Incorporation of [8−14C]Adenine into Cytokinins in Pea Fruits

J. PlantPhysiol. Vol. 141. pp. 380-382 {1993}

Short C0111111Ul1icatiol1

Incorporation of [8-14C]Adenine into Cytokinins in Pea Fruits

J. VAN STADEN and F. E. DREWES UN/FRD Research Unit for Plant Growth and Development, Botany Department, P.O. Box 375, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received May 12, 1992 . Accepted September 17, 1992

Summary

Following application of [8-1~C]adenine to developing seeds and pod walls of pea fruits radioactivity was found coincident with the retention time of trans-zeatin after HPLC fractionation. Various chemical treatments and chromatographic procedures provided additional evidence for the presence of zeatin.

Key words: Pisum sativum, seeds, pod wall, cytokinin, biosynthesis, zeatin. Abbreviations: ADE = adenine; ADO = adenosine; HPLC = high performance liquid chromatography; iP = iso-pentenyladenine; [9RJiP = iso-pentenyladenosine; [9R-MPJiP = iso-pentenyladenosineS'-monophosphate; [9RJZ = ribosylzeatin; [9R-MPJZ = ribosylzeatin-S'-monophosphate; TLC = thin layer chromatography; Z = trans-zeatin. Introduction

Young developing seeds and fruits are rich sources of endogenous cytokinins (Van Staden and Davey, 1979). There has been much debate as to the origin of these cytokinins and whether the developing seed is a site of cytokinin biosynthesis (Van Staden, 1983). Despite many experiments this matter has not yet been resolved. The majority of experiments, using both monocotyledonous and dicotyledonous species, led to findings which did not support the developing seed as an active site of cytokinin biosynthesis (Van Staden and Button, 1978; Krechting et al., 1978; Van Staden and Choveaux, 1981; Van Staden and Forsyth, 1981; Van Staden and Vos, 1989). In most labelled experiments [14C]adenine was used as precursor. The inconclusive data obtained could be due to the difficulty of culturing isolated seeds, or that too little radioactive compound was used to show effective incorporation, particularly as adenine plays a central role in plant metabolism. Using [14C]adenine Hocart and Letham (1990) reported that in germinating maize embryos incorporation was into zeatin nucleotide ([9R-MPJZ) while in germinating lupins Nandi et al. (1988) reported incorporation into dihydrozeatin riboside and its nucleotide. To date no incorporation of adenine into developing seeds or fruits have been reported and it remains a moot point as to whether these organs are sites of cytokinin biosynthesis or alternatively strong areas for its mobilization. © 1993 by Gustav Fischer Verlag, Stuttgart

Materials and Methods Seeds of Pisum sativum cv. Onward were sown in sterilized soil. They were fed weekly with 2 gL -I Multifeed and watered daily. After two months the plants commenced flowering. All flowers were labelled as they flowered so as to ultimately allow for the use of fruits of similar age in feeding experiments. Eight-day-old fruits were removed from the plants and the developing seeds excised from the pods in such a way that the funiculus and a small portion of the pod walls remained attached to the seed. This was done to ensure better uptake during subsequent incubation. Both the seeds and sections of the pod walls were incubated in 4 mL of water containing [S-14C]adenine ([S-14C]ADE) (Amersham International, specific activity 1.96GBqmmol- l ) for 6h. At the completion of incubation the plant material was boiled immediately in 5 mL SO % methanol for 3 minutes whereafter it was ground in a glass homogenizer and then filtered through Whatman No. 1 filter paper. The extracts were taken to dryness and resuspended in 1 mL SO % HPLC grade methanol and filtered successively through 0,45 and 0.2 ~m DynaGard filters. The final extracts were taken to dryness in a vacuum centrifuge and resuspended in 500 ~L SO % HPLC methanol. These seed and pod wall extracts were separated by semi-preparative HPLC using a Hypersil5 ODS column (5 ~m, C 18 , 10 x 250 mm) fitted to a Varian 5000 instrument using a 0.2 M acetic acid: methanol solvent system (5-50% over 90 min). The acetic acid was buffered to pH 3.5 with triethylamine. The flow rate was 3 mL min -I. One minute samples (3 mL) were collected and 400 ~L of each sample removed for the detection of radioactivity. Four mL Ready Solv EP scintillation fluor was added to each sample and the radioactivity detected using a Beckman DB 3S00 scintillation counter.

381

Cytokinin biosynthesis in pea fruits For TLC, standards and 50 ilL aliquots of the 2600 ilL remaining of the samples collected following semi-preparative HPLC, were spotted onto Merck 60 F2'>4 silica gel plates. They were separated using the upper phase of n-butanol: NH 40H: water (6: I: 2 v/v). The chromatograms were divided into 10Rf sections, which were eluted and aliquots of the eluant were counted to determine the radioactivity associated with each Rf. Following semi-preparative HPLC, 250 ilL of relevant fractions were removed and 125 ilL thereof treated with KMn04 (Miller, 1965) to determine whether the side chains of the putative cytokinins were saturated. Hundred ilL each of the treated and untreated samples were then filtered through a 0.2 Ilm Millipore filter and samples were then separated by analytical HPLC using a Supelcosil LC-18-DB column (CI8, 51l, 4.6 x 250 mm). The buffer system

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used was the same as that previously used for semi-preparative HPLC, but the flow rate was reduced to 1 mL min -I.

Results

Most of the radioactivity recovered from both the seeds and pod wall matarial co-chromatographed with ADE following semi-preparative HPLC (Fig. 1 A) and TLC (Fig. 1 B). The second largest radioactive peak detected had similar chromatographic properties to ADO (Figs. 1 A and C). The third peak of radioactivity detected had a similar retention time to Z (Fig. 1 A). When an aliquot was subjected to TLC

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Fig. 1: Radioactivity detected in the seeds (e--e) and pod wall (6- - - - -6) extracts obtained from 8-day-old fruits after having been incubated with [8- H C]ADE for 6 h (A). Samples (500 ilL) were fractionated by semi-preparative HPLC at a flow rate of 3 mlmin-I. Aliquots of 400 III were used for the detection of radioactivity. The retention times of authentic standards are indicated as bars. TlC separation was performed using aliquots of the respective peaks indicated by arrows and their radioactive profiles determined (B, C, D) .

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382

J. VAN STADEN and F. E. DREWES

the radioactivity was found coincident with authentic Z (Fig. 1 D). Fractionation of aliquots of this peak using analytical HPLC yielded one peak coincident with 90 % of the radioactivity which co-chromatographed with Z (Fig. 2). Upon treatment with KMn04 98 % of the radioactivity shifted from this retention time. Expressed as a percentage of the total radioactivity recovered by semi-preparative HPLC the peak co-eluting with Z accounted for 2.2 % of the radioactivity associated with the seeds and 5 % in the case of the pod walls. In the seeds 78 % of the radioactivity still coeluted with ADE and in the pods, 59 %. No radioactivity was detected at the retention times of [9R-MP]Z, iP, [9R]iP or [9R-MP]iP (Fig. 1 A). Discussion

Using [8- 14C]ADE it was shown that this purine is incorporated into a compound which co-chromatographed with Z in germinating maize embryos. In an earlier study on maize it was suggested that incorporation is into [9R-MP]Z (Hocart and Letham, 1990). Nandi et al. (1988) suggested that in germinating lupin seeds incorporation is into [9R]DHZ and [9R-MP]DHZ. There are therefore either considerable inconsistencies in the results and/or more than one route whereby incorporation can occur (Letham and Palni, 1983; Einset, 1991). This paper is the first to provide some evidence for the incorporation of ADE into cytokinins in developing seeds. Many earlier studies where low levels of radioactivity was used were negative (Van Staden and Choveaux, 1981; Van Staden and Vos, 1989). The present results do not provide a clear indication as to the biosynthetic route

as proposed by Letham and Palni (1983) and/or Einset (1991). Acknowledgements

The Foundation for Research Development is thanked for financial support.

References EINSET, J. W.: In: Biotechnology in Agriculture and Forestry. (BAJAJ, Y. P. S. (ed.)), Vol. 17. High-Tech and Micropropagation 1. pp. 190-201. Springer-Verlag, Berlin (1991). HOCART, D. H. and D. S. LETHAM: J. Exp. Bot. 41, 1525-1528 (1990).

KRECHTING, H. C. J. H., A. VARGA, and J. BRUINSMA: Z. Pflanzenphysiol. 87, 91-93 (1978). LETHAM, D. S. and L. M. S. PALNI: Annu. Rev. Plant Physiol. 34, 163-197 (1983).

MILLER, C. 0.: Proc. Natl. Acad. Sci. USA, 54, 1052-1058 (1965). NANDI, S. K., L. M. S. PALNI, D. S. LETHAM, and J. S. KNYPL: J. Exp. Bot. 39, 1649-1665 (1988). VAN STADEN, J.: Physiol. Plant. 58, 340-346 (1983). VAN STADEN, J. and J. BUTTON: Z. Pflanzenphysiol. 87, 129 -135 (1978).

VAN STADEN, J. and N. A. CHOVEAUX: Z. Pflanzenphysiol. 104, 395-399 (1981).

VAN STADEN, J. and J. E. DAVEY: Plant Cell Environ. 2, 93-106 (1979).

VAN STADEN, J. and C. FORSYTH: J. Plant Physiol. 124, 299-308 (1986).

VAN STADEN, J. andJ. E. Vos: J. Plant Physiol. 135, 114-116 (1989).