Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Different properties of the inward rectifying potassium conductance of aleurone protoplasts from dormant and non-dormant barley grains

Isogenic dormant and non-dormant barley grains provide a useful system to study the molecular mechanisms of grain dormancy and the role of plant hormones in this process. As ion fluxes are associated with dormancy-related plant hormone responses, we compared the properties of the inward rectifying potassium conductance in aleurone protoplasts isolated from dormant and non-dormant Triumph grains and in germinating Himalaya grains. Maximal conductance, voltage dependency of steady-state activation, activation and deactivation kinetics were studied in the whole-cell patch-clamp configuration. Activation and deactivation time courses were single exponential. No differences in the above described properties were found between the protoplasts isolated from non-dormant Triumph and Himalaya grains. However, the maximal conductance (corrected for cell size) in protoplasts from dormant Triumph grains was much smaller (65%), and activation time constants were much larger as compared to protoplasts from non-dormant grains. No differences were found in the deactivation kinetics in the three different types of protoplasts. The half-maximal activation potential was slightly more negative in protoplasts from dormant grains than from non-dormant grains.     

Abscisic acid in developing grains of wheat and barley genotypes differing in grain weight

Two genetically related wheat lines growing in cabinets were given different temperatures during grain filling, and abscisic acid (ABA) was measured in whole grains by gas chromatography with an electron-capture detector. Three genetically related barley lines grown in the field were assayed for ABA content in endosperm and embryo fractions separately by radiommunoassay.Maximum grain growth rate and final weight per grain of the two wheat lines differed by 50–60% at low temperature and 30–40% at high temperature. During grain development two peaks in ABA level were observed at low temperature but only one at high temperature. At times when differences in grain growth rate between genotypes and between temperature treatments were large, the corresponding differences in ABA concentration were small. In barley, one line (Iabo 14) had 30% heavier grains than the other two (Onice and Opale). Endosperm ABA concentrations showed no clear differences between genotypes until grain filling was nearly complete. Embryo ABA levels were up to 10-times greater than those in the endosperm, with Opale having significantly less ABA in the embryo than the other two cultivars.Our experiments did not provide evidence for a causal relationship between ABA levels during grain filling and grain growth rate or final weight.Abbreviations ABA Abscisic acid - DAA days after anthesis - DW dry weight - FW fresh weight     

Abscisic acid content and growth of spring barley plants treated with triazoles

Effects of triazolium compounds (paclobutrazol, uniconazole, and azovit), applied under laboratory and greenhouse conditions, on growth of barley plants (Hordeum vulgare L., cvs. Moskovskii 2 and Zazerskii 85) and ABA content in their tissues were studied for the first time. At various application techniques, the retardants suppressed growth of axial organs, promoted thickening and strengthening of lower internodes, and caused a considerable increase in ABA content. These changes were observed already on the next day after spraying plants with retardants and maintained for two weeks after the spraying treatment. At the same time, the retardant action sustained for 42 days when the retardants were applied to soil. The triazoles had positive effects on plant productivity characteristics, such as the number of filled grains in the main spike, total weight of grain per plant (grain collected from the main and lateral spikes), and the weight of 1000 grains. It was concluded that the retardant effect of triazoles on spring barley plants is related to elevated ABA content in plant tissues over a prolonged growth period.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 108–114.Original Russian Text Copyright © 2005 by Chizhova, Pavlova, Prusakova.     

Changes in water relations and endogenous abscisic acid content of wheat and barley grains and embryos during development

Abstract. Immature cereal embryo development can be controlled by in vitro culture on media containing ABA, or by media of low osmotic potential. To assess the possible in vivo roles of these factors, endogenous ABA levels and water relations of embryos and grains of wheat ( Triticum aestivum L.) and barley ( Hordeum vulgare L.) were determined during development. ABA concentrations remained consistent with those required to inhibit precocious germination in vitro of early stage embryos but not of more mature embryos. With increasing maturity, a difference in water potential developed between grain and embryo, suggestive of an in vivo role for water status in controlling the development of the embryo.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

A rule-based model of barley morphogenesis, with special respect to shading and gibberellic acid signal transduction

BACKGROUND AND AIMS: Functional-structural plant models (FSPM) constitute a paradigm in plant modelling that combines 3D structural and graphical modelling with the simulation of plant processes. While structural aspects of plant development could so far be represented using rule-based formalisms such as Lindenmayer systems, process models were traditionally written using a procedural code. The faithful representation of structures interacting with functions across scales, however, requires a new modelling formalism. Therefore relational growth grammars (RGG) were developed on the basis of Lindenmayer systems. METHODS: In order to implement and test RGG, a new modelling language, the eXtended L-system language (XL) was created. Models using XL are interpreted by the interactive, Java-based modelling platform GroIMP. Three models, a semi-quantitative gibberellic acid (GA) signal transduction model, and a phytochrome-based shade detection and object avoidance model, both coupled to an existing morphogenetic structural model of barley (Hordeum vulgare L.), serve as examples to demonstrate the versatility and suitability of RGG and XL to represent the interaction of diverse biological processes across hierarchical scales. KEY RESULTS: The dynamics of the concentrations in the signal transduction network could be modelled qualitatively and the phenotypes of GA-response mutants faithfully reproduced. The light model used here was simple to use yet effective enough to carry out local measurement of red:far-red ratios. Suppression of tillering at low red:far-red ratios could be simulated. CONCLUSIONS: The RGG formalism is suitable for implementation of multi-scaled FSPM of plants interacting with their environment via hormonal control. However, their ensuing complexity requires careful design. On the positive side, such an FSPM displays knowledge gaps better thereby guiding future experimental design.     

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.     

The effect of genotype and environment on the fatty acid content of barley (Hordeum vulgare L.) grains

Abstract A comparison was made of the content of total and some individual fatty acids in grains of nine barley varieties grown at six sites in Belgium. The varieties represented six- and two-rowed winter types and two-rowed spring types. The results showed that the winter types contain more linolenic acid (C18 : 3) than spring types and that six-rowed barleys have less total fatty acids than two-rowed barleys, due mainly to a low concentration of palmitic (C16:0), oleic (CI8 : 1) and linoleic (C18 : 2) acids. Analysis of variance showed that fatty acid content is affected by both the genotype and the environment and multiple regression analysis suggested that weather conditions before and after flowering affected lipid composition.     

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.     

Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid

Viable, long-lived, gibberellic acid (GA₃)-responsive protoplasts have, for the first time, been isolated from aleurone layers of mature wild oat (Avena fatua L.) grain. More than 90% of the cells of aleurone layers are recovered as protoplasts, and these respond to treatment with GA₃ in essentially the same manner as the tissue from which they were derived. Protoplasts become vacuolate during incubation in vitro and, although not dependent upon GA₃, vacuolation is markedly stimulated by the hormone. Amylase and ribonuclease (RNase) are produced and secreted only in the presence of GA₃ and only after lag periods of 3 d and 4 d respectively. The amounts of amylase produced and secreted are proportional to GA₃ concentrations as low as 1.61·10^-13 M. With increasing concentrations of mannitol in the culture medium both vacuolation and the GA₃-induced production and secretion of enzymes are inhibited progressively, the latter being precluded by 0.6 M to 0.7 M mannitol.Abbreviations GA₃ gibberellic acid₃ - RNase ribonuclease     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Polyamine catabolism in dormant embryos of the spindle tree (Euonymus europaeus L.) and in dormancy break obtained after treatment with gibberellic acid

Previously we showed that dormancy break of spindle tree embryos after gibberellic acid (GA₃) treatment was followed by an increase in arginine decarboxylase (ADC) activity (Béranger-Novat N. et al., Plant Sc. 102: 139–145, 1994). These results indicated that arginine decarboxylase pathway mediate hormone-induced growth responses in spindle tree embryos. In the present investigation we show that in GA₃-treated embryos diamine oxidase (DAO) increases immediately after putrescine content and the increase in DAO activity paralleles the accumulation of putrescine at the beginning of the culture (before the visible appearance of the radicle). In this system polyamine oxidase (PAO) increases immediately after DAO activity and follows closely the increase in spermidine content. These results demonstrate a direct correlation between the biosynthesis and oxidation of putrescine and spermidine. At every stage of development DAO and putrescine levels are lower than spermidine and PAO levels. Dormant embryos can be distinguished from GA₃-treated embryos by a complete lack of putrescine accumulation. In dormant embryos compared to GA₃-treated embryos DAO changed more or less in parallel and on the whole seemed to follow the same content and distribution, but the kinetics of the activation of DAOs were different in dormant embryos with a delay of 1.5 day for the first and 1 day for the second peak. During the first days of culture at least up to 4 days the distribution of spermidine and PAO in GA₃-treated embryos followed the same pattern observed in dormant embryos, but the levels of spermidine and PAO were greatly reduced in dormant embryos. On the other hand the kinetics of the activation of PAOs were different in dormant embryos with a delay of 1 day. The results suggest that dormant embryos are deficient in their ability to synthesize polyamines efficiently and support the view that spermidine catabolism (via PAO pool) is limiting in untreated embryos during the first days of culture.     

Novel approaches for examining the effects of differential soil compaction on xylem sap abscisic acid concentration, stomatal conductance and growth in barley (Hordeum vulgare L.)

Novel techniques were devised to explore the mechanisms mediating the adverse effects of compacted soil on plants. These included growing plants in: (i) profiles containing horizons differing in their degree of compaction and; (ii) split-pots in which the roots were divided between compartments containing moderately (1·4 g cm ^{? 3}) and severely compacted (1·7 g cm ^{? 3}) soil. Wild-type and ABA-deficient genotypes of barley were used to examine the role of abscisic acid (ABA) as a root-to-shoot signal. Shoot dry weight and leaf area were reduced and root : shoot ratio was increased relative to 1·4 g cm ^{? 3} control plants whenever plants of both genotypes encountered severely compacted horizons. In bartey cultivar Steptoe, stomatal conductance decreased within 4 d of the first roots encountering 1·7 g cm ^{? 3} soil and increased over a similar period when roots penetrated from 1·7 g cm ^{? 3} into 1·4 g cm ^{? 3} soil. Conductance was again reduced by a second 1·7 g cm ^{? 3} horizon. These responses were inversely correlated with xylem sap ABA concentration. No equivalent stomatal responses occurred in Az34 (ABA deficient genotype), in which the changes in xylem sap ABA were much smaller. When plants were grown in 1·7 : 1·4 g cm ^{? 3} split-pots, shoot growth was unaffected relative to 1·4 g cm ^{? 3} control plants in Steptoe, but was significantly reduced in Az34. Excision of the roots in compacted soil restored growth to the 1·4 g cm ^{? 3} control level in Az34. Stomatal conductance was reduced in the split-pot treatment of Steptoe, but returned to the 1·4 g cm ^{? 3} control level when the roots in compacted soil were excised. Xylem sap ABA concentration was initially higher than in 1·4 g cm ^{? 3} control plants but subsequently returned to the control level; no recovery occurred if the roots in compacted soil were left intact. Xylem sap ABA concentration in the split-pot treatment of Az34 was initially similar to plants grown in uniform 1·7 g cm ^{? 3} soil, but returned to the 1·4 g cm ^{? 3} control level when the roots in the compacted compartment were excised. These results clearly demonstrate the involvement of a root-sourced signal in mediating responses to compacted soil; the role of ABA in providing this signal and future applications of the compaction procedures reported here are discussed.     

Movement of fluorescein into isolated caryopses of wheat and barley

Abstract. The movement of fluorescein, a symplastic fluorescent tracer, into isolated caryopses of wheat and barley is described. The dye followed the pathway to the endosperm which has been proposed previously from anatomical studies, namely a movement from the phloem, through cells of the pigment strand and nucellar projection, followed by a radial spread of the dye from the endosperm cavity into the starchy endosperm. By contrast, the fluorochromes calcofluor white M2R and ANS remained confined to the apoplast and failed to cross the 'xylem discontinuity' at the base of the caryopses.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.