The integrity of metaphase chromosomes of Haplopappus gracilis (Nutt.) gray isolated by flow cytometry

Metaphase chromosomes were obtained from cell suspension cultures of Haplopappus gracilis (Nutt.) Gray using a mass isolation procedure, and were subsequently sorted by flow cytometry. The integrity of the sorted chromosomes, isolated at neutral pH, was examined by electron microscopy and DNA analysis.Electron microscopy showed no significant damage in the DNA leaked out of the chromosomes at high pH. Sedimentation of chromosomal DNA in alkaline sucrose gradients showed the presence of high molecular weight single stranded DNA fragments (mean MW 4–20 × 10⁸ daltons).These characteristics indicate preservation of DNA integrity and general chromosome structure during the synchronization of the cell cycle, the preparation of protoplasts and the subsequent chromosome isolation procedures. Implications for the use of such chromosomes for genetic manipulation are discussed.     

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.     

Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray

Summary Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 m. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Effects of Drought Conditioning on Abscisic Acid Accumulation in the Leaf and Stomatal Response

The effects of a period of water stress (drought conditioning)on responses to a second (challenge) stress were examined inyoung vegetative rice (Oryza sativa L.) plants. Drought conditioningdid not affect the rate of subsequent stress development, nor,in a first experiment, did it influence relations between turgor(_p) and total () leaf water potential. However, conditioningdid extend the range of _p over which stomata remained open andsignificantly reduced the amount of ABA which accumulated inthe leaf at a given _p. The change in stomatal behaviour (stomataladjustment) was quantitatively accounted for by the change inleaf ABA accumulation. The reduction in ABA accumulation due to conditioning did notinvolve a change in the potential capacity to produce ABA, asABA accumulation in partially dehydrated detached leaves wasnot reduced by conditioning. It is suggested that effects ofconditioning on leaf ABA content in the intact plant involvechanges in the rate of ABA export from the leaf. Oryza sativa L, rice, drought conditioning, stomata, water stress, abscisic acid     

Effects of Atmospheric Humidity on Abscisic Acid Accumulation and Water Status in Leaves of Rice (Oryza sativa L.)

Rice (Oryza sativa L ) plants were grown in controlled environmentcabinets with either low (c 0 4 kPa) or high (c 1 6 kPa) atmosphericwater vapour pressure deficit (v p d) The capacity of detachedleaves to accumulate ABA in response to rapidly induced waterstress was increased when plants were grown at high v p d Highv p d significantly lowered solute potential (     

Modification of Leaf Size in Rice (Oryza sativa L.) and its Effects on Water Stress-induced Abscisic Acid Accumulation

The ability of detached leaves of the rice cultivars IR20 and63–83 and their F₂ progeny to accumulate ABA in responseto water stress is negatively correlated with leaf size. Itwas shown that this association was not an artifact of incubationconditions following the imposition of stress. Also, it waspossible to break the correlation by selecting plants in segregatingpopulations which differed in ABA yet had similar leaf size. In further experiments, leaf size was altered phenotypicallyby various treatments; either being increased by gibberellinapplication or periodic removal of tillers, or reduced by priorexposure to water stress or ABA. Although responses to thesetreatments were complex, the results demonstrated that leafsize and accumulation capacity were at least partially independent.It is suggested that the correlations observed previously inF₂ populations from the cross IR20 x 63–83 were a resultof genetic linkage. Oryza saliva L., rice, leaf size, abscisic acid, water stress     

Abscisic Acid Translocation and Influence of Water Stress on Grain Abscisic Acid Content

The movement of foliar applied [1-¹⁴C]abscisic acid (ABA) inwheat plants (Triticum aestivum L., cv. Kolibri) was investigatedat two stages of grain development (1000 grains, weight 19 and24 g dry matter). [1–¹⁴C]ABA seemed to be readily translocated within 12h into the developing grains as well as in other plant parts.A subsequent rapid metabolism took place leading to a decreasedactivity of the ABA-containing chromatogram fraction in theyounger plants 48 h after application. The metabolism seemodto be less intensive in the older grains, where the activityrunning with the ABA increased over 64 h. Treating the leaves of barley plants (Hordeum vulgare, L., cv.Union) 2 weeks after anthesis with a gentle stream of warm air(36° C) resulted in a significant increase in the ABA contentof all parts of the ear. The results mentioned above indicatethat this may be partially due to translocation from other partsof the plant such as the leaves.     

Effects of Water Stress and Abscisic Acid on Transpiration Regulation in Wheat

The transpiration response to recurrent light periods was studied'n water-stressed wheat seedlings. Seedlings were stressed by three methods: addition of mannitol to the root medium, root cooling and drving of the roots in air. All three methods induced almost equal effects on transpiration regulation during alternating dark and light intervals. Exogenous abscisic acid supplied to the shoots of excised plants had qualitatively the same effect as water stress. Water stress and ABA increased the time lapse between light-on and the onset of transpiration increase and lowered the amplitude of transpiration increase in light. Weak light introduced before strong light shortened the delay times.     

Effects of Ultraviolet and X-Ray Radiation on in vitro Cultivated Cells of Haplopappus gracilis

A cell strain of Haplopappus gracills was used for investigations of the effects of UV (2537 A) and X-ray irradiation. Mitotic inhibition and killing after UV exposure were studied. A survival curve of UV treated and then plated cells is presented. The LD₅₀ seems to be about 2000 erg. mm^?2 under the experimental conditions used. All types of chromosome aberrations are induced by UV irradiation, but the frequency is relatively low at doses which do not completely inhibit cell division. A mutant strain of chromosome type is isolated after UV treatment and then plating. Mitotic inhibition and killing after X-ray treatment were studied. A survival curve is presented and the LD₅₀ under the culture conditions used seems to be about 2000 R. The frequency of chromosome aberrations induced by X-rays is highly increased by aeration during X-ray treatment which indicates that some degree of cell anoxia exists in a cell suspension. There arr indications that chromosome aherrations may not cause growth inhibition to such an extent as is usually believed.     

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

Effects of Abscisic Acid and Water Stress on Development and Morphology of Wheat

Experiments were conducted to compare the effects of abscisicacid (ABA) and water stress treatments on leaf morphology andfloral development in a spring wheat. In one experiment injectionsof ABA or a control solution were given twice a week into thebase of the main stem for a period of 3 weeks. In a similarexperiment control plants were watered daily and treated plantswere subjected to water stress by watering only once a week.In both experiments the treated plants produced smaller leavesand fewer spikelets per ear. Analysis of epidermal morphologyusing polystyrene imprints of selected leaf blades from themain stem and a tiller of each plant showed that, compared withcontrol plants, both ABA and water stress decreased the meancell size, reduced the number of stomata per leaf, and increasedthe production of trichomes in all the leaves sampled. Datafor stomatal lengths and stomatal indices showed differencesbetween a main stem leaf and a tiller leaf which were consistentfor both experiments. It is concluded that ABA could mediatemany of the responses of wheat plants to prolonged water stress.The possible adaptive value of these responses is discussed.     

Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis

10⁻⁵M abscisic acid (ABA) completely inhibits germination or (if seeds deprived of integuments are used) embryo elongation in Haplopappus gracilis (Nutt.) Gray. Nevertheless, considerable rates of protein and RNA synthesis were found in embryos grown in abscisic acid, at least during the early hours after sowing. On the contrary, seeds grown in cycloheximide + fusicoccin (a powerful promoter of cell expansion), where protein synthesis is almost completely inhibited, show full protrusion of radicle, thus simulating a "germination" process. These results suggest that some of the most important events involved in seed germination, i.e. protein and RNA synthesis, and cell elongation which leads to radicle protrusion, may not necessarily be linked together and are possibly regulated by different control mechanisms. Moreover, when seeds or embryos are grown in abscisic acid + fusicoccin, protein synthesis is considerable, cell elongation is greater than in water controls at least for 12 h, and germination in its early stages appears to be normal; but DNA synthesis and cell division are not resumed, possibly since some other factor is required. All these findings propose a reevaluation of criteria for defining successful germination.     

Concentrations of Abscisic Acid and Indole-3-Acetic Acid in Soybean Seeds during Development

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in seed parts were determined during reproductive development of soybean plants (Glycine max [L.] Merr. cv `Chippewa 64'). The concentration of ABA and IAA changed independently in individual seed parts with time. Measurement of the level of ABA and IAA in whole seeds masked the changes which occurred in individual seed tissues. The concentration of ABA was generally highest and that of IAA was generally lowest in the embryonic axis of soybean seeds. In the testa, the IAA concentration was generally highest while the ABA concentration was generally the lowest compared to other parts of the seed.     

Abscisic Acid and Water Relations of Rice (Oryza sativa L.): Sequential Responses to Water Stress in the Leaf

Water stress was imposed by withholding water at an early vegetativestage from plants of two rice cultivars (IR20 and 63–83)grown in pots. As stress intensified the following sequenceof responses of the leaves was observed: (i) rise in abscisicacid (ABA) content, (ii) closure of stomata, (iii) initiationof leaf rolling. In both cultivars, turgor (_p) declined linearly with total waterpotential () of the leaf. Bulk leaf ABA content increased linearlyas _p declined, and attained twice the control (unstressed) levelfollowing a reduction in _p of about 0.12 MPa. Stomatal conductance exhibited a sigmoidal relationship to _p,declining abruptly when a particular ‘critical’_p was reached (threshold response). The critical potentialsvaried considerably between experiments, but were closely correlatedwith control potentials and with the potentials at which ABAconcentration doubled relative to controls. Leaf rolling was initiated at s near to zero _p. Increases inthe ratio of adaxial to abaxial conductance were associatedwith rolling. Variations in the above responses could be accounted for byvariations in the rate of stress development, which in termsof reduction ranged from 0.38 to 0.86 MPa day^–1. Fastdrying rates resulted in: (a) reduced osmotic adjustment, (b)increased amounts of ABA in the leaf at a given level of or_p, (c) an increase in the ABA concentration present at 50 percent stomatal closure, and (d) initiation of leaf rolling ata higher . Oryza sativa L., rice, water stress, stomata, leaf rolling, abscisic acid     

Triadimefon Protects Bean Plants from Water Stress through its Effects on Abscisic Acid

Triadimefon is a fungicide that has plant growth regulatingproperties. In beans (Phaseolus vulgaris L.) it significantlyreduced shoot weight, shoot length and leaf area, and rootsappeared whiter and thicker in the treated plants. Chlorophylland carotenoid levels were increased in the leaves, but triadimefondid not affect protein levels in either leaves or roots. Triadimefonreduced transpiration and protected the plants from drought.It increased leaf diffusive resistance indicating partial closureof the stomates, and treated plants maintained their water potentialswhile those of the controls declined. Osmotic potentials ofboth treated and control leaves fell, but values in the controlswere significantly lower than those from the treated plants.Three days after treatment with triadimefon in both water stressedand non-stressed plants the abscisic acid levels in the leavesof the treated plants were more than twice the levels of thecontrols. It appears therefore that the protection conveyedby triadimefon during water stress is mediated at least partially,via its effects on ABA levels in treated tissue. (Received October 12, 1985; Accepted January 8, 1986)     

Effects of Abscisic Acid and Water Stress on the Senescence of Detached Rice Leaves

The effects of abscisic acid (ABA) and water stress on senescence and enzyme activities of oxygen scavenging enzymes of detached rice leaves were compared. Exogenously applied ABA exhibited water stress-like effects by promoting senescence, by decreasing the activities of catalase, peroxidase, ascorbate peroxidase and superoxide dismutase. It seems that the effects of water stress on senescence and enzyme activities are possibly mediated through increased content of endogenous ABA. This revised version was published online in July 2006 with corrections to the Cover Date.     

Germination and Dormancy of Abscisic Acid- and Gibberellin-Deficient Mutant Tomato (Lycopersicon esculentum) Seeds (Sensitivity of Germination to Abscisic Acid,Gibberellin, and Water Potential)

Germination responses of wild-type (MM), abscisic acid (ABA)-deficient (sitw), and gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds to ABA, GA4+7, reduced water potential ([psi]), and their combinations were analyzed using a population-based threshold model (B.R. Ni and K.J. Bradford [1992] Plant Physiol 98: 1057-1068). Among the three genotypes, sitw seeds germinated rapidly and completely in water, MM seeds germinated more slowly and were partially dormant, and gib-1 seeds did not germinate without exogenous GA4+7. Times to germination were inversely proportional to the differences between the external osmoticum, ABA, or GA4+7 concentrations and the corresponding threshold levels that would either prevent ([psi]b, log[ABAb]) or promote (log[GAb]) germination. The sensitivity of germination to ABA, GA4+7, and [psi] varied widely among individual seeds in the population, resulting in a distribution of germination times. The rapid germination rate of sitw seeds was attributable to their low mean [psi]b (-1.17 MPa). Postharvest dormancy in MM seeds was due to a high mean [psi]b (-0.35 MPa) and a distribution of [psi]b among seeds such that some seeds were unable to germinate even on water. GA4+7 (100 [mu]M) stimulated germination of MM and gib-1 seeds by lowering the mean [psi]b to -0.75 MPa, whereas ABA inhibited germination of MM and sitw seeds by increasing the mean [psi]b. The changes in [psi]b were not due to changes in embryo osmotic potential. Rather, hormonal effects on endosperm weakening opposite the radicle tip apparently determine the threshold [psi] for germination. The analysis demonstrates that ABA- and GA-dependent changes in seed dormancy and germination rates, whether due to endogenous or exogenous growth regulators, are based primarily upon corresponding shifts in the [psi] thresholds for radicle emergence. The [psi] thresholds, in turn, determine both the rate and final extent of germination within the seed population.     

Effects of Exogenously Applied Gibberellin and Red Light on the Endogenous Levels of Abscisic Acid in Photoblastic Lettuce Seeds

Endogenous levels of ABA in photoblastic lettuce seeds weredecreased both by red-light irradiation and exogenously appliedgibberellin. Since red light increases endogenous levels ofGA₁ and exogenously applied ABA inhibits both red-light- andgibberellin-induced germination, a decrease in ABA may be importantfor germination. (Received August 27, 1993; Accepted November 5, 1993)