezing tolerance attained is dramatically different. Winter rye and winter wheat seedlings have the genetic potential to cold acclimate to −30°C and −25°C, respectively. In contrast spring cereal seedlings can only cold acclimate from −7 to −9°C. Genetically winter and spring cereals are similar except winter cereals must be vernalized to initiate the reproductive cycle. A strong association has been established between the degree of vernalization and the degree of freezing tolerance that can be achieved in cereal seedlings. The freezing tolerance, water potential and expression of dehydrin transcripts of seedlings of spring, non-vernalized and vernalized winter cereals was determined using both controlled environment chambers and natural conditions. Winter cereal seedlings rapidly acclimate in response to environmental cues whereas temperatures approaching 0°C are required to induce freezing tolerance in spring cereal seedlings. In contrast to non vernalized seedlings, vernalized seedlings of Puma rye and Norstar winter wheat only acclimate to the same level as spring cereals (−7 to −9°C). The water potential of non vernalized winter cereal seedlings rapidly decreases within 12 hours of exposure to hardening conditions. In contrast, there is little or no decrease in the water potential in spring and vernalized winter cereal seedlings. During the acclimation period, crown moisture content decreased in both vernalized and non vernalized winter seedlings and in spring seedlings, however the largest decrease occurred in the non vernalized seedlings. Northern analysis revealed significant accumulation of dehydrin transcripts in non vernalized seedlings, however there was only a transient increase in transcripts in the spring cereal seedlings. Little or no expression of dehydrin transcripts was detected in vernalized seedlings exposed to hardening conditions. In summary, non vernalized winter cereal seedlings have the ability to decrease their water potential and accumulate dehydrins upon exposure to cold hardening conditions. In contrast, vernalized winter cereal seedlings respond similar to spring cereal seedlings when exposed to low temperature hardening conditions.