Halophyte seed germination

Present evidence indicates that only the root systems of plants have been shown conclusively to synthesize cytokinins. Although most of these compounds are apparently exported to the shoot via the xylem, there are indications that more attention should be given to the possibility of translocation through the phloem. Within mature leaves the cytokinins derived from the roots are converted to inactive or storage forms by means of glucosylation. While it would appear that glucosylation could occur in all living plant cells whenever the cytokinins are no longer required for active growth, and could provide the plant with a potential reservoir of cytokinins, very little is known with regard to the transport and reutilisation of these compounds.

The synthesis, transport and metabolism of endogenous cytokinins

INTRODUCTION 331 OCCURRENCE OF DORMANCY 332 STRUCTURE AND ORGANIZATION 332 Seed Coat Efficts 332 G en eral and Mitoch ondrial Activity .... ... ... ... . .. .. ... ........ 333 Nu cleic Acids and Prot ein Syntheses . . . 334 HORMONE AND PHYTOCHROME ACTION . . . . . . . . . .. .. . . . . . . . . .. . . . . ..... . .. . . . .. .. .. . .. .. . .. .. . . . . .. .. . . 336 Abs cisic and Gibb er el ic Acids ... .. 336 Cyto kinins and Ethylene .... .... 339 Phytochrome Action ...... 341 METABOLIC REGULATION 342 APPLIED COMPOUNDS .. . . . . . . . . . .. . .. . . . . . . . . ... .... 345 TEMPERATURE EFFECTS 346 CONCLUSION 347

Dormancy in Seeds

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.

tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.

The discovery that cytokinins oppose the actions of seed germination inhibitors provided a powerful conceptual tool in designing new hypotheses and in interpreting old ones. By using highly specific gibberellin-mediated seed processes, it was shown that gibberellins, inhibitors and cytokinins have primary, preventive and permissive roles respectively in the control of seed germination. We can now explain for the first time that seed dormancy can occur not only in presence of inhibitors as was generally believed but in many cases in absence or presence of promotors such as gibberellins or cytokinins. Thus, the uniqueness of the hormonal model presented lies in the effective use of the concept of designated functions of hormones in the control of seed germination. Cytokinin-inhibitor antagonism is not limited to seed systems but occurs in other systems, as well, including lateral bud growth and growth of tissues in culture. Evidence presented supports the hypothesis that auxin is the primary stimulus in the control of bud growth and growth of tissues in culture while the roles of gibberellins, cytokinins and inhibitors are secondary. Gibberellin appears to act after auxin has initiated growth in these systems. The highly selective roles of hormones in plant systems explains such anomalous situations as continued rapid growth in presence of high levels of inhibitors and lack of growth in spite of the presence of large amounts of cytokinins, gibberellins or auxins. Cytokinin-inhibitor antagonism and selective actions of hormones have also been shown at the level of nucleic acid and enzyme synthesis suggesting that both transcriptional and translational mechanisms might be involved.

Primary, preventive and permissive roles of hormones in plant systems

Stratification of sugar maple seed resulted in high levels of cytokinins after 20 days. A further increase in the chilling period led to a decrease in cytokinins. Most of the cytokinin activity detected was due to compound(s) that co-chromatographed with zeatin.

The effect of stratification on endogenous cytokinin levels in seeds of Acer saccharum.

The present studies on changes in the levels of cytokinins, gibberellins, and germination inhibitors during stratification of Acer saccharum seeds suggest that the breaking of dormancy is a phasic process Stratification at 5 °C led to a marked increase in the butanol-soluble cytokinins after 20 d Further chilling resulted in a loss of cytokinin The majority of the detectable cytokinin activity co-chromatographed with zeatin Similarly, at day 40, acidic gibberellin-like substances increased and declined with further chilling A loss of acidic germination inhibitors was also detected during stratification The acidic fraction was shown to contain abscisic acid (ABA) by its behaviour on GLC and isomerization with ultra-violet light Determinations of endogenous levels of ABA by combined GLC and isotope dilution methods indicated a loss of 97-9 per cent ABA during stratification The data suggest that the breaking of seed dormancy at low temperature involves the initiation of an ordered sequence of events that include the changes in growth hormones necessary to determine the course of development for the removal of dormancy

Seed Dormancy in Acer CHANGES IN ENDOGENOUS CYTOKININS, GIBBERELLINS AND GERMINATION INHIBITORS DURING THE BREAKING OF DORMANCY IN ACER SACCHARUM MARSH

Dormant seeds of Acer pseudoplatanus L. contain two zones of inhibition on paper chromatograms in “10:1:1” as detected by the lettuce and cress seed germination, and the wheat coleoptile bioassays. One zone at Rf 0.6–0.8 was partitioned into ethyl acetate at acid pH and was shown to contain ABA by its behaviour on GLC and isomerization under ultra-violet light. The other zone at Rf 0.9 was detected only in the germination bioassays and was partitioned into ethyl acetate over a range of pH indicating the presence of one or more neutral compounds.

Seed dormancy in Acer: Endogenous germination inhibitors and dormancy in Acer pseudoplatanus L

Seed dormancy in Acer

Changes in germination inhibitors, cytokinins, and gibberellin-like substances during the breaking of coat-imposed dormancy of sycamore seeds at low temperature suggest that dormancy may be controlled by the presence of endogenous germination inhibitors. Stratification at 5 °C led to a greater loss of neutral inhibitor(s) from the embryo than did incubation at 20 °C. No marked differences were observed in the level of cytokinins and gibberellin-like substances between treatments. However, a gradual decrease in the level of cytokinins was observed during stratification. Whether a cytokinin-inhibitor interaction is involved in the control of dormancy of sycamore seeds, as was previously suggested, remains to be determined. INTRODUCTION Experiments with dormant sycamore (Acer pseudoplatanus L.) fruits indicated that fruits and seeds with the testa intact require a period of chilling at 5 °C to break dormancy, whereas isolated embryos germinated immediately at 20 °C on moist filter-paper without pretreatment (Webb and Wareing, 1972a). Results from leaching experiments suggested that dormancy was the result of the restric tion, by the testa, of the outward diffusion of germination inhibitors present in the embryo. Inhibitors extracted from dormant sycamore seeds have also been shown to inhibit the germination of non-dormant sycamore seeds at relatively low concentrations (Webb and Wareing, 19726). A comparison with the effects of applied abscisic acid (ABA) indicated that the level of endogenous ABA alone could not account for the inhibition observed by the seed extracts. Both ABA and neutral compounds present in the embryo appeared to be involved. Leaching treatments that removed dormancy also led to a decrease in the level of inhibitors present in the basic and neutral ethyl acetate fraction. Application of kinetin to dormant sycamore seeds increased germination 1 Present address : Great Lakes Forest Research Centre, Canadian Forestry Service, P.O. Box 490, Sault Ste. Marie, Ontario, Canada. 2 Present address : Botany Department, University of Natal, Pietermaritzburg, Republic of South Africa. This content downloaded from 40.77.167.104 on Sun, 24 Jul 2016 04:18:58 UTC All use subject to http://about.jstor.org/terms 742 Webb, van Staden, and Wareing—Seed Dormancy in Acer whereas gibberellic acid (GA3) had no effect. A similar response was obtained with lettuce seeds in which germination was inhibited by the basic and neutral ethyl acetate fraction obtained from dormant sycamore seeds. These results led to the suggestion that a possible inhibitor-cytokinin interaction may be involved in the dormancy of sycamore seeds (Webb and Wareing, 19726). Similarly, the work of Pinfield and Stobart (1972) supports this hypothesis. The results of Van Staden, Webb, and Wareing (1972) with Acer saccharum suggested that endogenous cyto kinins may play an important role in embryo-dormant seeds. Stratification led to a marked increase in the level of cytokinins and gibberellin-like compounds with a concomitant loss of ABA. In sycamore seeds which show a coat-imposed dormancy, the natural means of dormancy removal is by moist low-temperature after-ripening as the fruit over winters on the ground. Thus, it was of interest to examine the possible involvement of endogenous germination inhibitors and promoters during the breaking of coat imposed dormancy of sycamore seeds at low temperatures. MATERIALS AND METHODS Seed storage and germination conditions A. pseudoplatanus L. fruits were collected from single, open-grown trees in the Botany Gardens, Aberystwyth, in November 1970 and 1971. The fruits were air-dried to remove surface water only and stored in sealed containers at 2-5 °C. In these studies seeds were used within 2 months from the dated collection since it is difficult to store sycamore fruits in a dormant state without loss of viability (Jahnel, 1955). Seeds were germinated on filter-paper in Petri-dishes at 20 °C (Webb and Wareing, 1972a). Protrusion of the radicle through the covering structures was used as the criterion for germination. Stratification conditions Whole fruits were presoaked in distilled water for 24 h and placed in plastic bags. Any excess moisture was drained off and the sealed bags stored at 5 °C for approximately 80-100 d. At 10-d intervals the bags were opened and checked for moisture. Extraction and bioassay of growth regulators Germination inhibitors and gibberellin-like substances were extracted as previously described (Webb, Van Staden, and Wareing, 1972). The seed material was homogenized and extracted in 80 per cent redistilled methanol at 2-5 °C. The aqueous fraction was partitioned four times at pH 8-0 with redistilled ethyl acetate. The aqueous fraction was then adjusted to pH 2-5 and again partitioned with ethyl acetate. Cytokinins were extracted using the methods of Van Staden et al. (1972). The partitioned extracts were dried under vacuum at 35 °C and strip-loaded on to Whatman No. 1 chromatography paper. The chromatograms were developed in a descending manner in iso-propanol: ammonia iwater (10:1:1 v/v/v) in the case of extracts to be assayed for inhibitors or gibberellins, and in water-saturated sec butanol for extracts to be assayed for cytokinins. Germination inhibitors in the acidic ethyl acetate fraction and in the basic ethyl acetate fraction (which would also contain any neutral compounds) were determined using the lettuce-seed germination bioassay (Webb and Wareing, 19726). In all experiments recorded the results are the means of at least two bioassays seen at different times. The lettuce hypo cotyl bioassay was used to determine gibberellin-like activity (Loveys, 1970). Cytokinin activity was determined using the soybean callus bioassay (Miller, 1965). RESULTS Whole fruits from the 1970 seed crop were stratified at 5 °C and a second control sample was incubated at 20 °C (a temperature that normally does not lead to This content downloaded from 40.77.167.104 on Sun, 24 Jul 2016 04:18:58 UTC All use subject to http://about.jstor.org/terms Webb, van Staden, and Wareing—Seed Dormancy in Acer 743 Fig. 1. The effect of stratification on the level of acidic germination inhibitors in sycamore seeds. The equivalent of 0-5 g dry weight of i material in the acidic ethyl acetate fraction was chromatographed on paper in '10:1:1' and assayed with the lettuce seed germination bioassay. r

Seed Dormancy in Acer CHANGES IN ENDOGENOUS GERMINATION INHIBITORS, CYTOKININS, AND GIBBERELLINS DURING THE BREAKING OF DORMANCY IN ACER PSEUDOPLATANUS L.

D. P. Webb

Papers

The effect of stratification on endogenous cytokinin levels in seeds of Acer saccharum.

Seed Dormancy in Acer CHANGES IN ENDOGENOUS CYTOKININS, GIBBERELLINS AND GERMINATION INHIBITORS DURING THE BREAKING OF DORMANCY IN ACER SACCHARUM MARSH

Seed dormancy in Acer: Endogenous germination inhibitors and dormancy in Acer pseudoplatanus L

Seed dormancy in Acer

Seed Dormancy in Acer CHANGES IN ENDOGENOUS GERMINATION INHIBITORS, CYTOKININS, AND GIBBERELLINS DURING THE BREAKING OF DORMANCY IN ACER PSEUDOPLATANUS L.