Variation of the Levels and Transport of Cytokinins during Germination of Chick-pea Seeds

LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

Department of Plant Biology, Faculty of Biology, University of Salamanca, Salamanca, Spain

Received June 18, 1986· Accepted October 21,1986

Summary

Cytokinins detected in chick-pea seeds were present in the embryonic axes and first appeared in the cotyledons after 12 hours of germination.

The appearance and initial increase of cytokinins in cotyledons is common to both the free and bound forms. However, from 24 hours of germination onwards the disappearance of bound cytokinins is much faster than that of the free forms, suggesting transformation of the bound forms into free ones during this stage of germination.

The cytokinins detected in the cotyledons seem to originate from the embryonic axes, though there is no passage of these substances from the cotyledons to the axes.

The /3-glucosidase activity measured both in cotyledons and in embryonic axes, was seen to be positively correlated with the decrease in bound cytokinins and the increase in free forms, suggesting the transformation of cytokinin glycosides into their corresponding free forms.

Key words: Cicer arietinum, free cytokinins, bound cytokinins, endogenous /3-glucosidase, ger­mination, seeds, transport.

Introduction

A fundamental point in the study of germination and seedling growth is its regula­tion. A current hypothesis is that breakage of dormancy and the start of germination depend on the balance between endogenous growth inhibitors and promotors (Amen, 1968). According to this idea, the different hormones must have a specific function in the physiological processes leading to germination and seedling growth (Khan, 1971).

In the cotyledons of germinating seeds, the main role of the cytokinins seems to be the regulation of reserve mobilization (Gepstein and Ilan, 1979; Metivier and Paulilo, 1980). Moreover, that a decrease is detected in endogenous cytokinins in cotyledons excised from embryonic axes and incubated (Rybicka et aI., 1977) seems to indicate that these organs may depend on a continuous supply of these substances through the embryonic axis (Rybicka et aI., 1977).

Taking these considerations into account we carried out a study on the variations in the endogenous levels of cytokinins in cotyledons and embryonic axes of germinat­ing chick-pea seeds. Other important aspects of this work were to study the transport

Abbreviations: Z, zeatin; ZR, zeatin riboside; ZG, zeatin glucoside; GZR, glycosil zeatinin ri­boside; DHZ, dihydrozeatin; DHZR, dihydrozeatin riboside; 2iP, N 6-(,1?-isopentenyl)adenine; 2iPA, N 6-(d2-isopentenyl)adenine riboside; cot, cotyledon.

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142 LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

of endogenous cytokinins, both from the cotyledons towards the axis and vice versa, and to study endogenous /1-glucosidase activity. This was to discover whether there is any relationship between the activity of this enzyme at the different times of ger­mination and the mean levels of glycosidic and free cytokinins at the same times of the germination period.

Materials and Methods

Germination of seeds

Seeds of Cicer arietinum L. cv. Castellana «chick-pea» and of Cucumis sativus cv. Calahorra «cucumber>' were used. They were germinated and grown on a glass plate covered with filter paper, in the dark at 25°C (chick-pea) or 28 °C (cucumber) at 80% relative humidity.

Cytokinin Transport

After different germination times, the cotyledons and embryonic axes, were placed into the agar. They were then left for 24 hours in the dark at 25°C for the diffusion to take place. Then, the agar blocks were collected for the extraction, purification and evaluation of cytokinins ac­cording to Martin et al. (1986).

Levels of cytokinins in excised cotyledons

Fifty seeds were sown under the above mentioned conditions. Embryonic axes were re­moved before sowing (time 0) and at different times of germination. In this case the seeds were kept under incubation until the end of the study period.

Determination of (:J-glucosidase activity

The seeds were sown as before. Enzyme extraction was performed (McCreight et al., 1976), and (:J-glucosidase activity was determined spectrophotometrically using 3 mM p-nitrophenyl B­D glycoside as the reaction substrate. The p-nitrophenol released was measured at 400 nm.

Protein was measured according to the method of Bradford (1976).

Results

Variation in the content of cytokinins

The total cytokinins in the cotyledons (Fig. 1) reached a maximum at 24 hours and then decreased until they finally disappeared at 120 hours.

The results obtained from the studies of bound and free cytokinin levels in the cotyledons may be seen in Fig.2. As can be observed, a constant increase in both forms: free and bound cytokinins takes place during the first 24 hours of germina­tion; from this moment until the end of the process (120 hours) a decrease in both forms occurs, being more pronunced in bound cytokinins which are indetectable from 72 hours onwards.

The total cytokinin in the embryonic axes (Fig. 1) underwent a large drop up to 18 hours after germination and then increased up to 36 hours; except for a slight decrease between 36 and 48 hours, they then remained practically constant until the fifth day. It appears from Fig. 3 that the drop was much more prolonged in the bound that in the free forms. After 24 hours of germination, the levels of both types of cytokinins remained practically constant.

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Transformation and transport of cytokinins in chick-pea seeds 143

1400 }

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H:::; '~, 400 ~

~ cotyledons 200 -"-M ?

o

o 3 6 9 12 18 24 36 48 72 96 120 GERMINATION(hours)

Fig. 1: Variation in the content of total cytokinins in cotyledons 0--0, and embryonic axes .--. during the germination of Cicer arietinum L. seeds. Vertical bars indicate SE.

1.400

1.200

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Fig. 2: Total free cytokinins 0--0 and bound cytokinins .--. from cotyledons of Cicer arietinum L. seeds over five days of germination. Vertical bars indicate SE.

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144 LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

1400

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03 69 12 18 24 36 48 72 96 120 GERMINATION (hours)

Fig. 3: Total free cytokinins 0--0 and bound cytokinins e--e from embryonic axes of Cicer arietinum L. seeds over five days of germination. Vertical bars indicate SE.

In the cotyledons (Fig. 4) it was only possible to detect substances with cytokinin activity between 12 and 72 hours after germination. The study of the variation of these substances during germination and seedling growth reveals an increase in the levels of glycosil zeatin riboside and zeatin glucoside between 12-24 hours. Zeatin ri­boside increased slightly up to 18 hours, thereafter converting into its dihydroderiva­tive; between 24-36 hours a mixture of both cytokinins was present. The dihydro­zeatin riboside formed, later decreased until it finally disappeared at the end of the period studied (120 hours). Likewise, zeatin, which hardly decreased at all after its ap­pearance up to 18 hours, began to be converted into its dihydroderivative form 24 hours after germination onwards, and the latter was the only cytokinin which was present in seeds from 36 to 96 hours, when it became indetectable. Isopentenyl ad­enine riboside and isopentenyl adenine remained at fairly constant levels from their appearance up to 48 hours, when they begin to decrease until they practically disap­pear at 96 hours.

In the embryonic axes (Fig. 5) the same substances as those found in the cotyledons could be detected, yet in contrast they are already present in the dry seeds. Of interest were the low levels between 24 and 36 hours of glycosil zeatin riboside, zeatin glu­coside, and zeatin riboside and at 18 hours of zeatin, isopentenyl adenine riboside, and isopentenyl adenine. The decrease was followed by a new increase which in turn led to a gradual drop. Zeatin riboside and zeatin, were transformed into their dihy­droderivative forms during the late stages of the germination period.

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Transformation and transport of cytokinins in chick-pea seeds 145

1400

1.200

1.000

800

600

400

200

0

1.400

1200

?:~ 1000 ";0 :;:u u'-«!:: em .- '" eN .ii", .Be >--u

GZR

ZR 0 DHZR rra

ZG

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GERMINATION(hours)

Fig. 4: Cytokinins detected in the extracts from cotyledons as dependent on the stage of ger­mination. Vertical bars indicate SE.

Cytokinin Transport

As shown in Fig. 6 A no release of cytokinins from the cotyledons into agar could be detected, suggesting that cytokinins are not exported from cotyledons to axes. On the other hand (Fig. 6 A) between 6 and 24 hours, the passage of substances from the embryonic axes into agar is quite conspicuous, the amount of cytokinins detected being maximum 12 hours after germination; this coincides with the appearance of these substances in the cotyledons. During the two hours of maximum diffusion (9 and 12 hours of germination) showed that six cytokinins appear to be transported (Fig.6B).

Influence of the embryonic axes on the levels of endogenous cytokinins in cotyledons of Cicer arietinum L. seeds

In order to sengthen the evidence that the cotyledons are provided with cyto­kinins from the axis during germination and initial seedling growth one kind of ex-

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146 LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

GERMINATION(hours)

Fig. 5: Different cytokinins detected in the embryonic axes extracts during germination of Cicer arietinum L. seeds. Vertical bars indicate SE.

periment was carried out: on one hand the embryonic axis was excided at time ° (rest­ing seeds) and the isolated cotyledons incubated and on the other hand the axis was excided at different times of incubation and the subsequent change of cytokinins in the cotyledons followed. When the axis was excised at time 0, no cytokinins were de­tected in the cotyledons during the 120 hours of incubation studied. Upon excising the axis at 9 hours after germination (Fig. 7) a small increase was observed in the cyto­kinins detected after 12 hours of incubation; levels later decreased until they finally disappeared after 72 hours. The small increase observed after 12 hours is logical taking into account that the transport of cytokinins from the embryonic axis to the cotyledons begins after 6 hours of germination. Excision of the axis after 12 hours of germination (Fig. 7) led to a slight increase in the cytokinins detected during the fol­lowing 6 hours, after which they began to decrease until they finally disappeared after

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~ >~ .;: c u .-<{~

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1400

1200

1000

800

600

400

200

a

1400

1200

1000

800

600

400

Transformation and transport of cytokinins in chick-pea seeds 147

A

/".

/ / ~ embryonic axes

---~~;~-~Q/9-----m--~otYle~ons --- ~

3 6 9 12 18 24 36

GERMINATION (hours)

B

9 hours 12 hours

200 -[if- - - -- - - ------------ ---rf -- - ---------------------a 1!23456789 ~2345S789 FRACTIONS

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Fig_ 6: A) Variation throughout germination of the cytokinins detected in the agar receptor blocks: cotyledons acting as donor segments 0--0; embryonic axes acting as donor seg­ments e--e_ Vertical bars indicate SE. - B) Different cytokinins detected in the agar re­ceptor blocks, with the embryonic axes of Cicer anetinum L seeds germinated over 9 and 12 hours, acting as donor segments. Vertical bars indicate SE.

72 hours. When the embryonic axis was excised after 18 hours (Fig. 7) a small increase could be observed in cytokinin levels until 24 hours of incubation, very similar to that observed under normal conditions. From 24 hours onwards they decreased gradually until the cytokinins finally disappeared after 96 hours of incubation. Exci­sion of the axis after 24, 36, and 48 hours of germination (Fig. 7) led to a decrease in cytokinin levels; this was also very similar to the decrease observed in intact plants.

(3-glucosidase activity

The (3-glucosidase activity in chick-pea seed cotyledons throughout germination is shown in Fig. 8. The level of activity was maximal after 24 hours of germination and

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148 LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

00 3 6 912 18 24 36 48 72 96 120

INCUBATION TIME (hours)

Fig. 7: Changes in the levels of total cytokinins in cotyledons of Cicer arietinum L. Control e--e and after removal of the axis at 6 hours Jt..--Jt.., 9 hours ______ , 12 hours 0--0, 18 hours 1:::,.--1:::,., 24 hours 0--0, 36 hours *--* and 48 hours *--* of germina­tion. SE less than 0.8.

08

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GERMINATION (hours)

Fig. 8: /1-glucosidase activity detected in cotyledons 0--0 and embryonic axes e--e of Cicer arietinum L. seeds over 72 hours of germination. Vertical bars indicate SE.

remained fairly high during the second day, thereafter the activity dropped rapidly and disappeared altogether after 72 hours.

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Transformation and transport of cytokinins in chick-pea seeds 149

Fig. 8 also shows the variation of the levels of l3-glucosidase activity in embryonic axes of chick-pea during germination. Enzymatic activity may already be detected after 3 hours and increases until it reaches its highest level after 36 hours of germina­tion, followed by a considerable reduction after 48 hours.

Discussion

In the cotyledons of chick-pea seeds, the study of cytokinin levels during germina­tion and initial seedling growth, reveals the presence of cytokinins only 12 hours after germination. The appearance of cytokinins and the initial increase is common to both the bound and free forms. The decrease from 24 hours onwards is much faster for the bound substances, suggesting that they may be transformed, as suggested by Julin-Tegelman (1979). The appearance and increase of cytokinins coincide with the phase of mobilization of reserves in these seeds (data not published). It seems that the mobilization of reserves is related with the cytokinins transported to the cotyledons at the beginning of the germination process (Gepstein and Ilan, 1980).

In the embryonic axes, the cytokinins pre-existed and decreased during the initial 18 hours. The drop was much more prolonged in the bound forms. It coincided with the decrease in these substances between 18 and 36 hours and with a substantial in­crease in the levels of free cytokinins, suggesting the possible transformation of bound forms into free ones.

The fact that in both, cotyledons and embryonic axes, the decrease in bound cyto­kinins was much faster that that of the free forms suggests a transformation of the for­mer into the latter during germination. In view of the presence of glycosides as bound forms, we were prompted to study the l3-glucosidase activity in chick-pea seeds since, according to Burrows and Carr (1970), the best indication of the capacity of the tissues to hydrolyze and utilize cytokinin glycosides is provided by the tissues them­selves. In the cotyledons, the results, are comparable with those obtained on the variations in bound and free cytokinins, since the possible hydrolysis of glycosides is carried out between 24-72 hours when there is a high level of l3-glucosidase activity. In the embryonic axes too, the high levels of activity of this enzyme coincide with the maximum period of utilization of the bound forms (between 18 and 48 hours). These results although they could suggest the hydrolysis of cytokinin-glycosides by the 13-glucosidase, do not demonstrate that this is what is really occurring in the tissues, since we are measuring p-nitrophenylglucosidase not cytokinin glycosidase activity.

The results obtained from the studies of cytokinin levels in cotyledons and embryonic axis seem to show the involvement of these substances in the germination process, as suggested by Julin-Tegelman and Pinfield (1982) in some way regulating the mobilization of reserves (Ilan and Gepstein, 1981), and playing a part in the re­newal of growth of the embryonic axes (Dodd et ai., 1980).

The appearance of cytokinins in cotyledons after 12 hours coinciding with the de­crease in these substances in the axes and their increase in the axes from 18 hours after germination onwards, may be in relationship with the idea of a supply of cytokinins towards the cotyledons that has already been suggested by several authors (van Onck-

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150 LUISA MARTIN, ANA DIEZ, GREGORIO NICOLAS, and NIEVES VILLALOBOS

elen et aI., 1977; Gepstein and Ilan, 1979; van Staden et aI., 1982). The present study confirms the existence of such transport only from embryonic axes to cotyledons when the appearance and increase of cytokinins is detected in the cotyledons. Simi­larly, the results obtained when the influence of embryonic axes was studied, suggest that all the cytokinins present in the cotyledons of Cicer arietinum L. seeds stem from the embryonic axes, in accordance with the idea already suggested by other researches (Rybicka et aI., 1977; Hutton et aI., 1982), and that in these organs no synthesis of cy­tokinins occurs at any time during the germination period (van Staden et aI., 1982).

References

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VAN ONCKELEN, H. A., R. CAUBERG, and J. A. DE GREEF: Effect of light treatment and en­dogenous growth hormones on €X- and iJ-amylase activities in cotyledons of Phaseolus vul­garis L. Plant Cell Physiol., 18,1029-1040 (1977).

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