COMPARTMENTATJON AND FLUXES OF SUCROSE IN INTACT LEAF BLADES OF BARLEY

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1 New Phytol. (1986) 103, COMPARTMENTATJON AND FLUXES OF SUCROSE IN INTACT LEAF BLADES OF BARLEY BY S. C. FARRAR AND J. F. FARRAR School of Plant Biology, University College of North Wales, Bangor, Gwynedd LL57 2UW, UK {Accepted 21 March 1986) SUMMARY Sucrose in barley leaf blades can be considered as being compartmented between two types of pool: transport (including mesophyll cytosol and apoplast, and vascular tissue) and vacuolar. The relative sizes of these pools, and their changes over a light/dark cycle, have been estimated using "C labelling of the intact leaf and washout of '"C from leaf discs. Both types of pool increase during the photoperiod, the proportional distribution between them remaining the same; roughly one-fifth of the sucrose is in the transport pool. The rate-constants describing translocation, and loading and unloading of the vacuole, show diel changes, and fluxes of sucrose across the tonoplast are likely to be critical in controlling fluxes of photosynthetically fixed carbon within and out of barley leaves. The unloading of sucrose from vacuoles is probably the site where control is exercised; this conclusion is supported by a simple simulation model. Key words; Sucrose, compartmentation, vacuoles, barley, translocation. INTRODUCTION Sucrose is synthesized in the cytosol of mesophyll cells (Giersch et al., 1980); it is also found in the apoplast (Madore & Webb, 1981; Huber & Moreland, 1980), vacuoles (Avigad, 1982; Gerhardt & Heldt, 1984; Fisher & Outlaw, 1979), and phloem (Giaquinta, 1980; Kursanov, 1984; Raven, 1977). The rapid appearance of ["CJsucrose in vacuoles during photosynthesis in i^coj has been shown in protoplasts of barley, wheat and spinach (Kaiser &Heber, 1984; Kaiser, Martinoia & Wiemken, 1982; Giersch et al., 1980). The dynamic relationships between sucrose in cytosol and vacuole are poorly understood. Procedures involving protoplasts are of limited use in understanding the behaviour of intact leaves where each mesophyll cell is under positive hydrostatic pressure, is in symplastic connection with other cells and rapidly exports a large proportion of the sucrose it manufactures. In particular, in intact leaves the quantitative importance of the vacuole in sucrose fluxes, and the rate of turnover of sucrose during light/dark cycles, are little explored. In those species characterized by the storage of starch during the photoperiod, such as spinach (Huber, 1981) beet (Fondy & Geiger, 1982) soybean (Silvius, Chatterton & Kremer, 1979; Kagawa & Wong, 1985) and Vicia faba (Outlaw, Fisher & Christy, 1975) the available evidence indicates that vacuolar sucrose is a small component of leaf carbohydrate metabolism, whether considered by amount or by fiux. Those species that characteristically store sucrose (rather than starch) during the photoperiod have been less explored, but the evidence suggests that here vacuolar storage of sucrose is of prime importance. Gordon & Johnson (1984) have used a simulation model to indicate how vacuolar sucrose storage might be integrated into carbon fiuxes in barley leaves, and data of Owera, Farrar & Whitbread (1983), Sicher, Kremer & Harris (1984) and Farrar & Farrar (1985a) X/86/ S03.00/ The New Phytologist

2 646 S. C. FARRAR AND J. F. FARRAR are consistent with a vacuolar pool of sucrose playing a large role in barley leaves. Limits on the size of this pool have also been suggested as a result of pulse-labelling and steady-state labelling of barley leaves with '''CO^, and a second type of pool, called transport sucrose (since it will probably include cytosolic, apoplastic and phloem pools of sucrose), was distinguished from it (Farrar & Farrar, 1985b) > t 70 o g 60 I 50 o 40 c (a) [ r/1 - \ ^ \siope = A 1 \ t{h) ^ b) tran sport ol Photosynthesis vacuolar pool doiosi ^" 1 ' F, tronslocation t (h) Fig. 1. An idealized semilogarithmic plot of translocatory "C efflux from a leaf (a), and the two-compartment model used to interpret it (b). The curve describing isotope efflux is Y = Ae^''' + Be'", where k and / are exponential coefficients (h"') (the inset shows how k can be obtained by replotting the shaded area of the main curve), and A and H are the initial percentage of isotope associated with eaeh compartment. The two-compartment model is a first-order model so that (for example) the translocatory Bux F = K^ Q^, where K,,, is a rate constant (b"') and Q^ a mass (g). A compartmental analysis is needed to relate the parameters in (a) and (b), except that K^ = k; half-times are given by (j = K"' (see Moorby & Jarman, 1975). When very long labelling times are used, B ~ 100 and then K.^, ~ I (K l) and K,^ (K l)-. This paper attempts to measure the sizes and rates of turnover of the transport and vacuolar pools of sucrose in mature leaves of the sucrose-storing species, barley, during a single light/dark cycle. Isotopic techniques are used: leaves are pulse-fed "COj before attaching them to Geiger-Muller tubes to obtain continuous traces for efflux of '"C from the leaves; leaves are fed "CO.^ until isotopic equilibrium is achieved and then either efflux curves are recorded for intact leaves, or discs cut from the leaves are placed in unlabelled sucrose and the washout of i*c monitored. Rate constants derived from these procedures are used in simple simulations to check their validity. The model adopted to relate "C fluxes to sucrose compartmentation (Fig. 1) is similar to that of Moorby & Jarman (1975) and Bell & Incoll (1982). It has been shown that, in barley, the first two phases of efflux of ^''C from leaf blades in the light are entirely attributable to loss of sucrose (Farrar & Farrar, 1985b). MATERIALS AND METHODS Plant growths and ^^CO^ feeding Plants of barley {Hordeum distichum (L.) Lam. cv. Maris Mink) were grown

3 Compartmentation of sucrose in barley leaves 647 as described previously (Farrar & Farrar, 1985b) and experiments were performed on 16-d-old plants at 19 C and 700/tmol m^^ g-i for 16 h d^^. All experiments were performed on fully expanded second leaf blades. Whole leaves, and bands of leaves 1 x 1 cm, were fed ^*CO,, at constant specific activity as described previously (Farrar & Farrar, 1985b). Bands about 7 cm from the leaf tip were fed by passing the leaves through slits in clear acrylic tubing of 1 cm internal diameter and then sealing with silicone rubber. The duration of feeding varied from 5 min to 72 h, and the combination of air flow rate, concentration of and rate of injection of carrier carbonate were adjusted to give a concentration of COj of about 350 ppm. Ejflux of ^*C from, and assay of, ^*C-fed leaves Continuous monitoring of ^*C remaining in intact leaves was carried out by attaching the leaf to a Geiger Muller tube as described previously (Owera et al., 1983). Loss of isotope was monitored through the normal light/dark cycles for up to 48 h following the feeding of '*CO.^. Frequently, many were fed in parallel. Only one was monitored, and from the others, the '"COg-fed areas were cut out and placed immediately into 5 cm^ 95 % ethanol at 80 C. This extract was analyzed for total sugars and "C activity, and sugar chromatography was performed, as described previously (Farrar & Farrar, 1985b). Monitoring of effective leaf mass with a /^-gauge was performed as described previously (Farrar & Farrar, 1985b). Where leaves were to be fed "CO^ until all non-structural carbohydrate was at constant specific activity, labelling began when the plants were 14 d old, at which stage leaf two was just fully expanded. The monitoring of subsequent loss of '*C was carried out from 16 d through at least 24 h of the normal light/dark cycle. Washout of ^"C from leaf discs Leaves which had been fed ^^COj for 48 h to achieve constant speciflc activity as described above were removed quickly from the feeding chamber. Leaf discs were then cut from the fed portion of each leaf and sets of four discs threaded onto lengths of fine steel wire for ease of handling. Fach set of discs was then transferred sequentially for 7 h through a series of sucrose solutions (3 cm^ of 25 mol m"''' sucrose and 0-1 mol m^^ CaSO^) so that ^*C sugars in the leaf discs could exchange with [^^CJsucrose in the medium. A photon fluence rate of 36 ju,mo\ m~'~ s~^ (near the light compensation point for these leaves) was provided by fluorescent tubes and temperature monitored continuously with copper constantan thermocouples. After the last transfer the leaf discs were removed from the wires and dropped into 5 cm''' 95 % ethanol at 80 C. Aliquots of the medium in each washout vial, and of extracts of leaf and leaf disc material, were assayed for '^*C activity; the ethanol extracts were also analyzed for total soluble sugars and the media chromatographed. The accumulated loss of ^*C from the discs was calculated as a percentage of the "C activity present in the leaf discs initially (total '""C washed out + "C remaining in ethanol soluble sugars extracted from discs at end of washout) and this was plotted semilogarithmically with time. Other leaves were fed simultaneously to constant specific activity and monitored continuously with Geiger Muller tubes, to provide parallel efflux curves. Numerical analysis Data are presented as the mean of three to five replicates +SEM. Both the

4 648 S. C. FARRAR AND J. F. FARRAR washout and efflux curves were fitted to double exponential curves using a maximum likelihood program, or to multiple exponential curves graphically. Straight lines were fitted by eye for efflux curves from pulse fed plants and by linear regression for efflux curves from plants fed to constant specific activity, and to washout data. Simulations of a first-order, open, two-compartment model (Fig. 1), like that of Moorby & Jarman (1975) and Owera et al. (1983), and where only one compartment can exchange materials with the environment, were performed on a DEC system 10 mainframe computer with an iteration time of 10~^h. Compartmental analysis was employed to calculate rate constants from exponential coefficients, using the method of Moorby & Jarman (1975). RESULTS Effect of duration of ^'^CO^ feeding on subsequent efflux kinetics Bands of leaves were fed ^""COj for 5, 15 or 30 min, or 48 h, and the efflux of ' 'C from the fed area then monitored continuously with a Geiger-Muller tube. To ensure that self-absorption of /i?-radiation by the leaf was not changing, the change in effective leaf mass was monitored continuously with a /S-gauge; it remained nearly constant at 18mgcm^^ throughout light/dark cycles. All the curves show at least two exponential phases of loss of isotope during the light period, the first representing loss from transport, and the second from vacuolar, sucrose. The kinetics of '''C efflux were different when '''COj feeding times were changed (Fig. 2). The percentage of the '^'*C lost in the first phase (the transport pool) decreased as the feeding time increased. The percentage ^*C in transport sucrose has been determined for many efflux curves; for 5 min feeds, % of the ^''C was lost from transport sucrose; for 15 and 30 min feeds, and %, and for feeds of over 48 h, only 4+ 1 % of ^*C was in transport sucrose. These differences are due to the long times taken for isotopic equilibrium to be reached in pools that turn over slowly; when "^^COg is fed for a short time, readily accessible pools with rapid turnover will acquire a disproportionately large share of the '''C. Pulse feeds of '^COj lasting 5 min thus provide efflux curves in which transport sucrose represents a large proportion of the efflux, and so are particularly suitable for examining this pool; conversely, leaves fed '''COj for long periods have high labelling of, and so are suitable for examining, vacuolar sucrose. During the dark period, an increased rate of '"C efflux could be seen, especially in those plants fed for short periods (F"ig. 2). This represents the mobilization of '^''C labelled starch, and is not considered further in this paper. Specific activity and abundance of sugars after ^'^CO,^ feeding The carbohydrates in leaves fed for long periods should all be at the same specific activity if feeding has been sufficiently prolonged. Following 50 h feeding of ^"COj, and at all times in the photoperiod, the specific activities of starch, low molecular weight fructan, sucrose, glucose and fructose were closely similar, showing that isotopic equilibrium had indeed been achieved. The efflux curves from these leaves fed to a constant specific activity show a smaller percentage '^'*C lostthan when leaves were fed for short pulses (50+ 10% as opposed to %); the percentage by weight and ^*C activity for starch were 38 and 39 % respectively. This implies that the largest percentage of the ^^C remaining in these leaves after loss of activity from sucrose is in the form of starch; the other soluble sugars and

5 Compartmentation of sucrose in barley leaves Time into photoperiod (h) Fig. 2. Efflux of '"C from intact leaf blades of barley previously fed '"COj at constant specific activity for the times, ranging from 5 min to 48 h, indicated by the curves. Continuous monitoring of tbe leaf blades was begun 6 h into the photoperiod and was continued through a normal light-dark cycle. The hatched bar on the x-axis indicates the dark period. fructans will appear to be lost during the 'sucrose phases', as they have high turnover rates; there can only be about 10% of the ^^C in protein. Changes in effiux from the transport pool during the photoperiod monitored after feeding ^^CO^for 5 min Bands of leaves were fed '''COg for 5 min at different times during the photoperiod. Each efflux curve showed two exponential phases of loss of isotope during the light period, and the percentage of '''C in transport sucrose decreased with time into the photoperiod. The rate constant for loss of isotope from the transport pool (the first phase of efflux) decreased through the photoperiod [Fig. 3(a)], from 0-82 to 0-31 h~\ and so the ti for turnover of transport sucrose {Q{) must therefore rise from about 0-9 h to 2 h during the photoperiod. The size of the transport pool of sucrose {Q^) may be estimated using the relationship Q^ = 0'i^io, where 0 is flux out of the pool - in this case the translocation rate at the appropriate times into the photoperiod (data from Farrar & Farrar, 1985b, Fig. 2) and i^^,,, is the rate constant for this loss, equal to K taken from Figure 3 (a). Transport pool size is estimated to have increased with time into photoperiod [Fig. 3(b)], from 298 to 2310 mg sucrose m~2 leaf (Table 1). Changes in effiux from the vacuolar pool during the photoperiod, monitored after feeding ^*CO^for 48 h When leaves were fed ^^COj for a minimum period of 48 h, and loss of this ^*C then monitored continuously during the light/dark cycle, two exponential phases of i*c loss were seen during the light period. For these curves, transport sucrose only represents an extremely small percentage of the "C in the fed area, because with the attainment of isotopic equilibrium a far larger proportion of ^*C is found in vacuolar sucrose. The exponential coefficient, /, for loss of "C from vacuolar sucrose in the light increased during the photoperiod and a linear regression of the relationship between / and time was highly significant [Fig. 4 (a)], implying that the half-time for turnover of vacuolar sucrose fell from 69 h at the start to 12 h at the end of the photoperiod. The rate constant for loss from vacuolar sucrose (calculated using the relationship in the legend to Fig. 1) also increased

6 650 S. C. FARRAR AND J. F. FARRAR Time into photoperiod (h) Fig. 3. (a) The change through the photoperiod in the exponential coefficient {k) for the initial slope of the efbux curves shown in Fig. 4. The fitted regression is A = <, where ( is time in h into the photoperiod. (b) The change through the photoperiod in the size of the transport pool of sucrose (Q,), estimated as 0/k where k is the exponential coefficient from Figure (5(a) and 0 the experimentally determined translocation rates (Farrar & Farrar, 1985a,b). The fitted regression is Qi = <, where ( is time in h into the photoperiod. Table 1. Sucrose compartmentation in second leaf blades of barley : assessment from efflux curves Method Time into photoperiod (h) 0 Efflux from intact leaves fed "CO., for 5 min Effiux from intact leaves fed "CO.; for 50 h 5 16 Qtotai (mg m-') 1 ransport Pool ' Qx (mg m-2) (j(h) %»c Mean concentration (mol m ^), of sucrose ~ Vacuolar Pool ' Q, (mg m--') h (h) % "C Q2 % Q total Mean concentration (mol m~'), of sucrose Notes; Qiotai- figures from Farrar & Farrar (1985b). Mean sucrose concentrations calculated using the relative volumes of vacuole, and cytoplasm plus wall, given for wheat leaves hy Altus & Canny (1985), at 53 & 12% of 180 g HjOm--' barley leaf blades. Q.^ calculated by 0u,tai " Oi- % '"C is percentage of total "C in leaf associated with the pool indicated.

7 Compartmentation of sucrose in barley leaves U-Ub a 0-06 D ^ (o) i 0-02 < Time into photoperiod Fig. 4. (a) The change through the photoperiod in the exponential coefficient (/) for the second slope of the efflux curves such as in Figure 6. The fitted regression is / = ( is time in h into the photoperiod. (b) The change through the photoperiod in the rate constant (K.^^ for loss of sucrose from the vacuole to the transport pool (Q^ to ),)- Values of K^^ were derived by compartmental analysis (Moorby & Jarman, 1975, and Fig. 1) using the changing values of the exponential coefficient (/) shown in Figure 4(a). (h) Table 2. Sucrose compartmentation in second leaf blades of barley: assessment by washout of isotope from leaves previously labelled with ^^CO^for 50 h Time into photoperiod (h) 16 sucrose) (mg m ^) Apoplast, (i (min) Apoplast, % "C washed out Symplast, ti (h) Symplast, % "C washed out Mean concentration of sucrose in transport pool (mol m"'') Q, (transport sucrose) (mg m~'^) Qj (vacuolar sucrose) (mg m"'^) OJ as % Ot^uu Vacuolar pool, ti (h) Vacuolar pool, % '''C washed out Mean concentration of sucrose in vacuolar pool (mol m~') Mean sucrose concentrations were calculated from relative volumes given for wheat leaves (Altus & Canny, 1985) and Qi^to, is from Farrar & Farrar (1985b). during the photoperiod [Fig. 4(b)]. Pool sizes calculated from these data are given in Table 1. Washout experiments with leaf discs Leaves were fed '''COa to a constant specific activity and discs were cut rapidly from them. These discs were allowed to exchange labelled "C sugars with external unlabelled [^^CJsucrose in low light, to obtain washout curves showing the amount of ^^C remaining in the discs with time. Where the efflux began 5 h into the photoperiod the washout curves showed three exponential phases of ^*C loss (Fig. 5). Lines were fitted by linear regression, and two log linear pbases subtracted sequentially to obtain the rate constant and percentage ^''C lost for each of the three phases. The percentage of "C lost in these

8 652 S. C. FARRAR AND J. F. FARRAR 00; 80.oL O 20 ok -^ 10 k 0-2 i 0-4 ' O 'i i k i--i s Duration ot washout ( h ) 1 1 Fig. 5. The washout of "C from leaf discs cut from leaf hlades of barley which had been fed "COj for 50 h and were then allowed to exchange ["C]sucrose with ['^CJsucrose in an external bathing medium. The three exponential components of three replicate curves were determined graphically; the means±se for the three curves are shown. 100 i.;;^:^ Time ot washout or ettiux ( h) Fig. 6. The comparison between the washout of "C activity from leaf discs exchanging ["C]sucrose with ['^CJsucrose into an external bathing medium ( ) and the efflux curves from leaf blades on intact plants ( ) starting at 0, 5 or 16 h, as indicated by the curves, into the photoperiod after feeding the second leaves of barley plants with "COj for 50h. phases, identified by half-time, were 11-5 for the rapid phase {ti~2 min), 8-7 for the second {ti~n min) and 79-8 for the slowest (<i~25 h) (Fig. 5; Table 2). The media were chromatographed following the washout; approximately 20% of the '^''C was in glucose and fructose and the remainder in sucrose. When this washout experiment was repeated, but starting the efflux at either 0 or 16 h into the photoperiod, it was found that the percentage of "C lost in each phase of efflux was similar at each time (Table 2), perhaps the only difference being a greater percentage in the second phase at 0 h into the photoperiod. The i-times may only be compared within experiments due to the temperature differences between experiments (Table 2). The washout curves that started at 0, 5 and 16 h into the photoperiod were compared with curves showing ^''C efflux from intact leaves fed ^^COg simultaneously with those used for the washout experiments. The amount of ^*C remaining in the leaf discs after 4 h efflux is substantially lower than that remaining in the intact leaf-71% as opposed to 85 % (Fig. 6), since the rapid phases of loss seen in the washout experiment are

9 Compartmentation of sucrose in barley leaves 653 (a) E / \ 16 6 Time into photoperiod {h} Fig. 7. Changes through the photoperiod in (a) the sucrose content and (b) the translocation rate of second leaf blades of barley as determined experimentally ( ) (Farrar & Farrar, 1985a, b) and as predicted by simulation where k (the exponential coefficient for the first phase of efflux) and / (the exponential coefficient for the second phase of efflux) remain constant through the photoperiod ( ), where k is decreased through the photoperiod whilst / remains constant ( ) and where / is increased through the photoperiod whilst k remains constant ( ) not seen in efflux from intact leaves; the slopes for "C loss from vacuolar sucrose are similar in both. Numerical simulation of sucrose pool sizes A program simulating the model of Figure 1 (b) was used to test the effects of the magnitude of rate constants on the diurnal changes in pool sizes of sucrose. When the exponential coefflcients k and / were held constant during the photoperiod, simulated total leaf sucrose and translocation were dissimilar to observed values (Fig. 7). Allowing k alone to decrease, in the manner noted above, during the photoperiod resulted in no improvement, but wben / was allowed to rise as noted above, then the simulations more closely approximated the observed values (Fig. 7). These analyses also allowed Q^ to be estimated: it rose throughout the photoperiod. DISCUSSION Sucrose in mature barley leaves must occur in at least two types of pool, vacuolar and transport (tbe latter comprising cytoplasm and apoplast of mesophyll cells, and the sieve element - companion cell complex). Fach of the techniques used is most suitable for estimating one or more features of these two types of pool, and the data they have provided is summarized in Tables 1 and 2. Three features are notable: a large proportion (about 80%) of sucrose is vacuolar; the flux through this vacuolar pool is about 40 % of the carbon fixed in photosynthesis; and the flux through the vacuole changes, relative to translocation, during the photoperiod. Fach of the methods used here indicated that about 80 % of the sucrose was in vacuolar, and the remainder in transport, pools (Table 1). This is within the limits suggested earlier for barley (Farrar & Farrar, 1985b), and is similar to the 65% of sucrose in barley leaves found to be vacuolar by Wagner, Keller & Wiemken (1983); they used protoplast and vacuolar isolation, during which processes some vacuolar sucrose may have been lost. In starcb-storing species, vacuolar sucrose in leaves has been reported to comprise 20 to 40 % (sugar beet; Geiger et al., 1983)

10 654 S. C. FARRAR AND J. F. FARRAR to 80% (spinach at the end of the light period; Gerhardt & Heldt, 1984; cultured tobacco cells; Delmer, 1979) of the total. The amount of sucrose in vacuoles can be re-expressed as a concentration, using the water content of barley leaf blades (180 g m"^) and the proportion of tissue volume occupied by vacuoles (53 %) given by Altus & Canny (1985) for wheat leaves; values range from 45 to 124 mol m~^ depending upon time in the photoperiod. The mean concentration in the transport pool, calculated similarly, ranges from 23 to 180 mol m~^. This is unlikely to represent cytosolic concentration, since autoradiographic evidence suggests that a large proportion of the '^^C ascribable to the transport pool is associated with veins (Farrar & Farrar, unpublished). If phloem, containing sucrose at 500 mol m"^, occupies 1 % of barley leaf volume, 300 mg m"^ sucrose would be within it. Separate estimates of sucrose in cytosol, phloem and apoplast are badly needed. The minimal mean concentration of 25 mol m~^ in the transport pool at the end of the dark period is similar to the cytosolic concentration estimated for the roots of barley (Farrar, 1985). Cytosolic sucrose concentrations of 5 to 13niolm~^ (barley leaf mesophyll; Kaiser et al., 1982; spinach protoplasts; Giersch et al., 1980; Gerhardt & Heldt, 1984), 24 mol m"^ (wheat protoplasts; Giersch et al., 1980), and 76 mol m"'' (sugar beet taproot; Saftner, Daie & Wyse, 1983) have been reported. Sucrose concentrations in vacuoles have been reported to vary from 514 mol m-^ in sugar beet taproot (Saftner et al., 1983) to 1 to 45 mol m~^, depending on the time in the photoperiod, in spinach (Gerhardt & Heldt, 1984), spanning the range reported here for barley leaves. Wagner et al. (1983) suggest that sucrose is unique among the soluble sugars of barley leaves as it occurs in the cytosol at a concentration equal to or greater than that in the vacuole; this is consonant with the failure to find evidence for an energyrequirement for sucrose transport at the tonoplast, reported by Kaiser & Heber (1984), and with our finding that the proportion of sucrose in the vacuolar pool does not change appreciably during a diel cycle in spite of considerable changes in concentration. Saftner et al. (1983) and Willenbrink & Doll (1979) give evidence for energized vacuolar loading of sucrose in the storage tissue of beet taproot, which also occurs in vacuoles from sugarcane suspension cultures (Komor, Thom & Maretzki, 1982). The question of energization of sucrose transport at the tonoplast thus seems open (Reinbold & Kaplan, 1984). The rise in the rate consant K^^ for loss of sucrose from the vacuole that occurs in these barley leaves, at a time when vacuolar concentrations are still increasing, may reflect an active efflux of sucrose across the tonoplast, but critical evidence at the appropriate time in the diel cycle is needed. The rates of turnover of the transport and vacuolar pools are not the same. The vacuolar pool has a half-time of 12 to 30 h over most of the photoperiod; since vacuoles contain a high proportion of the sucrose in the leaf, tbis implies that the tonoplast may have a major role in controlling fluxes of sugars. Indeed about 40 % of photosynthetically fixed carbon passes through the vacuole, as sucrose, before its eventual export from the leaf (calculated as net storage of sucrose in the leaf X 0-8, the proportion of sucrose that is vacuolar, plus rate of loss of sucrose from the vacuole, given by vacuolar pool size x /Cji)- Work with protoplasts has shown rapid appearance of photosynthetically fixed ^''C in the vacuoles of several species, including barley (Kaiser & Heber, 1984; Kaiser et al, 1982; Giersch et al, 1980) and therefore appears relevant to the situation in vivo. Our rate of vacuolar loading of sucrose can be re-expressed as 4-4 mmol sucrose g chlorophyll"^ h"^, about half of that obtained in vitro by Kaiser & Heber (1984).

11 Compartmentation of sucrose in barley leaves 655 The transport pool has a half time of 03 to O^85 h; this represents the rate of passage of sucrose through the mesophyll, from the cells in which it was synthesized to the veins and loading into and redistribution between veins (Altus & Canny, 1985) as well as eventual translocation out of the leaf. By contrast, the shorter half-times of 0^2 to 0^3 h for the phase of washout ascribable to the symplast will be due solely to leakage across the plasmalemma and out of leaf discs. That the half-time for this process is shorter than for translocation implies that the export of sucrose from mesophyll cells will not be a rate-limiting step m translocation. Washout allows the approximate estimate that apoplastic sugars can account for nearly half the transport sucrose, although this will include loss from cells damaged by cutting: a critical estimate of apoplastic sugars and their exchange across the plasmalemma in vivo is badly needed. The sizes of both the transport and the vacuolar pools of sucrose, and their rates of turnover, vary during the photoperiod. The transport sucrose pool increases from about 300 mg m"^ (measured by any of the three techniques used) to 860 to 2300 mg m"2 (low value from washout; high value from feeding "CO2 for 5 min). This parallels the increase in rate of translocation from, and the activity of sucrose phosphate synthetase in, these leaves during the photoperiod (Farrar & Farrar, 1985a,b). It is thus possible that the rate of translocation is dependent on the size of the transport sucrose pool, but the relative contributions to this pool of phloem and of mesophyll cytosol need to be known. The vacuolar pool similarly increases, with the proportioning of sucrose between the two pools staying roughly similar. The concentration in this pool increases by 79 mol m^^ Since the solute potential of these leaves is -Ml MPa, equivalent to 460 osmol m~^ (Farrar, unpubl.), this diel change in just one solute accounts for about 15 % of the total solutes in these leaves, and raises major problems of turgor regulation, and of the balances of solutes between cytosol, vacuole and apoplast, not experienced by starch-storing species. The rate constants describing fluxes between these pools also show changes with time, implying that the machinery responsible for membrane and phloem transport of sucrose is under control that varies during the photoperiod. Changes in the exponential coefficients describing effiux have previously been shown in wheat (Bell & Incoll, 1982) and Vicia faba (Pearson, 1974). Bell & Incoll (1982) suggested on this basis alone that phloem loading and transport was becoming limiting, perhaps due to saturation of membrane carriers, with a resultant fall in K. Our data shows clearly that this is not the case: K falls whilst the rate of translocation rises (Farrar & Farrar, 1985b) and so any change in K is independent of phloem loading, and in any case takes place during an increase in efflux from the vacuole, indicated by the rising value of K^^. The importance of varying rate constants is demonstrated by simulation models predicting translocation rate and pool sizes; only when rate constants are allowed to vary do the simulations approach observed values. These simulations also emphasize that compartmental analysis based on first order rate kinetics and unvarying rate constants will be inappropriate for precise work. Also, the simulations allow the relative sensitivity of the rate constants K^^, (translocation) and i^t^, (vacuolar unloading) to be explored; it is clear that vacuolar unloading is the more important of the two in allowing simulated to approach real values. This again emphasizes the importance of the tonoplast in the control of carbon fluxes in these leaves. At the start of the photoperiod, the concentration of sucrose in the vacuole is low and there is considerable accumulation of sucrose in it, at a rate that exceeds the rate of translocation (Farrar & Farrar, 1985b). Perhaps the vacuole has more

12 656 S. C. FARRAR AND J. F. FARRAR immediate access to cytosolically produced sucrose than the more distant sieve element-companion cell complex and so obtains a preferential share of sucrose, whether or not transport across the tonoplast is energized. At this stage, rate ot accumulation by the vacuole is greatly in excess of rate of loss from it. As the photoperiod proceeds, there is an increase in the rate of translocation and a fall in the rate of net accumulation of sucrose (Farrar & Farrar, 1985b). At least part of the fall in accumulation is due to the rise in the rate-constant for export across the tonoplast; this value needs to be high to account for the sucrose lost during the night, and it appears that it rises not just in response to darkness but during the latter part of the photoperiod. The control of this is not understood. Part of the fall in sucrose accumulation may be due to the relatively high sucrose (and possibly total solute) concentration in the vacuole making further vacuolar loading less probable than phloem loading. ACKNOWLEDGEMENTS We gratefully acknowledge the financial support of the A.F.R.C. REFERENCES ALTUS, D. P. & CANNY, M. J. (1985). Loading of assimilates in wheat leaves. II. The path from chloroplast to vein. Plant, Cell & Environment, 8, AviGAD, G. (1982). Sucrose and other disaccharides. In: Plant Carbohydrates I. Intracellular Carbohydrates (Ed. hy F. A. Loewus & W. Tanner), Encyclopedia of Plant Physiology, vol. 13A, pp Springer, Berlin. BELL, C. J. & INCOLL, L. D. (1982). Translocation from the flagleaf of winter wheat in the field. Journal of Experimental Botany, 33, DELMER, D. P. (1979), Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cells. Plant Physiology, 64, FARRAR, J. F. (1985). Fluxes of carhon in roots of harley plants. New Phytologist, 99, FARRAR, S. C. & FARRAR, J. F. (1985a). Fluxes of carhon compounds in leaves and roots of barley plants. In: Regulation of Sources & Sinks in Crop Plants, Monograph 12 (Ed. hy B. Jeffcoat, A. F. Hawkins & A. Stead), pp British Plant Growth Regulator Group, Bristol. FARRAR, S. C. & FARRAR, J. F. (1985h). Carhon fluxes in leaf hlades of harley. New Phytologist, 100, FISHER, B. & OUTLAW, W. H. (1979). Sucrose compartmentation in the palisade parenchyma of Viciafaba L. Plant Physiology, 64, FoNDY, B. R. & GEIGER, D. R. (1982). Diurnal patterns of translocation and carhohydrate metabolism in source leaves of Beta vulgaris L. Plant Physiology, 70, GERHARDT, R. & HELDT, H. W. (1984). Measurement of suhcellular metabolite levels in leaves hy fractionation of freeze stopped material in non-aqueous media. Plant Physiology, 75, GEIGER, D. R., PLOEGER, B. J., Fox, T. C. & FONDY, B. R. (1983). Sources of sucrose translated from illuminated sugar heet source leaves. Plant Physiology, 72, GiAQUiNTA, R. T. (1980). Translocation of sucrose and oligosaccharides. In: Biochemistry of Plants, vol. 3 (Ed. hy J. Preiss), pp Academic Press, New York. GiERSCH, C, HEBER, U., KAISER, G., WALKER, D. A. & ROBINSON, S. P. (1980). Intracellular metaholite gradients and flow of carhon during photosynthesis of leaf protoplasts. Archives of Biochemistry and Biophysics, 205, GORDON, A. J. & JOHNSON, I. R. (1984). The control of photoassimilate supply from barley leaves during light and dark periods. In: Advances in Photosynthesis Research, vol. 4 (Ed. hy C. Syhesma), pp Junk, The Hague. HUBER, S. C. (1981). Inter- and intra-specific variation in photosynthetic formation of starch and sucrose. Zeitschrift fur Pfianzenphysiologie, 101, HUBER, S. C. & MORELAND, D. E. (1980). Efflux of sugars across the plasmalemma of mesophyll protoplasts. Plant Physiology, 65, KAGAWA, T. & WONG, J. H. H. (1985). Allocation and turnover of photosynthetically labelled ''COj in leaves of Glycine max L. Clark. Plant Physiology, 77, KAISER, G. & HEBER, U. (1984). Sucrose transport into vacuoles isolated from harley mesophyll protoplasts. Planta, 161,

13 Compartmentation of sucrose in barley leaves 657 KAISER, G., MARTINOIA, E. & WIEMKEN, A. (1982). Rapid appearance of photosynthetic products in tbe vacuoles isolated from barley mesophyll protoplasts by a new fast method. Zeitschrift fiir Pfianzenphysiologie, 107, KOMOR, E., THOM, M. & MARETZKI, A. (1982). Vacuoles from sugarcane suspension cultures Protonmotive potential difference. Plant Physiology, 69, KuRSANOV, A. L. (1984). Assimilate Transport in Plants. Elsevier, Amsterdam. MADORE, M. & WEBB, J. A. (1981). Leaf free space analysis and vein loading in Cucurbita pepo. Canadian Journal of Botany, 59, MOORBY, J. & JARMAN, P. D. (1975). Tbe use of compartmental analysis in tbe study of movement of carbon through leaves. Planta, 122, OUTLAW, W. H., FISHER, B. & CHRISTY, A. L. (1975). Compartmentation in Vicia faba leaves. 11. Kinetics of "C sucrose distribution among individual tissues following pulse labelling. Plant Physiology, 55, OWERA, S. A. P., FARRAR, J. F. & WHITBREAD, R. (1983). Translocation from leaves of barley infected with brown rust. New Phytologist, 94, PEARSON, C. J. (1974). Daily changes in carbon dioxide exchange and photosynthate translocation of leaves of Vicia faba. Planta, 119, RAVEN, J. A. (1977). H+ and Ca" in phloem and symplast: relation of relative immobility of the ions to the cytoplasmic nature of the transport paths. Nezo Phytologist, 79, REINHOLD, L. & KAPLAN, A. (1984). Membrane transport of sugars and aminoacids. Annual Review of Plant Physiology, 35, SAFTNER, R. A., DAIE, J. & WYSE, R. E. (1983). Sucrose uptake and compartmentation in sugar beet taproot tissue. Plant Physiology, 72, 1-6. SicHER, R. C, KREMER, D. F. & HARRIS, W. G. (1984). Diurnal carbohydrate metabolism of barley primary leaves. Plant Physiology, 76, SiLVius, J. E., CHATTERTON, N. J. & KREMER, D. F. (1979). Photosynthate partitioning in soybean leaves at two irradiance levels. Comparative responses of acclimated and unacclimated leaves. Plant Physiology, 64, WAGNER, W., KELLER, F. & WIEMKEN, A. (1983). Fructan metabolism in cereals: induction in leaves and compartmentation in protoplasts and vacuoles. Zietschrift fiir Pfianzenphysiologie, 112, WiLLENiiRiNK, J. & DOLL, S. (1979). Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissues. Kinetics and specificity of two sucrose uptake systems. Planta, 147, ANP 103

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