seasonal changes in levels of cytokinin-like compounds from douglas-fir xylem extrudate.full

8/13/2019 Seasonal Changes in Levels of Cytokinin-like Compounds From Douglas-Fir Xylem Extrudate.full

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Tree Physiology 4, l-8 (1988).0 1988 Heron Publishing-Victoria, Canada.

Seasonal hanges n levels of cytokinin-like compounds fromDouglas-fir xylem extrudateP. DOUMAS and J. B. ZAERRDepartment of Forest Science, College of Forestry, Oregon State University, Corvallis, Oregon97331, USAReceived February 23, 1987

SummaryHigh-performance l iquid chromatograp hy, immunoch romatograph y, and radioimmunoassa y wereused to identify cytokinin-l ike bases and glycosrdes in xyle m sap of Douglas-fir (Pseudotsuga men-ziesii (Mirb.) Fran ce). Isopentenyladenosine-type (isopentenyladenine and isopentenyladenosine) andzeatin-riboside type (zeatin, zeatin riboside, and dihydrozeatin riboside) cytokinins were detectedduring springtime. A glucosyl conjugate of zeatin riboside wa s also present in small amounts. Levelsof cytokinin-l ike compou nds varied throughout the spring but were general ly highest in late Apri l toearly May.

IntroductionCytokinins in the xylem sap of herbaceous and woody plants have been the subjectof several investigations (Morris et al. 1976, Van Staden and Davey 1976, 1979),but few workers have tried to identify the cytokinin-like substances that arepresent. Horgan et al. (1973) and Purse et al. (1976) identified zeatin (Z), zeatinriboside (ZR), and dihydrozeatin (DHZ) in the spring sap of sycamore (Acerpseudoplatanus L.). In a more recent study, Palmer and Wong (1985) found tendifferent cytokinins, including Z, ZR, and DHZ, as well as some conjugates suchas O-glucosyl zeatin, O-glucosyl-dihydrozeatin, and O-glucosyl-dihydrozeatin-riboside, in xylem exudate of Phaseolus vulgaris L. Many authors have suggestedthat 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 xylemexudate originate in the roots (Kende and Sitton 1967, Skene 1975). If the xylemsap is the bridge between the site of biosynthesis of cytokinins and the receptorsites, the analysis of cytokinins present in xylem sap may contribute to a betterunderstanding of their role in plant development.

The aim of this study was to determine if cytokinin-like compounds occur inxylem 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 immu-noaffinity techniques (columns of immobilized antibodies directed against differ-ent cytokinins) and radioimmunoassay to screen the cytokinin-like compounds.

Present address: S tatio n ddm tlior ation des Arbres Forestiers, INRA , Ardon, 4 5100 Orlkans,France.

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2 DOUMAS AND ZAERR

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

Materials and methodsXylem sap was extruded in spring 1981 from the terminal 2 m of freshly harvestedDouglas-fir trees growing near Corvallis, Oregon, by means of a pressure-chamber technique described by Zaerr (1982). The trees were approximately 4 mtall, and some were beginning to produce seed cones. Sap from trees harvested onthe 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 ammon-ium acetate buffer (40 mM pH 6.5, about 30-40 ml 100 ml- sap) that contained0.2 mg ml-i sodium diethyldithiocarbamate and 0.5 mg ml-l butylated hydroxy-toluene as anti-oxidants, and [3H]isopentenyladenosine (iPA) dialcohol as an inter-nal standard. The sap was applied directly onto tandem columns. The first con-tained a 20-ml bed volume of DEAE-cellulose (Whatman DE-52) and the second al-ml bed volume of IgG-cytokinin-specific immunoaffinity-cellulose (MacDonaldand Morris 1985). The immunoaffinity column contained equal amounts of anti-ZR and anti- iPA IgG. Recovery of standards from the immunoaffinity column wastypically 80 . The anti-ZR antibody cross-reacted with Z (48 ), DHZ (48 ),DHZR (64 ), isopentenyladenine (iP) (< O.l ), and iPA (< 0.1 ). The anti-iPA antibody cross-reacted with iP (98 ), Z (68 ), and ZR (72 ). The DEAE-cellulose and antibody-cellulose were packed separately into disposable polypro-pylene syringes. Before being used, both columns were equilibrated with 40 mMammonium acetate, pH 6.5. The sample was applied to the DEAE-cellulosecolumn at a rate of 1 ml min- i and then run directly through the immunoaffinitycolumn. The columns were washed according to MacDonald and Morris (1985).In this system, if any cytokinin nucleotides were present in the xylem sap, theywould remain on the DEAE-cellulose column. But because our previous work hadindicated few or no cytokinin nucleotides in Douglas-fir shoots, they were notincluded in the analysis reported here. Cytokinins were eluted from the immu-noaffinity column with HPLC-grade methanol (Baker) at 0.8 ml mini. Themethanolic extract was dried in V~CUO Savant Speed Vat concentrator) and sub-jected to high-performance liquid chromatography (HPLC) and radioimmunoassay(RIA).

The separation of cytokinins was achieved by HPLC with a Beckman model420 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 triethylammon-ium acetate buffer pH 3.35, were prepurified on a Cis-Millipore filter system. Thecolumn was eluted at 1 ml mini with the following gradient: initial conditions6 acetonitrile, 94 triethylammonium acetate buffer; 6 to 11 acetonitrile over15 min; 11 to 35 acetonitrile over 15 min. The sample was injected in 50 ~1 ofmethanol and triethylammonium acetate buffer (1: 1 v/v). Fractions were collected

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CYTOKININS IN DOUGLAS-FIR XYLE M EXTRUDATE 3

every 0.5 min, dried in vacua, and stored at - 20 C. Recovery from the extrac-tion and purification steps, as calculated from the internal standard, was about65 .

Appropriate fractions collected after HPLC were divided into two replicates andassayed by means of anti-ZR IgG with [3H]ZR dialcohol or anti-iPA IgG with[3H]iPA dialcohol. Radioimmunoassay conditions were described previously byMacDonald et al. (1981).

ResultsEvidence of cytokinin-like compoundsAn example of results from the analysis of xylem sap is shown in Figure 1. Thecomparison between the HPLC graph of immuno-affinity-purified compoundsfrom Douglas-fir xylem extrudate and the HPLC graph of an authentic standardmixture of different cytokinins (Figure 1A) revealed several cytokinin-like peaksin the xylem sap. Immunoaffinity techniques established that most of these peakscontained cytokinin-like compounds. Radioimmunoassay of individual fractionsof the HPLC eluate with ZR anti-serum in retention time-zone O-22 min, or iPAanti-serum in retention time-zone 22-34 min, confirmed the presence of at leastsix major peaks of cross-reactive material in the sap extract (Figure 1B). Five ofthese 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 withany cytokinin previously described. The retention time of this compound was 16.5min, between the Z peak and the DHZ peak on the standard HPLC graph (FigureIA). Immunoaffinity chromatography showed Zx to have a cytokinin-like struc-ture, as did the RIA. Enzymatic treatment and analysis by gas chromato-graphy-mass spectrometry indicated that Zx is a glucoside of zeatin riboside,where the glucosyl is attached to the ribosyl moiety (Doumas et al. 1986). Tayloret al. (1984) described a compound that could be identical with Zx. Details of theidentification of Zx wil l be presented elsewhere (Morris, J.W., P. Doumas, J.B.Zaerr and R.O. Morris, manuscript in preparation).Cytokinin levels in the springThe major portion of all measured cytokinin-like substances in Douglas-fir xylemextrudate was made up of three compounds, Z-, ZR-, and iPA-like compounds.Three other compounds, Zx-, DHZR-, and iP-like compounds, were also presentin the xylem extrudate at low levels.

The cytokinin-like substances were present in xylem extrudate throughout thespring but varied in concentration. The highest total concentration (the sum of ZRequivalents and iPA equivalents) occurred between Apr il 29 and May 5, and otherpeaks 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

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Figure 1. Puri f ication of cytokinins from Douglas-f ir xylem sap (Z = zeatin, DH Z = dihydrozeatin,CZ = cis-zeatin, ZR = zeatin riboside, D HZ R = dihydrozeatin riboside, iPA = isopentenyladeno-sine, iP = isopentenyladenine). The compound Zx elutes at 16.5 min.(A) HPLC -Chromatogram of immunoaff ini ty-puri fied cytokinin-l ike substances from Douglas-f irxylem sap. The retention t imes of authentic standard cytokinins are indicated by horizontal bars.(B) Histogram of radioimmunoassay of HPL C fractions of immunoaff ini ty-puri fied cytokinin-l ikesubstance s from D ouglas-fir xylem s ap.

Zx- and iP-like compounds were low throughout the sampling period (Figure 3).Individual cytokinin-like substances reached their highest concentrations on dif-ferent dates; for example, the maximum level of Z-l ike cytokinin occurred onApri l 9 and the maximum level of iPA-like cytokinin on April 30.

DiscussionThe highly efficient and selective immunoaffinity-column system producespurified extracts of cytokinins suitable for sensitive analysis (MacDonald and

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I I I I I I I I I I I25 29 2 6 IO 14 16 22 2630 4 6 16M AR C H APR IL M A Y

COLLECTION DATEFigure 2. Total cytokinin-l ike level (ZR-equivalents plus iPA-equivalents) in Douglas-fir xylem sap atdifferent collection dates during spring. V ertical bars indicate one standard deviation (n = 3 to 5).

Morris 1985). We applied this method to xylem extrudate of Douglas-fir to obtaincytokinin-like compounds that, after HPLC, would be suitably pure for RIA(MacDonald et al. 1981). Our results extend those of Morris et al. (1976) anddemonstrate not only the presence of cytokinin-like substances in xylem-sap extru-date from Douglas-fir but a pattern of changes in individual cytokinin-likesubstances during spring.

We detected and quantified two groups of cytokinin-like substances, the ZRtype (Z, ZR, DHZR, and Zx) and the iPA type (iP and iPA), in xylem extrudateduring the spring. This is the first report of iPA-type cytokinins in xylem sap ofconifers. These results agree with those of Cahill et al. (1986), who found similarcytokinins, 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 fromthe root to the shoot.

We have found Zx in several parts of Douglas-fir. It seems to be carried in thexylem 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 ofplant development (Davey and Van Staden 1976), season (Purse et al. 1976), andenvironmental conditions to which roots are subjected (Burrows and Carr 1969,Banko and Boe 1975). In addition, there is some evidence that cytokinin flux isindependent of exudate flux (Heindl et al. 1982). The lack of similarity between

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CYTOKININS IN DOUGLAS-F IR XYLEM EXTRUDATE 1

Figure 3. Individual cytokinin-l ike substan ces in Douglas-fir xylem sa p at different col lection datesduring spring. (A and B) ZR-cyto kinin equivalents, (C) iPA-cytokinin equivalents. V ertical barsindicate one standard deviation (n = 3 to 5).

cytokinin flux and the volume of root xylem exudate has been observed in othermaterial (Davey and Van Staden 1976, 1978). In Oregon, shoot growth inDouglas-fir commences in late March or early April and continues into July. Theincreases in cytokinin-l ike substances in xylem sap observed in late Apri l to earlyMay occur at a time when shoots are rapidly expanding. The accelerated rate ofxylem sap movement in the spring, along with the increased concentration ofcytokinin-like substances in the sap, indicate substantial movement of cytokininsfrom roots to shoots during the period of bud break and rapid extension growth ofshoots.

Lorenzi et al. (1975) described cytokinins in needles and buds of Piceasitchensis (Bong.) Cat-r. They found levels of ZR increased in mature needlesduring the spring, but decreased in buds and new needles. A cytokinin believed tobe zeatin-9-glucoside reached a maximum concentration in March. They did notreport finding iP-type cytokinins in needles or buds, even though their bioassaysystem (soybean callus) presumably was sensitive to both types of cytokinins.Although we measured cytokinin activity in xylem sap rather than in needles orbuds, our results do not agree entirely with their findings. The concentration ofcytokinin-like substances in xylem sap seems to be quite variable, but the trendspoint toward a general increase in the spring (Figure 2) rather than a decline asreported by Lorenzi et al. (1975). Differences between spruce and Douglas-fircould account for this discrepancy, but more likely cytokinin metabolism in theshoot changes the size of cytokinin pools there. The fate of the iP cytokinin-likecompounds in the xylem sap remains to be explained.

Other reports of seasonal variations of cytokinin content in xylem exudate showhigh levels in the spring and low levels in late fall and winter (Hewett and Wareing1974, Alvim et al. 1976). The changes appear to parallel physiological changes.Whether cytokinins cause these physiological changes is unknown.AcknowledgmentsThis research was supported in part by the US DI B ureau of Land Manag ement and the USD A ForestService under the auspices of the Southw est Oregon Forestry Inten sif ied R esearch (F IR) Program(Grant Oll l) and by OIC D/U SD A Travel Grant 4C5. P. Dou mas acknowledge s the support of theMinistere de la Recherche et de la Technologie, Departem ent Agriculture et Industries Agro-Alimentaires-Bois de la Mission Scientifique et Technique, which m ade possible his involvement inthis research. This is Paper 2210, Forestry Resea rch Laboratory, Oregon State Unive rsity, Corvall is.

ReferencesAlvim, R. , E.W . Hewe tt and P.F. Saunders. 1976. Seasonal variation in the hormone content ofwil low. I. Changes in abscisic acid content and cytokinin activity in the xylem sap. Plant Physiol.

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8 DOUMAS AND ZAERR

Banko, T.J . and A.A. Boe. 1975 . Effec t of pH, temperature, nutri t ion, ethephon and chlormequat onendogenous cytokinin levels of Coleus blumei Benth. J. Amer. Sot. Ho rt. Sci. 100:168-172.Burrows, W .J. and D.J . Cat-r. 1969. Effects of f looding the root system of sunflower plants on thecytokinin content in the xylem sap. Physiol. Plant. 22:1105-1112.Cahil l , D.M ., G.M . W este and B.R . Grant. 1986. Changes in cytokinin concentrations in xylemextrudate fol lowing infection o f Eucalyptus marginatu donn ex Sm with Phytophthoru cinnamonirands. Plant Physiol. 81:1103-1109.Chen , C. M., J.R . Ertl , S.M . Leisner and CC . Chang. 1985. Localization of cytokinin biosyntheticsites in pea plants and carrot roo ts. Plant Physiol. 78:510-513.

Da vey, J.E. and J. Van Staden. 1976. Cytokinin translocation: changes in zeatin and zeatin-ribosidelevels in the root exudate of tomato p lants during their developmen t. Planta 130:69-72.Da vey, J.E. and J. Van Staden. 1978. Cytokinin activity in Lupinus albus. I. Distribution in vegeta-tive and flowering plants. Physiol. Plant. 43:77-81.Dou mas , P., J.W . Morris, C. Chien, M . Bonne t-Masimbert and J.B. Zaerr. 1986. A possible relation-ship between cytokinin conjugate and flowering in Doug las-fi ,. In Proc. 9th North American ForestBiology W orkshop . Sti l lwater, Oklahoma . pp 285-296.Heindl, J.C ., D.R . Carlson, W.A . Brun and M.L . Brenner. 1982. Ontogenetic variation of fourcytokinins in soybean root pressure exudate. Plant Physiol. 70:1619-1626.

He wett, E.W . and P.F. Wareing. 1974. Cytokinin changes during chi ll ing and bud burst in woodyplants. In Mech anisms of Regulation of Plant Growth. Eds. R.L . Bieleski, A.R . Ferguson andM.M . Cresswel l . R. Sot. N.Z. Bul l. 12:693-701.Horgan, R., E.W. Hew ett , J .G. Purse, J.M . Horgan and P.F. Wareing. 1973. Ident if icat ion of acytokinin in sycam ore sap by gas chromatogra phy-mass spectro metry. Plant. Sci. Lett.1:321-324.Kende, H. and D. Sitton. 1967. The physiological signif icance of kinetin and gibberell in-l ike roothormones. 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.MacD onald, E.M .S., D.E . Akiyoshi and R.O . Morris. 1981. Combined high-performance l iquid

chromatography-radioimmunoa ssay for cytokinins. J. Chroma togr. 214:101-109.MacD onald, E.M.S . and R.O . Morris. 198 5. Isolation of cytokinins by immuno affinity chromato-graphy and analysis by high-performance l iquid chromatography radioimmunoa ssay. Method sEnzymol. 110:347-358.Morris, R.O ., J.B. Zaerr and R.W . Chapm an. 1976. Trace enrichment of cytokinins from Douglas-firxylem extrudate. Planta 131:271-274.Palmer, M.V. and O.C . Won g. 1985. Identif ication of cytokinins from xylem exud ate of Phuseolusvulguris L. Plant Physiol. 79:296-298.

Purse, J.G ., R. Horgan, J.M . Horgan and P.F. Wareing. 1976. Cytokinins of sycam ore spring s ap.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 TheDevelopment and Funct ion of Roots. Eds. J.G. Torrey and D.T. Clarkson. Academic Press, NewYork. pp 365-396.Taylor, J.S., M . Koshioka, R.P . Pharis and G.B. Swe et. 1984. Changes in cytokinins andgibberellin-like sub stan ces in Pinus rudiutu buds during lateral shoo t initiation and the chara cteriza-tion of ribosyl zeatin and a novel ribosyl zeatin glycoside. Plant Physiol. 74:626-631.Torrey, J.G. 1976. Root hormones and plant growth and developme nt. Ann. Re v. Plant Physiol.271435-459.Van Staden, J. and J.E. Dave y. 1976. Cytokinin translocation in xylem sap of herbaceous plants. Z.Pflanzenphysiol. 77:377-382.

Van Staden, J . and J.E. Dav ey. 1979. The synthes is, transport and metabolism of endogenouscytokinins. Plant Cell Environ. 2:93-106.Zaerr, J.B. 1982. Pressure chamber device to col lect xylem sap from fo rest trees. For. Sc i.28:219-222.

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