Plant Growth Regulation32: 157–160, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

157

Vascular transport of auxins and cytokinins in Ricinus

Dennis A. BakerDepartment of Biological Sciences, Wye College, University of London, Ashford, Kent TN25 5AH, UK(e-mail: d.baker@wye.ac.uk)

Key words:auxins, cytokinins,Ricinus, phloem sap, xylem sap, vascular transport

Abstract

Analyses of vascular saps supplying source and sink organs have demonstrated the presence of major endogenoushormones and/or their precursors. Indol-3yl-acetic acid, a number of gibberellins, cytokinins and abscisic acid,as well as the precursor for ethylene production have been found in these vascular saps, allowing the sites ofhormonal synthesis and putative target tissues to be deduced. Exogenously applied hormones are also readilyloaded into these vascular pathways and may be translocated over considerable distances from a point ofapplication. Observations such as these indicate a possible co-ordination system between source and sink regulatedby the synthesis and transport of endogenous hormones. It is widely accepted that the partitioning of assimilatesbetween photosynthetic source organs and utilising sink organs is regulated by endogenous plant hormones. Thekey intermediate steps involved in assimilate transport, such as phloem loading and unloading, have been shownto be responsive to applied hormones, although the role of endogenous hormones in these processes remainsessentially unresolved. Results of the analyses of vascular saps fromRicinus communis, which have been obtainedusing a range of physicochemical methods, are compared and contrasted with those obtained by the applicationof exogenous hormones or their precursors. These results are evaluated critically and interpreted in the light ofcurrent models of source:sink regulatory processes and the long-distance transport of auxins and cytokinins inhigher plants.

1. Introduction

Movement of hormones through the vascular tissuesof whole plants is a relatively neglected area ofhormonal physiology compared with the numerousstudies on short distance transport through isolatedplant segments. Whereas the xylem pathway is rela-tively easy to sample by collecting root pressureexudate, the phloem pathway has received little atten-tion, in part due to the difficulty of obtaining puresamples of phloem sap and, until relatively recently,the absence of reliable detection methods for preciseidentification and quantitation of these substances[12]. Phloem exudates have now been sampled froma number of plant species using a range of techniquesincluding aphid stylets severed by hand and morerecently by laser or radio frequency micro-cautery[9, 14]. However these methods are limiting in that

the volume of sap that can be collected is minimal(<5 µl h−1). Exudate collection from incisions intothe phloem of stems, petioles and fruits yield rela-tively large volumes, which allow the identificationand quantitation of phloem mobile hormones [12].Of these various collection methods the exudationof phloem sap from incisions into the inner bark ofRicinus communisresults in large volumes of purephloem sap which can be collected for several hours[10]. Root pressure exudate can be collected simultan-eously from the same plant allowing the endogenoushormones to be identified and subsequently quantifiedwithin the vascular pathways at any particular stageduring the growth of the plant.

Analyses of these vascular saps fromRicinusbyphysico-chemical or immunological techniques, havedemonstrated the presence of auxins [2, 3, 7, 11]and cytokinins [1, 4, 13, 19] at concentrations which

158

Table 1. IAA identified and quantified by GC-MS and freetryptophan in the vascular saps ofRicinus[3]

Sample IAA (ng ml−1 sap) Tryptophan (µg ml−1 sap)

Phloem 13.0 96.0

Xylem 0.32 0.38

evidence physiological activity in bioassays [10]. Thisimplies that they exert a direct influence in the long-distance regulation of source and sink.

2. Auxins

The transport of 3-indolyl-acetic acid (IAA) from sitesof synthesis to binding receptors in cells elsewherewithin a plant tissue or organ is widely believed toprovide the regulatory basis of spatial and temporalorganisation in plants. The prevailing view of IAAtransport is that IAA synthesised in young, metabol-ically active tissues is transported through a cell tocell pathway with a distinct polarity, which is basi-petal in shoots and acropetal in roots. Additionallythere is a vascular transport of free IAA throughthe phloem as evidenced by the detection by GC-MS analysis of endogenous IAA inRicinusphloemsap [1] (see Table 1) and the transport of14C-IAAfollowing synthesis from14C-tryptophan after applic-ation to mature leaves ofRicinus [5]. Some earlierreports of IAA detected moving through the xylemhave been shown to be the result of bacterial contamin-ation of the samples and, where care is taken to avoidcontamination, the amount of IAA detected in xylemsap is negligible (<1 ng ml−1) [2]. This virtual absenceof IAA in the xylem sap, leads to the inevitable conclu-sion that the free IAA present in the phloem sap isprovided by synthesis and export from the matureleaves. This corroborates the Sheldrake hypothesis[20] that the meristems are net importers rather thansynthesisers of IAA in higher plants. The widely heldview that the high IAA content detected in meristemsis evidence of a localised synthesis is based on a falselogic which has long prevailed in hormone physiology.Researchers in transport physiology accept that thehigh concentrations of sucrose in the root of sugarbeet do not implyde novosynthesis in that organ,but reflect accumulation of imported assimilates. Asimilar concept can be applied to IAA accumulation

in meristems and need to be interpreted in these termswhen long distance transport of IAA is considered [5].

There is compelling evidence that IAA is synthes-ised in mature leaves and is acid-trapped withinthe high pH phloem compartment (pH∼ 8.0 [10])wherein it is transported with the photoassimilates tosink tissues such as meristems, there providing thesource of IAA for extravascular polar transport [3].Clearly these observations need to be reconciled witha number of current investigations on short distancetransport of IAA which ignore the origin of the IAAdetected in the polar transport pathway. The latter is ofcourse essential for temporal and spatial developmentof apices but the IAA involved in this role is importedinto these sink tissues with the photoassimilates via thephloem pathway.

3. Cytokinins

It is widely accepted that roots are as the major sitesof cytokinin synthesis [21] and the subsequent trans-port of root-derived cytokinins through the xylemin the transpiration stream to the aerial parts ofplants exerts control over shoot development [18],probably through the regulation of sink activity andnutrient partitioning [16]. The induction of a cellwall acid invertase and of a glucose tranporter bycytokinins may provide a mechanism for the regula-tion of source:sink relations by cytokinins [8]. Otherplant organs in addition to the roots have been shownto be capable of converting radiolabelled cytokininprecursors to active cytokinins [6]. The phloempathway has been shown to be involved in the circu-lation of cytokinins within the plant [13] althoughthe source of the cytokinins detected in phloemsaps remains unresolved. Three possible origins ofcytokinins within phloem saps have been postulated[13]:1. Synthesis within mature leaves, loading into the

phloem and subsequent transport with the otherassimilates to various sinks. Direct evidence ofleaves as a source of cytokinins is still awaited.

2. Direct exchange from the xylem. This is unlikelyas the partition coefficients of naturally occur-ring cytokinins would not favour transfer fromthe acidic xylem to the alkaline phloem compart-ment. The principal cytokinins within the xylemand phloem saps are different [4] (see Table 2).

3. Recirculation of cytokinins received from the rootsvia the transpiration stream following loading into

159

Table 2. Cytokinins in the vascular saps ofRicinusidentified andquantified by GC-MS-selective ion-monitoring

Sample IP DHZ Z IPA DHZR ZR

Cytokinins detected (ng.ml−1 sap)

Phloem 27.7 nd 104 nd nd 5.1

Xylem nd nd 23.2 14.1 nd 58.7

Following BuOH partition and alkaline

phosphatase treatment

Phloem nd nd nd nd nd nd

Xylem nd nd nd 3.1 nd 14.6

IP – Isopentenyl adenine; DHZ – Dihydrozeatin; Z – Zeatin; IPAIsopentenyl adenosine; DHZR – Dihydrozeatin riboside; ZR –Zeatin riboside.

the phloem within mature leaves. This possibleorigin of cytokinins in the phloem sap is consistentwith the observed distribution of cytokinins tomajor sinks such as meristems and storage organsnot connected directly to the transpiration stream[18].

Detailed analyses of vascular saps by GC-MS(Table 2) confirm earlier reports that zeatin riboside isthe predominant cytokinin transported from the rootsvia the xylem and zeatin is the major transport formin the phloem. Additionally isopentenyladenine wasfound in the phloem and not in the xylem sap ofRicinuswhile isopentenyl adenosine was found in thexylem and not in the phloem sap from the plants.Studies by Komor et al. [15] have demonstrated thatmost free bases and ribosides of various cytokinins aretranslocated if they are presented to the phloem.

4. Conclusions

Transport of auxins and cytokinins occurs extensivelythrough the phloem of higher plants. Other hormonessuch as abscisic acid and gibberellins are also trans-ported through this pathway [12]. The mature leavesare the major source of these hormones. In the caseof IAA they are the primary source whereas in thecase of the cytokinins it appears likely that they arere-translocated after synthesis elsewhere in the plantand move into the mature leaves via the xylem stream.

The phloem mobile auxin and cytokininsundoubtedly play a key role in the integration ofsource:sink regulatory processes and root:shootinteractions during the growth and development of the

plant [17]. The precise nature of this role is a key areaof hormone physiology which requires resolution.

Acknowledgement

GC-MS-SIM analyses of cytokinins were conductedby Patrick Blake at HRI, East Malling, UK.

References

1. Allen JRF and Baker DA (1980) Free tryptophan and indole-3-acetic acid levels in the leaves and vascular pathways ofRicinus communisL. Planta 148: 69–74

2. Allen JRF, Greenway AM and Baker DA (1979) Determina-tions of indole-3-acetic acid in xylem sap ofRicinus communisL. using mass fragmentography. Planta 144: 299–303

3. Baker DA and Allen JRF (1988) Auxin transport in thevascular system. In: Kutacek M, Bandurski RS and KrekuleJ (eds) Physiology and Biochemistry of Auxins in Plants.Prague: Academia, pp 21–32

4. Baker DA and Allen JRF (1992) Cytokinin transport in thevascular system. In: Kaminek M, Mok DWS and ZazimalovaE (eds) Physiology and Biochemistry of Cytokinins in Plants.SBP Academic Publishing, pp 225–228

5. Borkovec V, Didehvar F and Baker DA (1994) The biosyn-thesis and translocation of14C-IAA in Ricinus communis.Plant Growth Regulation 15: 137–141

6. Chen C-M, Ertl JR, Leisner SM and Chang C-C (1985) Local-isation of cytokinin biosynthetic sites in pea plants and carrotroots. Plant Physiol 78: 510–513

7. Chino M, Hayashi H, Nakamura S, Oshima T, Turner H,Sabnis D, Borkovec V, Baker DA, Bonnemain JL and DelrotS (1991) Phloem sap, composition. In: Bonnemain JL, DelrotS, Lucas WJ and Dainty J (eds) Recent Advances in PhloemTransport and Assimilate Compartmentation, Ouest Editions.Nantes Cedex, France: Presses Académiques

8. Ehness R and Roitsch T (1997) Co-ordinated induction ofmRNAs for extracellular invertase and a glucose transporterin C. rubrumby cytokinins. Plant J 11: 539–548

9. Girousse C, Bonnemain J-L, Delrot S and Bournoville R(1991) Sugar and amino acid composition ofMedicago sativa:A comparative study of two collecting methods. Plant PhysiolBiochem 29: 41–48

10. Hall SM and Baker DA (1972) The chemical composition ofRicinusphloem exudate. Planta 106: 131–140

11. Hall SM and Medlow GC (1974) Identification of IAA inphloem and root pressure saps ofRicinus communisL., bymass spectrometry. Planta 119: 257–261

12. Hoad GV (1995) Transport of hormones in the phloem ofhigher plants. Plant Growth Regulation 16: 173–182

13. Kamboj JS, Blake PS and Baker DA (1998) Cytokinins in thevascular saps ofRicinus communis. Plant Growth Regulation25: 123–126

14. Kawabe S, Bukomorita T and Chino M (1980) Collection ofrice phloem sap from stylets of homopterous insect severed byYAG laser. Plant Cell Physiol 21: 1319–1327

15. Komor E, Liegl I and Schobert C (1993) Loading and trans-location of various cytokinins in the phloem and xylem of theseedlings ofRicinus communisL. Planta 191: 252–255

160

16. Kuiper D, Kuiper PJC, Lambers H, Schuit J and Stall M (1989)Effects of internal and external cytokinin concentration on rootgrowth and shoot to root ratio ofPlantago major sp. Pleio-spermaat different nutrient concentrations. Plant and Soil 111:231–237

17. Kuiper D (1993) Sink strength: established and regulatedby plant growth regulators. Plant, Cell and Environment 16:1025–1026

18. Letham DS (1978) Cytokinins. In: Letham DS, GoodwinPB and Higgins TJV (eds) Phytohormones and RelatedCompounds : A Comprehensive Treatise, Volume 1. Amster-dam: Elsevier, pp 205–263

19. Phillips DA and Cleland DF (1972) Cytokinin activity fromthe phloem sap ofXanthium strumariumL. Planta 102: 173–178

20. Sheldrake AR (1973) The production of hormones in higherplants. Biol Rev 48: 509–559

21. Van Staden J and Davey JE (1979) The synthesis, transportand metabolism of endogenous cytokinins. Plant Cell Environ2: 93–106