Planta (Berl.) 85, 111--117 (1969)

Sugar Uptake by the Grape Berry: A Note on the Absorption Pathway P. E. KRI]~DEMANN Division of Horticultural Researeh, C.S.I.R.O., Merbein Received August 30/l~ovember 8, 1968

Summary. 3H-glucose was fed to excised Sultana grape berries via their pedicels for up to 5 hours. Autoradiography showed that the label was distributed throughout the fruit within 1 hour. Microautoradiography of tissue sections taken at a number of points showed that within the pedicel the walls o~ cortical cells had become heavily labelled, suggesting that the cortical cell walls offered a diffusion pathway for the solutes entering the vascular system from the external aqueous solution. Transport along the pedicel was confined to the central vascular tissue with little radioactivity occurring in the cortical cells. Within the periearp, the vascular bundles and walls of nearby parenchyma cells had become heavily labelled, indicating that the labelled solute was present within the vicinity of cell walls. The general pattern of 3It-glucose accumulation by excised berries was similar to the deposition pattern of ~4C-labelledphotosynthate within attached fruit. Introduction The developing grape b e r r y represents a strong sink for carbohydrates (HAZE a n d WEAVER, 1962) a c c u m u l a t i n g s u b s t a n t i a l a m o u n t s of sugar (glucose a n d fructose) during the later stages of its d e v e l o p m e n t on the vine. The f r u i t when detached from the vine continues to absorb substrates supplied ~ s ~ q u e o u s solutions to the cut pedicel, a capacity which has been of use i n s t u d y i n g the metabolic fate of certain substrates (HA~])u 1967). The p a t h w a y of m o v e m e n t of labelled substrates into the detached b e r r y i s h o w e v e r n o t known. The present p a p e r provides some i n f o r m a t i o n on the a b s o r p t i o n p a t h w a y of all-glucose supplied to the pedicels of detached ~berries u n d e r l a b o r a t o r y conditions.

Material and Methods Berries were detached from potted vines of Viti8 vini/era L. cv. sultana (syn. sultanina, "Thompson's Seedless") grown in the glasshouse under natural daylength and with a diurnal temperature range of 20 to 35~ The fruit had recently undergone "colour change" (veraison) and had now entered their second growth phase, i.e. the period of rapid volume increase and sugar accumulation. 3H-glucose was supplied as 10 ~l of a 0.41 M solution (specific activity 1 mc/ml) in narrow bore soft rubber tubing to the pedicels of detached berries for up to 5 hours. Upon completion of the glucose feeding, longitudinal hand sections were taken from the midplane of the labelled fruit. Precautions were observed to minimise 8 Planta (Berl.), Bd. 85

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the spread of radioactivity due to the action of the razor. They included rinsing and blotting the tissue surface of the berry following sectioning to remove debris, as well as washing and drying the blade of the razor after each use. These hand sections were then freeze dried at room temperature and the mounted specimens exposed to X-ray film (Kodak Industrial X-ray film type AA) for a suitable period. Developing and fixation followed conventiona! procedures. The two remaining halves of sectioned fruit were snap frozen in isopentane held in a freezing mixture of acetone and dry ice. The frozen tissue was then dehydrated ("freeze-substituted') at --15 ~ in anhydrous ether over AlzO~ (LiiTTQE and WEIGL, 1965). The dehydrated tissue was transferred to xylene held at --15 ~ and raised slowly to room temperature for embedding in paraplast. Sections (10 ~.) were cut on to microscope slides and dewaxed with ether before coating with a thin film of nuclear track emulsion (Kodak NTB-3) using the technique previously described (KRIEDEig.~NN,1967) which minimises migration of water soluble label. After exposure at 5 ~ over anhydrous CaC12 for 10 days the emulsion was developed in Kodak dektol (15 ~ for 2 rain) and fixed in Kodak fixing bath F5 (15~ for 5 rain). After washing for 5 rain and subsequent drying, the sections were stained with toluidine blue (O'BRIEN et al., 1964) and mounted permanently in Canada balsam.

Results and Discussion

1. The Overall Pattern el Substrate Accumulation within the Berry. Figs. 1 a ~ f show l o n g i t u d i n a l sections of berries fed all-glucose for 1, 2 a n d 5 hours. The specimens are shown on t h e left (Figs. 1 a, c a n d e) a n d t h e i r c o r r e s p o n d i n g a u t o r a d i o g r a p h s on t h e r i g h t - h a n d side (Figs. 1 b, d a n d f)l. I n 1 h o u r t h e label h a d c o m p l e t e l y p e n e t r a t e d the berry, showing some localisation in the central a n d p e r i p h e r a l v a s c u l a r systems. The a c c u m u l a t i o n p a t t e r n r e m a h l e d u n c h a n g e d o v e r t h e ensuing 4 hours alt h o u g h t h e overall level of a c t i v i t y , as g a u g e d from t h e i m a g e i n t e n s i t y on the X - r a y film, showed a n increase. Some localisation of label also occurred in t h e zone b e t w e e n t h e p l a c e n t a a n d p e r i e a r p of t h e b e r r y (arrows in Figs. 1 a - - d ) . This region of t h e b e r r y is n o t served b y a n y m a j o r v a s c u l a r b u n d l e a n d a p p a r e n t l y acts as free space for s u b s t r a t e deposition. The s a m e general p a t t e r n of a c c u m u l a t i o n occurred when 14C labelled p h o t o s y n t h a t e e n t e r e d t h e berry. Figs. 1 g a n d h show a l o n g i t u d i n a l section a n d a u t o r a d i o g r a p h of a b e r r y a t a similar d e v e l o p m e n t a l stage to those used for t h e sit-glucose feeding, laC02 g e n e r a t e d from Ba14C03 (2 rag; specific a c t i v i t y , 1.5 mC/mM) h a d been supplied to a n e a r b y leaf 24 hours previously. I n this case (Fig. 1 h) the b e r r y showed a general d e p o s i t i o n of labelled p h o t o s y n t h a t e t h r o u g h o u t its tissues b u t a g a i n t h e r e were localised a c c u m u l a t i o n s of label in t h e v a s c u l a r tissue a n d in t h e zone s e p a r a t i n g t h e p l a c e n t a a n d p e r i c a r p of t h e berry. 2. all-Glucose Transport in the Pedicel el Excised Berries. T h e m o v e m e n t of ~H-glucose from t h e e x t e r n a l a q u e o u s solution across t h e 1. A key to the symbols used throughout the figures is given on page 113.

Fig. 1 a ~ h . The overall pattern of substrate deposition within the sultana berry. a, c and e; Longitudinal sections through berries supplied with 3H-glucose for 1, 2 and 5 hours, respectively. - - b, d and f: Corresponding autoradiographs. - g and h: Longitudinal section and autoradiograph of ~ sultana berry supplied with 14C labelled photosynthate trom an adjacent leaf. pl placenta, pc pericarp, E external medium originally bathing the pedicel, C cortical tissue, cv central vascular strand of the pedicel, P H phloem, x y xylem, V B vascular bundle, C W cell wall 8*

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P. E. KRI]~DENANN:Sugar Uptake by the Grape Berry

cortical cells of the pedicel towards the central vascular system resulted in high concentrations of labelled solute in the walls of cortical cells as shown in Fig. 2 a. This microautoradiograph was made from a transverse section of the pedicel towards the cut end. I n some regions of Fig. 2 a (arrows) the deposit of silver grains is more concentrated along the inner face of the cell wall. Such deposits could represent labelled solute in transit or simply adsorption of vacuolar contents on to the wall during dehydration. When the labelled solute reached the vascular system of the pedicel, movement down the pedicel into the berry was restricted to the central vascular strand (Fig. 2 b) with relatively little activity in the surrounding cortical tissue. A portion of Fig. 2 b viewed with planepolarised light is shown as Fig. 2 c where xylem elements can be more readily discerned by their positive birefringence. Although labelled solute is present in both xylem and phloem elements, the density of silver grains in the emulsion across the vascular bundle suggests a higher concentration of solute in the phloem than in the xylem.

3. Accumulation within the Pericarp o/ Excised Berries Fed all-Glucose. The distribution of labelled solute in the pericarp tissue is shown in Figs. 3 a and b. The two vascular bundles shown in Fig. 3 a represent part of the peripheral vascular system of the berry and they were located at the shoulder of the fruit near the central vascular strand. In Fig. 3 a silver grain deposits appear as a reticulate pattern which outlines the walls of parenchymatous cells of the pericarp. The significance of these deposits is more apparent in Fig. 3 b which shows an enlarged view of the vascular bundle outlined at the top of Fig. 3 a. In Fig. 3b the lumen of the large cell adjacent to the vascular bundle shows a virtual absence of labelled solute compared to the level in the vicinity of the cell walls. I n view of the wide distribution of silver grain deposits in Fig'. 3a the present antoradiograph (Fig. 3 b) could represent evidence that labelled solutes moving out of the vascular system have entered the pericarp by diffusing around the periphery of the parenehyma cells rather than by accumulating into the lumen of cells surrounding the vascular bundle and then moving from cell to cell. This situation compares with that for the pedicel (Fig. 2a) where the walls of cortical cells were thought

Fig. 2 a--c. a Microautoradiograph of a transverse section through the outer cortical tissue in the pedicel of an excised sultana berry 5 hours after supplying 8H-glucose to that same region of the pedicel. In some regions (arrows) a heavy deposit of silver grains corresponds to the inner face of cell walls. - - b Microautoradiograph of a longitudinal section through the pedicel of an excised sultana berry 5 hours after supplying aH glucose to its cut end. - - c The area outlined in b when viewed under plane-polarised light

Fig. 2 a - - c

Fig. 3a and b. Microautoradiographs of sections of periearp tissue from an excised sultana berry 5 hours after supplying 8I-I-glucose through its pedicel. Area outlined in a is shown under higher magnification in b

:P. E. KRIEDEMANN: Sugar Uptake by the Grape Berry

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to offer a diffusion p a t h w a y for t h e labelled solute m o v i n g f r o m t h e e x t e r n a l aqueous solution to t h e v a s c u l a r s y s t e m of t h e pedicel. Studies on solute m o v e m e n t in other tissues h a v e also suggested t h a t cell walls can offer a diffusion p a t h w a y ( C o ~ A C ~ a n d L~MAY, 1963; Lf3TTGE, 1966; BDDUL~'E, 1967, I~ItIEDEMA~N, 1967). The m i e r o a u t o r a d i o g r a p h s p r e s e n t e d here (Figs. 2 - - 3 ) lend a d d i t i o n a l s u p p o r t to this suggestion. Grateful acknowledgement is made to Dr. J. V. POSSINO~A~and to my colleagues for their criticism of the manuscript and to Mrs. E. T6RSKFALVufor technical assistance. References

BIDDULPIt, S. F. : A microautoradiographie study of Ca~5and S~5 distribution in the intact bean root. Planta (Berl.) 74, 350--367 (1967). CO,MACK, R. G.H., and P. LEMAu Sugar in the intercellular spaces of white mustard roots. J. exp. Bet. 14, 232--236 (1963). HALE, C.R., and R. J. WEAVER: The effect of developmental stage on direction of translocation of photosynthate in Vitis vini/era. Hilgardia (Berkeley, Calif.) 88, 89--131 (1962). ttA~ny, P. J. : Sucrose breakdown and synthesis in the ripening grape berry. Aust. J. biol. Sci. 20, 4 6 5 ~ 7 0 (1967). KP*IEDEMA~, P. ; Sugar uptake and translocation in the castor bean seedling. III. An autoradiographic study of the absorption pathway. Planta (Berl.) 78, 175--180 (1967). LfdTTGE, U. : Investigations on the physiology of the glands of carnivorous plants. V. Microautoradiographic investigations of chloride secretion by the gland tissue of Nepenthes. Planta (BEE.) 68, 269--285 (1966). - - , u. J. WEmL: Zur Mikroautoradiographie wasserlSslicher Substanzen, Planta (Berl.) 64, 28--36 (1965). O'BI~IEN, T. t)., N. FEDER, and M. E. McCuLLY: Polychromatic staining of plant cell walls by Toluidine Blue 0. Protoplasma (Wien) ~9, 368--373 (1964). Dr. P. E. KRIEDEMANN C.S.I.R.O. Division of Horticultural Research Merbein, Victoria, 3505, Australia

Sugar uptake by the grape berry: A note on the absorption pathway.

(3)H-glucose was fed to excised Sultana grape berries via their pedicels for up to 5 hours. Autoradigraphy showed that the label was distributed throu...
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