The physiological role of old stolon material in white clover (Trifolium repens L.)

Transcription

1 Neiv Phytol. (1992), 122, The physiological role of old stolon material in white clover (Trifolium repens L.) BY D. F. CHAPMAN^ AND M. J. ROBSON^ ^Ag Research, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand ^AFRC Institute of Grassland and Environment Research, Hurley, Maidenhead, Berks SL6 5LR, United Kingdom (Received 28 February 1992; accepted 26 March 1992) SUMMARY Clonal plants of white clover {Trifolium repens L,), consisting of a single stolon plus roots and either three fully unfolded leaves or one fully unfolded leaf at the stolon apex, were grown in a controlled environment and sampled three times (after 7, 49 and 85 d growth) to examine the effects of increasing respiratory demand imposed by an increasing mass of old stolon material upon carbon allocation patterns and plant morphology. Where the total pool of available carbohydrate remained more-or-less constant over time, greater C allocation to stolon tissue was associated with reduced allocation to new leaf growth between d 7 and d 49. Rapid death of old stolon material began about d 70, perhaps as the result of withdrawal of C previously supplied to stolon tissue by leaves at the apex. Current assimilate no longer needed to maintain this old stolon material was, in part, reallocated to the apex region. Stolon death was greater when stolons were covered rather than fully illuminated, reflecting the elimination of direct CO.^ assimilation by stolon tissue (estimated to be 12-22% as efficient as leaves in assimilating COj, on a surface area basis) and accentuation of the carbohydrate deficit. Respiration accounted for an estimated 32 o of C fixed by leaves on d 85 when stolons were covered, compared to 25 % when stolons were fully illuminated. The relevance of these results to the seasonal changes in plant morphology in pastures, and the effects of stolon burial, are discussed. Key words: White clover {Trifolium repens), carbon allocation, stolons, photosynthesis, senescence. INTRODUCTION positions > 8 from the stolon apex) constitutes, ^,,,,., nearer 60 o of the total stolon pool m grazed pastures Stolon tissue comprizes at least half of the total shoot (M. J. M. Hay, personal communication), weight of white clover plants in grazed pastures Old stolon may nevertheless perform some im- (Brock et al., 1988). Pastures may contain several portant functions; some well-established roots orighundred kilograms of stolon per hectare (Hay e^ a/., inate from older nodes, and these are physically 1987), and total harvestable yield of white clover can connected to the active stolon regions (nodes 1-8) by be related to stolon weight per unit area (Lambert et old stolon material. Given the relatively open al., 1986). Much of the stolon material present in vascular anatomy of the clover stolon (Devadas & pastures is quite old and plays no obvious role in Beck, 1972; Thomas, 1987), and evidence for growth. Hay, Newton & Thomas (1991) found that unrestricted translocation of carbohydrate from 50 % of all stolon nodes in a white clover population single sources to multiple sinks throughout clover were located at nodal positions > 8 from the stolon plants (Chapman, Robson & Snaydon, 1991), old apex; most stolon branches first emerge from their stolon may also provide connecting pathways for the enclosing stipule when at nodal positions < 8 transfer of growth resources among plant modules (Chapman, 1983; Hay et al., 1991) and most leaves (such as the main stolon apex and stolon branches, are found at nodes 1-4 in a grazed pasture (Brock ef after Barlow 1989). Thus, old stolon may enable al., 1988). The weight of individual internodes functional integration of spatially separated plant increases with increasing distance from the apex up modules, with possible benefits for whole-plant to about node 7 so that, on a weight basis,' old' stolon growth and survival in heterogeneous environments (hereafter defined as encompassing all nodes at such as grazed pastures (Hartnett & Bazzaz, 1983;

2 54 D. F. Chapman and M. y. Robson Pitelka & Ashmun, 1985; Hutchings & Bradbury, of the medium-large leaved T. repens cv. Blanca RvP 1986; Marshall, 1990). Old stolon also acts as a by transplanting stolon cuttings directly from stock storage organ for reserve carbohydrate (Moran, plants growing in the glasshouse into Perlite in long, Sprague & Sullivan, 1953; Boiler & Nosberger, narrow growth containers (2-8 dm'' volume). Transplants consisted of the stolon tip plus the youngest 1983; Hay et al, 1989). In evaluating the overall role of old stolon material 2-3 internodes, and included one developing leaf and in whole-plant functioning, the costs of maintaining the youngest visible nodal root. Plants received small a large stolon pool must also be considered. For amounts of a full-strength nutrient solution (conexample, there is an energy requirement witbin the taining nitrogen) two days after transplanting. One stolon (Harris, Rbodes & Mee, 1983), presumably week after transplanting, plants were inoculated with met from respiration of current, leaf-derived photo- Rhizobium bacteria; nutrient solutions used theresynthate, or carbohydrate stored within the stolon, after contained all essential macro- and microalthough stolons themselves are able to fix some nutrients except N at the following concentrations: COg, Harris et al (1983) recdrded net CO^ as- Ca 4-2 mm, K 3-7 mm, S 1-1 mm, P 1-2 mm, Mg similation rates of stolons in excess of stolon 1-1 mm, Na 5-1 mm, Fe 0-2 mm, Mn 9-0/^M, Cu respiratory requirements at 130 V^ m ^ PAR, but at 0-3/^M, B 46-0/tM, Zn 0-8/<M. MO 0-4/^M and Co 16 W m"'^ net assimilation and respiration rates were 0-1 fim. Plants were transferred from the glasshouse to a about equal. In any event, self-sufficiency in energy growth room after 51 d, and treatments were imsupply can be largely ruled out because most stolon material in grazed pasture is buried beneath the soil posed 64 d later (115 d after transplanting). surface (Hay, Brock & Fletcher, 1983; Hay et al, Throughout the pre-treatment period, plants were 1987). Since the respiratory cost to the whole plant maintained with a maximum of three fully unfolded of maintaining stolon tissue will depend greatly on leaves per apex (main stolon plus branches) by the amount of stolon present, situations might arise clipping and removing the oldest leaf when a new where the burden imposed upon the pool of available leaf unfolded. This practice restricted leaf numbers assimilate by stolon maintenance affects carbo- per apex to the range commonly found in grazed hydrate supply to other sinks. Consequent changes pastures; three leaves represents the upper limit of in carbohydrate allocation patterns to maintain this range (e.g. Chapman et al, 1984; Chapman, balanced plant growth could profoundly alter plant 1986). Environmental conditions provided by the structure and functioning. The hypotheses under growth room were: 23/18 C day/night temperature, examination here assume that intra-plant compe- 500/imol m"^ s~^ photon flux density, 12 h phototition exists between sinks for resources, and that period and 70 % relative humidity. trade-offs in resource allocation occur among sinks to maintain homeostatic growth in situations where Treatments resource supply for continued maximum growth becomes limiting (Mooney & Chiariello, 1984; Five replicate plants of all combinations of two Bazzaz et al, 1987); these trade-offs usually favour defoliation treatments and two stolon covering the organs which acquire the most strongly limiting treatments were allocated to each of three sample dates. Defoliation treatments were: {a) lenientresource (Thornley, 1972; Bloom, Chapin & maximum of three fully unfolded leaves retained on Mooney, 1985). the main stolon apex, and {b) severe - leaving one The aim of this work was to examine the interplay fully unfolded leaf. Stolon covering treatments, between the amount of stolon tissue supported by simulating stolon burial, were: (a) stolon uncovered clover plants and the photosynthetic capacity - t h e entire length of the main stolon was fully (' source size') of the plant. Changes in the amount of illuminated on the surface of the growing medium; carbohydrate provided by leaves at the main stolon (6) stolon covered - all stolon behind the oldest leaf apex for old stolon tissue were monitored over time was covered by black polythene tubing cut lengthto assess the demand imposed upon a more-or-less ways and held firmly in place by metal pins. These constant pool of current assimilate by an increasingly treatments were maintained by clipping the oldest large mass of stolon. Carbohydrate fluxes into leaf on a stolon when a new leaf unfolded, and adventitious roots were monitored simultaneously, periodically moving the polythene tube forward to and the time course of death of old stolon and roots cover new stolon material. Plants were sampled 7, 49 was established. The effects of a simulated stolon and 85 d after treatments were applied. burial treatment were also examined. One week before treatments were imposed, all existing stolon branches were cut and removed from MATERIALS AND METHODS plants; thereafter, new branches were also excised as Plant culture they emerged from the enclosing stipule. Branch The experiment was conducted at Hurley, southern removal was adopted to simplify plant structure, and England. Plants were cloned from a single genotype to eliminate carbohydrate flow from branches to

3 Role of old stolon material in white clover DayO Day 7 Day 49 Day 85 1:2:3:4 : 10: Figure \. Description of stolon dissection categories (sections numbered 1-10) at three sampling dates (d 7, d 49 and d 85), with reference to the original plant structure (day 0), and to the position of leaves on the apex. sinks on the main stolon and vice-versa (Chapman, Robson & Snaydon, 1992), processes which might have obscured the effects of variation in source size per plant imposed by manipulation of the number of leaves on the main stolon apex. The plants therefore consisted of one major source of carbohydrate (leaves on the main stolon), and three principal sinks - the main stolon apex, stolon tissue of the main stolon, and adventitious roots attached to the main stolon. We expected the intra-plant relationships under examination to emerge more clearly in these simplified plants than in plants where branches were present. The main stolon on some plants was up to 80 cm long by the end of the experiment. To allow unrestricted stolon elongation, additional containers of Perlite were joined to the original (35 cm long) when necessary, providing a continuous surface of moist Perlite. Measurements The area of all leaves removed during the routine clipping treatments was measured, and the laminae were dried and weighed. One day before each designated sample date, the net photosynthetic capacity (Pjj) of the first fully unfolded leaf on each plant was measured in the growth room using a portable gas exchange system (Analytical Development Co., Hoddesdon, Herts, U.K.). On the sample date, all unfolded leaves on each plant were fed "CO,^ by sealing leaves individually in small plastic bags and injecting 10 ml of a standard gas mixture containing 7-25 k Bq C through the wall of the bag using a syringe. Leaves remained sealed for 2 min during ^^CO,^ feeding. Twenty-four hours later, plants were removed intact from the growth container and dissected into the following categories: (1) all unfolded (='''C-fed) leaves, including laminae plus petiole; (2) apex of the 55 main stolon (from the youngest unfolded leaf to the stolon tip, and including all developing ( = folded) leaves); (3) stolon tissue; and (4) all roots. Categories (1) and (2) were analyzed for " C content without further dissection. Stolon material was partitioned for ^*C analysis as shown in Figure 1. Stolon laid down before treatment imposition on day 0 was divided into four sections at all three samplings. Sections 1-3 were of equal length, and comprised all material basal to the oldest unfolded leaf. Section 4 consisted of the two internodes between the oldest and youngest unfolded leaf as they were positioned on day 0. Sections 5 and 6 were of equal length to each other, as were sections 8 and 9. An exception occurred on day 7, when section 5 represented all new stolon material produced since day 0 (about three internodes). Sections 7 and 10 both consisted of two internodes. The root material associated with each section of stolon was also separated for ^^C analysis. Plant material was dried overnight in a 100 C oven, weighed, combusted (Canberra Packard B306 sample oxidizer) and the released ^*C counted in a scintillation counter. The length of dead stolon tissue per plant was measured on days 63, 70, 77 and 84. Stolon tissue was considered dead when it had turned brown and lost all rigidity. Direct CO^ fixation by stolons Five additional replicate plants of each of two treatment combinations (leniently defoliated/stolon uncovered, and severely defoliated/stolon uncovered) were fed 9167 k Bq '""COg for four minutes in a large perspex chamber (0-38 m^), then harvested immediately to compare '*C fixation by stolon tissue with that of the leaves. This was carried out 90 d after treatment imposition, on plants where the main stolon was about 72 cm and 43 cm long for lenient and severe defoliation treatments respectiv ely. Feeding was conducted outdoors in full sunlight on a clear, warm day in April. After '*C feeding, leaves (laminae plus petiole) were removed, and stolon tissue from the oldest fully unfolded leaf (or the equivalent node, for severely defoliated plants) to the base of the plant was divided into five equal-length sections. Leaf and stolon material was dried at 100 C for 24 hours and subsequently analysed for i*c content. The surface area of stolon per plant (cm^) was estimated as 0-5 yr.r./where r = 0-15 cm for leniently defoliated plants and 0-10 cm for severely defoliated plants, and / = stolon length (cm). Data analysis Data were analyzed by ANOVA mostly as a randomized block design of 3 sample dates x 2

4 56 D. F. Chapman and M. J. Robson defoliation treatments x 2 stolon covering treatments, after transformation to logarithms or arcsin values (for percentages) to normalize distributions where necessary. Respiratory losses were estimated for the d 85 sampling only by difference between the amount of " C fed to each plant and the amount remaining in the whole plant 24 h later. This procedure probably overestimated true respiration; leaves were unlikely to have assimilated all the " C fed to them because the CO.^ compensation point of Cg species imposes a limit on net COj fixation. RESULTS Leaf morphology and photosynthesis Leniently defoliated plants produced an average of 32-6 new leaves per plant during the full 85 days of treatment imposition (mean leaf appearance interval = 2-6 d), whereas severely defoliated plants produced only 24-6 new leaves (mean leaf appearance interval = 3-5 d). Covering stolons had no effect on the rate of new leaf production. Chronological changes in leaf morphology are obtained by plotting characteristics of the leaves removed during the routine clipping of plants to maintain defoliation treatments (Fig. 2). Mean leaf area (Fig. 2a) increased nearly 3-fold in the lenient defoliation treatment over the period d 0-d 60, and then decreased over the last quarter of the experiment. Mean leaf area changed relatively little over time in the severe defoliation treatment. Leaves were larger in the lenient defoliation treatment than in the severe treatment ( P < 0-001), and when the stolon was covered compared to fully-illuminated (P < 0-01). Similar chronological trends and defoliation treatment effects were seen for leaf weight (Fig. 26). However, mean leaf weight did not differ between stolon covering treatments and thus the specific leaf area (Fig. 2c) was greater when stolons were covered compared to uncovered (P < 0-001), i.e. leaves were much thinner when the stolon was covered than when the stolon was uncovered. sample date and defoliation treatment for both stolon and root weight (P < 0-001; Fig. 3). Stolon weight increased steadily between d 7 and d 85 in the lenient treatment but, in the severe treatment, stolon mass increased between d 7 and d 49 then decreased between d 49 and d 85. Root weight increased between d 7 and d 49 in the lenient treatment, but did not change between d 49 and d 85. In the severe treatment, however, root mass declined by 63 % between d 49 and d 85. Covering the stolon did not infiuence stolon or root weights, either alone or in combination with defoliation treatment. Stolon length differed substantially between defoliation treatments on d 49 and d 85. On d 49, leniently defoliated stolons were, on average, 462 mm long whereas severely defoliated stolons averaged only 366 mm. Leniently defoliated stolons produced 317 mm of new stolon material between do and d 49 (6-5 mm d"'), versus 214 mm produced on severely defoliated stolons (4-9 mm d"^). Differences between the defoliation treatments were even more marked on d 85 (769 mm versus 289 mm total stolon length, respectively), refiecting continued restriction of new leaf production and internode lengths at the stolon apex in the severe treatment plus accelerated death of older basal stolon compared to the lenient treatment (see below). Covering stolons did not infiuence stolon elongation rate. Death of stolon tissue occurred earlier, and proceeded more rapidly, in severely-defoliated plants than in leniently-defoliated plants (Fig. 4). Initially, at least, severely defoliated plants where the stolon was covered suffered greater losses of stolon material than severely defoliated plants where the stolon was uncovered. '*C allocation patterns Percent export did not differ between defoliation or stolon shading treatments (overall mean = 60%). There was a small difference among sample dates (58-2, 63-1 and 58-7% for d 7, d 49 and d 85 Rates of net photosynthesis were greater after respectively, P < 0-05, LSD = 4-2). Overall, the proportion of exported " C allocated 49 d (9-12//mol CO., m"'s"') and 85 days (9-59 /^mol CO., m"^ s"') than after 7 days (8-48 //mol to the main stolon apex did not differ among sample CO2 m"^ s '; main effect of sample date significant at dates, but allocation to stolon tissue increased, while P<0-01), and in the lenient defoliation treatment allocation to roots decreased, between d 7 and d 49 than in the severe defoliation treatment (9-44 vs (Fig. 5; P < for stolon and root). Since these //mol CO,, m ^' s"^ respectively, P < 0-01). Covering categories constituted all the major plant sinks (there the stolon had no effect on Pj,,, and there were no were no branches), changes in allocation to stolon material must have been accompanied by more-orsignificant interactions between treatments. less equal but opposite changes in allocation to root material, since export to the apex was relatively Stolon and root weights constant. Thus there was a relatively simple tradewhen meaned across treatments, total stolon weight off between stolon and root material in C supply over increased between all sample dates, while total root the course of the experiment. Interactions between sample date and defoliation weight increased between d 7 and d 49, but did not treatment in C allocation to sinks were observed change significantly between d 49 and d 85. However, significant interactions occurred between (P < 0-01, and 0-05 for apex, stolon and root

5 Role of old stolon material in white clover 57 1 o (0 d) eat ai LU).n. ra 53 c olo 1200 Y (a) ^~~7] d I b 1 c a 1H 1 1 / Day 7 Day 49 Day ra E O O CC 800 Y ib) b JB 0- a 3 / 7 I 1 c ^H Day 49 Sample date d /A/ / Day 85 Figure 3. Effect of defoliation treatment on total weight (mg) of stolon tissue {a), and roots {b) at three sampling dates (d 7, d 49 and d 85)., Lenient defoliation; 0, severe defoliation. Means with a common letter are not significantly different. Bar is LSD at P = (0 E _ c _o o ^ 100 a Days Figure 2. Changes with time in {a) area (cm^), {b) weight (mg), and (c) specific area (cm'^ g"^) of leaves clipped continuously from plants sampled on d 85, D D, Lenient defoliation - stolon uncovered; O O, lenient defoliation - stolon covered; A A. severe defoliation-stolon uncovered; V V, severe defoliationstolon covered. Arrows indicate sample dates. T3 CC CU o o JZ *-/ u> c Days Figure 4. Effect of defoliation and stolon covering treatments on rate of stolon death (mm of dead stolon per plant), # #, Lenient defoliation - stolon uncovered;, lenient defoliation - stolon covered; A A> severe defoliation - stolon uncovered;, severe defoliation - stolon covered. Bars are standard errors of means. respectively. Fig. 6). For the apex, severe defoliation resulted in decreased C supply between d 7 and d 49, with no change between d 49 and d 85 (Fig. 6«); no difference between sample dates was seen in the lenient treatment. Carbon allocation to stolon increased between d 7 and d 49 in the severe treatment, but did not differ between d 49 and d 85 (Fig, 66); allocation to stolon increased steadily over the whole experiment in the lenient treatment. Export to roots in the severe treatment decreased between d 7 and d 49, but remained constant between d 49 and d 85 (Fig. 6c) whereas the proportion of C exported to roots in the lenient treatment decreased throughout the experiment.

6 58 D. F. Chapman and M. J. Robson Apex Stolon Roots Figure 5. Overall mean percentage of exported ^*C moving to the apex, and to all stolon and root material, at three sampling dates:, d 7; 0, d 49 and S, d 85. Bars are LSD at P= 0-05, o Q. X 50-< Day 7 Day 49 Day 85 Day 7 Day 49 Day 85 "C export to individual stolon segments and associated root material The distribution of '^C among individual sections of stolon and associated root is shown for lenient and severe defoliation treatments (meaned for + stolon covering) at each sample in Figure 7. Initially (d 7), the proportion of exported "C which moved to stolon tissue decreased progressively in a basipetal direction, irrespective of defoliation treatment. At this early stage, the highest proportion of exported '*C among the root fractions in the lenient treatment was found in the very oldest roots - this was easily the largest of the 5 separated root fractions. In the severe treatment, however, youngest and oldest roots drew most of the ^*C which moved to roots, even though the youngest roots comprized a very small proportion of total root weight. On d 49, a very difterent pattern of distribution was found. Old stolon and root material drew little C, even though there was still a substantial amount of root material near the base and all stolon tissue was still intact and, apparently, alive. A substantial new section of roots was established under lenient defoliation (c. 340 mg spread over about a 28 cm length of stolon behind the oldest leaf) and this received virtually all the ^*C exported to the root fraction. On d 49, there was a clear concentration of exported C within the root and stolon material produced since d 7, and substantial withdrawal of support for the older material. On d 85, however, the ^*C distribution pattern resembled the original pattern (d 7) more than the pattern observed on d 49. There was a wider spread of C along the stolon, among all stolon and root tissue formed in the preceding 78 days. In the lenient treatment, no C whatsoever moved to the oldest root and stolon material even through this material was, superficially, still alive. In the severe treatment, most of this older material had died (Fig. 7) (c) S 1 im//, Day 7 P Day 49uDay 85 Sample date Effect ()f defoliation treatment on the percentage Figure 6. of exported "C moving to {a) the apex, {h) all stolon tissue, and (c) all root material, at three sampling dates:, lenient defoliation; 0, severe defoliation. Bars are interaction LSD at P = 005, 1 Respiration On d 85, greater estimated respiratory losses were recorded for severely defoliated plants than for leniently defoliated plants, and for plants where the stolon was covered compared to those where the stolon was fully illuminated (Table 1), There was no interaction between defoliation and stolon covering treatments. Direct C fixation by stolons Plants in the lenient defoliation treatment had an estimated surface area of stolon equal to about half the measured area of all three leaves, whereas severely defoliated plants had similar surface area of

7 Role of old stolon material in white clover 59 mg mg (%) mg ' ' 40^-' 35'-^ 148 Day mg 200 Figure 7 Percentage of exported ''C moving to individual sections of stolon ( ) and associated roots (0) at three sample dates (d 7, d 49 and d 85) in two defoliation treatments [lenient (= L) and severe (= S)]. Root weights (D in mg) and the length of stolon (values, in mm), appropriate to each section of stolon are also indicated, as is the position of leaves at the apex. Broken lmes at d 85 indicate sections of stolon partially dead^ The three oldest stolon sections in the severe defoliation treatment died and decayed completely between d 49 and d 85. stolon and leaf (Table 2). Stolon material assimilated 6-2 % and 22-9 % as mucb "CO^ as leaves for the respective treatments. Using P^ data for d 85, and allowing for tbe differences between stolon surface area and leaf area, tbe net pbotosyntbetic capacity of stolon tissue is estimated at 1-18/imol CO2 m ^s for tbe leniently defoliated plants and 2-00 fimo\ CO, m"^ s"* for tbe severely defoliated plants at 500 //mol m PAR and 23 C air temperature (= 11-8% and 21-9% of P^ rate of leaves, respec-

8 60 D. F. Chapman and M. J. Robson Table 1. Effects of defoliation and stolon covering treatments on the estimated percentage of ^*C fixed by leaves which was respired over 24 h {d 85 sampling) Treatment Defoliation Stolon covering Lenient Severe Uncovered Covered Respiration (%) Significance P = P < 0-05 Table 2. Mean {±standard errors of mean, n = 5) stolon length, stolon surface area, leaf area and assimilation by stolon and leaves during ^^CO^ feeding of whole plants Defoliation treatment Total stolon length (cm) Estimated total stolon surface area (cm^) Total leaf area (cm-^) Total '"C fixed by stolon (k Bq) (A) Total '^C fixed by leaves (k Bq) (B) Stolon fixation as a percentage leaf fixation (A/B)x 100 Lenient Severe 71-6-f ± tively). Clearly, the contribution of stolons to whole-plant CO^ assimilation relative to the contribution of leaves was much greater in the severe defoliation treatment than in the lenient treatment. Younger sections of stolon (nearest the stolon apex) assitnilated more C than older sections of equivalent length. DISCUSSION By maintaining a constant number of leaves per plant, it was anticipated that the pool of carbohydrate available for the three main sinks (apex, stolon and roots) to utilize would also remain more-or-less constant over time. However, this was not the case in the lenient defoliation treatment because the area of individual leaves increased greatly over the first 60 days (Fig. 2 a), resulting in a large increase in total source size per plant. Initially, this increase in leaf area was probably a consequence of re-allocation of carbon previously translocated to branches among the remaining sinks. The main stolon apex region of white clover plants has a high carbohydrate demand associated with cell division and expansion (Chapman et al, 1991), and could be expected to attract excess C more strongly than less-active sinks, such as non-elongating stolon material. The massive size which leaves reached in this treatment (up to 15 cm^) and the high rate of leaf appearance (every 2-6 d) shows that there were few restrictions on leaf growth and also, presumably, the growth and maintenance of stolon and root material. Under severe defoliation, however, leaf area did remain relatively constant, at least after about d 15 (Fig. 2a). Since Pf^ and % export of carbon also changed little during the experiment, this treatment allowed an evaluation of the effects of an increasing mass of stolon (and root) material on the carbon economy and morphology of plants utilizing a constant-size pool of available carbohydrate. Our aim was to search for a point in the growth of the plant when the respiratory burden imposed by an increasing mass of old stolon triggered changes in carbon allocation patterns within the plant to maintain homeostatic growth which, in turn, result in the death of material which no longer received C from leaves. There was strong circumstantial evidence for the existence of such a critical point in the growth of the severely defoliated plants, and the ensuing changes in plant morphology are relevant to the explanation of morphological changes occurring in clover populations in pastures. The evidence suggests that this critical point occurred around d 55 d 60, Certainly, death of old stolon material increased rapidly after d 63, especially in those plants where the stolon was covered (Fig. 4). Other data also support this argument. Firstly, stolon mass (Fig, 3 a) and carbon allocation to stolon (Fig. 6 b) both increased between d 7 and d 49, showing that stolon material represented an increasing burden upon C supply, but this burden decreased between d 49 and d 85, due presutnably to stolon death. Secondly, the marked increase in carbon allocation to stolon between d7 and d 49 (from 22% to 38%) was associated with decreased export to both the apex and roots (Fig. 6 a, c). Given that the pool of available carbon was about the same size on d 49 as on d 7, these changes in proportionate supply would have

9 Role of old stolon material in white clover 61 been paralleled by changes in the quantity of C Brock et al., 1988), when clover is under competitive moving to sinks. Thirdly, on d 49, about 10% of the stress from faster-growing companion grasses like total carbon exported from the single unfolded leaf perennial ryegrass, and is also being grazed on the stolon apex moved to the very oldest stolon frequently (Clark et al, 1984). and root fractions which subsequently died from d 63 on (Fig. 7). This carbon was therefore available for re-allocation to other sinks, and may have been ACKNOWLEDGEMENTS sufficient to restore C supply to the apex and The authors are grateful to Helena Tompkins and staff of therefore maintain steady-state leaf growth. the controlled environment facility. Hurley, for technical It is difficult to separate the importance of old assistance, and to the New Zealand Department of stolon and old root material in this chain of events. Scientific and Industrial Research for the Study Award Death of both components often occurs con- granted to D, F,C, currently, but it is not possible to determine cause and effect in stolon and root death. Here, stolon death was visible and measurable while root death was not, so interpretation is biased toward stolon REFERENCES death at the key process. However, during the first BARLOW, P, W, (1989), Meristems, metamers and modules and the development of shoot and root systems. Botanical Journal of half of the experiment (d 7-d 49), it was stolon mass the Linnean Society 100, , (Fig. 3 a) and carbon allocation to stolon (Fig. 66) BAZZAZ, F. A., CHIARIELLO, N, R,, COLEV, P. D. & PITELKA, L. F. which were increasing in severely defoliated plants, (1987). Allocating resources to reproduction and defense, BioScience 37, not root mass (Fig. 3 b) and C allocation to roots (Fig..A.. J,, CHAPIN, F, S. & MOONEY, H..\. (1985). Resource 6c) which were actually decreasing. Therefore, it BLOOM, limitation in plants - an economic analogy. Annual Review of seems that demands of the stolon component were Ecology and Systematics 16, generating pressure on the utilization of available BoLLER, B, C. & NosBEHGER, J. (1983), Effects of temperature and photoperiod on stolon characteristics, dry matter partitioning carbon and driving the changes in C allocation and and non-structural carbohydrate concentration of two white plant morphology which occurred in the second half clover ecotypes. Crop Science 23, BROCK, J. L., HAY, M. J. M., THOMAS, V. J. & SEDCOLE, J. R. of the experiment. (1988). Morphology of white clover {Trifolium repens L.) plants Superimposed within the sequence of events in pastures under intensive sheep grazing. Journal of Agricultural Science {Cambridge) 111, described above were several effects of covering the stolon. The fact that covering the stolon influenced CHAPMAN, D. F. (1983). Growth and demography of Trifolium repens stolons in grazed hill pastures, ^ourwa/ of Applied Ecology the specific area of leaves (Fig. 2 c) provides a strong 20, indication that this treatment did influence the CHAPMAN, D. F. (1986). Development, removal and death of white clover leaves under 3 grazing managements in hill carbon economy of plants, since specific leaf area is country. New Zealand Journal of Agricultural Research 29, extremely sensitive to alterations in the source-sink 39^7. ratio of clover plants (Chapman, Robson & Snaydon, CHAPMAN, D, F,, CLARK, D. A., LAND, C.. ^. & DYMOCK, N. (1984). Leaf and tiller or stolon death of Lolium perenne, 1990). When fully illuminated, the stolon was clearly Agrostis spp. and Trifolium repens in set-stocked and rotationally capable offixingbiologically-significant quantities of grazed hill pastures. New Zealand Journal of Agricultural carbon (Table 2). Elimination of this source of Research 27, carbon could therefore have hastened the onset of C CHAPMAN, D. F,, ROBSON, M. J. & SNAYDON, R. W, (1990). Short-term effects of manipulating the source: sink ratio of stress and stolon death, especially in the severelywhite clover {Trifolium repens) plants on export of carbon from, defoliated plants where C assimilated by the stolon and morphology of, developing leaves. Physiologia Plantarum 80, represented a larger fraction of total (i.e. leaf plus CHAPMAN, D. F,, ROBSON, M. J. & SNAYDON, R. W. (1991). stolon) assimilation than in leniently-defoliated Quantitative carbon distribution in clonal plants of white clover plants. The stolon death rate data in Figure 4 {Trifolium repens)-. source-sink relationships during undisturbed gro-wth. Journal of Agricultural Science {Cambridge) 116, support this (compare severely defoliated/uncovered and severely defoliated/covered combinations). The CHAPMAN, D. F., ROBSON, M. J. & SNAYDON, R. W, (1992). respiration data collected on d 85 (Table 1) show- Physiological integration in the clonal, perennial herb Trifolium that covering stolons placed a significantly higher repens L. Oecoiogia 89, CLARK, D. A., CHAPMAN, D. F"., LAND, C. A, & DYMOCK, N. respiratory burden upon carbon fixed by leaves. (1984). Defoliation of Lolium perenne and Agrostis spp. tillers Burial of stolon material in the field (Hay, 1983 ; Hay and Trifolium repens stolons in set-stocked and rotationally et al. 1983, 1987) could therefore be expected to grazed hill pastures. New Zealand Journal of Agricultural Research 27, increase the dependence of stolon tissue on carbon DEVADAS, C. & BECK, C. B. (1972). Comparative morphology of supply from leaves to meet respiratory requirements. the primary vascular systems in some species of Rosaceae and When demand for assimilate from other sinks is Leguminosae. American Journal of Botany 59, high, and source size per plant is restricted (e.g, by HARRIS, W., RHODES, I. & MEE, S. S. (1983). Observations on environmental and genotypic influences on the overwintering of high grazing pressure), accelerated stolon death white clover. Journal of Applied Ecology 20, might result. Such an interpretation fits the observed HARTNETT, D. C. & BAZZAZ, F. A. (1983). Physiological integration among intraclonal ramets in Solidago canadensis. sharp increase in stolon death and plant fragmenecology 64, tation in pastures in spring (Hay et al., 1983; HAY, M. J. M. (1983). Seasonal variation in the distribution of

10 62 D. F. Chapman and M. y. Robson white clover (Trifotium repens L,) stolons among 3 horizontal strata in 2 grazed swards, Netv Zealand Journal of Agricultural Research 26, 29-34, HAY, M, J, M,, BROCK, J, L, & FLETCHER, R, H, (1983), Effect of sheep grazing management on distribution of white clover stolons among 3 horizontal strata in ryegrass/white clover swards. New Zealand Journal of Experimental Agriculture 11, , HAY, M, J, M,, CHAPMAN, D, F,, HAY, R, J, M,, PENNELL, C, G, L,, WOODS, P, W, & FLETCHER, R, H, (1987), Seasonal variation in the vertical distribution of white clover in grazed swards. New Zealand Journal of Agricultural Research 30, 1-8, HAY, M, J, M,, CHU, A, C, P., KNIGHTON, M, V. & WEWALA, S, (1989), Variation with season and node position in carbohydrate content of white clover stolons. Proceedings of the XVI International Grassland Congress, , HAY, M, J, M,, NEWTON, P, C, D, & THOMAS, V, J, (1991), Nodal structure and branching of Trifolium repens in pastures under intensive grazing by sheep. Journal of Agricultural Science (Cambridge) 116, , HUTCHINGS, M, J, & BRADBURY, I, K, (1986), Ecological perspectives on clonal perennial herbs, BioScience 36, , LAMBERT, M, G,, CLARK, D, A,, GRANT, D, A, & COSTALL, D, A, (1986), Influence of fertiliser and grazing management on North Island moist hill country, 3, Performance of introduced and resident legumes. New Zealand Journal of Agricultural Research 29, 11-21, MARSHALL, C, (1990), Source-sink relations of interconnected ramets. In: Clonal Growth in Plants: Regulation and Function (Ed, by J, van Groenendael & H, de Kroon), pp, 23^1, SPB Academic Publishing, The Hague, MOONEY, H, A, & CHIARIELLO, N, R. (1984), The study of plant function-the plant as a balanced system. In: Perspectives on Plant Population Ecology (Ed, by R, Dirzo & J, Sarukhan), pp, , Sinaner Associates Inc, Sunderland, Massachusetts, MORAN, C, H,, SPRAGUE, V, G, & SULLIVAN, J, T, (1953), Changes in the carbohydrate reserves of Ladino white clover following defoliation. Plant Physiology 28, Abl^lA. PITELKA, L, F, & ASHMUN, J, W, (1985), Physiology and integration of ramets in clonal plants. In: Population Biology and Evolution of Clonal Organisms (Ed, by J, B, C, Jackson, L, W, Buss & R, E, Cook), pp, , Yale University Press, New Haven, THOMAS, R, G, (1987), The structure of the mature plant. In: White Clover (Ed, by M, J, Baker & W, M, Williams), pp, 1-29, CAB International, Wallingford, THORNLEY, J, H, (1972), A balanced quantitative model for rootshoot ratios in vegetative plants. Annals of Botany 36, ,

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