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Seed dormancy is a critical mechanism that delays germination until environmental conditions are favorable for growth. Plant hormones gibberellin (GA) and abscisic acid (ABA) have long been recognized as key players in regulating dormancy and germination. Recent data have increased interest in brassinosteroid (BR) hormones that promote germination by activating GA downstream genes and inactivating ABA signaling. Exposure of imbibed seeds to low temperature (cold stratification) is widely used to release seed dormancy and to improve germination frequency. However, the mechanism by which cold stratification overcomes the inhibitory role of ABA is not completely understood. In the present study, we show delayed germination of seeds of the BR insensitive mutant, bri1-5, that was largely reversed by treatment with fluridone, an inhibitor of ABA biosynthesis. In addition, the bri1-5 seeds were markedly less sensitive to the cold stratification release of dormancy. These results suggest that BR locates upstream of ABA signaling and downstream of cold stratification signaling in dormancy and germination pathways. Consistent with this notion, BR biosynthetic genes, DWF4 and DET2, were upregulated by cold stratification. The transcripts of the GA biosynthesis gene, GA3ox1, and cold responsive genes, CBF1 and CBF2, increased in response to cold stratification in wild type seeds but not in bri1-5 seeds. Conversely, transgenic seeds overexpressing BRI1 germinated more rapidly than wild type in the absence of cold stratification. Thus, we propose that BR signaling plays a previously unrecognized role in the cold stratification pathway for seed dormancy and germination.

The methionine residues of proteins are the preferred targets of oxidation by reactive oxygen species resulting in the formation of methionine sulfoxide (MetSO), which impairs protein function. Methionine sulfoxide reductase A and B (MSR) catalyze the reduction of the MetSO S and R epimers back to Met residues, respectively. The roles of abscisic acid (ABA) and nitric oxide (NO) on the transcript levels of methionine sulfoxide reductase (MSR; EC 1.8.4.6) in the roots of 2-d-old etiolated rice (Oryza sativa L.) seedlings exposed to NaCl were examined. The OsMSR transcript levels increased upon exposure to NaCl, which increased as the NaCl concentrations increased. Fluridone (Flu) pretreatment inhibited the increases in ABA and NO contents and the OsMSRA4, OsMSRA5, OsMSRB1.1, OsMSRB3 and OsMSRB5 transcripts by NaCl, while ABA application reversed the effects of Flu. Flu did not affect the OsMSRA2 and OsMSRB1.2 transcripts. The application of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), to NaCl-treated roots inhibited the increase in all of the OsMSRs transcripts with the exception of OsMSRB1.2. Treatment with the NO donor sodium nitroprusside (SNP) increased all the OsMSRs transcripts. The inhibitory effect of Flu on the increase of the OsMSRA4, OsMSRA5, OsMSR1.1, OsMSRB 3, and OsMSRB5 transcripts in the NaCl-treated roots was reversed by SNP. cPTIO inhibited the expression of all the OsMSR genes. The OsMSRA2.1 and OsMSRB1.2 transcripts can be increased by SNP. The Flu-inhibited internal ABA contents cannot be recovered by treatment with cPTIO or SNP. In addition, NaCl-induced NO production can be divided into ABA-dependent and ABA-independent routes. Therefore, the ABA-dependent NO route regulated the expression of OsMSRA4, OsMSRA5, OsMSRB1.1, OsMSRB 3, and OsMSRB5 in the NaCl-treated rice roots, while the ABA-independent NO pathway modulated OsMSRA2.1, and the ABA-independent and NO-independent pathway modulated OsMSRB1.2 expression in response to NaCl treatment.

Inhibition of lettuce plant growth under increased planting density was accompanied by accumulation of abscisic acid (ABA) in the shoots of competing plants. To check causal relationship between these responses we studied the effect of decreased synthesis of ABA on growth indexes and hormonal balance of lettuce plants under elevated density of their planting (one (single) or three (competing) plants per pot). Herbicide fluridone was used to inhibit ABA synthesis. Preliminary experiments with single plants showed that presence of fluridone in the soil solution at rather low concentration (0.001mg/L) did not affect either chlorophyll content or growth rate of shoots and roots during at least one week. Treatment of competing (grouped) plants with this concentration of fluridone prevented both accumulation of ABA and competition induced growth inhibition. These results confirm important role of this hormone in the growth inhibiting effect of increased planting density. Furthermore, as in the case of ABA, fluridone prevented allocation of indoleacetic acid (IAA) to the shoots of competing plants likely contributing to leveling off the increase in the ratio of leaf area to their mass that is characteristic effect of shading in the dense plant populations. The results suggest involvement of ABA in allocation of IAA in competing plants. Application of fluridone did not influence the concentration of cytokinins in the shoots, whose level was decreased by competition either in fluridone treated or control (untreated with fluridone) plants. Accumulation of ABA in the shoots of competing plants accompanied by inhibition of their growth and the absence of either accumulation of ABA or inhibition of their growth in fluridone treated grouped plants confirms importance of ABA synthesis for growth response to competition.

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.

The physiological and biochemical bases of seed dormancy in Lolium rigidum (annual ryegrass) are largely unknown, and study of this process is complicated by the outcrossing nature of the species and the strong influence of environment on seed dormancy. In order to identify heritable biochemical factors contributing to seed dormancy in L. rigidum, seeds from a field-collected population were used to select sub-populations with consistently low or high seed dormancy over four generations. Low-dormancy seeds showed constitutive alpha-amylase activity prior to imbibition, higher concentrations of polyphenols and cis-zeatin, and lower abscisic acid and cis-zeatin riboside concentrations than high-dormancy seeds. Selection for high dormancy was associated with a reduction in response to dark-stratification for 21d at 20 degrees C (an effective means of releasing dormancy in the original, unselected population) over successive generations, but fluridone remained effective in breaking dormancy. Crossing of low- and high-dormancy populations indicated that dormancy level was not dependent upon the maternal genotype of the seed, and that the constitutive alpha-amylase activity and high seed anthocyanin concentrations characteristic of the low-dormancy populations were not correlated to high basal germination ability.

Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.

Summary Crassulacean acid metabolism induction by water deficit, osmotic or ionic stress, or abscisic acid (ABA) treatment is mediated by a calcium-dependent signaling pathway. To understand the relationship between stress signals and ABA and between the downstream signaling events activated by these stimuli, ABA content was determined in detached Mesembryanthemum crystallinum leaves exposed to osmotic stress (dehydration or mannitol), ionic stress (NaCl) or treated with exogenous ABA. Dehydration and exogenous ABA application led to large increases in ABA accumulation, whereas mannitol treatment caused only a small increase in ABA accumulation. NaCl exposure failed to induce a significant change in ABA accumulation, yet resulted in CAM induction as measured by the increased accumulation of transcripts of Ppc1, which encodes a CAM-specific isoform of phosphoenolpyruvate carboxylase (PEPC). Pretreatment of detached leaves with the ABA biosynthesis inhibitor, Fluridone, failed to inhibit Ppc1 transcript accumulation following stress induction. Taken together, these results suggest that stress-induced CAM gene expression may occur via both ABA-dependent and ABA-independent signaling pathways. Additional pharmacological investigations suggest a role for phosphoinositides in the CAM-induction signaling process. Pre-treatment of detached leaves with cycloheximide inhibited Ppc1 transcript accumulation in response to abiotic stresses and ABA application demonstrating that signaling and/or transcriptional activation events leading to the induction of CAM gene expression are dependent upon protein synthesis.

Summary The effects of salt stress and abscisic acid (ABA) on the expression of betaine aldehyde dehydrogenase (BADH) were determined in sorghum ( Sorghum bicolor L.) plants. BADH mRNA expression was induced by salinity, and the timing coincided with the observed glycinebetaine (betaine) accumulation. The leaf water potential in the leaves of the sorghum plants was significantly affected by salinity. In response to salinity, betaine, ABA, Na and Cl accumulations increased 6-, 16-, 90-, and 3-fold, respectively. In the leaf disks from unsalinized plants incubated on NaCl, or ABA solution, the BADH mRNA level was lower than in the ABA-treated disks. Exogenous application of the ABA biosynthetic inhibitor fluridone to the NaCl-treated disks reduced the ABA accumulation and BADH mRNA levels compared with NaCl-treated leaves. The results indicate that the salt-induced accumulation of betaine and BADH mRNA coincides with the presence of ABA.

Summary A dual role of abscisic acid (ABA) in photoperiodic flowering of Pharbitis nil , cv. Violet was studied. ABA given before an inductive dark treatment lowered the flowering response without influencing the critical night length and the most effective time of a night break. ABA given after a dark treatment accelerated the transport of flowering stimulus and enhanced flower initiation response at shoot apex. Endogenous ABA level in cotyledons was determined by gas chromatography-selected ion monitoring. There was no remarkable difference between ABA levels under short-day and long-day conditions. Fluridone, an inhibitor of ABA biosynthesis, given before and during an inductive dark treatment affected flowering response only slightly. The results indicate that exogenous ABA inhibits the early phase of the flowering without influencing the time-measuring process, and promotes both transport of the flowering stimulus and flower-initiation process, although endogenous ABA plays little role in the regulation of flowering.

Summary Detached spikes from soft white winter wheat ( Triticum aestivum L.) cultivars that produce caryopses with high dormancy levels (Brevor) or low dormancy levels (Greer) were cultured from anthesis in basal media and media amended with fluridone or fluridone plus ABA. Effects of fluridone and exogenous ABA on kernel development and dormancy acquisition and maintenance were assessed by measuring fresh and dry mass of embryos and caryopses, ABA content in embryos and caryopses, and kernel post-desiccation dormancy levels. Caryopses and embryos exposed to fluridone accumulated very little ABA. Detached spike culture increased ABA concentration and reduced fresh and dry mass in both caryopses and embryos. Fluridone did not affect fresh and dry mass. Addition of ABA to culture media slightly reduced media uptake by detached spikes. During desiccation, ABA and dry matter decreased in caryopses whereas embryos continued to accumulate both. After 2d, 95 % of Greer kernels exposed to fluridone germinated while only 80 % of Brevor kernels were germinated after 4 d. Culture of detached spikes in media amended with fluridone and ABA induced higher dormancy levels in Brevor than in Greer. Results from this study support the hypothesis that ABA may be involved in acquisition of dormancy as well as maintenance of dormancy in wheat kernels.

Summary Accumulation of abscisic acid (ABA) in thalli of Riccia fluitans is reduced by a factor of two after 4 days of treatment with 5 μmol/L fluridone. This inhibition, however, cannot be taken as a convincing argument against the direct biosynthetic pathway of ABA. When radioactive abscisic acid is fed to the thalli, phaseic acid (PA) and dihydrophaseic (DPA) acid are formed, similarly as in higher plants. This degradation step is inhibited by tetcyclacis, an inhibitor of P-450 cytochrome monooxygenases. Additionally, an unidentified less polar ABA metabolite appears, indicating a degradation step that may be specific for Riccia . An efflux compartmental analysis with radioactive ABA has been performed with submerged and terrestrial thalli of Riccia fluitans . In both types of thalli ABA concentrations are markedly higher in the cytoplasm than in the vacuole. ABA fluxes across the plasmalemma are higher in the submerged form whereas in the terrestrial form fluxes across the tonoplast are higher. Cytoplasmic ABA of the submerged form can be decreased, both by degradation and by release to the surrounding medium. After conversion to the terrestrial form, however, ABA translocation into the vacuole seems to become more important.

Summary Abscisic acid (ABA) and proline were evaluated during in situ development of anthers and pollen grains of Nicotiana tabacum . No conjugated forms of ABA were detected. The maximum accumulation of free ABA in the whole anther occurred at the mid-binucleate stage (MB), almost 95 % of the ABA being located in the pollen; this ABA distribution was not observed in the microspore (Mi) or mature (Ma) stages of the anther, in which the sporophytic tisue provided more ABA than did the pollen. A maximum in proline accumulation was also found at the MB stage of the anther; however, at the Ma pollen stage (when the ABA was scarcely detectable) the proline accumulation was maximum. The germination of the pollen grain was inhibited by exogenous ABA and stimulated by fluridone, a compound that inhibits carotenoid biosynthesis. The results of the present work suggest an important role for ABA in maturation and germination, as well as in the elongation of the pollen tube of N. tabacum .

Summary Cell suspension cultures of oilseed rape ( Brassica napus L. cv. Kasan) showed a transient increase in cellular abscisic acid (ABA) content at the beginning of the exponential growth phase, with a maximum after 5 days of subculture. In proportion to the endogenous levels, ABA was excreted into the culture medium. Treatment with 10 -4 mol·L -1 fluridone, an inhibitor of ABA biosynthesis, suppressed the rise in ABA levels almost completely. Thus, ABA might be produced in response to osmotic stress resulting from subculturing the cells in fresh medium with higher sugar and mineral concentrations. This hypothesis was supported by the finding that endogenous ABA accumulation correlated with increasing osmotic stress induced by mannitol (from -0.39 to -0.74MPa) in the medium. Concomitantly, cellular water content and the osmotic potential of the cell sap decreased. Application of the triazole-type growth retardant BAS 111 .. W also increased the amount of endogenous ABA, but without changes in the osmotic potential of the medium. The compound is thought to inhibit ABA catabolism to phaseic acid. In contrast, the cyclohexanetrione-type retardants prohexadione calcium and LAB 198 999 did not alter ABA contents significantly. Dose response curves obtained 5 days after treatment with BAS 111 .. W revealed that ABA accumulation in the cells was accompanied by rising potassium and water levels and osmotic potential of the cell sap. The latter effect could be explained by a restricted uptake of sucrose and/or its hydrolysis products glucose and fructose, in BAS 111 .. W treated suspension cells. Calculated on a cell number and cell dry weight basis, ABA, potassium and water content increased to approximately 210, 160 and 140%, respectively, of the control values after treatment with 10 -5 to 10 -4 mol·L -1 BAS 111.. W. A significant increase was also found in magnesium while calcium was only slightly elevated and sodium levels remained unchanged. The changes in the potassium and water contents following the elevated endogenous ABA levels mediated by BAS 111 .. W were thus opposite to those observed when ABA was increased in response to osmotic stress. The specific effects of ABA on these parameters may thus be elucidated without interference from the effects of simultaneous water stress. It is speculated that retardant-caused ABA accumulation influences endogenous potassium, water and sucrose levels and the osmotic potential of the cell sap.

Summary The effects of cytokinins, fluridone, cefotaxime, activated charcoal (AC), abscisic acid (ABA) and silver nitrate (AgNO3) on plant regeneration from 2-month-old callus of perennial ryegrass (Lolium perenne L.) were determined. Numbers of embryogenic sectors in calli increased when the medium was amended with 60 to 200 mg/L cefotaxime. Numbers of regenerated plants increased when the medium was amended with 0.5 mg/L BA, kinetin or zeatin and 2% activated charcoal. Nearly all of these plants were albino. The frequency of green-plant regeneration increased when calli were pretreated with 60 mg/L cefotaxime for 6 weeks prior to placement on regeneration media with 0.5 mg/L fluridone and any of the cytokinins mentioned above. In the absence of the cefotaxime pretreatment, only albino plants were produced, regardless of the fluridone treatment. Protoplasts isolated without agitation and plated by the mixed-nurse method had a plating efficiency of about 3%. Liquid cell suspensions that produced embryos were established from these colonies. Upon transfer to solid regeneration medium only albino plants were produced.

Summary Induction of peroxidase isozymes during shoot enhancement on cotyledon explants of loblolly pine by abscisic acid (ABA) was studied using native polyacrylamide gel electrophoresis (PAGE). Unique peroxidase bands were detected when cotyledon explants were cultured in the presence of exogenous ABA. The number of peroxidase bands increased with the length of exposure to ABA. Explants exposed to fluridone (FLU), an inhibitor of ABA biosynthesis, produced fewer bands showing peroxidase isozyme banding patterns that did not vary regardless of the length of treatment. Four weeks of FLU treatment produced the same peroxidase isozyme band pattern as the ABA-treated explants if exogenous ABA was present in the media for the first two weeks of treatment, but not after.

We describe the involvement of abscisic acid (ABA) in the control of differential growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold dilution of the optimal concentration of nutrient solution inhibited shoot growth, while root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation in favour of the roots. This suggests the involvement of ABA in the redirecting of growth in favour of roots under limited nutrient supply. The ABA content was greater in shoots and growing apical root parts of starved plants than in nutrient sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem apparently contributed to the accumulation of ABA in the apical part of the roots. Thus, partitioning of growth between roots and shoots of wheat plants limited in mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA may originate in the shoots, where its synthesis is stimulated by the loss of leaf turgor.

Summary Embryogenic cell suspensions were established from callus cultures initiated from sections of young leaf tissues of Saccharum officinarum L. (sugarcane). Protoplasts isolated from the cell suspensions were cultured in modified KM or CSK media supplemented with 50 ml coconut milk, 500 mg casein hydrolysate, 1 mg 2,4-D and 0.5 mg BA (per litre). Cell wall regeneration, sustained cell divisions and colony formation were observed in the same culture medium. Dimethyl sulfoxide (1 %) promoted colony formation. Somatic embryos were produced upon transfer of protocolonies to agar-based MS medium plus 1 % activated charcoal and 0.25 – 1 mg · 1 −1 2,4-D, with or without cytokinins. The embryos were germinated in 0.5 mg −1 1 −1 BA and produced several roots and a coleoptile. When placed on MS medium containing 0.5 mg ·1 1 −1 each of BA and fluridone, some of the embryos grew into plantlets with green leaves. Several green plants were grown successfully in soil.

Summary Leaves of Canola rapeseed ( Brassica napus L. cv. Westar) and sugar beet ( Beta vulgaris L. cv. Prima Hill) shoot cultures grown on MS ( Murashige and Skoog, 1962 ) medium supplemented with 0.01-100 µM fluridone, (1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyll-4(1 H)-pyridinone) for 28 days showed a decline in levels of carotenoids and chlorophyll, compared to the control. Shoot cultures of Brassica napus growing on 1-100 µM fluridone also acquired symptoms of vitrification. After one subculture of 28 days on fluridone-free medium, both shoot cultures treated with 0.01-10 µM of fluridone regained normal (close to control) levels of carotenoids and chlorophyll, and the leaves of canola lost excess water. Complete recovery of tissue treated with 100 µM fluridone was achieved in both species only after a second subculture on fluridone-free medium. Abscisic acid content, measured by means of an enzyme-linked immunosorbent assay, showed no direct relationship to the level of carotenoids in either species.