MODIFICATION OF RESPIRATION AND CARBOHYDRATE STATUS OF BARLEY ROOTS BY SELECTIVE PRUNING

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1 Phytol. i^9s6) 102, 5\3-52\ 513 MODIFICATION OF RESPIRATION AND CARBOHYDRATE STATUS OF BARLEY ROOTS BY SELECTIVE PRUNING BY J. F. FARRAR AND C. L. JONES School of Plant Biology, University College of North Wales, Bangor, Gwynedd LL57 2UW, UK {Accepted 10 December 1985) SUMMARY Barley plants were pruned selectively to give root systems with relative growth rates that varied two-fold. Rates of elongation of the seminal root axes were unaffected. Respiration rate was highest in the fast-growing roots, but there was no change in the proportion of carbon entering the root that was subsequently lost in respiration. Soluble sugar contents were greater in fast than in slow growing plants, but the rate of uptake of exogenously supplied sucrose and glutamine was highest where root growth rate was reduced by pruning of the shoot. Turnover of sugars in the vacuole was slower in roots with reduced growth rate. Key words: Barley, roots, respiration, sugars. INTRODUCTION The respiration rate of plant tissues has been related to both demand for respiratory energy {by coupled, adenylate-limited, respiration) and to substrate - specifically carbohydrate - supply. It has been suggested that any attempt to relate respiration rate to carbohydrate status should assess cytosolic, rather than total, sugars, since these alone will be readily available as substrates {Farrar, 1985b). This paper reports a series of experiments in which the growth rate, respiration rate and carbohydrate status of barley root systems still attached to the shoot were altered by selective pruning, as a means of investigating relationships between these variables. Cereal roots are particularly suitable for this type of investigation. Their respiration has been studied {Yemm, 1965), and the compartmentation and fluxes of carbohydrates in barley roots has been described {Farrar, 1985a). It has been suggested that barley root respiration can be carbohydrate-limited {Pitman, Mowat & Nair, 1971), but this may only occur in excised roots, as even at the end of the dark period attached roots show no evidence of carbobydrate-limited respiration {Farrar, 1981; Farrar & Farrar, 1985a). The seminal roots of a single plant are quite uniform, in spite of their differing developmental origin; excision of one or more seminals thus changes the amount, but not the morphology, of the root system. Such partial root pruning results in an increased relative growth rate of the remaining root system {Crossett, Campbell & Stewart, 1975) and, under certam conditions, the retention of high elongation rates by seminal axes whilst those of control axes fall {Rahman, Aspinall & Paleg, 1975). Such experiments have been used to suggest that substrate availability determines root growth {Rahman et al., 1975) although firm evidence is lacking. Any limitation due to substrate is unlikely to be due to tbe ability of the phloem to transport it, since seminal roots of wheat X/86/ $03.00/ The New Phytologist

2 514 J. F. F A R R A R AND C. L. J O N E S are capable of mass transfer in the phloem ten-fold higher than they normally sustain (Passioura & Ashford, 1974). Since they also show no secondary thickening cereal roots provide a readily manipulated system in which long-distance transport 15 unlikely to complicate interpretations relating to meristematic growth. M A T E R I A L S AND M E T H O D S Seedlings of barley {Hordeum distichum (L.) Lam. cv. Maris Mink) were grown in aerated solution culture as described previously (Farrar & Farrar, 1985b) in a T I T H ' W ^ '? i ^ T T^ ^"^^^ P^"'"^^ "^" ^ " f^«"^fluorescenttubes for 16 h d. When 8 d old, plants were assigned randomly to one of three treatmentscontrols, which remained intact; root-pruned, in which all roots except for one seminal were removed; shoot-pruned, in which all leaf blades except the first were removed^ Daily over the next 7 d pruning was repeated as necessary Atter 7d of treatment, plants were harvested or assayed 5 to 8h into the photoperiod. For morphological measurements, the length of each seminal and nodal root axis was measured and the root systems oven dried at 80 C before weighing. Respiration rate of freshly excised roots was measured polarographicallv as oxygen uptake in Clark-type electrodes, with the roots in 50 mol m - MES QOor'.K ', on^^^/f ' T ' ' ^^'^ extracted by two successive treatments with 90o/o ethanol at 80 C and measured by the phenol-sulphuric acid method of Dubois et al (1956). The rates of uptake of sucrose and glutamine by excised \T^^ Z l T^ "'''S'.'n ' ' ^^ ^ ^^ P^^^^"^ ^^^"^ 150 mg f.wt of root in \ F^.K^' containing 0-1 mol m - CaSO, and 70 kbq of nn l o n T 3U-^^C]glutamine (Radiochemical Centre, Amersham) in 04 out of t h T ^^/P^^^^^^ly ^«ld carrier for 15 min and then washing isotope out of the free space for 10 mm before dropping the roots into hot 90% ethanol Both an aliquot of the ethanol extract, and the extracted roots, were counted by liquid scintillation in an LKB-Wallac L.S. spectrometer using Aquasol as scintillant; the Aquasol cleared the root material giving high counting efficiencies ^ our replicates were used per substrate. The compartmentation of soluble sugars between apoplast plus cytoplasm on the one hand, and vacuoles on the other, was assessed by isotope washout as described previously (Farrar, 1985a), except that here the first leaf blades only of al treatments were fed - C O, for 50 h to produce uniform specific activity o solub e sugars in the root. Then single seminal roots were excised and transferred serially through a series of tubes containing 5 cm^ of 25 mol m-^ sucrose at 24 C IrLhs' of 0/ soirhl - r "' "'" ' " ^ ' " ^ ' " ' ' '"""^^'^ ^^ ^^^"^^ scintillation and d r l n TK ««l"ble i^c remaining in the tissue against time of washout were drawn. These showed a log-linear portion, ascribed to washout from the vacuole Tfn T T f K? J ^''' ''? ^'""^^^ ' ^P^P^^^^ P^"«^yt ««i superimposed on ^for the first hour (Farrar 1985a). The slope and intercept of the vacuole portion were measured for each of three replicates per treatment. RESULTS Root growth Over the ages 7 to 16 d, root systems gained dry matter exponentially with rthetemint ' f ^~''T^' ' '"^^ "^^ attributable to seminal r l ^. 1). 1 he semmal axes elongated at a mean rate of 20 7 mm d-\ a rate in excess

3 Respiration and carbohydrates in roots 515 of that achieved by the nodal roots, which grew at 17-5 mm d~^ for about 4 d after their first appearance and more slowly thereafter. Both pruning treatments resulted in large changes in root growth; in each case a large fall in the relative growth rate of the root system resulted in a reduced mass of seminal roots, but this reduction had quite different causes (Table 1). Whereas Plant age (d Fig. 1. Growth of root systems of barley plants in aerated solution culture; (a) increase in length of seminal (#) and adventitious (O) axes, and (b) increase in dry weight of seminal (#), and adventitious (O) roots, on single plants, and total root weight (dashed line). Table 1. Growth of roots of barley plants which had been root-pruned {all roots except one seminal removed) or shoot-pruned (all leaf blades except the first removed) for 7 d previously Control Root-pruned Shoot-pruned Dry weight of all seminal roots (41) (mg per plant) Dry weight per seminal root (mg) (08) Length of seminal axis (mm) (63) Dry weight of adventitious roots (0 3) (mg plant"*) Length of adventitious axis (mm) Relative growth rate of all seminal roots (d~') Relative growth rate of each seminal (d~^) 35-3± ±21 6 0±0-7 81± ± ±l ± Before treatment values given in parentheses. The relative growth rates are means for the 7 d of treatment. in shoot-pruned plants, the growth in mass of each seminal was reduced below that of the control, it was increased in the sole remaining seminal of root-pruned plants so that relative growth for this seminal root was 150% of the control; its final weight was three times that of the control and five times that of each seminal on the shoot-pruned plants (Table 1). Since the rate of elongation of the seminal axis was quite unaffected by treatment, all of these differences are attributable to changes in the growth of first-order laterals, and it was clear that the amount of

4 J. F. FARRAR AND C. L. JONES b) lateral root growth was greatest on the root-pruned, and least on the shoot-pruned p ants (F.g^ 2), Adventitious (nodal) roots were removed from the root-pruned T T' V ^^ appeared, and shoot-pruning seemed not to aflfect their growth Metabolism of roots from pruned plants Respiration rate per unit weight was significantly greater than that of controls in the faster-growing seminal root from root pruned plants, and lower in the s ow-growmg roots of shoot-pruned plants (Table 2). The rate of uptake of sucrose from solutions of 0-4 mmol m- sucrose was greatest in roots from shoot-pruned plants; the seminal from root-pruned plants also took up sucrose faster than controls. Incorporation into 90% ethanol-insoluble material of ^^c from this endogenous sucrose was greatest in roots from shoot-pruned plants, but this may d^utiorof " r' rt in^.''^r^r'^^ P^ ^^ ^" ^^^^^ ^^^^^ causing^compkratively I'tle dilut on of ^^C (Table 2). Glutamine uptake only differed from controls in the shoot-pruned plants: here it increased to 150%, and incorporation into protein (assumed equivalent to 90 % ethanol insoluble material) to 250 % of control

5 Respiration and carbohydrates in roots 517 Table 2. Rates of respiration, and sucrose and glutamine uptake, by seminal roots of 15-d barley plants given selective pruning 7 d earlier Controls Roots pruned to one seminal Shoots pruned to one leaf blade Respiration rate (nmol O2 g f.wt~' "') (nmol O2 g d.wt"^) Sucrose uptake (Bq mg~^) Incorporation of ^*C from sucrose into insoluble material (%)* Glutamine uptake (pmol mg~^ h~*) Incorporation of glutamine into protein (pmol mg~^ h~^)* ± ±2-2 42±2 244 ±9 9-0 ± ±2-3 49± ± ± ± ±5 374 ± ±0-6 * In calculating these figures, no allowance has been made for dilution of isotope in endogenous pools. Table 3. Soluble carbohydrate status of seminal roots of 15-d barley plants given selective pruning 7 d earlier. Control Roots pruned to one seminal Shoots pruned to one leaf blade Soluble sugar content (mg g~' d.wt) Soluble sugar **C in vacuolar phase of washout (%)* Non-vacuolar soluble sugar (mg g-i)t Rate-constant for loss of sugar from vacuole (h~*)* Steady-state flux of sugar across tonoplast (mg g~^ root h~^);i: ± ± ± ± * Obtained directly from curve of '*C remaining in tissue versus time, in washout experiments. t The product of soluble sugar content and fraction of "C that was non-vacuolar in a washout experiment. X Rate-constant times vacuolar sugar content. Soluble carbohydrate status of roots from pruned plants When only one seminal root was left on a pruned plant, its content of soluble carbohydrate was twice that of controls; conversely, shoot-pruning resulted in seminal roots of these plants containing slightly less sugar than controls (Table 3). Washing experiments showed that the proportion of this sugar that was vacuolar was unchanged by pruning, but since the total amount of sugar was changed, the sizes of both the vacuolar and the non-vacuolar sugar pools were altered by pruning treatment; thus non-vacuolar (apoplastic and cytosolic) sugar was at double the control value in the surviving seminal of root-pruned plants (Table 3). Root-pruning did not change the rate of washout of ^^C from the vacuolar pool - expressed here as a rate constant - but shoot pruning did, reducing it to about two-thirds, and thus indicating a slower turnover of vacuolar sugars in these plants (Table 3). The product of this rate-constant and vacuolar sugar content gives the fiux of sugar across the tonoplast in steady-state; this was greater in root-pruned plants than in the controls, and lower than controls in shoot-pruned plants (Table 3).

6 5^8 JF. FARRAR AND C. L. JONES Table 4. Carbohydrate budgets for a 24-h period of seminal roots of 15-d old barley plants given selective pruning 7 d earlier Control Root-pruned Shoot-pruned Net carbohydrate gain* (g g * d 1) Respiratory loss of carbohydrate! (gg-'d-i) Carbohydrate imported into roott 0-49 o-71 C\A-i. (gg ' d ') Respiratory loss import * From relative growth rate. t Measured. X Net gain + respiratory loss, and assuming negligible leakage of organic compounds from roots. Carbohydrate budgets for seminal roots of selectively pruned plants Approximate carbohydrate budgets were calculated for roots of plants subject to all three treatments (Table 4). Net carbohydrate gain was equated with relative growth rate, and carbohydrate import into the root given by adding to this the respiration rate given in Table 2 after conversion to the appropriate units The amount of carbohydrate imported daily into each unit of root was twice as great m the surviving seminal of root-pruned plants than in seminals of shoot-pruned plants, with controls intermediate. In spite of this difference in rate of import the proportion of carbohydrate imported that was lost in respiration was unchanged at about 35 % (Table 4). These calculations assume negligible leakage of organic material from the roots, an assumption supported by the very low rates of i^c loss to the rooting medium recorded for these plants (Farrar, 1985a). DISCUSSION In terrns of sugar content and rate of turnover of vacuolar sucrose, the seminal roots of these plants respond markedly to pruning treatments, whereas the shoots are much less affected (Farrar & Farrar, 1985a). At least some of the responses appear to be confined to the first-order laterals, as it is the elongation of these to which the large changes in growth can be attributed. Nutrient supply (May Chapman & Aspinall, 1965; May et al., 1967; Hackett, 1972; Drew Drew' baker & Ashley, 1973) and root decapitation (Hackett, 1971) as well as selective pruning (Rahman, Aspinall & Paleg, 1975) have previously been shown to affect first-order laterals rather than the seminal axis; ; these effects were partly due to. r^^l{ r^a^l{ ^ u ^^'}^^ ^}^ ' '^^ exti extension rate, of f laterals l l per seminal axis. Crossett et al. (1975) showed a small increase in axis elongation after selective pruning. Jackson (1983) has collated data which show the relative insensitivity of the elongation rate of primary root axes to chemical treatments. Indeed, the cells in the meristern of a fast-elongating root have rates of division and growth as fast as any reported for plant cells (J. F. Farrar, unpublished) and so it is not surprising that response to treatment is seen in first-order laterals, whose meristems are supporting lower rates of root elongation under control conditions. Since the rate ot growth of the surviving seminal on root-pruned plants is greater than that of

7 Respiration and carbohydrates in roots 519 individual seminals on the control, but less than that of the whole root systems of the controls although supplied by the same amount of shoot, it is possible that in this treatment growth of the root is limited by the capacity of the root to grow - a function of the number and potential growth rate of meristems. The differences in growth rate induced by pruning are accompanied by changes in root metabolism. The rate of uptake of exogenously supplied sucrose is greater in controls than in the fast-growing seminal remaining on root-pruned plants, but a higher proportion is not incorporated into insoluble material, nor is the rate of glutamine uptake increased, in this treatment. Conversely, roots with a low growth rate consequent on pruning the shoot show rates of uptake of sucrose and of glutamine considerably greater than those of controls, and a higher proportion of the ^^C in each is incorporated into insoluble material, presumably mainly walls and protein. These changes are consistent with, but do not prove, substrate limitation to root growth in those plants whose shoots were pruned to one leaf blade, and with no such limitation in the controls or in root-pruned plants. Rates of respiration per unit weight of root closely followed rates of growth, being higher than controls in the fast-growing and lower in the slow-growing treatment. This is consistent with the proposition that a proportion of respiration is associated with the processes of growth; relationships between relative growth rate and respiration have been found in a number of species (Farrar, 1985b; Ryle, 1984). In addition to the costs of root growth, respiration will here be providing energy for uptake and transport of ions for the growing shoot. Interestingly, the same proportion (about 35 %) of the carbohydrate entering the root is consumed in respiration; thus pruning seems to have little effect on the efficiency of carbohydrate use in growth. It has been suggested that some root respiration (a considerable proportion in some species) represents, not energy-generation for processes of metabolism and transport, but wasteful overflow of excess carbohydrate ; such ' energy overflow' is equated with cyanide-resistant respiration by the alternative pathway (Lambers, 1982). It is difficult to reconcile these results with the ' energy overflow' hypothesis, since a more efficient use of respiratory substrate would be expected in the slow-growing, substrate-limited, roots of shoot-pruned plants than in the carbohydrate-rich, sink-limited roots of root-pruned plants, but no such difference is found. Nor is the ' energy overflow' hypothesis consistent with the lack of stimulation of barley root respiration by exogenous sucrose (Farrar, 1981). These barley roots show some CN-resistant but SHAM-inhibited respiration (Bingham & Farrar, unpublished) and the relationships between terminal oxidase and pruning treatment will be the subject of a forthcoming paper. Cyanide-resistant (alternative path) respiration may permit a rapid flux of carbon skeletons through the respiratory network (Moore & Rich, 1980; Farrar, 1985b) which may be of importance in tissues, such as growing roots, with high rates of biosynthesis. Respiration rates in these treatments also follow closely soluble sugar content, which is highest in root-pruned plants with high rates of growth. Although it has been argued that the supply of carbohydrate limits the rate of respiration in many plants tissues, the balance of evidence is that carbohydrate content is not usually a direct regulator of respiration rate (Farrar, 1985b); it would thus be unwise to ascribe a causal connection to the correlated in between sugar content and respiration rate, especially since the localization of fast respiration and of sugars may be different, both in terms of tissues (meristems versus mature root), and in terms of subcellular compartmentation. It is clear that respiration and cytosolic (non-vacuolar) sugar content are correlated, but the localization of this

8 52O J. F. F A R R A R A N D C. L. J O N E S sugar relative to that of actively respiring cells is unknown. That fluxes, rather than content, of sugars are altered may be of considerable significance: here we hate shown a reduced flux of sugars across the tonoplast (a longer half-time for sugars in the vacuole) m the slow-growing roots of shoot-pruned plants; since the half-time of cytosolic sugar in barley roots is only ^ 0-5 h (Farrar, 1985a) the vacuole mtv act as a major buffer for supply of sugar to the cytosol, and so changes in flux ac^osl the torioplast could have a major role in control of root carbon fluxes Whilst all experiments involving the pruning of plants are complex in inter mtolt m m ' t'h' '^^'"^^^^^ «- - between shoots and roots that are modified (Herold 1980), these surgically manipulated plants seem to oflfer an excellent system for investigating the control of respiration and growth rates, and futur papers will explore the inhibitor- and substrate-sensitivity of this system further ACKNOWLEDGEMENTS We gratefully acknowledge the flnancial support of the A.F.R.C, and thank Stell farrar for her participation in the washout experiments. REFERENCES R. N., CAMPBELL, D. J. & STEWART H F nq?';^ r,^^., Plant & Soil ^^EWART, H. E. (1975). Compensatory growth m cereal root systems. CROSSETT, B:B;P DuBois, M., GiLLEs, K. A., HAMILTON J K ^ HE»OLD, A.^(, 980). ReguWon of photosynthesis by sink-.ctivi,y - the miss,n, link. Ne. Phy.olo,is.. 86, > * " "

9 Respiration and carbohydrates in roots 521 PITMAN, M. G., MOWAT, J. & NAIR, H. (1971). Interactions of processes for accumulation of salt and sugar in barley plants. Australian Journal of Biological Sciences, 24, RAHMAN, L., ASPINALL,, D. & PALEG, L. G. (1975). Quantitative studies on root development. Ill Further observations on growth in the seedling phase. Australian Journal of Plant Physiology 2 425^34 RYLE, G. J. A. (1984). Respiration and plant growth. In: The Physiology and Bioc'hemistry of Plant Respiration (Ed. by J. M. Palmer), pp Cambridge University Press, Cambridge. ' YEMM, E. W. (1965). Respiration of plants and their organs. In: Plant Physiology IVA (Ed by F C Steward) pp Academic Press, New York. } h

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