Tree Physiology 4, l-8 (1988).

0 1988 Heron Publishing-Victoria,

Canada.

Seasonalchangesin levels of cytokinin-like compounds from Douglas-fir xylem extrudate

P. DOUMAS
Department 97331, USA

and J. B. ZAERR
Science, College

of Forest

of Forestry,

Oregon

State University,

Corvallis,

Oregon

Received February 23, 1987

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Summary High-performance liquid chromatography, immunochromatography, and radioimmunoassay were used to identify cytokinin-like bases and glycosrdes in xylem sap of Douglas-fir (Pseudotsuga menziesii (Mirb.) France). Isopentenyladenosine-type (isopentenyladenine and isopentenyladenosine) and zeatin-riboside type (zeatin, zeatin riboside, and dihydrozeatin riboside) cytokinins were detected during springtime. A glucosyl conjugate of zeatin riboside was also present in small amounts. Levels of cytokinin-like compounds varied throughout the spring but were generally highest in late April to early May.

Introduction Cytokinins in the xylem sap of herbaceous and woody plants have been the subject of several investigations (Morris et al. 1976, Van Staden and Davey 1976, 1979), but few workers have tried to identify the cytokinin-like substances that are present. Horgan et al. (1973) and Purse et al. (1976) identified zeatin (Z), zeatin riboside (ZR), and dihydrozeatin (DHZ) in the spring sap of sycamore (Acer pseudoplatanus L.). In a more recent study, Palmer and Wong (1985) found ten different cytokinins, including Z, ZR, and DHZ, as well as some conjugates such and O-glucosyl-dihydrozeatinas O-glucosyl zeatin, O-glucosyl-dihydrozeatin, riboside, in xylem exudate of Phaseolus vulgaris L. Many authors have suggested that roots are the major site of biosynthesis of cytokinins (Short and Torrey 1972, Torrey 1976, Chen et al. 1985), and there is evidence that cytokinins from xylem exudate originate in the roots (Kende and Sitton 1967, Skene 1975). If the xylem sap is the bridge between the site of biosynthesis of cytokinins and the receptor sites, the analysis of cytokinins present in xylem sap may contribute to a better understanding of their role in plant development. The aim of this study was to determine if cytokinin-like compounds occur in xylem extrudate of Douglas-fir (Pseudotsuga menziesii (Mirb.) France) and, if so, to measure the extent of the change in their levels during spring. We used immunoaffinity techniques (columns of immobilized antibodies directed against different cytokinins) and radioimmunoassay to screen the cytokinin-like compounds.
Present France. address: Station ddmtlioration des Arbres Forestiers, INRA, Ardon, 45100 Orlkans,

DOUMAS

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Because preliminary analysis of Douglas-fir shoots failed to detect the presence of cytokinin nucleotides, these nucleotides were not evaluated in this study. Materials and methods

Xylem sap was extruded in spring 1981 from the terminal 2 m of freshly harvested Douglas-fir trees growing near Corvallis, Oregon, by means of a pressurechamber technique described by Zaerr (1982). The trees were approximately 4 m tall, and some were beginning to produce seed cones. Sap from trees harvested on the same date was combined, frozen on dry ice, and stored at - 80 C. Before extraction and purification, the sap was adjusted to pH 6.5 with ammonium acetate buffer (40 mM pH 6.5, about 30-40 ml 100 ml- sap) that contained 0.2 mg ml-i sodium diethyldithiocarbamate and 0.5 mg ml-l butylated hydroxytoluene as anti-oxidants, and [3H]isopentenyladenosine (iPA) dialcohol as an internal standard. The sap was applied directly onto tandem columns. The first contained a 20-ml bed volume of DEAE-cellulose (Whatman DE-52) and the second a l-ml bed volume of IgG-cytokinin-specific immunoaffinity-cellulose (MacDonald and Morris 1985). The immunoaffinity column contained equal amounts of antiZR and anti-iPA IgG. Recovery of standards from the immunoaffinity column was typically 80%. The anti-ZR antibody cross-reacted with Z (48%), DHZ (48%), DHZR (64%), isopentenyladenine (iP) (< O.l%), and iPA (< 0.1%). The antiiPA antibody cross-reacted with iP (98%), Z (68%), and ZR (72%). The DEAEcellulose and antibody-cellulose were packed separately into disposable polypropylene syringes. Before being used, both columns were equilibrated with 40 mM ammonium acetate, pH 6.5. The sample was applied to the DEAE-cellulose column at a rate of 1 ml min- i and then run directly through the immunoaffinity column. The columns were washed according to MacDonald and Morris (1985). In this system, if any cytokinin nucleotides were present in the xylem sap, they would remain on the DEAE-cellulose column. But because our previous work had indicated few or no cytokinin nucleotides in Douglas-fir shoots, they were not included in the analysis reported here. Cytokinins were eluted from the immunoaffinity column with HPLC-grade methanol (Baker) at 0.8 ml mini. The methanolic extract was dried in V~CUO(Savant Speed Vat concentrator) and subjected to high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The separation of cytokinins was achieved by HPLC with a Beckman model 420 system fitted with a C18 reverse-phase column (Altex Ultrasphere-ODS, 250 X 4.6 mm, 5 p,m) and a Beckman model 160 UV-detector monitoring at 260 nm. The HPLC solvents, Baker HPLC-grade acetonitrile and 40 mM triethylammonium acetate buffer pH 3.35, were prepurified on a Cis-Millipore filter system. The column was eluted at 1 ml mini with the following gradient: initial conditions 6% acetonitrile, 94% triethylammonium acetate buffer; 6 to 11% acetonitrile over 15 min; 11 to 35% acetonitrile over 15 min. The sample was injected in 50 ~1 of methanol and triethylammonium acetate buffer (1: 1 v/v). Fractions were collected

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every 0.5 min, dried in vacua, and stored at - 20 C. Recovery from the extraction and purification steps, as calculated from the internal standard, was about 65%. Appropriate fractions collected after HPLC were divided into two replicates and assayed by means of anti-ZR IgG with [3H]ZR dialcohol or anti-iPA IgG with [3H]iPA dialcohol. Radioimmunoassay conditions were described previously by MacDonald et al. (1981). Results Evidence of cytokinin-like compounds
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An example of results from the analysis of xylem sap is shown in Figure 1. The comparison between the HPLC graph of immuno-affinity-purified compounds from Douglas-fir xylem extrudate and the HPLC graph of an authentic standard mixture of different cytokinins (Figure 1A) revealed several cytokinin-like peaks in the xylem sap. Immunoaffinity techniques established that most of these peaks contained cytokinin-like compounds. Radioimmunoassay of individual fractions of the HPLC eluate with ZR anti-serum in retention time-zone O-22 min, or iPA anti-serum in retention time-zone 22-34 min, confirmed the presence of at least six major peaks of cross-reactive material in the sap extract (Figure 1B). Five of these peaks, which co-eluted with Z, ZR, dihydrozeatin riboside (DHZR), iPA, and iP, had retention times, respectively, of 15.5, 18.5, 20.5, 29, and 31.5 min. We also detected an unknown compound (designated Zx) that does not elute with any cytokinin previously described. The retention time of this compound was 16.5 min, between the Z peak and the DHZ peak on the standard HPLC graph (Figure IA). Immunoaffinity chromatography showed Zx to have a cytokinin-like structure, as did the RIA. Enzymatic treatment and analysis by gas chromatography-mass spectrometry indicated that Zx is a glucoside of zeatin riboside, where the glucosyl is attached to the ribosyl moiety (Doumas et al. 1986). Taylor et al. (1984) described a compound that could be identical with Zx. Details of the identification of Zx will be presented elsewhere (Morris, J.W., P. Doumas, J.B. Zaerr and R.O. Morris, manuscript in preparation). Cytokinin levels in the spring The major portion of all measured cytokinin-like substances in Douglas-fir xylem extrudate was made up of three compounds, Z-, ZR-, and iPA-like compounds. Three other compounds, Zx-, DHZR-, and iP-like compounds, were also present in the xylem extrudate at low levels. The cytokinin-like substances were present in xylem extrudate throughout the spring but varied in concentration. The highest total concentration (the sum of ZR equivalents and iPA equivalents) occurred between April 29 and May 5, and other peaks occurred on April 9 and April 22 (Figure 2). Between April 29 and May 5, increases in concentrations of compounds like Z, ZR, DHZR, and iPA (Figure 3) contributed to the high total cytokinin-like level at this time. Concentrations of

DOUMAS

AND ZAERR

DHZ

DHZR

iPA

iP

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8-

ZR

,PA

6-

Z 4-

1 DHZR iP

ZX o(
IO RETENTION

,+7

20 TIME (min) 30

nh

Figure 1. Purification of cytokinins from Douglas-fir xylem sap (Z = zeatin, DHZ = dihydrozeatin, CZ = cis-zeatin, ZR = zeatin riboside, DHZR = dihydrozeatin riboside, iPA = isopentenyladenosine, iP = isopentenyladenine). The compound Zx elutes at 16.5 min. (A) HPLC-Chromatogram of immunoaffinity-purified cytokinin-like substances from Douglas-fir xylem sap. The retention times of authentic standard cytokinins are indicated by horizontal bars. (B) Histogram of radioimmunoassay of HPLC fractions of immunoaffinity-purified substances from Douglas-fir xylem sap. cytokinin-like

Zx- and iP-like compounds were low throughout the sampling period (Figure 3). Individual cytokinin-like substances reached their highest concentrations on different dates; for example, the maximum level of Z-like cytokinin occurred on April 9 and the maximum level of iPA-like cytokinin on April 30. Discussion The highly efficient and selective immunoaffinity-column system produces purified extracts of cytokinins suitable for sensitive analysis (MacDonald and

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25

29

IO

14 APRIL

16

22

2630

6 MAY

16

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Figure 2. Total cytokinin-like level (ZR-equivalents plus iPA-equivalents) in Douglas-fir xylem sap at different collection dates during spring. Vertical bars indicate one standard deviation (n = 3 to 5).

Morris 1985). We applied this method to xylem extrudate of Douglas-fir to obtain cytokinin-like compounds that, after HPLC, would be suitably pure for RIA (MacDonald et al. 1981). Our results extend those of Morris et al. (1976) and demonstrate not only the presence of cytokinin-like substances in xylem-sap extrudate from Douglas-fir but a pattern of changes in individual cytokinin-like substances during spring. We detected and quantified two groups of cytokinin-like substances, the ZR type (Z, ZR, DHZR, and Zx) and the iPA type (iP and iPA), in xylem extrudate during the spring. This is the first report of iPA-type cytokinins in xylem sap of conifers. These results agree with those of Cahill et al. (1986), who found similar cytokinins, including the iPA type, in the xylem extrudate of Eucalyptus species. Compounds like iP and iPA were also found in roots and shoots of Douglas-fir (Doumas et al. 1986), which suggests that they are transported in xylem sap from the root to the shoot. We have found Zx in several parts of Douglas-fir. It seems to be carried in the xylem sap and to accumulate in vegetative shoots before and during bud break (Doumas et al. 1986, Morris et al., in preparation). Levels of cytokinins in root exudate have been shown to fluctuate with stage of plant development (Davey and Van Staden 1976), season (Purse et al. 1976), and environmental conditions to which roots are subjected (Burrows and Carr 1969, Banko and Boe 1975). In addition, there is some evidence that cytokinin flux is independent of exudate flux (Heindl et al. 1982). The lack of similarity between

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IO

15-

o OHZR

. ZR

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Figure 3. Individual cytokinin-like substances in Douglas-fir xylem sap at different collection dates during spring. (A and B) ZR-cytokinin equivalents, (C) iPA-cytokinin equivalents. Vertical bars indicate one standard deviation (n = 3 to 5).

cytokinin flux and the volume of root xylem exudate has been observed in other material (Davey and Van Staden 1976, 1978). In Oregon, shoot growth in Douglas-fir commences in late March or early April and continues into July. The increases in cytokinin-like substances in xylem sap observed in late April to early May occur at a time when shoots are rapidly expanding. The accelerated rate of xylem sap movement in the spring, along with the increased concentration of cytokinin-like substances in the sap, indicate substantial movement of cytokinins from roots to shoots during the period of bud break and rapid extension growth of shoots. Lorenzi et al. (1975) described cytokinins in needles and buds of Picea sitchensis (Bong.) Cat-r. They found levels of ZR increased in mature needles during the spring, but decreased in buds and new needles. A cytokinin believed to be zeatin-9-glucoside reached a maximum concentration in March. They did not report finding iP-type cytokinins in needles or buds, even though their bioassay system (soybean callus) presumably was sensitive to both types of cytokinins. Although we measured cytokinin activity in xylem sap rather than in needles or buds, our results do not agree entirely with their findings. The concentration of cytokinin-like substances in xylem sap seems to be quite variable, but the trends point toward a general increase in the spring (Figure 2) rather than a decline as reported by Lorenzi et al. (1975). Differences between spruce and Douglas-fir could account for this discrepancy, but more likely cytokinin metabolism in the shoot changes the size of cytokinin pools there. The fate of the iP cytokinin-like compounds in the xylem sap remains to be explained. Other reports of seasonal variations of cytokinin content in xylem exudate show high levels in the spring and low levels in late fall and winter (Hewett and Wareing 1974, Alvim et al. 1976). The changes appear to parallel physiological changes. Whether cytokinins cause these physiological changes is unknown.
Acknowledgments This research was supported in part by the USDI Bureau of Land Management and the USDA Forest Service under the auspices of the Southwest Oregon Forestry Intensified Research (FIR) Program (Grant #Olll) and by OICD/USDA Travel Grant #4C5. P. Doumas acknowledges the support of the Ministere de la Recherche et de la Technologie, Departement Agriculture et Industries AgroAlimentaires-Bois de la Mission Scientifique et Technique, which made possible his involvement in this research. This is Paper 2210, Forestry Research Laboratory, Oregon State University, Corvallis. References Alvim, R., E.W. Hewett and P.F. Saunders. 1976. Seasonal variation in the hormone content of willow. I. Changes in abscisic acid content and cytokinin activity in the xylem sap. Plant Physiol.
511476476.

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Banko, T.J. and A.A. Boe. 1975. Effect of pH, temperature, nutrition, ethephon and chlormequat on endogenous cytokinin levels of Coleus blumei Benth. J. Amer. Sot. Hort. Sci. 100:168-172. Burrows, W.J. and D.J. Cat-r. 1969. Effects of flooding the root system of sunflower plants on the cytokinin content in the xylem sap. Physiol. Plant. 22:1105-1112. Cahill, D.M., G.M. Weste and B.R. Grant. 1986. Changes in cytokinin concentrations in xylem extrudate following infection of Eucalyptus marginatu donn ex Sm with Phytophthoru cinnamoni rands. Plant Physiol. 81:1103-1109. Chen, C.M., J.R. Ertl, S.M. Leisner and CC. Chang. 1985. Localization of cytokinin biosynthetic sites in pea plants and carrot roots. Plant Physiol. 78:510-513. Davey, J.E. and J. Van Staden. 1976. Cytokinin translocation: changes in zeatin and zeatin-riboside levels in the root exudate of tomato plants during their development. Planta 130:69-72. Davey, J.E. and J. Van Staden. 1978. Cytokinin activity in Lupinus albus. I. Distribution in vegetative and flowering plants. Physiol. Plant. 43:77-81. Doumas, P., J.W. Morris, C. Chien, M. Bonnet-Masimbert and J.B. Zaerr. 1986. A possible relationship between cytokinin conjugate and flowering in Douglas-fi,. In Proc. 9th North American Forest Biology Workshop. Stillwater, Oklahoma. pp 285-296. Heindl, J.C., D.R. Carlson, W.A. Brun and M.L. Brenner. 1982. Ontogenetic variation of four cytokinins in soybean root pressure exudate. Plant Physiol. 70:1619-1626. Hewett, E.W. and P.F. Wareing. 1974. Cytokinin changes during chilling and bud burst in woody plants. In Mechanisms of Regulation of Plant Growth. Eds. R.L. Bieleski, A.R. Ferguson and M.M. Cresswell. R. Sot. N.Z. Bull. 12:693-701. Horgan, R., E.W. Hewett, J.G. Purse, J.M. Horgan and P.F. Wareing. 1973. Identification of a cytokinin in sycamore sap by gas chromatography-mass spectrometry. Plant. Sci. Lett. 1:321-324. Kende, H. and D. Sitton. 1967. The physiological significance of kinetin and gibberellin-like root hormones. Ann. N.Y. Acad. Sci. 144:235-243. Lorenzi, R., R. Horgan and P.F. Wareing. 1975. Cytokinins in Pica sitchensis Carriere: identification and relation to growth. Biochem. Physiol. Pflanz. 168:333-339. MacDonald, E.M.S., D.E. Akiyoshi and R.O. Morris. 1981. Combined high-performance liquid chromatography-radioimmunoassay for cytokinins. J. Chromatogr. 214:101-109. MacDonald, E.M.S. and R.O. Morris. 1985. Isolation of cytokinins by immunoaffinity chromatography and analysis by high-performance liquid chromatography radioimmunoassay. Methods Enzymol. 110:347-358. Morris, R.O., J.B. Zaerr and R.W. Chapman. 1976. Trace enrichment of cytokinins from Douglas-fir xylem extrudate. Planta 131:271-274. Palmer, M.V. and O.C. Wong. 1985. Identification of cytokinins from xylem exudate of Phuseolus vulguris L. Plant Physiol. 79:296-298. Purse, J.G., R. Horgan, J.M. Horgan and P.F. Wareing. 1976. Cytokinins of sycamore spring sap. Planta 132:1-8. Short, K.C. and J.G. Torrey. 1972. Cytokinins in seedling roots of pea. Plant Physiol. 49:155-160. Skene, K.G.M. 1975. Cytokinin production by roots as a factor in the control of plant growth. In The Development and Function of Roots. Eds. J.G. Torrey and D.T. Clarkson. Academic Press, New York. pp 365-396. Taylor, J.S., M. Koshioka, R.P. Pharis and G.B. Sweet. 1984. Changes in cytokinins and gibberellin-like substances in Pinus rudiutu buds during lateral shoot initiation and the characterization of ribosyl zeatin and a novel ribosyl zeatin glycoside. Plant Physiol. 74:626-631. Torrey, J.G. 1976. Root hormones and plant growth and development. Ann. Rev. Plant Physiol. 271435-459. Van Staden, J. and J.E. Davey. 1976. Cytokinin translocation in xylem sap of herbaceous plants. Z. Pflanzenphysiol. 77:377-382. Van Staden, J. and J.E. Davey. 1979. The synthesis, transport and metabolism of endogenous cytokinins. Plant Cell Environ. 2:93-106. Zaerr, J.B. 1982. Pressure chamber device to collect xylem sap from forest trees. For. Sci. 28:219-222.

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