Endogenous Plant Hormones in the. Xylem Sap. of Grapevines during Development

J. Japan. Soc. Hort. Sci. 47(2) : 181-187. 1978. Changes in Endogenous Plant Hormones in the Xylem Sap of Grapevines during Development Yoshiyuk1 NIIMI and H1rotaka TORIKATA Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464 Summary Endogenous plant hormone levels in the xylem sap of the grapevine were determined at different stages of development to demonstrate the relationship between top growth arid endogenous plant hormones. (1) The growth of the new shoot was a typical double sigmoid curve. The inception of the second growth was at a full bloom stage. The growth of roots increased rapidly after the shoot growth stopped. (2) Cytokinin activities in the xylem sap were present in n-butanol (Free) and aqueous (Bound) fractions, In each fraction there were at least two major peaks of cytokinin activities. (3) The free cytokinins increased from April to July, and thereafter decreased rapidly. The bound cytokinins were maintained at high levels from May to June as flowers opened. Gibberellin-like activity in the xylem sap was present in May and increased in June as flowers opened and fruits developed, and thereafter decreased rapidly. Auxin and absccisic acid-like activities in the xylem sap were very low during their development. These results lead to the suggestion that gibberellin and cytokinin synthesized in roots may play an important physiological role in controlling the growth of the shoot and flower clusters. Introduction The presence of several plant hormones in the xylem sap of woody and herbaceous plants have been demonstrated on a number of occasions (2, 5, 6, 9, 12, 28, 31). In the xylem sap of the grapevine, cytokinin-like and gibberellin-like substances were present (15, 29, 30). These results had led to the suggestion that cytokinins and gibberellins synthesized in roots might play an important physiological role in controlling the top growth. Owing to rich xylem sap, grapevines are well suited to sap analysis. The present paper describes the changes in endogenous plant hormone levels in the xylem sap of grapevine in relation to the top growth. Materials and Methods Three years old cuttings of grapevines (Vitis labruscana Bailey cv. Delaware) were grown in the pot (30 cm in diameter) under Received for publication August 9, 1977 natural conditions. On the day when plants were transffered to the greenhouse, they were cut off at the base of their shoots, and sterilized vinyl tubes were connected to the stumps in order to collect the xylem sap. The sap collected from three grapevines for 48 hrs was filtered and then stored until further analysis. The average dry weight of root and whole plant of those three grapevines was determined. Extraction of endogenous hormones(fig. 1): (1) Auxin, ABA (abscisic acid) and GAs (gibberellins) Sap samples wese reduced in vacuo, adjusted to ph 2.5 with 1 N HC1 and then partitioned three times against ethyl acetate. The ethyl acetate fraction was reduced in quantity in vacuo and partitioned three times against 2% sodium bicarbonate. The basic aqueous fraction was then adjusted to ph 2.5 with 1N HC1 and again partitioned three times against ethyl acetate. The acidic ethyl acetate fraction was separated by paper chromatography. 181

182 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE Fig. 1. Flow diagram showing procedures for extraction and separation endogenous plant hormones in the xylem sap of the grapevine. of (2) Cytokinins The aqueous fraction, after extracted with ethyl acetate, was adjusted to ph 8.4 with 1N NaOH and partitioned three times against water-saturated n-butanol. The n-butanol fraction was reduced to dryness and dissolved in a small amount of 70% ethanol. While, the aqueous fraction was also reduced to dryness and dissolved in a small amount of 35% ethanol. Separation of endogenous hormones: (1) Paper chromatography Each extract was streaked on Toyo filter No. 51A paper strips. Ascending chromatography was carried out for 14 hrs. A solvent system of isopropanol, ammonia and water (10:1:1, v f v) was used for separation of auxin, ABA and GAs. For cytokinins, n-butanol, acetic acid and water (12:3:5, v/v) was used. The dried chromatograms were divided into 10 equal strips. Endogenous hormone-like substances were eluted from the strip with water for bioassay. (2) Column chromatography The aqueous fraction of sap sample was reduced to 1.5m1 and passed through a Sephadex LH-20 column (1.8 X 45 cm). Cytokininlike substances were subsequently eluted with 35% ethanol at a flow rate of 13 m1jhr (1). Each fraction (6m1) was dried up. The dried sample was dissolved in 5m1 of water for bioassay. Bioassay of endogenous hormones: Auxin and ABA-like activities in the extracts were determined by the slightly modified Avena coleoptile straight growth test (24, 25). Activities of cytokinin-like substance (14) and gibberellin-like substance (4) were determined by the radish cotyledon assay and the barley endosperm assay, respectively. Results Separation of cytokinins: As shown in Fig. 2, cytokinin-like activities in the xylem sap of a grapevine were present in both n-butanol and aqueous fractions. Each fraction has two major peaks of cytokinin-like activities. In the n-butanol Fig. 2. Histograms showing the distribution of cytokinlike activities on a paper chromatogram of both aqueous (Bound) and n-butanol (Free) fractions. Cytokinin-like activity was determined by the radish cotyledon assay..

NIIMI AND TORIKATA : ENL OGENOUS PLANT I-ORMONES IN THE XYLEM SAP OF GRAPE 183 Fig. 3. The distribution of cytokinin-like activities on a Sephadex LH-20 column fractionation of aqueous fraction of the xylem sap. The column was eluted with 35% ethanol. Cytokinin-like activity was determined by the radish cotyledon assay. Fig. 4. Changes in the growth (Whole plant and the volume of xylem sap of the during their development. and Root) grape vine fraction, cytokinin-like activities were present at Rf 0-0.4 (A) and 0.6-0.9 (B). The latter peak was cochromatographed with zeatin and zeatin riboside. In the aqueous fraction, cytokinin-like activities were present at Rf 0-0.2 (a) and 0.2-0.5 (b). Separation of cytokinin-like substances in the aqueous fraction by a Sephadex LH-20 column chromatography is demonstrated in Fig. 3. Cytokinin-like activity present in the 9th-16th fractions was obviously different from zeatin and zeatin riboside. Changes in the volume of xylem sap accompanyed by growth: Fig. 4 shows a relationship between the plant growth and the volume of xylem sap during Fig. 5. Cumulative shoot during growth curve of. the grape their development. the development. The xylem sap began to translocate in April, just before bud sprouting, and exudation of the sap increased from May to August. Thereafter, the volume of xylem sap decreased rapidly from August to November. The dry weight of the whole plant rapidly increased from May (after bud sprouting) to August, and this phenomenon was similar to the changes of the volume of the xylem

184 JOURNAL OF THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE Fig. 6. Changes in gibberellin-like activity in the xylem during their development. Gibberellin-like activity the barley endosperm bioassay. sap of the grapevine was determined by Fig. 7. Changes in cytokinin-like activity (Free) in the xylem sap of the grape vine during their development. S-S Rf 0.2-0.5 (A). 0-0 Rf 0.6-0.8 (B) sap. The dry weight of roots, however, rapidly incresed from July, which the shoot growth had already ceased. Shoot growth of grape vines showed a typical double sigmoid curve (Fig. 5). After bud sprouting, the first growth till full bloom was expressed as shoot growth I (SG-I) and the second growth as shoot growth II (SG-II). Changes in endogenous hormoues in the xylem sap of the grape vine: Auxin and ABA-like activities in the acidic Fig. 8. Changes in cytokinin-like activity (Bound) in the xylem sap of the grape vine during their development. S-S Rf 0-0.3 (a), 0-0 Rf 0.3-0.5 (b) ethyl acetate fraction were very low during the vine development. Gibberellin-like activity in the same fraction was first noticed in May, and increased in June as flowers opened and fruits developed, and thereafter decreased rapidly (Fig. 6). Fig. 7 shows cytokinin-like activities in the n-butanol fraction (Free). The activities in both eluates of Rf 0.3-0.5 (A) and 0.6-0.9 (B) increased from April to July and thereafter decreased rapidly. On the other hand, cytokinin-like activities (Bound)

NIIMI AND TORIKATA : ENDOGENOUS PLANT HORMONES IN THE XYLEM SAP OF GRAPE 185 in the aqueous fraction are shown in Fig. 8. The activities in both eluates of Rf 0-0.2 (a) and 0.2-0.5 (b) markedly increased at full bloom, and decreased once in July and thereafter maintained at high levels. Discussion Skene (29) have shown gibberellin-like activity in the bleeding sap of two seedless varieties of Vitis vinifera by the barley endosperm bioassay, and compared with levels for other parts of the plant, mainly roots and leaves. Gibberellin-like activity in the xylem sap of the grapevine was detected from May and increased in June when shoot growth was very great, and thereafter decreased rapidly. Shoot growth of the grapevine showed typically double sigmoid curve, and divided into two phases (SG-I, SG-II). This phenomenon may suggest the growth of SG-I depends on the carbohydrate reserves from preceding year and that of SG-II mainly on the photosynthate of newly developing leaves of the grapevine. A close relationship between the shoot growth of Stage-II and the high activity of gibberellin-like substance in the xylem sap of the grapevine leads to the suggestion that gibberellin synthesized in roots may play an important physiological role in controlling the shoot growth in addition to the carbohydrate reserves and the photosynthate of newly developing leaves. On the other hand, it is widely recognized seedless grape berries induced by GA-application only once before full bloom does not show a good growth thereafter. The presence of gibberellin in immature seeds of grapevines (3,10) and other tree fruits (11, 17, 22) suggests gibberellins synthesized in seeds may also control the growth of grape berries in that stage. The presence of cytokinin-like activity in the bleeding sap of a sunflower plant (9) and a grapevine (11, 15) was reported. Cytokinin-like activity was present in the xylem sap of the grapevine and high activity of cytokinin-like substances was observed from full bloom stage to the early growth stage of grape berries. Mullins (19, 20) observed that the inflorescences on grape cuttings failed to develop in the absence of roots. The retention of the inflorescences on the cuttings resulted from the presence of roots. In the absence or roots, however, application of synthetic cytokinins to the base of unrooted cuttings or directly to the emergent inflorescences promoted inflorescence growth. Negi and Olmo (23) and other investigators (7, 8) have shown application of synthetic cytokinins to the male flower cluster on the male plant of Vitis species induced typical hermaphroditic flowers instead of the usual male flowers. Pool (26) also reported that a cytokinin requirement for pistil development of hermaphroditic flowers was demonstrated by an in vitro culture technique. These results suggest that cytokinins synthesized in roots may play an important role in controlling the growth of the flower cluster and floral organ. One of two types of cytokinins in the xylem sap of grapevines was n-butanol extractable (Free) and the other was not n-butanol extractable (Bound). Davey and van Staden (5) showed the major translocational form of cytokinins (n-butanol extractable) in the xylem sap of tomato plant was zeatin riboside. That of the grapevines may be bound cytokinins. But the relationship between the bound and the free cytokinins was not clear. Further work is needed to clarify this point. Pool and Powell (27) investigated the growth of new shoots excised from grapevines by using the aseptic culture technique. Sustained shoot development was observed only when cytokinins were present in the media. While, the relationship between the shoot growth (S G-II) and cytokinin-like activities in the xylem sap of the grapevine was also observed in this experiment. These evidence suggests cytokinins synthesized in roots, in addition to gibberellin, may also play a physiologically important role in controlling the shoot growth of a grapevine. The levels of natural cytokinins and auxin in the xylem sap of apple are normally at a maximum at the time of full bloom and falls to zero when shoot growth ceases (16). While, auxin activity in the xylem sap of the grapevine was very low during their development. The presence of ABA in the xylem sap of sycamore (18) and willow determined by gas chromatography (13) was reported.

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