Albert J. Robertson

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

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 [mu]M abscisic acid (ABA) without prior heat treatment had a 87% survival rate, as determined by regrowth analysis, following exposure to 42.5[deg]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 [mu]M 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[deg]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[deg]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[deg]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.

Comparison of viability tests for assessing cross-adaptation to freezing, heat and salt stresses induced by abscisic acid in bromegrass (Bromus inermis Leyss) suspension cultured cells

Several viability assays were compared to determine the most sensitive and appropriate method for estimating the freezing, heat and salt tolerance of Bromus inermis Leyss cells cultured with or without 75 μM abscisic acid (ABA) for 4–7 days at 25°C. The sensitivity and reliability of individual viability tests depended on the type of stress applied and degree of injury. Regrowth, amino acid (AA) leakage and fluorescein diacetate (FDA) staining all gave comparable estimates of freezing tolerance. Triphenyl-tetrazolium chloride (TTC) reduction assays slightly overestimated freezing tolerance, but was most convenient. Polypeptide leakage from freeze-thawed cells, as determined by SDS-polyacrylamide electrophoresis (SDS-PAGE), revealed that protein leakage only occurred in ABA-treated cells frozen to −21°C or colder in contrast to control cells which leaked proteins following freezing to −5°C. The most sensitive test for assessing heat tolerance was regrowth, followed by FDA staining, when TTC. TTC tests overestimated heat tolerance compared with the other tests. The degree of overestimation was greater for heat tolerance estimates than for freezing tolerance estimates. It was partially improved by washing the cells prior to TTC assays. AA leakage tests were not appropriate for assessing heat tolerance, due to the erroneously high values of A280 readings obtained. For estimating salt tolerance, TTC assays were most convenient and were in close agreement with regrowth measurements whereas FDA staining tended to overestimate it. In general, while regrowth was most sensitive and reliable, TTC was most convenient. All viability assays consistently showed that ABA induced cross-adaptation to freezing, heat and salt stresses in bromegrass cells without a prior exposure to any of these stresses.

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.

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 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.

Differential Stress Tolerance and Cross Adaptation in a Somaclonal Variant of Flax

Summary Andro flax, a tissue-culture-derived, salt-tolerant selection of McGregor flax, was compared to McGregor for heat and frost tolerance and for germination and emergence rate. To estimate heat and frost tolerance following a controlled artificial stress, the growth of excised shoots was compared with electrolyte leakage from excised leaves and with regrowth of intact plants. Excised shoots of Andro were more heat tolerant than McGregor shoots obtained from plants grown on a salt gradient in the field. The excised-leaf test overestimated heat tolerance in comparison to the excised-shoot test. Preconditioning plants grown at 15°/5 °C (light/ dark) by slow heating (5 °C h -1 ) dramatically increased their heat tolerance with Andro being superior in heat tolerance to McGregor. In field and controlled environment studies, both cultivars were similar in frost tolerance. The excised-shoot test agreed with whole plant freeze tests, whereas the excised-leaf test underestimated freezing tolerance. The germination and emergence rates of Andro were compared to McGregor at seeding depths of 2 and 4 cm over a temperature range of 5 to 15 °C. Andro germinated at a faster rate than McGregor between 5 and 8 °C but not between 10 and 15 °C. Andro emerged earlier than McGregor at both 8 and 15 °C when sown at a 4 cm depth. The above results suggest that selection for one form of stress (e.g. salinity) may also result in increased tolerance to other stresses, e.g. heat and ability to germinate at low temperatures.

Oxidation of the 8'-position of a biologically active abscisic acid analogue

Abstract The metabolism of a biologically active abscisic acid (ABA) analogue, (+)-(1′ S ,2′ S )-2′,3′-dihydroabscisic acid [(+)-4-(1 E ,3 Z )-(

The Effect of Prolonged Abscisic Acid Treatment on theGrowth, Freezing Tolerance and Protein Patterns of Bromus inermis (Leyss) Cell Suspensions Cultured at either 3° or 25°C

Summary The growth, freezing tolerance and two-dimensional protein profiles of bromegrass (Bromus inermisLeyss) cell cultures were investigated following repeated exposure to 75 μM abscisic acid (ABA) for 5 weeks. Following prolonged (36 d) exposure to ABA, freezing tolerance was enhanced and fresh-mass gain was reduced compared to a 10-d ABA treatment. Also, fifteen additional ABA-responsive proteins were detected after 5 weeks of ABA treatment that were not detected in cells treated for 10 d. In addition, intensity changes occurred in several ABA-responsive proteins that were detected after 10-d incubation periods. Two polypeptides (26 and 29 kD) initially responsive to ABA decreased over 5 weeks of treatment. Transfer of ABA-treated cells to culture media lacking ABA for 14 d resulted in a 22°C decrease in freezing tolerance and increased growth, but there was no detectable change in the ABA-responsive polypeptides. Abscisic acid treatment at 3°C increased freezing tolerance at an accelerated rate and to a greater level than 3°C alone, but less so than ABA treatments at 25°C. However, the same ABA-responsive polypeptides detected at 25°C were present in cells treated with ABA at 3°C. These observations suggest that synthesis and accumulation of ABA-responsive polypeptides are only one component involved in freezing tolerance and other factors must be involved.