Planta
Planta (Berl.)128, 9 3 - 1 0 0 (1976)
9 by Springer-Verlag 1976
Source, Sink and Hormonal Control of Translocation in Wheat I.F. Wardlaw and L. Moncur CSIRO Division of Plant Industry, P.O. Box 1600 Canberra, Australia
Summary. An analysis of the pattern of movement of 14C-labelled flag leaf assimilates in wheat (Triticum aestivum I. c.v. Gabo) during grain development, indicated that the greater the requirement for assimilates by the ear the more rapid was the speed of movement of these through the peduncle to the ear and also the lower their concentration. Experiments with [14C] indoleacetic acid ([a4C]IAA) suggested that auxin production by the grains was not responsible for the control of assimilate translocation through the peduncle. Limiting the supply of available assimilates by shading the lower parts of the plant, did not significantly alter the speed of movement of ~4C-photosynthate through the peduncle, wfiile severing half of the vascular tissue in the peduncle altered the pattern of movement of a4C to the ear and enhanced the speed of movement of 14C through the remaining functional conducting tissue. These results are discussed in relation to the mechanism of translocation.
Introduction
In an earlier paper (Wardlaw, 1965) data were presented, which indicated that the speed of movement of 14C_labelled flag leaf assimilates, through the peduncle to the ear of wheat, varied with the number of developing grains. This observation was confirmed in more detail later (see Wardlaw, 1974 a) and a similar association between speed of movement and demand for assimilates also appears to apply to sugarcane (Hartt and Kortschak, 1964) and tomato (Moorby et al., 1974). It was considered that a more detailed analysis of both the effect of sink size and the availability of assimilates on the movement of 14C-photosynthate through the vascular tissue, could be helpful in distinguishing between the different proposed mechanisms of translocation, as well as establishing some of the characteristics of the transport system. However any sink size control
of assimilate movement could be an indirect one, a complication that is evident, for example, in the possible role of auxins formed in growing organs, in regulating the movement of assimilates through the transport system (Seth and Wareing, 1967; Patrick and Wareing, 1973). Thus the present study, on the effect of assimilate supply and demand on translocation in wheat during grain development, included an examination of the movement of [14C]indoleacetic acid ([ t,C]IAA) in the stem and the effect of IAA on the movement of flag leaf assimilates.
Materials and Methods a) General. Wheat plants (Triticum aestivum c.v. Gabo) were grown singly in 12-cm pots, containing a mixture of equal parts of perlite and vermiculite, in a naturally lit glasshouse of the Canberra Phytotron. Air temperature was controlled at 21~ for 8h of the daylight period and 16~ for the remainder of the 24h cycle, with natural daylength extended to 16 h by low intensity incandescent lamps. All plants were supplied with standard nutrient solution in the morning and with water each afternoon. Tillers were removed 5 weeks after sowing, at anthesis and again just before specific experimental treatments, l0 to 20 days after anthesis, in order to facilitate handling and to restrict water use. For treatment all plants were transferred to an artificially lit (LBH) cabinet (Morse and Evans, 1962) set at 21~ and a light intensity of 3500 f.c. (96 w m - 2 visible). b) Sink Size. The rate o f import of dry matter into the ear of wheat, through the peduncle, was estimated from the difference between the rate of dry weight accumulation by the ear and net photosynthetic rate of the ear ( I m p o r t = e a r dry wt i n c r e a s e - e a r net photosynthesis). Dry weights were determined for 8 to 10 replicates at the start and the end of a 48 h period in continuous light. For determination of net photosynthetic rate, ears (still attached to the plant) were enclosed in a vertical perspex (plexiglass) assimilation chamber 5 x 2 cm in cross section. The differential in CO2 concentration of an air stream, before and after passing over an ear, was determined with a Grubb-Parsons infrared gas analyser (model SB2), calibrated with W6sthoff gas mixing pumps. Air flow rates were such that the maximum difference in COa concentration was no greater than 30 ppm by volume and ranged from 1 to 2 litres per rain. Generally
94 5 replicates were measured at various times throughout the 48 h light period. c) 14C-assimilate Labelling and Translocation. l~COz was simultaneously supplied to the mid section of the flag leaf blades from a group of 12 to 14 plants, with the blades placed across a perspex assimilation chamber (5 cm wide x 2 cm high). The 14COz, containing 1001xCi of 14C, was generated from 100mg Ba 14CO3 with the addition of 50% lactic acid, giving an initial concentration of 2000 ppm CO2 by volume. Following a 10 min exposure to 14CO2 in the light (3000 f.c.), in which the gas was recirculated through a peristaltic pump at 7 l/min, excess 14CO2 was absorbed by sodalime and the leaves were freed from the assimilation chamber. For speed of movement (velocity) measurements individual plants were harvested at 10 or 20 rain intervals after the 14CO2 uptake was complete. To determine the distribution of ~4C-activity, the flag leaf sheath and stem, both above and below the flag node, were divided into 4 cm lengths, fixed to an aluminium planchet with adhesive, oven dried and the surface radioactivity was measured with an end-window gas-flow Geiger tube. The mean speed of 14Cassimilate translocation through the peduncle (upper stem internode) to the ear, was estimated by determining the time of arrival of the a4C profile at each 4 cm stem length, based on the time at which half the peak activity was attained (c.f. Wardlaw 1965). A comparative estimate of the 14C-activity entering the ear was made by measuring the radioactivity of 4 grains taken from the basal florets of 4 central spikelets. d) Application and Distribution of 14C-labelled IAA. Carboxyllabelled IAA (34ndolyl [1-14C]acetic acid; 52mCi/mmol), at 5-10 mg/1, was either applied in 1.5% agar blocks to the ends of isolated 9 mm stem segments to study polarity of movement (cf. Thimann and Wardlaw 1963), or in 0.7% agar in a 1 ml reservoir replacing the ear at the top of the peduncle. The distribution of 14C-activity in the stem was determined, at various time intervals after the application of [14C]IAA, from surface counts of oven dried (80~ C) lengths of stem. To check whether the ~4C-activity transported through the stem was still associated with IAA, extracts were prepared from 10 cm lengths taken 15 h after the application of [~4C]IAA to the top of the peduncle. Each length was ground in liquid nitrogen and extracted in methanol in the dark at 3~ for 24 h, the extract was then evaporated to dryness and taken up in 0.5 M phosphate buffer at pH8. Chlorophylls and lipids were removed by partitioning into petroleum ether, after which the pH was adjusted to 2.5 with 5N HC1 and the IAA was partitioned into ethyl acetate. From 80 to 95% of the total t4C-activity was retained in the final ethyl acetate fraction. Samples from the latter were subsequently chromatogrammed on Whatman No. 1 paper strips using isopropanoh ammonia: water (8 : 1 : 1, v/v/v) as solvent.
Results
Sink Size Control of Translocation Fig. 1 summarizes the results of three experiments in which the speed of movement of 14C_labelled assimilates, through the peduncle to the ear, was compared in plants with intact ears and ears from which grains had been removed 24 hours earlier. A line joins each treatment within a particular experiment and comparisons should not be made between experiments, because growth and vascular development can be expected to vary between plants with seasonal light and other differences in the open glasshouse. From these results
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I.F. Wardlaw and L. Moncur: Translocation in Wheat
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it can be seen that, as the demand for assimilates (ear import) was decreased their speed of movement through the peduncle also decreased. In all three experiments the decrease in speed of movement was relatively greater than the decrease in demand, i.e. halving the demand more than halved the speed of movement. To establish how rapidly translocation responds to a change in assimilate demand by the sink, grains were removed from the ears of selected plants at various times before and after the assimilation of 14CO2 by the flag. In Fig. 2 a a comparison is shown of the rise in 14C-activity in the basal half of the peduncle, between intact controls and plants in which the grains were removed from the ear 30 min after the application of ~4CO2 to the flag leaf blade. In this experiment the effect of sink removal was detected along the path of transport 40 minutes after the assimilation of ~4CO2, i.e. 10 min after grain removal, and the differences were very similar to those obtained when degraining was carried out 24 h prior to the application of 14CO2. To confirm the above results a second experiment was undertaken in which grains were removed from the ears of one set of plants 40 min after 14CO 2 uptkae by the flag leaf. A comparison of the ~4C-distance profiles along the peduncle, at various time intervals (Fig. 2b), indicated that the effect of grain removal was evident in the base of the peduncle within a period of 10 min (50 rain after 14CO2 uptake), and at the top of the peduncle (30 cm from the flag node) within 40 rain (80 min after 14CO2 uptake), i.e. as the 14C was reaching the ear. In the previous experiments variation in the requirement for flag leaf assimilates was established by
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Fig. 2. (a) The effect of grain removal on 14C-assimilate movement into the basal half of the peduncle, with time from the start of 14COz assimilation by the flag blade. The closed triangles ( , ) indicate control plants with intact ears, the open triangles (zx) plants in which half the grains were removed from the ear 30 minutes after the start of 14COz Assimilation (b) The effect of grain removal 40 rain after the start of a4CO2 assimilation by the flag leaf blade, on ~4C-distribution in the peduncle at variou various times from the start of t 4CO2 assimilation. The closed triangles (A) indicate control plants, the open triangles (•) plants with half their grains removed
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Fig. 4. The movement of [14C]IAA through 9 mm wheat stem lengths isolated from the peduncle, as affected by polarity and temperature. (Polarity is estimated from the difference in (a) counts, or (b) % 14C, between comparable sections when the [14C]IAA source agar block was placed at either the basipetal or acropetal end of the section
the flag n o d e (Fig. 4 a). This p o l a r m o v e m e n t , a l t h o u g h still evident, was v e r y m u c h r e d u c e d at low t e m p e r a ture (3 ~ C) even after a l l o w i n g for t e m p e r a t u r e effects on the u p t a k e o f I A A b y the stem f r o m the a g a r s u p p l y b l o c k s (Fig. 4b). F u r t h e r evidence for an effect o f t e m p e r a t u r e on auxin t r a n s p o r t was o b t a i n e d b y r e p l a c i n g the e a r with
96
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Fig. 5. The movement of [~4C]IAA down the peduncle of wheat as affected by a reduction in temperature from 21~ C (so]id line) to 1.5~ C (broken line) over a 10 cm length of stem just below the uptake zone
a small reservoir containing 1 ml of [14C][AA in agar (10 mg/l) and following the distribution of 14C down the peduncle with time (Fig. 5). In control plants, at cabinet temperature (21 ~ C), the ~4C profile moved down the peduncle at a speed of just under 1 cm/h, but in view of the limited replication this must be considered a very rough estimate. Cooling a 10 cm length of stern to 1.5 ~ C just below the site of I A A application, hence avoiding the problem of temperature effects on uptake, resulted in a marked depression of m o v e m e n t of ~4C through the cold zone and a greater accumulation of ~4C at the top of the peduncle. A check was made on the nature of the ~4C-label moving through the stem 15 h after the application of [~4C]IAA to the top of the de-eared peduncle. In 5 replicate samples a methanol extract was shown by c h r o m a t o g r a p h y to contain a single peak of activity, that coincided with both cold marker I A A and the activity of the original [~4C]IAA. Removing the ear and replacing it with plain agar, 4 h prior to a4CO 2 assimilation, reduced the m o v e m e n t of ~4C-photosynthate from the flag leaf to the top of the peduncle, but increased the accumulation of ~4C at the base of the peduncle and the stem below the
Fig. 6. Stem accumulation of 14C-labelled flag leaf assimilates as affected by ear removal ( o - -- o), replacing the ear with IAA 5 mg/l (o - o), or IAA in conjunction with a 10 cm cold block (1~ C) placed immediatelybelow the site of IAA application ( x ) in comparison with intact controls (A--A)
flag leaf node, over a period of 4 hours (Fig. 6). Replacing the ear with I A A (5 rag/1 in agar), in comparison with the plain agar, significantly enhanced the accumulation of 14C-photosynthate in both the top of the peduncle and the lower parts of the stem. The effect of I A A on the accumulation of flag leaf assimilates in the stem below the flag node was prevented by the insertion of a 10 cm cold jacket (1.5 ~ C) on the peduncle just above the junction with the sheath, i.e. between the source of I A A and the node. There was however still evidence for a stimulation of 14C-photosynthate m o v e m e n t to the top of the peduncle in response to IAA.
The Effect of Varying Assimilate Supply Assimilate supply available to the ear was reduced by shading the parts of the plant below the position of the flag leaf ligule, either 1 or 5 days prior to the assimilation of a~CO2 by the flag blade and in the latter case a reduction i n grain weight was apparent with shading. In neither case was there any evidence that shading significantly altered the m o v e m e n t of 14Cphotosynthate from the flag leaf, through the peduncle, to the grain (Fig. 7), although there was
I.F. Wardlaw and L. Moncur: Translocation in Wheat
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Fig. 7~ :m(l b. The effect of reducing assimilate sttpply by shading the lower parts of the plant, on the movement of ~4C-photosynthate from the flag leaf to the grain. Solid circles o - - - e indicate control plants, open circles o - - o plants in which the lower parts were shaded. (a) 1 day's equilibration. (b) 5 days equilibration
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some evidence for greater m o v e m e n t into the lower parts o f the stem. Effectively limiting the n u m b e r o f direct vascular connections between the n o d e and the ear, by severing half the peduncle just above the flag leaf sheath, resulted in a distinct change in the pattern o f m o v e m e n t
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o f 1~C t h r o u g h the peduncle to the ear (Fig. 8). There was little difference in the activity/time profiles at the base of the peduncle (2 cm f r o m the flag node), but at the top of the peduncle the rise in activity c o m m e n ced earlier in the severed system, and this rise continued for a longer time at a lower rate, yielding a m u c h flatter profile than in the intact system. A plot o f time to half peak activity against distance (Fig, 8 - i n s e t ) , indicates that the speed o f m o v e m e n t o f assimilate t h r o u g h the peduncle was enhanced by severing. The accelerated m o v e m e n t o f 14C-assimilates t h r o u g h the severed peduncle is supported by the ob-
98
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Fig. 10a and b. The effect of restricting transport through the peduncle on the movement of laC-assimilates down the stem from the flag leaf. (So[id lines,-control plants; broken lines,-p[ants with petiole half blocked). (a) node to 4cm below node (b) 8 to t2cm below node servation (Fig. 9) that the initial accumulation of 14C in the grain was also enhanced in response to severing. Severing the peduncle, although having no discernible effect on movement of :4C-assimilates from the flag leaf into the stem immediately below the flag node (Fig. 10), did enhance the overall movement into the lower parts of the stem. This result gives some indication that the total mass transfer of assimilates to the ear was at least partially restricted by severing the peduncle, although the accumulation of dry material by the ear over 48 hours was not noticeably altered by severing.
Discussion When the requirement for dry matter (demand) by the ear of wheat was increased, the resulting enhancement of mass transfer through the peduncle was assod a t e d with an increase in the speed of movement of ~4C-photosynthate. In fact there was a consistent overcompensation in the speed of movement with increasing demand, such that doubling the demand was associated with about a fourfold increase in speed of movement. Thus the increase in demand, would also appear to result in a decrease in the concentration of sugar being transported per unit volume of phloem. One possible interpretation of the resnlts is that the resistance to the transfer of assimilates into the stem bundles was relatively high and that the resistance to sugar movement through the peduncle was relatively low, a concept in keeping with the model presented by Tyree et al., (1974). There is evidence from this and earlier work (Wardlaw, 1965), indicating that the transfer of photosynthate from the leaf traces to the stem traces below a node is restricted. Thus, although
altering ear demand had a big effect on the movement of ~4C-photosynthate through the peduncle, no measurable effect could be seen on the movement of ~4Cphotosynthate out of the flag leaf. The assimilate supply available to the stem traces leading to the ear is complicated by the pattern and nature of the vascular connections (Wardlaw, 1965; Patrick, 1972). With an increased demand, some compensation in supply occurs with a greater transfer of photosynthate to the ear from the lower parts of the plant, i.e, there is an increase in the source loading area supplying the ear, with the ear effectively competing against an alternative sink in the roots and young tillers. There are also additional reasons for believing that the resistance to sugar movement through the peduncle is of little importance in regulating the movement of photosynthate to the ear. In previous experiments (Wardlaw, 1974b) it was shown that the translocation of both :4C-photosynthate and 32P043- to the ear was insensitive to a drop in temperature from 21 ~ C down to 1~ C, over a 10 cm length of peduncle. If resistance to flow had been a significant factor, the increased viscosity resulting from the reduction in temperature should have been reflected in the pattern of movement of tZC-photosynthate. In the current experiments cutting the peduncle, just above the junction with the flag leaf sheath, effectively halved the vascular pathway available for the transport of assimilates to the ear, but had no detectable effect on dry weight accumulation by the ear over the subsequent 48 hours in continuous light. In an experiment with Sorghum, Fischer and Wilson (1975) also found that severing half the peduncle at anthesis had little effect on grain development in this species. There was however some evidence for wheat, that the movement of photosynthate past the cut was restricted, in that more 14C
I.F. Wardlawand L. Moncur: Translocationin Wheat was observed to accumulate in the lower parts of the stem with cutting. Mason and Maskell (1928) found that the rate of movement of photosynthate per unit area of phloem, in cotton, was substantially increased, when part of the bark was removed to restrict the channel of transport. They associated this response with an increased sugar concentration gradient, which would also explain the greater period and slower rate of accumulation of 14C-photosynthate with time, along the length of the wheat peduncle, in response to cutting. However cutting was also shown to enhance the speed of movement of l~C_photosynthate through the remaining functional vascular tissue of the peduncle. Thus the increased specific mass transfer of photosynthate through the restricted vascular pathway may be accounted for by an increase in both the concentration and speed of movement of assimilates being translocated. Perhaps it is significant that there is no evidence for' P-protein' in the sieve tubes of grasses, where these have been investigated (Evert et al., 1971 ; Singh and Srivastava, 1972). However the observation by Geiger et al. (1973) that there was little change in the concentration of sugars, in the sieve elements, along the path of transport in sugar beet, does suggest that the resistance to sugar movement in this species, which does contain some "P-protein" (Giaquinta and Geiger, 1973), is also comparatively low. A lower concentration ofpho.tosynthate in the sieve tubes, under a high demand situation, would presumably favour loading of assimilates into the phloem of the leaf, or in these experiments into the stem traces, and reduce the amount of lateral transfer and storage as the sugars are translocated. Unfortunately little direct information is available on the role of concentration gradients in regulating the movement of assimilates, in and out of the sieve tube system. Limiting the supply of carbohydrate available for translocation to the ear, by shading the plant below the flag leaf blade (an area expected to supply 30 to 40% of the grain requirements), although enhancing the movement of 1~C-photosynthate from the flag leaf downwards in the stem, did not significantly alter the speed of movement of 14C through the peduncle to the ear. These findings contrast with the observation by Moorby et al. (1974), that in maize the speed of movement of a~C-photosynthate through the leaf was significantly reduced in darkness. However in an earlier experiment on Loliurn t e m u l e n t u m (Evans and Wardlaw 1966), the speed of movement of ~4C-photosynthate down through the leaf was not apparently changed by a reduction in light intensity from 3000 f.c. to 60 f.c. and presumably also a reduction in the supply of available assimilates. One possible explanation for the present results, in the context of a pressure flow mechanism, is that the concentration gradient
99
between the source and sink was maintained with lower concentrations at both ends of the system. However this can only be speculation an a more careful experimentation is needed on the effect of assimilate supply on translocation. It may be argued that the observed patterns of photosynthate movement in response to variations in assimilate demand, could occur in a metabolically controlled mechanism such as electro-osmosis, or contractile microtubules, if these were regulated by a supply of auxin from the centres of growth (c.f. Wardlaw, 1974a). The expected source of auxin in this instance would be the developing grain, but data obtained by Wheeler (1972) indicated that auxin levels were quite low during the period of maximum grain growth in wheat and only reached a peak as the grain matured. The results of the present experiments also suggest that auxins were not involved in the effect of sink size on translocation. The accumulation of flag leaf assimilates in the base of the peduncle, a distance of at least 30 cm from the ear, was reduced within 10 min of grain removal. This would appear to be too rapid a response to be consistent with auxin control, speed of auxin movement being about 1 cm/h (Vardar 1968), with a considerable lag in the time needed to clear residual auxin following removal of the source (c.f. Scott and Briggs, 1960); an estimate of speed that also appears to apply to basipetal movement through the wheat peduncle. In these experiments the demand for assimilates by the ear was not only decreased by selective grain removal, but also increased by preventing ear photosynthesis with a DCMU spray. In the latter case the speed of assimilate movement through the peduncle was increased without altering the rate of grain growth and presumably auxin production, which further suggests that there was not an overriding hormonal control of transport. With the use of [14C]IAA it was possible to demonstrate a strongly polar, basipetal movement of auxin in the peduncle, i.e. directed away from the ear. Thus in the grass stem, as in the grass leaf (Sheldrake 1972) the direction of movement was towards the intercalary meristem at the node and hence towards the morphologically youngest tissue. For wheat, as in other systems (Vardar 1968), the transport of IAA was considerably reduced by a temperature drop from 21~ down to 3~ C. However, although the movement of IAA through the stem away from the ear was found to be sensitive to low temperature, the movement of photosynthate in the opposite direction was shown in earlier experiments (Wardlaw, 1974b) to be insensitive to temperature in this range, and this further suggests that auxin movement was not closely associated with the transport of assimilates in wheat. These results parallel those of TIBA, which can stop basipetal trans-
100
port of IAA out of the shoot apex, but not the movement of IAA or sugar through the phloem transport system in the reverse direction (Morris, Kadir and Barry, 1973; Goldsmith et al., 1974). The observed relation between ~4C-photosynthate movement and changes in the supply and demand for assimilates, the lack of suport for any direct hormonal control of translocation, and the failure to inhibit translocation at low temperatures in wheat, all appear to fit a mechanism of translocation, such as that of pressure flow first described by Mfinch (cf. Crafts and Crisp, 1971), which is fully dependent on a source sink concentration gradient and only indirectly dependent on pathway metabolism. However, the involvement of surface flow in translocation, either as an alternative, or in conjunction with pressure flow, should not be discounted, at least until it can be shown that functioning sieve tubes are unrestricted by the structural continuum present as "P-protein" or endoplasmic reticulum in many sieve elements (Wardlaw, 1974a).
References Crafts, A.S., Crisp, C.E. : Phloem transport in plants. San Francisco : Freeman 197t Evans, L.T., Wardlaw, I.F. : Independent translocation of 14C-labelled assimilates and of the floral stimulus in Lolium temulentum. Planta (Berl.) 68, 310-326 (1966) Evert, R.F., Escherich, W., Eichhorn, S.E.: Sieve plate pores in leaf veins of Hordeum vulgare. Planta (Berl.) 100, 262-267 (1971) Fischer, K.S., Wilson, G.L. : Studies of grain producion in Sorghum bicolor (L. Moench). III The relative importance of assimilate supply, grain growth capacity and transport system. Aust. J. Agric. Res. 26, 11-23 (1975) Geiger, D . R , Giaquinta, R.T., Sovonick, S.A., Fellows, R.J. : Solute distribution in sugar beet leaves in relation to phloem loading and translocation. Plant Physiol. 52, 585-589 (1973) Giaquinta, R.T., Geiger, D.R. :Mechanism of inhibition of translocation by localized chilling. Plant Physiol. 51, 372-377 (1973) Goldsmith, M.H., Cataldo, D.A., Karn, J., Brenneman, T., Trip, P.: The rapid non-polar transport of auxin in the phloem of intact Coleus plants. Planta (Bed.) 116, 301-317 (1974)
I.F. Wardlaw and L. Moncur: Translocation in Wheat Hartt, C.E., Kortschak, H.P.: Sugar gradients and translocation of sucrose in detached blades of sugar cane. Plant Physiol. 39, 460-474 (1964) Mason, T.G., Maskell, E.J. : Studies on the transport of carbohydrates in the cotton plant. II. The factors determining the rate and the direction of movement of sugars. Ann. Bot. 42, 571~536 (1928) Moorby, J., Troughton, J.H., Currie, B.G. : Investigations of carbon transport in plants. II. The effects of light and darkness and sink activity on translocation. J. exp. Bot. 25, 937-944 (1974) Morris, D.A., Kadir, G.O., Barry, A.J. : Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-Triiodobenzoic acid. Planta (Ber.) 110, 173-182 (1973) Morse, R.N., Evans, L.T.: Design and development of CERES, an Australian phytotron. J. Agric. Engng. Res. 7, 128-140 (1962) Patrick, J.W.: Vascular system of the stem of the wheat plant. II. Mature state. Aust. J. Bot. 20, 49-63 (1972) Patrick, J.W., Wareing, P.F. : Auxin-promoted transport of metabolites in stems ofPhaseolus vulgaris L.J. exp. Bot. 24, 1158-1171 (1973) Scott, T.K., Briggs, W.R. : Auxin relationships in the Alaska pea (Pisum sativum). Amer. J. Bot. 47, 492-499 (1960) Seth, A.K., Wareing, P.F. : Hormone-directed transport of metabolites and its possible role in plant senescence. J. exp. Bot. 18, 65-77 (1967) Sheldrake, A.R. : Polar auxin transport in leaves of monocotyledons. Nature 238, 352-353 (1972) Singh, A.P., Srivastava, L.M. : The fine structure of corn phloem. Canad. J. Bot. 50, 839446 (1972) Thimann, K.V., Wardlaw, I.F.: The effect of light on the uptake and transport of indoleacetic acid in the green stem of the pea. Physiol. Plant. 16, 368-377 (1963) Tyree, M.T., Christy, A.L., Ferrier, J.M. : A simpler iterative steady state solution of Mfinch pressure flow systems applied to long and short translocation paths. Plant Physiol. 54, 589~00 (1974) Vardar, Y., (Ed.) : The transport of plant hormones. Amsterdam: North-Holland PuN. Co. 1968 Wardlaw, I.F. : The velocity and pattern of assimilate translocation in wheat plants during grain development. Aust. J. biol. Sci. 18, 269~81 (1965) Wardlaw, I.F.: Phloem transport: Physical, chemical, or impossible. Ann. Rev. Plant Physiol. 25, 515-539 (1974a) Wardtaw, I.F. : Temperature control of translocation. In : "Mechanisms of regulation of plant growth", pp. 533-538. Ed. : R.L. Bieleski, A,R. Ferguson, M.M. Cresswell. Bull. 12, Roy. Soc., N.Z. (1974b) Wheeler, A.W.: Changes in growth-substance contents during growth of wheat grains. Ann. Appl. Biol. 72, 327-334 (1972)
Received 1 June," accepted 10 September 1975
Planta (Berl.)128, 9 3 - 1 0 0 (1976)
9 by Springer-Verlag 1976
Source, Sink and Hormonal Control of Translocation in Wheat I.F. Wardlaw and L. Moncur CSIRO Division of Plant Industry, P.O. Box 1600 Canberra, Australia
Summary. An analysis of the pattern of movement of 14C-labelled flag leaf assimilates in wheat (Triticum aestivum I. c.v. Gabo) during grain development, indicated that the greater the requirement for assimilates by the ear the more rapid was the speed of movement of these through the peduncle to the ear and also the lower their concentration. Experiments with [14C] indoleacetic acid ([a4C]IAA) suggested that auxin production by the grains was not responsible for the control of assimilate translocation through the peduncle. Limiting the supply of available assimilates by shading the lower parts of the plant, did not significantly alter the speed of movement of ~4C-photosynthate through the peduncle, wfiile severing half of the vascular tissue in the peduncle altered the pattern of movement of a4C to the ear and enhanced the speed of movement of 14C through the remaining functional conducting tissue. These results are discussed in relation to the mechanism of translocation.
Introduction
In an earlier paper (Wardlaw, 1965) data were presented, which indicated that the speed of movement of 14C_labelled flag leaf assimilates, through the peduncle to the ear of wheat, varied with the number of developing grains. This observation was confirmed in more detail later (see Wardlaw, 1974 a) and a similar association between speed of movement and demand for assimilates also appears to apply to sugarcane (Hartt and Kortschak, 1964) and tomato (Moorby et al., 1974). It was considered that a more detailed analysis of both the effect of sink size and the availability of assimilates on the movement of 14C-photosynthate through the vascular tissue, could be helpful in distinguishing between the different proposed mechanisms of translocation, as well as establishing some of the characteristics of the transport system. However any sink size control
of assimilate movement could be an indirect one, a complication that is evident, for example, in the possible role of auxins formed in growing organs, in regulating the movement of assimilates through the transport system (Seth and Wareing, 1967; Patrick and Wareing, 1973). Thus the present study, on the effect of assimilate supply and demand on translocation in wheat during grain development, included an examination of the movement of [14C]indoleacetic acid ([ t,C]IAA) in the stem and the effect of IAA on the movement of flag leaf assimilates.
Materials and Methods a) General. Wheat plants (Triticum aestivum c.v. Gabo) were grown singly in 12-cm pots, containing a mixture of equal parts of perlite and vermiculite, in a naturally lit glasshouse of the Canberra Phytotron. Air temperature was controlled at 21~ for 8h of the daylight period and 16~ for the remainder of the 24h cycle, with natural daylength extended to 16 h by low intensity incandescent lamps. All plants were supplied with standard nutrient solution in the morning and with water each afternoon. Tillers were removed 5 weeks after sowing, at anthesis and again just before specific experimental treatments, l0 to 20 days after anthesis, in order to facilitate handling and to restrict water use. For treatment all plants were transferred to an artificially lit (LBH) cabinet (Morse and Evans, 1962) set at 21~ and a light intensity of 3500 f.c. (96 w m - 2 visible). b) Sink Size. The rate o f import of dry matter into the ear of wheat, through the peduncle, was estimated from the difference between the rate of dry weight accumulation by the ear and net photosynthetic rate of the ear ( I m p o r t = e a r dry wt i n c r e a s e - e a r net photosynthesis). Dry weights were determined for 8 to 10 replicates at the start and the end of a 48 h period in continuous light. For determination of net photosynthetic rate, ears (still attached to the plant) were enclosed in a vertical perspex (plexiglass) assimilation chamber 5 x 2 cm in cross section. The differential in CO2 concentration of an air stream, before and after passing over an ear, was determined with a Grubb-Parsons infrared gas analyser (model SB2), calibrated with W6sthoff gas mixing pumps. Air flow rates were such that the maximum difference in COa concentration was no greater than 30 ppm by volume and ranged from 1 to 2 litres per rain. Generally
94 5 replicates were measured at various times throughout the 48 h light period. c) 14C-assimilate Labelling and Translocation. l~COz was simultaneously supplied to the mid section of the flag leaf blades from a group of 12 to 14 plants, with the blades placed across a perspex assimilation chamber (5 cm wide x 2 cm high). The 14COz, containing 1001xCi of 14C, was generated from 100mg Ba 14CO3 with the addition of 50% lactic acid, giving an initial concentration of 2000 ppm CO2 by volume. Following a 10 min exposure to 14CO2 in the light (3000 f.c.), in which the gas was recirculated through a peristaltic pump at 7 l/min, excess 14CO2 was absorbed by sodalime and the leaves were freed from the assimilation chamber. For speed of movement (velocity) measurements individual plants were harvested at 10 or 20 rain intervals after the 14CO2 uptake was complete. To determine the distribution of ~4C-activity, the flag leaf sheath and stem, both above and below the flag node, were divided into 4 cm lengths, fixed to an aluminium planchet with adhesive, oven dried and the surface radioactivity was measured with an end-window gas-flow Geiger tube. The mean speed of 14Cassimilate translocation through the peduncle (upper stem internode) to the ear, was estimated by determining the time of arrival of the a4C profile at each 4 cm stem length, based on the time at which half the peak activity was attained (c.f. Wardlaw 1965). A comparative estimate of the 14C-activity entering the ear was made by measuring the radioactivity of 4 grains taken from the basal florets of 4 central spikelets. d) Application and Distribution of 14C-labelled IAA. Carboxyllabelled IAA (34ndolyl [1-14C]acetic acid; 52mCi/mmol), at 5-10 mg/1, was either applied in 1.5% agar blocks to the ends of isolated 9 mm stem segments to study polarity of movement (cf. Thimann and Wardlaw 1963), or in 0.7% agar in a 1 ml reservoir replacing the ear at the top of the peduncle. The distribution of 14C-activity in the stem was determined, at various time intervals after the application of [14C]IAA, from surface counts of oven dried (80~ C) lengths of stem. To check whether the ~4C-activity transported through the stem was still associated with IAA, extracts were prepared from 10 cm lengths taken 15 h after the application of [~4C]IAA to the top of the peduncle. Each length was ground in liquid nitrogen and extracted in methanol in the dark at 3~ for 24 h, the extract was then evaporated to dryness and taken up in 0.5 M phosphate buffer at pH8. Chlorophylls and lipids were removed by partitioning into petroleum ether, after which the pH was adjusted to 2.5 with 5N HC1 and the IAA was partitioned into ethyl acetate. From 80 to 95% of the total t4C-activity was retained in the final ethyl acetate fraction. Samples from the latter were subsequently chromatogrammed on Whatman No. 1 paper strips using isopropanoh ammonia: water (8 : 1 : 1, v/v/v) as solvent.
Results
Sink Size Control of Translocation Fig. 1 summarizes the results of three experiments in which the speed of movement of 14C_labelled assimilates, through the peduncle to the ear, was compared in plants with intact ears and ears from which grains had been removed 24 hours earlier. A line joins each treatment within a particular experiment and comparisons should not be made between experiments, because growth and vascular development can be expected to vary between plants with seasonal light and other differences in the open glasshouse. From these results
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I.F. Wardlaw and L. Moncur: Translocation in Wheat
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it can be seen that, as the demand for assimilates (ear import) was decreased their speed of movement through the peduncle also decreased. In all three experiments the decrease in speed of movement was relatively greater than the decrease in demand, i.e. halving the demand more than halved the speed of movement. To establish how rapidly translocation responds to a change in assimilate demand by the sink, grains were removed from the ears of selected plants at various times before and after the assimilation of 14CO2 by the flag. In Fig. 2 a a comparison is shown of the rise in 14C-activity in the basal half of the peduncle, between intact controls and plants in which the grains were removed from the ear 30 min after the application of ~4CO2 to the flag leaf blade. In this experiment the effect of sink removal was detected along the path of transport 40 minutes after the assimilation of ~4CO2, i.e. 10 min after grain removal, and the differences were very similar to those obtained when degraining was carried out 24 h prior to the application of 14CO2. To confirm the above results a second experiment was undertaken in which grains were removed from the ears of one set of plants 40 min after 14CO 2 uptkae by the flag leaf. A comparison of the ~4C-distance profiles along the peduncle, at various time intervals (Fig. 2b), indicated that the effect of grain removal was evident in the base of the peduncle within a period of 10 min (50 rain after 14CO2 uptake), and at the top of the peduncle (30 cm from the flag node) within 40 rain (80 min after 14CO2 uptake), i.e. as the 14C was reaching the ear. In the previous experiments variation in the requirement for flag leaf assimilates was established by
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the flag n o d e (Fig. 4 a). This p o l a r m o v e m e n t , a l t h o u g h still evident, was v e r y m u c h r e d u c e d at low t e m p e r a ture (3 ~ C) even after a l l o w i n g for t e m p e r a t u r e effects on the u p t a k e o f I A A b y the stem f r o m the a g a r s u p p l y b l o c k s (Fig. 4b). F u r t h e r evidence for an effect o f t e m p e r a t u r e on auxin t r a n s p o r t was o b t a i n e d b y r e p l a c i n g the e a r with
96
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a small reservoir containing 1 ml of [14C][AA in agar (10 mg/l) and following the distribution of 14C down the peduncle with time (Fig. 5). In control plants, at cabinet temperature (21 ~ C), the ~4C profile moved down the peduncle at a speed of just under 1 cm/h, but in view of the limited replication this must be considered a very rough estimate. Cooling a 10 cm length of stern to 1.5 ~ C just below the site of I A A application, hence avoiding the problem of temperature effects on uptake, resulted in a marked depression of m o v e m e n t of ~4C through the cold zone and a greater accumulation of ~4C at the top of the peduncle. A check was made on the nature of the ~4C-label moving through the stem 15 h after the application of [~4C]IAA to the top of the de-eared peduncle. In 5 replicate samples a methanol extract was shown by c h r o m a t o g r a p h y to contain a single peak of activity, that coincided with both cold marker I A A and the activity of the original [~4C]IAA. Removing the ear and replacing it with plain agar, 4 h prior to a4CO 2 assimilation, reduced the m o v e m e n t of ~4C-photosynthate from the flag leaf to the top of the peduncle, but increased the accumulation of ~4C at the base of the peduncle and the stem below the
Fig. 6. Stem accumulation of 14C-labelled flag leaf assimilates as affected by ear removal ( o - -- o), replacing the ear with IAA 5 mg/l (o - o), or IAA in conjunction with a 10 cm cold block (1~ C) placed immediatelybelow the site of IAA application ( x ) in comparison with intact controls (A--A)
flag leaf node, over a period of 4 hours (Fig. 6). Replacing the ear with I A A (5 rag/1 in agar), in comparison with the plain agar, significantly enhanced the accumulation of 14C-photosynthate in both the top of the peduncle and the lower parts of the stem. The effect of I A A on the accumulation of flag leaf assimilates in the stem below the flag node was prevented by the insertion of a 10 cm cold jacket (1.5 ~ C) on the peduncle just above the junction with the sheath, i.e. between the source of I A A and the node. There was however still evidence for a stimulation of 14C-photosynthate m o v e m e n t to the top of the peduncle in response to IAA.
The Effect of Varying Assimilate Supply Assimilate supply available to the ear was reduced by shading the parts of the plant below the position of the flag leaf ligule, either 1 or 5 days prior to the assimilation of a~CO2 by the flag blade and in the latter case a reduction i n grain weight was apparent with shading. In neither case was there any evidence that shading significantly altered the m o v e m e n t of 14Cphotosynthate from the flag leaf, through the peduncle, to the grain (Fig. 7), although there was
I.F. Wardlaw and L. Moncur: Translocation in Wheat
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some evidence for greater m o v e m e n t into the lower parts o f the stem. Effectively limiting the n u m b e r o f direct vascular connections between the n o d e and the ear, by severing half the peduncle just above the flag leaf sheath, resulted in a distinct change in the pattern o f m o v e m e n t
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o f 1~C t h r o u g h the peduncle to the ear (Fig. 8). There was little difference in the activity/time profiles at the base of the peduncle (2 cm f r o m the flag node), but at the top of the peduncle the rise in activity c o m m e n ced earlier in the severed system, and this rise continued for a longer time at a lower rate, yielding a m u c h flatter profile than in the intact system. A plot o f time to half peak activity against distance (Fig, 8 - i n s e t ) , indicates that the speed o f m o v e m e n t o f assimilate t h r o u g h the peduncle was enhanced by severing. The accelerated m o v e m e n t o f 14C-assimilates t h r o u g h the severed peduncle is supported by the ob-
98
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Fig. 10a and b. The effect of restricting transport through the peduncle on the movement of laC-assimilates down the stem from the flag leaf. (So[id lines,-control plants; broken lines,-p[ants with petiole half blocked). (a) node to 4cm below node (b) 8 to t2cm below node servation (Fig. 9) that the initial accumulation of 14C in the grain was also enhanced in response to severing. Severing the peduncle, although having no discernible effect on movement of :4C-assimilates from the flag leaf into the stem immediately below the flag node (Fig. 10), did enhance the overall movement into the lower parts of the stem. This result gives some indication that the total mass transfer of assimilates to the ear was at least partially restricted by severing the peduncle, although the accumulation of dry material by the ear over 48 hours was not noticeably altered by severing.
Discussion When the requirement for dry matter (demand) by the ear of wheat was increased, the resulting enhancement of mass transfer through the peduncle was assod a t e d with an increase in the speed of movement of ~4C-photosynthate. In fact there was a consistent overcompensation in the speed of movement with increasing demand, such that doubling the demand was associated with about a fourfold increase in speed of movement. Thus the increase in demand, would also appear to result in a decrease in the concentration of sugar being transported per unit volume of phloem. One possible interpretation of the resnlts is that the resistance to the transfer of assimilates into the stem bundles was relatively high and that the resistance to sugar movement through the peduncle was relatively low, a concept in keeping with the model presented by Tyree et al., (1974). There is evidence from this and earlier work (Wardlaw, 1965), indicating that the transfer of photosynthate from the leaf traces to the stem traces below a node is restricted. Thus, although
altering ear demand had a big effect on the movement of ~4C-photosynthate through the peduncle, no measurable effect could be seen on the movement of ~4Cphotosynthate out of the flag leaf. The assimilate supply available to the stem traces leading to the ear is complicated by the pattern and nature of the vascular connections (Wardlaw, 1965; Patrick, 1972). With an increased demand, some compensation in supply occurs with a greater transfer of photosynthate to the ear from the lower parts of the plant, i.e, there is an increase in the source loading area supplying the ear, with the ear effectively competing against an alternative sink in the roots and young tillers. There are also additional reasons for believing that the resistance to sugar movement through the peduncle is of little importance in regulating the movement of photosynthate to the ear. In previous experiments (Wardlaw, 1974b) it was shown that the translocation of both :4C-photosynthate and 32P043- to the ear was insensitive to a drop in temperature from 21 ~ C down to 1~ C, over a 10 cm length of peduncle. If resistance to flow had been a significant factor, the increased viscosity resulting from the reduction in temperature should have been reflected in the pattern of movement of tZC-photosynthate. In the current experiments cutting the peduncle, just above the junction with the flag leaf sheath, effectively halved the vascular pathway available for the transport of assimilates to the ear, but had no detectable effect on dry weight accumulation by the ear over the subsequent 48 hours in continuous light. In an experiment with Sorghum, Fischer and Wilson (1975) also found that severing half the peduncle at anthesis had little effect on grain development in this species. There was however some evidence for wheat, that the movement of photosynthate past the cut was restricted, in that more 14C
I.F. Wardlawand L. Moncur: Translocationin Wheat was observed to accumulate in the lower parts of the stem with cutting. Mason and Maskell (1928) found that the rate of movement of photosynthate per unit area of phloem, in cotton, was substantially increased, when part of the bark was removed to restrict the channel of transport. They associated this response with an increased sugar concentration gradient, which would also explain the greater period and slower rate of accumulation of 14C-photosynthate with time, along the length of the wheat peduncle, in response to cutting. However cutting was also shown to enhance the speed of movement of l~C_photosynthate through the remaining functional vascular tissue of the peduncle. Thus the increased specific mass transfer of photosynthate through the restricted vascular pathway may be accounted for by an increase in both the concentration and speed of movement of assimilates being translocated. Perhaps it is significant that there is no evidence for' P-protein' in the sieve tubes of grasses, where these have been investigated (Evert et al., 1971 ; Singh and Srivastava, 1972). However the observation by Geiger et al. (1973) that there was little change in the concentration of sugars, in the sieve elements, along the path of transport in sugar beet, does suggest that the resistance to sugar movement in this species, which does contain some "P-protein" (Giaquinta and Geiger, 1973), is also comparatively low. A lower concentration ofpho.tosynthate in the sieve tubes, under a high demand situation, would presumably favour loading of assimilates into the phloem of the leaf, or in these experiments into the stem traces, and reduce the amount of lateral transfer and storage as the sugars are translocated. Unfortunately little direct information is available on the role of concentration gradients in regulating the movement of assimilates, in and out of the sieve tube system. Limiting the supply of carbohydrate available for translocation to the ear, by shading the plant below the flag leaf blade (an area expected to supply 30 to 40% of the grain requirements), although enhancing the movement of 1~C-photosynthate from the flag leaf downwards in the stem, did not significantly alter the speed of movement of 14C through the peduncle to the ear. These findings contrast with the observation by Moorby et al. (1974), that in maize the speed of movement of a~C-photosynthate through the leaf was significantly reduced in darkness. However in an earlier experiment on Loliurn t e m u l e n t u m (Evans and Wardlaw 1966), the speed of movement of ~4C-photosynthate down through the leaf was not apparently changed by a reduction in light intensity from 3000 f.c. to 60 f.c. and presumably also a reduction in the supply of available assimilates. One possible explanation for the present results, in the context of a pressure flow mechanism, is that the concentration gradient
99
between the source and sink was maintained with lower concentrations at both ends of the system. However this can only be speculation an a more careful experimentation is needed on the effect of assimilate supply on translocation. It may be argued that the observed patterns of photosynthate movement in response to variations in assimilate demand, could occur in a metabolically controlled mechanism such as electro-osmosis, or contractile microtubules, if these were regulated by a supply of auxin from the centres of growth (c.f. Wardlaw, 1974a). The expected source of auxin in this instance would be the developing grain, but data obtained by Wheeler (1972) indicated that auxin levels were quite low during the period of maximum grain growth in wheat and only reached a peak as the grain matured. The results of the present experiments also suggest that auxins were not involved in the effect of sink size on translocation. The accumulation of flag leaf assimilates in the base of the peduncle, a distance of at least 30 cm from the ear, was reduced within 10 min of grain removal. This would appear to be too rapid a response to be consistent with auxin control, speed of auxin movement being about 1 cm/h (Vardar 1968), with a considerable lag in the time needed to clear residual auxin following removal of the source (c.f. Scott and Briggs, 1960); an estimate of speed that also appears to apply to basipetal movement through the wheat peduncle. In these experiments the demand for assimilates by the ear was not only decreased by selective grain removal, but also increased by preventing ear photosynthesis with a DCMU spray. In the latter case the speed of assimilate movement through the peduncle was increased without altering the rate of grain growth and presumably auxin production, which further suggests that there was not an overriding hormonal control of transport. With the use of [14C]IAA it was possible to demonstrate a strongly polar, basipetal movement of auxin in the peduncle, i.e. directed away from the ear. Thus in the grass stem, as in the grass leaf (Sheldrake 1972) the direction of movement was towards the intercalary meristem at the node and hence towards the morphologically youngest tissue. For wheat, as in other systems (Vardar 1968), the transport of IAA was considerably reduced by a temperature drop from 21~ down to 3~ C. However, although the movement of IAA through the stem away from the ear was found to be sensitive to low temperature, the movement of photosynthate in the opposite direction was shown in earlier experiments (Wardlaw, 1974b) to be insensitive to temperature in this range, and this further suggests that auxin movement was not closely associated with the transport of assimilates in wheat. These results parallel those of TIBA, which can stop basipetal trans-
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port of IAA out of the shoot apex, but not the movement of IAA or sugar through the phloem transport system in the reverse direction (Morris, Kadir and Barry, 1973; Goldsmith et al., 1974). The observed relation between ~4C-photosynthate movement and changes in the supply and demand for assimilates, the lack of suport for any direct hormonal control of translocation, and the failure to inhibit translocation at low temperatures in wheat, all appear to fit a mechanism of translocation, such as that of pressure flow first described by Mfinch (cf. Crafts and Crisp, 1971), which is fully dependent on a source sink concentration gradient and only indirectly dependent on pathway metabolism. However, the involvement of surface flow in translocation, either as an alternative, or in conjunction with pressure flow, should not be discounted, at least until it can be shown that functioning sieve tubes are unrestricted by the structural continuum present as "P-protein" or endoplasmic reticulum in many sieve elements (Wardlaw, 1974a).
References Crafts, A.S., Crisp, C.E. : Phloem transport in plants. San Francisco : Freeman 197t Evans, L.T., Wardlaw, I.F. : Independent translocation of 14C-labelled assimilates and of the floral stimulus in Lolium temulentum. Planta (Berl.) 68, 310-326 (1966) Evert, R.F., Escherich, W., Eichhorn, S.E.: Sieve plate pores in leaf veins of Hordeum vulgare. Planta (Berl.) 100, 262-267 (1971) Fischer, K.S., Wilson, G.L. : Studies of grain producion in Sorghum bicolor (L. Moench). III The relative importance of assimilate supply, grain growth capacity and transport system. Aust. J. Agric. Res. 26, 11-23 (1975) Geiger, D . R , Giaquinta, R.T., Sovonick, S.A., Fellows, R.J. : Solute distribution in sugar beet leaves in relation to phloem loading and translocation. Plant Physiol. 52, 585-589 (1973) Giaquinta, R.T., Geiger, D.R. :Mechanism of inhibition of translocation by localized chilling. Plant Physiol. 51, 372-377 (1973) Goldsmith, M.H., Cataldo, D.A., Karn, J., Brenneman, T., Trip, P.: The rapid non-polar transport of auxin in the phloem of intact Coleus plants. Planta (Bed.) 116, 301-317 (1974)
I.F. Wardlaw and L. Moncur: Translocation in Wheat Hartt, C.E., Kortschak, H.P.: Sugar gradients and translocation of sucrose in detached blades of sugar cane. Plant Physiol. 39, 460-474 (1964) Mason, T.G., Maskell, E.J. : Studies on the transport of carbohydrates in the cotton plant. II. The factors determining the rate and the direction of movement of sugars. Ann. Bot. 42, 571~536 (1928) Moorby, J., Troughton, J.H., Currie, B.G. : Investigations of carbon transport in plants. II. The effects of light and darkness and sink activity on translocation. J. exp. Bot. 25, 937-944 (1974) Morris, D.A., Kadir, G.O., Barry, A.J. : Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-Triiodobenzoic acid. Planta (Ber.) 110, 173-182 (1973) Morse, R.N., Evans, L.T.: Design and development of CERES, an Australian phytotron. J. Agric. Engng. Res. 7, 128-140 (1962) Patrick, J.W.: Vascular system of the stem of the wheat plant. II. Mature state. Aust. J. Bot. 20, 49-63 (1972) Patrick, J.W., Wareing, P.F. : Auxin-promoted transport of metabolites in stems ofPhaseolus vulgaris L.J. exp. Bot. 24, 1158-1171 (1973) Scott, T.K., Briggs, W.R. : Auxin relationships in the Alaska pea (Pisum sativum). Amer. J. Bot. 47, 492-499 (1960) Seth, A.K., Wareing, P.F. : Hormone-directed transport of metabolites and its possible role in plant senescence. J. exp. Bot. 18, 65-77 (1967) Sheldrake, A.R. : Polar auxin transport in leaves of monocotyledons. Nature 238, 352-353 (1972) Singh, A.P., Srivastava, L.M. : The fine structure of corn phloem. Canad. J. Bot. 50, 839446 (1972) Thimann, K.V., Wardlaw, I.F.: The effect of light on the uptake and transport of indoleacetic acid in the green stem of the pea. Physiol. Plant. 16, 368-377 (1963) Tyree, M.T., Christy, A.L., Ferrier, J.M. : A simpler iterative steady state solution of Mfinch pressure flow systems applied to long and short translocation paths. Plant Physiol. 54, 589~00 (1974) Vardar, Y., (Ed.) : The transport of plant hormones. Amsterdam: North-Holland PuN. Co. 1968 Wardlaw, I.F. : The velocity and pattern of assimilate translocation in wheat plants during grain development. Aust. J. biol. Sci. 18, 269~81 (1965) Wardlaw, I.F.: Phloem transport: Physical, chemical, or impossible. Ann. Rev. Plant Physiol. 25, 515-539 (1974a) Wardtaw, I.F. : Temperature control of translocation. In : "Mechanisms of regulation of plant growth", pp. 533-538. Ed. : R.L. Bieleski, A,R. Ferguson, M.M. Cresswell. Bull. 12, Roy. Soc., N.Z. (1974b) Wheeler, A.W.: Changes in growth-substance contents during growth of wheat grains. Ann. Appl. Biol. 72, 327-334 (1972)
Received 1 June," accepted 10 September 1975
