Planta (Berl.) 81, 64--70 (1968)

Inhibitor Content of Phloem and Xylem Sap Obtained from Willow (Salix viminalis L.) Entering Dormancy M. R. Bow~N a n d G. V. HeAD Botany Department, University College of Wales, Aberystwyth l~eceived February 22, 1968

Summary. I t is shown that there is one growth inhibitor in the phloem sap and two in the xylem sap of willow (S. viminalis L.). The concentration of the inhibitor in the phloem sap, (+)-abscisie acid, increases as the plants enter dormancy. This is Mso shown for (+)-abscisic acid in the xylem sap, hut the concentration of the second inhibitor decreases in a reciprocal maimer. Introduction This w o r k was carried o u t w i t h a view to d e t e r m i n i n g how t h e i n h i b i t o r c o n t e n t of p h l o e m a n d x y l e m sap changes as willow (S. viminali8 L.) grown u n d e r n a t u r a l conditions in t h e field enter d o r m a n c y . P r e v i o u s w o r k on t h e i n h i b i t o r c o n t e n t of t h e x y l e m sap of S./ragilis L. b y DAVlSON (1965) i n d i c a t e d t h a t seasonal v a r i a t i o n s can occur, t h e highest level of i n h i b i t i o n being recorded in bioassays p e r f o r m e d on s a m p l e s o b t a i n e d from willow during t h e d o r m a n t period. R e c e n t w o r k on e x u d e d p h l o e m sap of S. viminalis L. has shown t h a t a p o t e n t g r o w t h inhibitor, ( + ) - a b s c i s i c acid, is p r e s e n t (I{OAD, 1967). The level of this i n h i b i t o r was f o u n d to be g r e a t e r u n d e r short d a y s t h a n u n d e r long days. I n view of these results i t seemed t h a t it w o u l d be v a l u a b l e to s t u d y t h e i n h i b i t o r c o n t e n t of b o t h t r a n s p o r t s y s t e m s s i m u l t a n e o u s l y d u r i n g t h e p e r i o d from t h e s t a r t of leaf senescence to t h e onset of d o r m a n c y .

Materials and Methods Willow (S. viminalis L.) were grown in the Botany Gardens, University College of Wales, Aberystwyth. Samples of xylem and phloem sap were obtained from two year old material at about two weekly intervals between 1st October and 1st December, 1965. The technique of BOLLAt~D(1953) was employed for the collection of xylem sap in the manner described by DAVlSON (1965). Lengths of stem 1 metre in length and free from side branches were cut from the plants and immediately extracted in the laboratory. The volume of sap used at each sampling was 20 ml. Phloem sap was obtained as honeydew from the aphid Tuberolachnus salignus (GMELII~). The aphids were caged in perspex boxes lined with aluminium foil on stems growing a few degrees from the vertical position. The boxes were covered with polythene sheets to protect them from wind and rain damage. Any honeydew

Inhibitor Content of Phloem and Xylem Sap of Willow

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produced by the aphids was collected directly on the aluminium foil. The collection period lasted four days, after which time the honeydew was dried and stored at --18~ until the end of the final collection. Both honeydew and xylem sap were extracted in the manner described by D)~wso~ (1965). Weighed aliquots of honeydew were dissolved in 100 ml of distilled water and the xylem sap was used without the addition of further water. The samples were taken to pH 2.6 with HC1 and extracted with three times their volume of freshly redistilled ether. The ether fraction was reduced to dryness and then line loaded on to 10 cm wide strips of Whatman No. 1 chromatography paper. The chromatograms were allowed to run 30 cm in the solvent systems given later in the text, dried and cut into the ten Rf zones. Each chromatogram segment was placed in a 4.5 cm petri dish and eluted for twelve hours with 1 ml of buffered sucrose. Bioassays were then performed using ten pieces of 10 mm long subapical segments of wheat coleoptile var. 'Atle'. Results showing the mean growth of the coleoptiles in each dish are presented as histograms, the darkened areas indicating growth significantly different from the controls at P ~ 1 per cent. Results and Discussion I t can be seen f r o m t h e d a t a p r e s e n t e d in Fig. 1 t h a t t h e x y l e m sap c o n t a i n e d t w o v e r y active g r o w t h inhibitors. A t t h e t i m e of t h e first collection ( l s t October) an i n h i b i t o r was p r e s e n t r u n n i n g a t t h e s o l v e n t front in i s o p r o p a n o l : a m m o n i a : w a t e r 1 0 : 1 : 1 (v/v), which decreased in a c t i v i t y d u r i n g successive collections u n t i l on 1st D e c e m b e r i t was c o m p l e t e l y a b s e n t . The second inhibitor, r u n n i n g a t t h e fi-inhibitor zone (BENNET-CLARK a n d KEFFORD, 1953) was n o t p r e s e n t a t the first collect i o n b u t m a d e its a p p e a r a n c e a t t h e second s a m p l i n g a n d increased in a c t i v i t y d u r i n g t h e r e m a i n d e r of t h e collection period. I n p h l o e m sap collected b e t w e e n 1st O c t o b e r a n d t h e beginning of N o v e m b e r , one p e a k of i n h i b i t i o n was d e t e c t e d a t R f 0 . 6 - - 0 . 7 which a g a i n increased in a c t i v i t y in successive collections (Fig. 2). A f t e r 3rd N o v e m b e r t h e m e a n air t e m p e r a t u r e d r o p p e d too low for t h e a p h i d colonies to be m a i n t a i n e d in t h e field a n d c o n s e q u e n t l y after this d a t e no f u r t h e r h o n e y d e w collections were m a d e . T h e h o n e y d e w s a m p l e s were also r u n in o t h e r solvent s y s t e m s to confirm (a) t h e r e was a single i n h i b i t o r p r e s e n t a n d (b) t h e l~fs of t h e i n h i b i t o r in t h e p h l o e m sap c o r r e s p o n d e d to those of t h e i n h i b i t o r detect e d b y DAvIsoN in x y l e m sap of S./ragilis L. The results of DAWSON are p r e s e n t e d below, a n d t h e results o b t a i n e d w i t h t h e p h l o e m sap are shown in Figs. 2 a n d 3. I t is shown t h a t t h e r e was close a g r e e m e n t in t h e Rfs of t h e i n h i b i t o r s o b t a i n e d from t h e two sources.

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M . R . BOWEN a n d G. V. HOAD:

At the time the first honeydew samples were collected, the leaves of the willow were green and showed no marked visible signs of senescence. B y the beginning of November, all the leaves had turned yellow and about A

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Fig. l. W h e a t coleoptile assay of chromatographed acid fractions of 20 ml of xylem sap obtained from S. viminalis L. The dates of sampling and the number of hours

of light on each of these days is indicated one-third had abscinded. I t is interesting to note t h a t although the leaves were senescing at this time, they were still exporting solutes into the phloem on which the aphids were feeding. This was shown b y bringing leafy shoots into the laboratory and illuminating the leaves when held

Inhibitor Content of Phloem and Xylem Sap of Willow OCT 19

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68

1Y[.1~. Bow]~l~ and G. V. HO~D:

in an atmosphere containing 20~C of 14C02. Samples of honeydew collected 3 hrs after the administration of the 1~C02 from aphids situated below the last leaf gave a count at least 20 times more than the background level, confirming the export of solutes from the leaves. No attempt was made to determine the nature of the 14C-labelled compounds present.

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Fig. 3. Wheat coleoptile assay of acid fractions of 150 mg dry wt. of S. salCgnus honeydew ehromatographedin three different solvent systems These results raise several questions about the chemical nature of the inhibitors present, the significance of the change in their concentration in the sap, and their site of synthesis within the plant. The inhibitor present in the phloem sap has been positively identified by optical rotatory dispersion measurements as (+)-abscisie acid (HEAD, 1967), but the inhibitor present at the/~-inhibitor zone in the xylem sap extracts has not been identified by this method. However, data obtained using thin

Inhibitor Content of Phloem and Xylem Sap of Willow

69

layer and gas-liquid chromatographic techniques indicate t h a t the major part of the activity of the fi-inhibitor zone can be accounted for by the presence of (d-)-abseisic acid (L~TOCr B o w ~ and SAC?ODORS, unpublished data). The identity of the other inhibitor from the xylem sap (Fig. 1) running at the solvent front has not been established. When compared with the data of DAVISON, the results obtained in the present work with xylem sap show one marked difference, namely in the presence of the second inhibitor running at the solvent front. This inhibitor was not found by DavIsoN, but this worker only chromatographed samples obtained during August when the willow were dormant. The data he presented on the seasonal variation in inhibitor levels were obtained with material which was not chromategraphed, t t is quite possible t h a t ~he presence of a second inhibitor in the samples at other times of the year could have passed unnoticed. The presence of (d-)-abscisic acid in both xylem and phloem sap would seem to indicate t h a t there are two possible sites of synthesis, the roots and the mature leaves. The latter of these two is to be favoured as previous workers have shown t h a t mature leaves are the sites of photoperiodie perception and t h a t when the daylength under which certain plants are grown is reduced the level of an inhibitor, presumably (d-)-abscisie acid, in the mature leaves rises very quickly (EAGLES and WA~S,I~G, 1964). (d-)-abscisie acid could then be exported to the apical region of the stem and to the root system from where it is re-exported in the transpiration stream. The absence of the other inhibitor in the phloem sap precludes the leaves as the site of synthesis of this compound. I t s presence in the xylem stream alone would seem to indicate t h a t the roots are the site of synthesis. Finally, it has to be established whether the changes in the concentration of the inhibitors in the sap are a true reflection of the rate of their synthesis in the plant. I t has been stated that over the period t h a t the experiment was conducted there was a marked senescence and abscission of the leaves under the influence of decreasing daylength. This will affect a number of processes which are of relevence here. A loss of leaves will (a) reduce the number of possible sites of synthesis of (d-)-abscisic acid; (b) it will cause a reduction in the total volume of sap exported from the leaves and transloeated down the stem; and (c) it will lead to a lessening in transpiration flow. The loss of the xylem sap inhibitor at the solvent front in successive collections is presumably a reflection of reduced synthesis within the roots due to a loss in root activity with the onset of dormancy. Factors (a) and (b), because they occur simultaneously, should not influence the levels of abscisie acid detected in the sap to any great extent, and therefore it is suggested t h a t the levels of abseisie

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Bows.it and HeAD : Phloem and Xylem Sap of Willow

acid d e t e r m i n e d d i r e c t l y reflect t h e r a t e of synthesis w i t h i n t h e leaves. I f this is a c c e p t e d t h e n t h e b u i l d u p of abscisic acid in t h e x y l e m sap m u s t also be due to increased synthesis w i t h i n t h e leaves, a l t h o u g h i t m a y be e x a g g e r a t e d b y a r e d u c e d t o t a l v o l u m e of sap flow in t h e t r a n s p i r a t i o n stream. Thanks are due to the Science Research Council for the provision of a studentship to. M. R. B. References BS~NN~T-CLAR~,T. A., and N. P. KV,lVFORD: Chromatography of the growth substances in plant extracts. Nature (Lend.) 171, 645--647 (1953). BOLLARD,E. G. : The use of tracheal sap in the study of apple tree nutrition. J. exp. Bet. 4, 363--368 (1953). DAwso~r R. ~ . : Some properties of a plant growth inhibitor present in xylem sap of woody species. Aust. J. biol. Sci. 18, 475--486 (1965). EAGLS.S,C. F., and P. F. WAR]~ING: The role of growth substances in the regulation of bud dormancy. Physiol. Plantarum (Kbh.) 17, 697--709 (1964). HEAD, G. V. : (d-)-abscisin II, ((-4-)-dormin) in phloem exudate of willow. Life Sci. 6, 1113--1118 (1967). Dr. G. V. HeAD Pomology Department University of Bristol Research Station Long Asthon, Bristol. (U.K.)

Mr. ~ . R. Bow~N Botany Department University College of Wales Aberystwyth. (Wales, U.K.)

Inhibitor content of phloem and xylem sap obtained from willow (Salix viminalis L.) entering dormancy.

It is shown that there is one growth inhibitor in the phloem sap and two in the xylem sap of willow (S. viminalis L.). The concentration of the inhibi...
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