Onset of desiccation tolerance during development of the barley embryo

We have investigated events which take place in the developing barley (Hordeum vulgare L.) embryo during its acquisition of desiccation tolerance. Excised embryos are capable of precocious germination as early as 8 d after pollination (DAP). At this age, however, they are not capable of resisting a desiccation treatment which induces a loss of 96–98% of their initial water content. At 16 DAP the embryos germinate despite the drastic drying treatment. The pattern of in-vivo and in-vitro proteins synthesized by the developing embryos from 12 DAP (desiccation-intolerant) and 16 DAP (desiccation-tolerant) were compared. A set of 25–30 proteins was identified which is denovo synthesized or enhanced during the developmental period leading to desiccation tolerance. Abscisic acid (ABA; 100 M) applied in vitro for 5 d to 12-DAP embryos induces desiccation tolerance and represses a subset of polypeptides preferentially associated with 16-DAP embryos. During in vitro culture of barley embryos ABA stimulates the appearance of a set of proteins and prevents the precocious germination allowing embryogenesis to continue in vitro. It also suppresses a set of germination-related proteins which appear 4 h after the incubation of the dissected embryo on a germination medium without ABA. Almost all mRNAs remain functional for translation when isolated embryos are dried at the desiccation-intolerant and tolerant stages of embryo development.Abbreviations ABA abscisic acid - DAP days after pollination - GM germination medium - poly(A)RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Observations on dividing plastids in the protonema of the moss Funaria hygrometrica Sibth.

The process of division was investigated in the different types of plastids found in the tip cell of the protonema of Funaria hygrometrica Sibth. There were no structural changes in the envelope membranes of any of the plastid types during the initial stage of division. As the process of constriction advanced, thylakoids were locally disintegrated and sometimes starch grains in the isthmus were locally dissolved. In the isthmus, tightly constricted plastids were characterized by an undulating envelope and an increasing number of vesicles. After three-dimensional reconstruction of electronmicrographs a distinct filamentous structure was observed in the plane of division outside the plastid but close to the envelope. At different stages of division the constricted regions were partly surrounded by one or a few filaments. The roundish plastids in the apical zone were accompanied by single microtubule bundles, and the spindle-shaped plastids in the cell base were surrounded by single microtubules and microtubule bundles. A model of co-operation between microtubules and the filamentous structure in the division process is discussed.A preliminary report was presented at the Tagung der Deutschen Botanischen Gesellschaft und der Vereinigung für Angewandte Botanik, Hamburg, September 1986     

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D₂O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.Abbreviations DMSO dimethylsulfoxide - CB cytochalasin B Dedicated to Prof. Dr. A. Pirson on the occasion of his 70. birthday     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine     

Abscisic acid and the induction of desiccation tolerance in the extremely xerophilic liverwortExormotheca holstii

Abscisic acid (ABA) induces formation of a set of proteins in the xerophilic liverwortExormotheca holstii. Some of them have immunological properties similar to the dehydrins of desiccated corn embryos and the desiccation-related proteins ofCraterostigma plantagineum. The fluctuations of endogenous ABA during cycles of desiccation and rehydration seem to be sufficiently high to indicate a role for ABA as a stress hormone and there by as an endogenous inductor of stress-related protein synthesis. Desiccation tolerance disappears when thalli are cultivated for a longer period under well-watered conditions; such thalli are not able to increase stress-dependent ABA biosynthesis sufficiently, or to form the desiccation-related proteins unless they are treated with external ABA. The rehydrated thalli cannot recover from a rapid water loss, while ABA-treated, non-hardened thalli regain their photosynthetic activity within two hours.Abbreviations ABA abscisic acid - F_o initial fluorescence yield - F_m maximum fluorescence yield - Q_A primary quinone receptor of PSII We are grateful to Deutsche Forschungsgemeinschaft for financial support (SFB 251, TP3, Graduiertenkolleg Ka 456/5-1), to Prof. E.W. Weiler (Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, Bochum, Germany), Dr. D. Bartels (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) and Dr. T.J. Close (Department of Botany and Plant Science, University of California, Riverside, Calif., USA) for generous gifts of immunochemicals for ABA assay and antibodies 6–19, 37-31 and Rb-2b, to Miss. B. Dierich for skilful technical assistance and to Mrs. E.M. Arnold, Omaruru, Namibia for the generous supply ofExormotheca thalli.     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Experiments with inhibitors and with centrifugation

Summary Colchicine treatment ofFunaria caulonemata, usually does not inhibit initiation of a side branch or its incipient elongation but does prevent movement of chloroplasts and the nucleus into the outgrowth. After colchicine and after cytochalasin B treatment side branches are formed about at the normal age of the cells; because of the inhibition of the apical cell they arise at an abnormal position,i.e., not in the third but in the second cell of a filament. After D₂O treatment the organelles are dislocated toward the basal cross wall. The site of side branch formation is then obviously determined by the position of the nucleus. Cells with an irreversibly reversed longitudinal polar axis can be found; by centrifugation in proximal direction the sites of side branch initiation likewise are displaced into the proximal region of the cell, especially if the remigration of the nucleus is inhibited by colchicine. High concentrations of Ca²⁺ ions induce the formation of side branch cells, without any outgrowth. The calcium ionophore A 23 187 influences the position of the nucleus and of the side branch only slightly. After these various treatments intercalary divisions frequently occur. The role and interrelationship of the nucleus and peripheral cytoplasm in establishing and maintaining the polar axes, and the role of microtubules are discussed.     

Identification of further <Emphasis Type="Italic">Craterostigma plantagineum cdt</Emphasis> mutants affected in abscisic acid mediated desiccation tolerance

The resurrection plant (Craterostigma plantagineum) is desiccation tolerant. However, callus derived from this plant, when propagated in vitro, requires exogenously applied abscisic acid (ABA) in order to survive desiccation. Treatment of callus tissue with ABA induces most of the genes that are induced by dehydration in the whole plant. This property has been exploited for the isolation of mutants that show dominant phenotypes resulting from the ectopic expression of endogenous genes induced by the insertion of a foreign promoter. Here we describe new T-DNA tagged Craterostigma desiccation-tolerant (cdt) mutants with different molecular and physiological characteristics, suggesting that different pathways of desiccation tolerance are affected. One of the mutants, cdt-2, constitutively expresses known osmoprotective Lea genes in callus and leaf tissue. Further analysis of this mutant revealed that the tagged locus is similar to a previously characterised gene, CDT-1, which codes for a signalling molecule that confers desiccation tolerance. The nature of the T-DNA insertion provides insight into the mechanism by which the CDT-1/2 gene family functions in ABA signal transduction.     

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

Side branch formation and orientation in the caulonema of the moss,Funaria hygrometrica: Normal development and fine structure

Summary The regular branching of theFunaria caulonema filaments is partly related to rhythms in nuclear and cell division. The formation and development of the branches were studied by light and electron microscopy with particular attention directed to the distribution of microtubules and the polar organization of the cytoplasm. The new side branch breaks through the wall of the mother cell. The site of branch development is determined by the position of the nucleus of the mother cell. In protonemata which grow in vertically placed Petri dishes gravity influences the position of nuclei and side branches, and also the direction of oblique cross walls in the caulonema filaments to a certain extent.     

Calcium Ameliorates Effects of Lead in Protonema of Funaria hygrometrica Hedw.

Two-days-old in vitro grown protonemata of Funaria hygrometrica Hedw. were treated with a mixture PbCl₂ (4 M Pb²⁺) and CaCl₂ (16 M Ca²⁺) (Ca+Pb) for 48 h. The results were compared with the control: distilled water (H₂O) and the solution of PbCl₂ (4 M Pb²⁺) (Pb). Protonemata treated with Ca+Pb were longer and contained more cells than those treated with Pb. Moreover, a lower number of cells showed apical cell deformations typical for lead toxicity: swollen tips and wall thickenings at the apex. If deformations were present they were not as extended as in Pb. In comparison with the control, however, protonemata treated with Ca+Pb were shorter, contained a lower number of cells and some apical cells in this material were altered. It can be concluded that the presence of calcium partially neutralised toxic effects of lead in Funaria hygrometrica protonemata cells.     

Tip-localised H+-fluxes and the applicability of the acid-growth hypothesis to tip-growing cells: Control of chloronemal extension in Funaria hygrometrica by auxin and light

The role of tip-localised H⁺ secretion in regulating chloronemal tip growth in the moss Funaria hygrometrica Hedw. was investigated. pH was monitored with pH microelectrodes placed close to the cell surface while the rate of extension growth was manipulated by illumination and by the application of indole-3-acetic acid. Growth stimulations were accompanied by acidification of the external solution; this acidification was most pronounced at the growing tip. The timing and extent of acification external to the tip correlated well with the magnitude and time course of growth stimulations. The maintenance of both growth and H⁺ efflux under CO₂-free conditions indicated that neither photosynthetic nor respiratory CO₂ metabolism were involved. Artificially acidifying the nutrient solution rapidly but transiently stimulated elongation in both white light and darkness. Furthermore, the stimulation of elongation caused by white light was inhibited if the nutrient solution was buffered strongly near neutrality. We conclude that the acid growth hypothesis is applicable to tip growth in Funaria and that light and exogenous indole-3-acetic acid act at least in part by stimulating localised H⁺-ion efflux.Abbreviations D darkness - IAA indole-3-acetic acid - WL white light     

Cytokinin effect on branching and bud formation in Funaria

Cytokinins have two different effects on protonemata of Funaria hygrometrica. They induce branching of unbranched caulonemata and bud formation. Branching occurs after treatment with pico-molar concentrations of cytokinins whereas bud formation requires micro-molar concentrations. Both processes are therefore independently stimulated by cytokinins.Abbreviation BA N⁶-benzyladenine     

Cytokinin activation and redistribution of plasma-membrane ion channels in Funaria

I have investigated changes in electrical current across the plasma membrane that occur during cytokinin-induced bud formation in Funaria hygrometrica Hedw., using a non-intrusive vibrating microelectrode. Before cytokinin treatment the target caulonema cells have maximal inward current at the nuclear region. After cytokinin treatment inward current increases twofold along the length of the cell. Within minutes, however, current decreases at both the nuclear zone and the proximal end while increasing at the distal end of target cells, preceding and predicting the presumptive division site. Inward current at the distal end falls to resting levels after establishment of a bulging growth zone, and remains low around developing buds. This current is blocked by gadolinium nitrate, a Ca²⁺-uptake inhibitor, indicating a Ca²⁺ component of the current. The polarity of the target cells can be disrupted by microfilament inhibitors and cytokinin-induced buds form over the nucleus, halfway along the length of the cell. I suggest that cytokinin activates plasma-membrane ion channels which are subsequently redistributed to the distal ends of target cells by a microfilament-dependent process. Cytokinin-induced concentration of ion channels over presumptive bud sites may be envisioned to exert spatial control of cytoplasmic ion concentrations and stimulate bud formation by establishing a new growth zone, directing nuclear migration, and stimulating cell division.Abbreviations BA 6-benzyladenine - [Ca²⁺]_i intracellular calcium-ion concentration     

Desiccation tolerance acquisition in developing beech (Fagus sylvatica L.) seeds: the contribution of dehydrin-like protein

The acquisition of desiccation tolerance (DT) in developing beech (Fagus sylvatica L.) seeds and the role of a dehydrin protein in this process were investigated. DT was determined by measurement of electrolyte leakage and germination capacity after drying to 10–12% moisture content (MC). In addition to mass maturity, the presence of heat-stable proteins, dehydrin accumulation and the peak of ABA content were measured in relation to the acquisition of DT. Mass maturity was achieved at 16 weeks after flowering (WAF). The germination capacity increased from 8% at 12 WAF to 80–90% after 16 WAF. Cell membrane integrity, measured as a decrease in electrolyte leakage after desiccation, was acquired at 16 WAF. Additionally, the ratio of heat-stable to soluble proteins was the highest at 16 WAF. One dehydrin-like protein with a molecular mass 44 kDa, named DHN44, was detected in embryonic axes at 16 WAF and in cotyledons at 17 WAF, and its gradual accumulation was observed in mature seeds. With regard to the acquisition of DT, the strongest correlations were detected between electrolyte leakage, DHN44 accumulation, and the percentage of heat-stable proteins. These results suggest that developing beech seeds become tolerant to desiccation at 16 WAF. The effect of desiccation and ABA treatment on DHN44 synthesis was tested before (14 WAF) and after the DT acquisition (18 WAF). Depending on the maturation stage desiccation and ABA treatment can induce or enlarge DHN44 expression.     

Comparative ecophysiology ofLarix kaempferi (Lamb.) Carr. andAbies veitchii Lindl. II. Mechanisms of higher drought resistance of seedlings ofL. kaempferi as compared withA. veitchii

The underlying mechanisms of drought resistance in seedlings ofLarix kaempferi as compared with those inAbies veitchii were investigated. First, seedlings of the two species were desiccated in air of differing water potential, and their survival rates were compared to detect any difference in desiccation tolerance between them. Contrary to differences in their drought resistance,A. veitchii showed higher survival. Next, the shoot heights and root depths of the two species were compared to detect any difference in desiccation avoidance. Although the shoot height differed little between the two species, the roots ofL. kaempferi seedlings were much deeper than those ofA. veitchii seedlings, and the death rate under progressing drought was well correlated with the root extensibility of each species. It was thus concluded thatL. kaempferi seedlings are able to avoid desiccation by extending their roots in deep soil faster thanA. veitchii seedlings, thereby resisting to drought. This paper is dedicated to Prof. Toshiro Saeki to mark his fruitful career in plant ecology.     

Growth,cell wall formation and differentiation in the protonema of the moss,Funaria hygrometrica: Effects of plasmolysis on the developmental program and its expression

Summary Cultivation ofFunaria protonemata under plasmolytic or slightly subplasmolytic conditions initially causes a cessation of growth which is accompanied by a transient disappearance (or strong reduction in frequency, respectively) of putative cellulose synthesizing particle rosettes in the plasma membrane. Simultaneously, the formation and exocytosis of cell wall materialsecreting Golgi vesicles is slowed down. The latter process does not become apparent for several hours, though the reduction in activity can be proved indirectly. As a consequence of the imbalance between exocytosis, cell wall material accumulates in the plasmolytic space, generally at the cell tip. This indicates that the pattern of local, polar deposition of cell wall formation and cell elongation, membrane debris as well as wall material is maintained for some time. Later, however, the whole protoplast may become covered by new wall layers. Potentially growing filament tips and the distal region of nontip cells increase in diameter after longer cultivation in subplasmolytic conditions. It is suggested that normal wall growth results from a softening of the existing wall, its stretching and simultaneous stabilization by the apposition of new wall layers. We believe that the swelling is caused by a change in the equilibrium between the obviously less affected softening process and the imperfect stabilization by new wall layers because the wall layers which are formed at reduced turgor pressure are looser than normal and may have a changed composition.Kinetin-induced buds do not develop under plasmolytic conditions. Instead, spiral filaments are formed which readily give rise to buds when the osmotic value of the (kinetin-containing) medium is normalized. The results show that plasmolysis affects the expression of the developmental program rather than its initiation or maintenance.     

Unequal distribution of nuclear pores in oryzalin-induced mini-nuclei in protonema cells of the mossFunaria hygrometrica: influence of the nucleolus

Summary Mini-nuclei, formed in tip cells ofFunaria caulonemata after oryzalin treatment, have unequally distributed nuclear pores. The region of the nuclear envelope near the nucleolus, in a distance of up to 3 m, is devoid of pores. In other areas pores occur with a high frequency.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane