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

In seed formation the role of ethylene has received little attention. The data available on zygotic embryogenesis suggest an association of the ethylene biosynthetic pathway and seed maturation. Over the course of dicot embryogenesis, ACC-oxidase mRNA can be expressed in the cotyledons and embryonic axis. However, as maturation proceeds, cotyledonary ACC-oxidase expression disappears. In some seeds that develop primary dormancy, ethylene synthesis can be among the prerequisites for breaking dormancy. Moreover, the persistence of dormancy may be related to the difficulty of the embryonic axis to produce the necessary ethylene levels or to low tissue sensitivity. The use of inhibitors of ethylene biosynthesis or its action has provided data implicating an ethylene requirement for seed dormancy or germination in some species. However, the role of ethylene in germination remains controversial. Some authors hold that gas production is a consequence of the germination process, while others contend that ethylene production is a requirement for germination. Furthermore, among seeds that require ethylene, some are extremely sensitive to the gas, while others require relatively high levels to trigger germination. Recent studies with Xanthium pennsylvanicum seeds suggest that � cyanoalanine-synthase is involved in ethylene-dependent germination. In addition, regulation of the partitioning of S-adenosyl-L-methionine (AdoMet) between the ethylene vs polyamine biosynthetic pathways may be a way of controlling germination in some seeds. Such regulation may also apply to the reversal of seed thermoinhibition, which can occur when polyamine synthesis is inhibited, thereby strongly channelling AdoMet towards ethylene. The biological models and approaches that may shed additional light on the role of ethylene during seed germination are presented.

Ethylene in seed formation and germination

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

Trials were carried out to investigate the effects of light and temperature on germination of Rumex obtusifolius L. After several months of storage, seeds gradually lost dormancy and became photosensitive. Thermal optima for germination were between 20 °C and 25 °C in light or in darkness. At lower temperatures there was a greater demand for light, so that the greatest differences in germination percentage (between low and high temperatures) were found within the 10-15 °C temperature range. The calculated thermal minimal (x-intercept method) in light and darkness were 8.3 °C and 6.1 °C respectively. Daily temperature fluctuation increased germination even after seed irradiation with far-red light, suggesting a lower demand for the far-red-absorbing form of phytochrome. Seed burial inhibited germination in proportion to depth; however, germination inhibition was independent of seed phytochrome photo-equilibrium, which had been diversified by seed pretreatment with light Seedlings did not emerge when seeds were buried > 8 cm deep. Recovery of ungerminated seeds showed that excessive burial did not impede seedling emergence but rather prevented seed germination. However, this induction of dormancy was lost once germination processes were activated (24-48 h at 20 °C) that made germination irreversible. Temperature was also involved in inhibition, and low temperature ( < 15 °C) induced the least inhibition. This is discussed in terms of processes of respiration and fermentation in buried seeds.

Light, temperature and burial depth effects on Rumex obtusifolius seed germination and emergence

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

Exposure of Rumex obtusifolius seed to red light resulted in a rapid increase in extractable cytokinins. This increase can be reversed if the red light treatment is immediately followed by far-red irradiation. In germinating seed hardly any cytokinin activity could be detected in the butanol and aqueous extracts. An increase in the amount of activity present in the petroleum ether extracts of these seeds was, however, observed. The present findings suggest that cytokinin activity in light-sensitive seeds could be under the control of phytochrome.

The effect of light on endogenous cytokinin levels in seeds of Rumex obtusifolius

The cytokinin content of Xanthium strumarium L. plants decreased markedly when they were exposed to short days (SD). There was a significant decrease in the content of the butanol-soluble cytokinins of the mature leaves after only 5 SD cycles, and after 10 SD there was no significant cytokinin activity in butanol extracts; the changes in the young leaves were less marked. Most of the cytokinin activity in mature leaves appears to be present in the aqueous fraction, whereas in young leaves most activity occurs in the butanol-soluble fraction. SD treated plants produced less root exudate than LD plants, but there were no significant differences in the amounts of cytokinin in the root exudates from LD and SD plants collected over an equivalent time period. The cytokinin levels of SD-induced leaves remained low even when transferred back to LD. The observed differences in cytokinin levels did not appear to be the result of photosynthetic differences. Exposure of detached leaves to LD or SD did not result in differences in cytokinin content. It is not clear whether the observed changes in cytokinin levels in the leaves under SD are involved in the flowering response, but they may be causally related to a reduced chlorophyll content observed in SD-induced leaves.

The Effect of Photoperiod on Levels of Endogenous Cytokinins in Xanthium strumarium

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.

J. Van Staden

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

The effect of light on endogenous cytokinin levels in seeds of Rumex obtusifolius

The Effect of Photoperiod on Levels of Endogenous Cytokinins in Xanthium strumarium

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