Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern <Emphasis Type="Italic">Cyathea delgadii</Emphasis> Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.     

The effect of 9-(2,3-dihydroxypropyl) adenine (DHPA) on seedling roots ofVicia faba L. in comparison with adenine,adenosine and some cytokinins

3-day-old seedlings ofVicia faba L. were put onto a diluted Knop’s solution containing the tested substances within the intentional concentration range and left there for 7 days. In comparison with controls in plain nutrient solution, the DHPA treated plants revealed a suppression of shoot and root development. The main root growth was less sensitive than root branching. 8 h exposure was sufficient to gain a distinct effect. Microscopically the block of mitosis and/or cytokinesis, the formation of binucleate cells and local tissue damage were revealed. Under the given conditions, the cytokinins (BAP, (9R)BAP, iP, (9R)iP) proved to be much more powerful inhibitors than DHPA, whereas adenine and adenosine were less impairing.     

Changes in cell ultrastructure and endogenous abscisic acid and indole-3-actic acid concentrations in Fatsia japonica leaves under polyethylene glycol-induced water stress

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA = abscisic acid - IAA = indole-3-acetic acid - PEG = polyethylene glycol - = leaf water potential - TEM = transmission electron microscope     

The <Emphasis Type="Italic">trans</Emphasis> and <Emphasis Type="Italic">cis</Emphasis> zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.     

The cytogenetic effect of products of cytosine radiolysis: Isobarbituric acid and dialuric acid

The cytogenetic effect of two radiolytic cytosine products, i.e. of isobarbituric acid and of dialuric acid has been studied on a system of resting meristem ofVicia faba L. on chromosomal level. Both compounds produced in a concentration 10^-3 and 10^-4 M chromosomal aberrations with a relatively low frequency, about 4 aberrations per 100 anaphases after 12 h of treatment. Among the aberration types chromosomal and chromatid breaks and minutes pre-dominated.     

Variations in abscisic acid, indole-3-acetic acid and zeatin riboside concentrations in two Mediterranean shrubs subjected to water stress

Water stress induced an increase in endogenous concentrations of ABA in Lavandula stoechas L. plants to 13100 pmol ABA g^–1 FW, which may contribute to the maintenance of water relations between the second and the third day of water stress treatment. After the third day, a sharp decrease in ABA levels was observed to 2630 pmol ABA g^–1 FW, together with a decrease in water content and water potential and a loss of plant response to water stress. Water deficit did not induce an increase in endogenous ABA concentration, which remained at 514 pmol ABA g^–1 FW in Rosmarinus officinalis L., which is more sclerophyllous than L. stoechas. Nevertheless, the relative water content of Rosmarinus officinalis L. after seven days of water stress decreased more than 40% and reached values of –3.2 MPa. R. officinalis showed lower levels of ABA, but significantly higher levels of IAA and ZR than L. stoechas (4 times and 6 times respectively in well watered-plants). The increase in ABA levels is not a common mechanism in these two Mediterranean shrubs which survive under water stress conditions.Abbreviations ABA abscisic acid - d days of water stress treatment - DW dry weight - FW fresh weight - IAA indole-3-acetic acid - RP Reversed Phase - RWC relative water content - TW turgid weight - WC water content - ZR zeatin riboside - water potential     

The effect of high concentrations of growth substances on water uptake

Changes in water uptake immediately following the application of high concentrations of 2-methyl-4-chlorophenoxyacetic acid (MCPA) into the root medium of whole plants ofPisum sativum andVicia faba were investigated potometrically under controlled conditions. It was found that concentrations between 10^?2 and 10^?5 m bring about a sudden, pronounced and lasting inhibition of water uptake. Its rate depends directly on MCPA concentration. At 10^?2 m, sudden inhibition of water uptake becomes apparent as early as 10 min after application. With decreasing concentrations the appearance of inhibition is retarded but the inhibition is still sudden and well-pronounced. The inhibition of water uptake observed after MCPA application to the root medium of whole plants agrees with the sudden striking reduction in transpiration intensity observed byAllerup (1964) after the application of similar concentrations of 2,4-dichlorophenoxyacetic acid. The presence of MCPA in the nutrient medium causes inhibition of water uptake even in cut plants. Its rate again depends on the concentration of the growth substance. The inhibition does not appear here as quickly and is not as sudden and pronounced. High concentrations of MCPA during application to over-ground plant organs do not bring about immediate inhibition of water uptake by the root.     

Effect of abscisic acid on symbiotic nitrogen fixation activity in the root nodules of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 µM ABA, we obtained mutants that not only showed increased root nodule number, but also enhanced nitrogen fixation. The mutant was designated enf1 (enhanced nitrogen fixation 1) and was confirmed to be monogenic and incompletely dominant.In long-term growth experiments with M. loti, although some yield parameters were the same for both enf1 and wild-type plants, both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared than those traits in wild-type seeds and plants. The augmentation of the weight and N content of the enf1 plants most likely reflects the increased N supplied by the additional enf1 nodules and the concomitant increase in N fixation activity.We determined that the endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than that of wild-type seedlings. When wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), which results in reduced ABA content, the N fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide (NO) in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation, but also nitrogen fixation activity by decreasing NO production in nodules.Key words: Lotus japonicus, symbiotic nitrogen fixation, nitric oxide, ABA, root nodulePhytohormones are known to be important for regulating the number of nodules established on the root of legumes.¹ For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads.^2–4 In contrast, cytokinin is a positive regulator of nodulation. The cytokinin insensitive mutant hit1 (loss-of-function) of Lotus japonicus and the snf2 (gain-of-function) mutants of Medicago truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia.^5–7Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes.^8–11 Recently, Medicago truncatula overexpressing abi1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA signaling pathway,^12,13 was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype.¹⁴In this study, we isolated a Lotus japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and a lowered sensitivity to ABA, and proceeded to carry out its characterization. This mutant, named enf1 (enhanced nitrogen fixation 1) exhibit enhanced symbiotic N fixation activity. Most legume N fixation activity mutants, such as ign1, sen1 and sst1, are Fix-.^15–17At first, to obtain ABA-insensitive or low-sensitive mutants of Lotus japonicus, we treated Miyakojima MG20 with EMS to induce base substitutions randomly in the genome. M3 seeds were sown on an agar-solidified medium containing 70 µM ABA, a concentration that inhibits the germination of wild-type MG20 seeds. M4 plants obtained by the screening were inoculated with rhizobia (Mesorhizobium loti MAFF303099), and the number of nodules per plant was counted 35 days after inoculation (DAI). Plant No. 12 not only formed more root nodules than did the wild-type MG20 plants, but surprisingly it also exhibited increased nitrogen fixation activity per plant. Both mutant phenotypes were stably inherited in the M4 and M5 generation. Back-crossing mutant No. 12 to wild-type MG20 yielded 153 F2 progeny from which a line that showed the highest N fixation activity and more nodules per plant was derived. This line was designated enf1 (enhanced nitrogen fixation 1).At 28 DAI, the number of nodules formed on enf1 roots was approximately 1.7 times greater than that of MG20, and the N fixation activity per enf1 plant was elevated 1.8 times over that of the wildtype plants. Because the N fixation activity per unit of enf1 nodule weight was also increased 1.7 times, we concluded that the increased N fixation activity was not solely due to the enhanced number of root nodules.The endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than those of wild-type seedlings. ABA is believed to regulate early nodulation stages negatively by inhibiting Nod factor signaling, bacterial infection, and nodule initiation.^14,18 Elongated ITs were more common in enf1 root hairs at later stages of development (8–12 DAI). Furthermore, ITs were detected in nodule primordia more frequently in enf1 compared to MG20. These results suggest that the earliest stages of nodule development are not as strongly inhibited in enf1 as they are in wild-type MG20.Because enf1 had a low endogenous ABA concentration, we hypothesized that the decrease in ABA concentration caused the elevation of N fixation activity. To test this hypothesis, we treated wild-type plants at 28 DAI with 20 µM abamine, a specific inhibitor of ABA synthesis.¹⁹ After a three day-treatment period, acetylene reduction activity was measured. Such short treatment periods of abamine are not expected to induce new nodule development. Wild-type plants treated with abamine had a reduced endogenous ABA concentration in roots, to about one-fourth of the level of control plants. However, N fixation activity was elevated to about 170% over the non-treated controls (Fig. 1A and B). This result phenocopies enf1, which shows decreased endogenous ABA concentration as well as elevated N fixation activity. These results strongly suggest that the decrease in endogenous ABA concentration in enf1 was responsible for the increased levels of N fixation activity. Applying 0.5 µM ABA did not result in a further increase in N fixation activity even though the endogenous ABA concentrations are presumed to increase (Fig. 1A and B).Open in a separate windowFigure 1Effects of ABAconcentration on nitrogen fixation activity. M. loti-inoculated plants were grown for 28 days on vermiculite-filled pots supplied with B & D medium. Plant roots 28 DAI were treated with 0.5 µM ABA, 20 µM abamine, with both ABA and abamine, or were untreated (B & D medium control), respectively, for 3 days. (A) ARA per nodule weight. (B) ABA concentration in root. At least 15 plants were used in acetylene reduction assay. Four different plants were used for the measurement of ABA concentration and 3 repeats were performed. Error bars indicate the standard error, and the significance of differences between untreated control and treated values was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (three comparisons in four groups), *p < 0.05, **p < 0.01.Nitric oxide (NO) is known as a strong inhibitor of N fixation activity,²⁰ as well as a signal component in ABA signaling pathway.^21,22 NO production in root nodules formed by enf1 21 DAI and 28 DAI was examined by using the fluorescent dye diaminofluorescein-FM (DAF-FM), a NO specific detector, and relative fluorescence unit (RFU) values were estimated. The RFU values of enf1 nodules 21 DAI were clearly decreased compared with that of MG20; this trend was more obvious at 28 DAI. Moreover, the effect of reduced ABA concentration caused by treatment with abamine on NO production was analyzed (Fig. 2). When nodules formed on the roots of 28-d-old plants were treated, the RFU value of the enf1 mutant was almost the same for (−) abamine and (+) abamine-treated, whereas, the RFU value of abamine-treated MG20 plants was significantly reduced compared to untreated MG20 (Fig. 2). These results strongly suggest that decreased production of NO caused by the low concentration of ABA in enf1 nodules was responsible for the increase in N fixation activity.Open in a separate windowFigure 2NO production in nodules. Quantification of nitric oxide in nodules that were treated with abamine. Nodules on the root of 28-day-old plants were treated with 20 µM abamine for 3 days. Relative fluorescent units (RFU) per nodule fresh weight at 515 nm, normalized against MG20 plants, are shown. The data represent the average ± standard error of 3 independent experiments derived from nodules of 6 to 8 plants. The significance of differences among the four groups was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (six comparisons in four groups) and the different letters refer to significant differences at p < 0.01.Until now, the majority of symbiotic mutants that have been described represents loss of or defects in root nodule formation.^6,23,24 Many of these mutants induce nodules that are Fix-.^15–17 Although reports of mutants that show increased root nodule number^25–28 or spontaneous root nodule formation exist,^7,29 reports concerning mutations where N fixation activity is elevated without deleterious effects on plant growth and development are limited. One exception is the L. japonicus rdh1 mutant, which also exhibits a hypernodulation and enhanced nitrogen fixation phenotype.³⁰In this report, we have shown that mutating the ENF1 gene leads to an elevation of N fixation activity without accompanying adverse growth effects. In long-term growth experiments, some yield parameters were the same for both enf1 and wild-type plants, but both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared to those parameters in wild-type seeds and plants. These results strongly suggest that more nitrogen is fixed in the enf1 mutant than in wild-type plants. Therefore, this gene should be an important target for molecular breeding. We have determined that ENF1 gene is inherited in a monogenic and incompletely dominant manner. Our future work will identify the gene responsible for these positive growth effects.     

Reduced abscisic acid content is responsible for enhanced sucrose accumulation by potassium nutrition in vegetable soybean seeds

In order to understand the physiological mechanism of potassium (K) application in enhancing sugar content of vegetable soybean seeds, pot experiments were conducted in 2014 and 2015 with two vegetable soybean (Glycine max L. Merr.) cultivars (c.v. Zhongkemaodou 1 and c.v. 121) under normal rate of nitrogen and phosphorus application. Three potassium (K) fertilization treatments were imposed: No K application (K0), 120 kg K₂SO₄ ha^?1 at seeding (K1), and 120 kg K₂SO₄ ha^?1 at seedling?+?1% K₂SO₄ foliar application at flowering (K2). Contents of indole-3-acetic acid (IAA), gibberellins (GA), cytokinins (ZR) and abscisic acid (ABA) in seeds were determined from 4 to 8 weeks after flowering. K fertilization increased the contents of IAA, GA, ZR, soluble sugar, sucrose and fresh pod yield, but reduced ABA content consistently. When the contents of soluble sugar and sucrose reached the highest level at 7 weeks after flowering for the 2 cultivars, the contents of IAA、GA、ZR all reached the lowest level in general. The content of ABA in seed was negatively correlated with the sucrose content (P?<?0.01, r = ?0.749**, ?0.768** in 2014 and ?0.535**, ?0.791** in 2015 for c.v.121 and c.v. Zhongkemaodou 1 respectively). The changes in ratio of the ABA to (IAA?+?GA?+?ZR) from 4 to 8 weeks after flowering affected by K application were coincident to the changes of sucrose accumulation. The reduced ratio of ABA/(IAA?+?GA?+?ZR) affected by K nutrition particularly reduced abscisic acid content plays a critical role in enhancing sucrose content, which might be a partial mechanism involved in K nutrition to improve the quality of vegetable soybean.     

Dynamics of growth and the content of endogenous phytohormones during kidney bean scoto-and photomorphogenesis

The dynamics of growth and the contents of free and bound endogenous IAA, gibberellins (GA), cytokinins (zeatin and its riboside), and ABA in kidney bean plants (Phaseolus vulgaris L., cv. Belozernaya) grown in darkness or in the light was studied. Phytohormones were quantified in 5–15-day-old plants by the ELISA technique. Plant growth and phytohormone content were shown to depend on plant age and the conditions of illumination. During scotomorphogenesis, changes in the biomass and hypocotyl length were highly correlated with the content of GA, whereas during photomorphogeneses, these parameters were correlated with the content of zeatin. In darkness, epicotyl growth displayed a positive correlation with the content of GA, whereas in the light, the correlation was negative. Growth characteristics of the primary leaves were shown to correlate with IAA in darkness and with GA and zeatin in the light. At a low concentration of cytokinins in illuminated leaves, cell divisions occurred, whereas, at the higher cytokinin concentrations, cell expansion occurred. The highest content of GA was characteristic of leaves in the period of growth cessation. ABA accumulated during active leaf and root elongation and biomass increment in the light and during hypocotyl growth in darkness. After plant illumination, the ratio of auxins to cytokinins increased in bean roots and decreased in their epicotyls. Thus, light changed the developmental programs of bean plants, which was manifested in the changed rate and duration of growth of various organs (root, hypocotyl, epicotyl, and leaf). Some mechanisms of light action depended on the contents of IAA, ABA, GA, and cytokinins and the ratios between these phytohormones. Differences between scotonorphogenesis of mono-and dicotyledonous plants are discussed in relation to the levels of phytohormones in them.     

Changes in the level of endogenous cytokinins in apical buds ofChenopodium rubrum L.

CCC (2-chloroethyl)trimethylammonium chloride applied to plants ofChenopodium rubrum during floral induction led to an increase in the level of endogenous cytokinins in the apical buds. Application of gibberellic acid or indole-3-acetic acid at concentrations reversing the effect of CCC reduced the level of cytokinins. After simultaneous treatment with both CCC and one of the growth substances this reduction was less pronounced. From the comparison bf the present results, as well as of those published in previous papers it follows that in apical buds ofChenopodium rubrum there exists a mutual interaction between gibberellins and cytokinins. Under certain conditions both these groups of hormones may substitute for each other in flowering. IAA seems to affect flowering by regulating the level of both gibberellins and cytokinins.     

Effects of Chlorocholine Chloride on Phytohormones and Photosynthetic Characteristics in Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

Effects of chlorocholine chloride (CCC) on phytohormones and photosynthetic characteristics of Zhongshu 3, a potato (Solanum tuberosum L.) variety widely cultivated in south China, were studied by foliar CCC application on 24 and 28 days after emergence, that is, at the tuber initiation stage. It was found that on 42 days after emergence, that is, at the tuber bulking stage, spraying CCC increased indolacetic-3-acid (IAA) and zeatin (Z) contents but decreased abscisic acid (ABA) content in leaves. The content ratios of IAA/Z, IAA/ABA, Z/ABA, and (IAA + Z)/ABA in leaves treated with CCC were higher than those of the control. CCC plays a prominent regulating role in the photosynthesis of Zhongshu 3. The net photosynthetic rate (P _n), stomatal conductance (G _s), intercellular CO₂ concentration (C _i), and transpiration rate (T _r) of treated leaves were superior to those of controls at the tuber bulking stage. CCC markedly increased tuber yield and quality. The contents of sucrose and starch in tubers treated with CCC increased at the end of the vegetation period, whereas the contents of reducing sugars and solanine decreased. CCC at 2.0 g L⁻¹ was found to be the most effective concentration. Collectively, the results of this research identify phytohomonal metabolism and photosynthetic physiology of potato leaves as processes affected early after application of CCC resulting in significantly improved increases in tuber yield and quality.     

Changes in the level of endogenous gibberellins and auxins in apical buds ofChenopodium rubrum L. after application of growth substances reversing the effect of (2-chlorethyl)-trimethylammonium chloride (CCC) on flowering

The application of CCC at concentrations inhibiting flowering ofChenopodium rubrum reduces the level of endogenous gibberellins in the apical buds of the plants. The effect of CCC may be reversed by appropriate concentrations of gibberellin (GA-), indole acetic acid (IAA) or kinetin. Kinetin applied to the apical bud during floral induction reduced the level of endogenous gibberellins similarly as CCC and if both CCC and kinetin were applied simultaneously their action was additive. On the other hand IAA applied under the same conditions increased the level of endogenous gibberellins and after joint application of CCC and IAA their level was the same as in untreated control plants. After application of CCC during floral induction the level of endogenous auxins did not change markedly but an active substance “x” appeared on the chromatograms of indole compounds. This substance was found also after simultaneous application of GA- and CCC but not after joint application of CCC and kinetin. If follows from our results that the same morphological phenomenon (flowering) can take place in plants considerably differing as to their level of endogenous growth substances. The ratio of different growth substances is obviously more important than the actual level of the single substances.     

Endogenous growth regulator content in suspension cells of different male and female genotypes of asparagus

The endogenous levels of abscisic acid (ABA), indoleacetic acid (IAA) and of three cytokinins (transzeatin riboside, tZR; isopentenyladenosine, IPA; dihydrozeatin riboside, DHZR) were assayed in suspension cell cultures of male and female plants of asparagus by an immunological method. Assays were carried out on two different materials:

Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh.

Mutant lines of Arabidopsis thaliana (L.) Heynh., which are characterized by symptoms of withering and the absence of seed dormancy, showed much lower levels of endogenous abscisic acid (ABA) in developing seeds and fruits (siliquae) than the wild type. Reciprocal crosses of wild type and ABA-deficient mutants showed a dual origin of ABA in developing seeds. The genotype of the mother plant regulated a sharp rise in ABA content half-way seed development (maternal ABA). The genotype of the embryo and endosperm was responsible for a second ABA fraction (embryonic ABA), which reached much lower levels, but persisted for some time after the maximum in maternal ABA. The onset of dormancy correlated well with the presence of the embryonic ABA fraction and not with the maternal ABA. Dormancy developed in both the absence and presence of maternal ABA in the seeds. In this respect maternal ABA resembled exogenously applied ABA which did not induce dormancy in ABA-deficient seeds. However, both maternal and applied ABA stimulated the formation of a mucilage layer around the testa, which could be observed during imbibition of the mature seeds. In the mature state, ABA-deficient seeds germinated in the siliquae on the plant, but only when the atmosphere surrounding the plant was kept at high relative humidity. In younger stages germination in siliquae occurred after isolation from the plants and incubation on wet filter paper. Therefore, it seems that limited access to water is the primary trigger for the developmental arrest in these seeds.     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

Identification and measurement of indoleacetic and abscisic acids in the cambial region of Picea sitchensis (Bong.) Carr. by combined gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified by combined gas chromatography-mass spectrometry (GCMS) in fractions obtained by diffusion and extraction from bark peelings of Sitka spruce. A procedure is described for the quantitative analysis of IAA and ABA levels in the same extract using the GCMS technique of single-ion current monitoring. This procedure was used to measure the diffusible, free, and bound fractions of IAA and ABA in the cambial region of Sitka spruce throughout one year; the range in concentration for these fractions was 0.06–0.30, 0.46–3.85, and 0.04–0.20 g/g oven-dry weight, respectively, for IAA, and 0–0.08, 0.03–2.21, and 0.13–0.66 g/g oven-dry weight, respectively, for ABA. Movement in the cambial region was found to be polar for endogenous IAA and nonpolar for endogenous ABA. Recoveries of [¹⁴C]IAA internal standards showed that 73–99.5% of the IAA was lost during purification, and that there could be up to 5-fold differences in recovery between purifications, indicating that IAA loss shold be measured in quantitative analyses.Abbreviations ABA aoscisic acid - GCMS combined gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PVP polyvinylpyrrolidone - SICM single ion current monitoring - TMS trimethylsilyl