Regulation of Assimilate Partitioning in Soybean: Initial Effects ...

Plant Physiol. (1987) 83, 341-348 0032-0889/87/83/0341/08/$01.00/0

Regulation of Assimilate Partitioning in Soybean1 INITIAL EFFECTS FOLLOWING CHANGE IN NITRATE SUPPLY Received for publication May 14, 1986 and in revised form September 26, 1986

J. KEVIN VESSEY* AND DAVID B. LAYZELL Department ofBiology, Queen's University, Kingston, Ontario, Canada, K7L 3N6 ABSTRACT Increased concentrations of nitrate in a nutrient solution (2, 5, and 10 millimolar KNO3) were correlated with increased shoot:root ratios of non-nodulated soybeans (Glycine max [L.] Meff.) grown in sand culture. While altering the pattern of C and N partitioning, the N treatments did not affect whole plant photosynthesis over the study period. To determine the mechanism responsible for the observed changes in assimilate partitioning, detailed C and N budgets were worked out with plants from each N treatment over three consecutive 4-day periods of midvegetative growth. The information for the C and N budgets from the 2 and 10 millimolar N03- treatments was combined with data on the composition of xylem and phloem exudates to construct a series of models of C and N transport and partitioning. These models were used to outine a 'chainreaction' of cause-and-effect relationships that may account for the observed changes in assimilate partitioning in these plants. The proposed mechanism identifies two features which may be important in regulating the partitioning of N and other nutrients within the whole plant. (a) The concentration of N in the phloem is highly correlated with the N concentration in the xylem. (b) The amount of N which cycles through the root-from phloem imported from the shoot to xylem exported by the root-is regulated by the root's requirement for N: only that N in excess of the root's N requirements is returned to the shoot in the xylem. Therefore, roots seem to have the highest priority for N in times of N stress.

The interdependence of photosynthate supply from shoots and mineral N acquisition by roots has been recognized as an important relationship in determining the rate and pattern of whole plant growth in both empirical studies (23) and in theoretical models (5). In general, a decrease in the availability of combined N to roots decreases the shoot to root ratio and the overall rate of plant growth (25). However, the mechanism responsible for altering the pattern of C partitioning in response to decreased concentrations of combined N has not been clearly identified. Brouwer (4) suggested that under N deficiency enhanced root growth relative to shoot growth was the result of a greater proportion of absorbed N being retained by roots and never being translocated by the xylem to the shoots. Alternatively, other researchers have suggested that the decrease in shoot to root ratio due to suboptimal concentrations of combined N was due to the redistribution of shoot N to the roots as mediated by the effects of growth regulators (25). In particular, the decrease in root cytokinin synthesis and shoot cytokinin levels in response

to N stress has been associated with this hypothesis (26). The present study describes the effects of changes in the nitrate supply on C and N transport and partitioning over a 12 d period of growth in non-nodulated soybeans. Budgets of C and N partitioning were combined with information of xylem and phloem sap composition to construct models of C and N transport similar to those described previously (15, 21, 22). Finally, these models have been used to develop a hypothesis to explain the mechanism through which a decrease in nitrate supply alters photosynthate partitioning and plant growth in soybean before N becomes limiting to C fixation.

MATERIALS AND METHODS Plant Culture. Soybean seeds (Glycine max [L.] Merr. cv Harosoy 63) of a uniform size were surface sterilized in 0.3% (v/ v) NaClO for 5 min and planted (1 seed per pot) in sterilized silica sand in 700 cm3 plastic pots which could be sealed for in situ root and shoot gas exchange analysis. In comparison with growth of soybeans in larger containers, these pots did not restrict root growth over the 33 d study period. The plants were grown in a controlled environment chamber (model PGW36, Controlled Enviroment Ltd. Winnipeg, Manitoba) with 14 h light/ 10 h dark and with day/night temperatures of 25°C/19°C. Light irradiance during the light period was 450 (±50) Amolm-2.s-' from a combination of cool white fluorescent and incandescent lamps. Plants were rotated around the chamber every 2nd d to ensure even plant growth within the population. Concentration of CO2 was kept at near-ambient levels (350 ± 40 Ml L-') by flushing the growth chambers with temperature controlled outside air. The minimum RH was 70% (higher following watering). Plants were watered twice daily (0800 and 1600 h) with 250 ml (1.5 times the holding capacity of the pots) of a nutrient solution (pH 6.5) containing 2 mm KNO3, 0.5 mM K2SO4, 0.6 mM K2HPO4, 0.25 mM KH2PO4, 0.25 mM MgSO4, 0.25 mM MgCl2, 0.75 mm CaCl2, 37 AM Fe (II) as Fe-sequestrene 330 (CIBA-Geigy Canada Ltd., Mississauga, Ont.) 2.25 AM H3BO3, 1.0 liM MnCl2, 0.35 ,AM ZnSO4, 0.4 AM CuSO4, 0.1 Mm CoSO4, and 0.3 AM Na2MoO4 for the first 7 DAP.2 From 8 to 21 DAP, this solution was supplemented to 5 mm KNO3. From 21 to 33 DAP plants were divided into three populations (about 50 plants per population) which received either the basal nutrient solution or the basal nutrient solution supplemented to 5 or 10 mM KNO3. During this 12 d period an equal concentration of K+ was maintained in the solutions by addition of the required concentration of K2SO4. The 10 mM NO3- treatment was considered to be the optimal for plant growth. Plants fed concentrations of 20 mM NO3- showed signs of stress following 1 week of treatment (data not shown). Gas Exchange Analyses. Over the period 19 to 33 DAP, CO2

' Supported by an National Sciences and Engineering Research Council (Canada) Operating Grant to D. B. L. and an NSERC Pc stgraduate 2Abbreviations used: DAP, days after planting; Lv, leaves; SP, stems + petioles; Rt, roots; SHAM, salicylhydroxamic acid. Scholarship to J. K. V. 341

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Plant Physiol. Vol. 83, 1987 evolution was monitored continuously from the roots (day and construct empirically-based models of N transport between root night) and from the shoots (night only) in plants connected to a and shoot of plants exposed to each N treatment (15, 21, 22). 12 channel open gas exchange system as described previously Models were constructed for the 2 and 10 mm plants over the (30). At any one time, root respiration was monitored from two periods 26 to 29 DAP and 30 to 33 DAP. In constructing these plants from each of the three populations and, at night, dark models it was assumed (a) that the xylem and phloem streams respiration of shoots was determined from one plant from each are the only conduits of long distance transport, and solutes population. Individual plants were on the gas exchange system moving through these streams move with water by mass flow for a maximum of 2 d before being replaced with fresh plants (17), (b) that the ratio of C:N in the xylem and phloem exudates selected randomly from the population. Specific rates of gas are representative of that moving in the xylem and phloem of exchange were calculated for each plant using dry weight values intact plants (12), and (c) that N supply to the roots in the estimated from the dry weight of the plant at harvest and the phloem which was in excess of that required by the roots was relative growth rate of the population. These specific rates were recycled to the shoots in the xylem. Statistics. Analysis of variance and least significant difference applied to the population's mean dry weight of the organ group to obtain a measure of the tissue respiration rate of the average tests were used to compare the mean dry weight of plants from the three treatments. All other data were compared using the plant within the population. Plant Harvesting. Approximately 13 plants were harvested Student t test. All tests were carried out at the 95% confidence from each population during the morning of the 21, 25, 29, and level, and results are expressed as means ± SE. 33 DAP. The harvested plants did not receive nutrient solution on the mornings they were harvested. Plants were divided into RESULTS leaves, stems + petioles, and roots (roots were rinsed with deionPlant Growth and C Partitioning. At the end of the 12 d ized H20 after being removed from the pots), and these plant fractions were dried and weighed. The leaf area of each plant was experimental period, no significant differences were observed in measured using a Zeiss MOP-3 digitizer. The plant parts of five whole plant dry weight between treatments (Fig. IA). However, plants from each population were finely ground, and the C and plants which were fed 5 and 10 mM NO3- displayed shoot to N content of the tissues measured with a CHN elemental analyzer root ratios which were higher than similar ratios in plants fed 2 (Leco Corporation, model 600). Aqueous extracts of the dried mM NO3-, and the magnitude of the differences increased over plant material were analyzed for tissue NO3-N content using a the 12 d study period (Fig. 2). The higher shoot:root ratios in the N03 specific ion electrode and a millivolt meter (models 93-07 plants fed 5 and 10 mM NO3-, compared with those fed 2 mM and 81 1, respectively, Orion Research). The difference between NO3-, resulted from higher rates of dry matter accumulation in total N and N03-N was used as an estimate of the reduced N the shoots and, more significantly, to lower rates of root growth in these plants (Fig. 1 A). At 33 DAP, the mean leaf number and content of each plant part. mean leaf area per plant were slightly lower (but not significantly) measplants was intact potted loss from Water Transpiration. in the plants fed 2 mm NO3- than in those fed 5 or 10 mM NO3ured gravimetrically twice a day for each 24 h period of the not shown). (data study. Transpiration during the light period accounted for more The C content of dry matter ranged from 33 to 41%, and than 90% of the water loss by the plants over each 24 h d. within each organ type, no significant differences were observed Xylem and Phloem Sap Collection and Analysis. Xylem sap among treatments or harvest dates. Therefore, the pattern obexudates were collected from root stumps of all plants on harvest served for accumulation with time (Fig. 1B) reflected that for days as previously described (15). Collections were made during dry matterCaccumulation (Fig. 1A). C lost in dark respiration the morning and collection times were restricted to less than 30 from shoots at night showed no consistent differences among min. No differences were observed between N treatments in the rate of xylem exudation. The xylem sap of 3 to 4 plants was I pooled and analyzed for ureides according to the method of Vogels and Van der Drift (see Layzell and LaRue [ 15]); for NO3 z by the Cd-Cu reduction technique (28); and for amino acids by HPLC (8). EDTA-phloem sap exudates were collected from the severed 0 end of entire intact shoots of plants from the 2 and 10 mm treatment over a 2 h period in midmorning at 27 and 33 DAP (15). The exudates were analyzed for total sugars by a modified 2mM lOmM 5 50mM anthrone method (9) and for amino-N by a modified ninhydrin z method (12). These results were combined with a previous, more /Lv-_ /Lv-/Lv complete study of phloem sap composition in soybean (15) to I~ estimate the C:N ratio of the solutes within the exudates. Since NL-J() tI RiItI previous studies with legumes (19) have shown there to be little OL] L)a,-u day/night variation in the C:N ratio of phloem sap, phloem n w1 C NITROGEN U.3 0 mM 2mM 5mM exudates that were collected during the light period were assumed to be representative of the C:N ratio of the sap over the entire Z 0.2 Lv 24 h day. Since 90% of whole plant transpiration, and therefore I Lv xylem transport, was found to occur during the light period (see 0N 0.1 above), xylem exudates during the light period were considered zCY$ 0^-. to be representative of that moving through the xylem over the 29 33 21 29 33 25 29 33 21 25 21 25 entire 24 h day. Previous studies (DB Layzell, TA LaRue, unDAYS AFTER SOWING published data; 19) have found no diurnal variation in the C:N ratio of xylem exudates. FIG. 1. Cumulative plot of dry weights (A), carbon (B), and nitrogen Model of N Transport. The data on C, N, and NO3- accu- (C) of leaves (Lv), stems + petioles (SP, shaded), and roots (Rt) for mulation in plant parts and C lost in respiration were combined soybeans grown on three levels of N03- over a 12 d period. Bars represent with information on the xylem and phloem sap compositions to ±2 SE. -

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REGULATION OF ASSIMILATE PARTITIONING IN SOYBEANS 2.7 3: o 2.5

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Table I. Shoot and Root Specific Respiration Rates Respiratory losses of CO2 from shoots (night only) and roots (day and night) of soybean plants fed 2, 5, or 10 mm NO3- over three consecutive 4 d periods of vegetative growth. Each value represents the mean ± SE. N03- Concentration Growth Period 2 mM 5 mM 10 mM Shoot Night Respiration

Mgmol C02/g dry wt- h 57 ± 11

58±2 46±2

68 ± 5 73±2 51 ±4

76 ± 8 64±2 42±6

Root Respiration

gumol C02/g dry wt- h 22-25d 26-29 d 30-33d

SP

sp

RtR

RtR

RtR

313±6 314 ± 14 240± 10

335±6 313 ± 15 260±7

361 ±7 334 ± 9 300±4

treatments. However, respiration from roots during the day and night increased with increasing NO3- nutrition such that in the final period (30-33 d inclusive), roots of the plants fed 10 mm NO3- displayed specific respiratory activities of 20% higher than those in plants fed 2 mm NO3- (Table I). The rates for C accumulation in dry matter were added to measurements of C lost in dark respiration of roots (day and night) and shoots (night only) to obtain a measure of the net photosynthetic rate and the C partitioning pattern of the plants from each treatment over the entire 12 d study period (Fig. 3A).

While more C was fixed in the plants fed 10 mM NO3-, the differences were not significant at the 95% confidence level and no differences were found among the specific net photosynthetic rates on either a leaf area or a dry weight basis (data not shown). There was a slight decrease in the proportion, but not in the amount of C partitioned to roots with increasing NO3- concentrations, and C partitioning to root dry matter declined and root respiration increased with increasing concentrations of NO3-. N Partitioning. Over the 12 d study period, the total N increment in the root, stems + petiole, and leaf tissues was significantly higher (P > 0.05) than that in similar organ groups in plants which received higher levels of NO3- (Figs. lC and 3B). With increasing NO3- supply, the proportion of N accumulated by root tissue decreased from 35% of the whole plant N increment at 2 mm NO3-, to 27% of the N increment in plants fed 10 mm NO3- (Fig. 3B). The total N content of the tissues was divided into two components: a NO3- storage pool (Fig. 4B) and a reduced N component (Fig. 4C). In root tissues, the N03 -N component accounted for up to 50% of total N content of the tissues but only a maximum of 7 and 14% of the total N content of the leaf and stem tissues, respectively (Fig. 4B). Expressed per g dry weight, the N03 -N content of the tissues decreased with time in

Rt

Rt

0BNITROGEN 0..2-T

33

DAYS AFTER PLANTING FIG. 2. Shoot to root dry weight ratios for soybeans grown on to 2 (A), 5 (U), or 10 (0) mM NO3- over a 12 d period. Bars represent ±2 SE.

22-25 d 26-29d 30-33d

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TREATMENT (mM NO-) FIG. 3. Carbon expenditure (A) in respiration in shoots (StR) and roots (RtR) and in dry weight in leaves (Lv), stems + petioles (SP), and roots (Rt) and nitrogen accumulation (B) in leaves (Lv), stems and petioles (SP) and roots (Rt) for soybeans grown on three levels of N03over a 12 d period. Bars represent two times the combined SE of all components.

the 2 and 5 mm NO3- fed plants, but remained high in the plants fed 10 mm NO3- (Fig. 4B). Calculation of the reduced N content of the tissues revealed declining levels with time in leaves at 2 mM NO3- and stems + petioles at 2 and 5 mm NO3-, but levels of reduced N in root tissues were not affected by the N03treatments (Fig. 4C). Xylem Sap Composition. The concentation of N-containing compounds in the xylem sap exudate decreased with time in the 2 mm NO3- fed plants, was constant in the 5 mM NO3- fed plants and increased in the 10 mm NO3- fed plants (Fig. 5). Asparagine represented the major N-containing compound in the xylem sap. Nitrate accounted for only 6 to 26% of the N composition of the xylem exudate. Since the concentration of amino acids in the xylem exudates were similar to those described previously (15), estimates of organic C content were used from this earlier study to obtain an estimate of the xylem sap C:N ratio (Table II, item

4e). C:N Ratio of Phloem Sap. In the 10 mM NO3- fed plants the C:N weight ratio ofthe phloem EDTA exudates feeding the roots were only 10:1 and 1:1 compared with those from the 2 mM NO3- fed plants which were from 19:1 to 27:1 (Table II, item 5a). While generally lower than the C:N ratios which have been reported for lupin (21,22), the values obtained here are similar to those reported for phloem sap exudate from petiole and fruit tips in soybean ( 15). Transpiration Rates. Mean transpiration rates for the 2 and 10 mM NO3- treated populations over 4d periods are given in Table II, item 3. The whole plant transpiration rates were not different between treatments except over the last 4 d when the 2 mM NO3- fed plants transpired less than the 5 or 10 mm N03fed plants. Expressed per dm2, rates of transpiration were similar in all treatments (data not shown). DISCUSSION C Assimilation and Partitioning. Although small differences among the NO3- treatments were observed in whole plant net photosynthesis (Fig. 3A), no differences were found in the specific net photosynthetic rate (dry weight or leaf area basis). Reductions in leaf area development at low NO3- concentrations have previously been reported (25), but decreases in specific photosynthetic rates are generally associated with severe limitations of

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FIG. 4. Total-N (A), nitrate-N (B), and reduced-N (C) accumulation for leaves (Lv), stems + petioles (SP), and roots (Rt) for soybeans grown on 2 (A), 5 (U), or 10 (0) mm NO3- over a 12 d period. Inserts in (B) magnifies the ordinate axis. Bars represent ±2 SE.

z

DAYS AFTER PLANTING (3) in the root of the 10 mM N03- compared to the 2 and 5 mM NO3- fed plants. Estimates of these costs will be presented and discussed later. An attempt was made to determine whether greater activity of inefficient alternative chain respiration in the high NO3- treatments could account for the greater rates of respiration (14). However, no alternative chain activity (SHAM sensitive respi-

other

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N0

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25

29

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DAYS AFTER PLANTING

FIG. acids,

5. Cumulative concentrations of N in asparagine, other amino

N03--N,

and ureides in xylem sap for soybeans grown on three

levels of NO3- over a 12 d period.

NO3- supply (16). Even though leaf N concentrations in the 2 and 5

mMv NO3- fed plants decreased to 58 and 89%, respectively, mrM NO3- fed

of the N concentration in the shoots of the 10

plants, no effect of N treatment was observed in the specific net photosynthetic rate. This result indicated that these plants did not undergo a severe N stress during the study period (11).

Presumably with time the N concentration in the leaves of the plants fed 2 or 5

mMs

NO3- would decrease to a point at which

the rate of specific net photosynthesis would be limited. Although the plants in the study did not undergo severe N stress, highly significant differences were observed in the pattern

of photosynthate partitioning to root and shoot. After 12 d of

N03- treatment, the shoot:root ratio in the 2 mM NO3- treatment declined to a value of 1.8 while in the plants provided with 5

and 10

mMi NO3-,

the ratio increased to 2.2 and 2.6, respectively

(Fig. 2). The lower shoot:root ratio in the plants fed 2 mM

N03-

than in those fed 5 or 10 mM NO3- was attributed to (a) the ability of roots to attract a slightly greater proportion of whole plant photosynthate (Fig. 3A), and (b) a lower specific activity of root respiration (Fig. 3A, Table I). The latter factor was attributed to the increased energetic cost associated with

N03-

reduction

grown

soybean although alternative chain capacity (CN resistant respiration) was present in these tissues (data not shown). N Uptake and Partitioning. At the start of the treatment period the NO3- pool accounted for less than 14% of the N of shoot tissues, but 50% of the total N in the root. Over the time course of the study, the reduced N content declined with time in the stems + petioles of the 2 and 5 mM treatments, and in the leaves of the 2 mM treatment. We concluded that the supply rate of NO3- in these treatments was inadequate to maintain maximal NO3- uptake rates. Since no differences between treatments were observed in the reduced N content of the root tissues, an order of priority (lowest to highest) for N supply to plant organs may be proposed as follows: stem and petioles, leaves, and then roots. That roots were least affected by limitations in NO3- supply may also be an important factor in determining the effect of N supply on the shoot:root ratio in these plants. This will be discussed in more detail later. Models of C and N Transport. Empirically based models of C and N transport were constructed for the plants fed 2 and 10 mM NO3- over the growth periods 26 to 29 and 30 to 33 DAP (Fig. 6; Table II). Since the N increment in the plants fed 10 mm NO3- was 3.1 to 3.3 times greater than that in the 2 mM N03 fed plants, it is not surprising that the models indicated that these plants transported in the xylem and phloem more N than did the 2 mm NO3- fed plants. For example, in the 10 mm plants, N transport to the shoot in the xylem was 2.5 and 3.7 times greater than that in the 2 mm plants for the growth periods 26 to 29 and 30 to 33 DAP, respectively (Table II, item 6b). Similarly, N transport from the shoot to the root in the phloem was, in the plants fed 10 mM NO3-, 2.0 and 3.0 times greater over the 26 to 28 and 30 to 33 DAP periods, respectively, than that in the plants fed 2 mm NO3- (Table II, items 7a, b). Despite the greater flow of N to the roots in the phloem of the 10 mm plants, C transport was, at most, only 23% greater than that in the 2 mm treatment (Table II, item 6a) and the model indicated that most

REGULATION OF ASSIMILATE PARTITIONING IN SOYBEANS

345

Table II. Components of C and N Transport Models Aspects of carbon and nitrogen utilization by plant parts, transport fluid composition, and modeled N flux and NO3- reduction for 2 and 10 mm NO3- fed plants over two time periods. 10 mM 2 mM Item 30-33 d 26-29 d 30-33 d 26-29 d 1. N Increment (mg N/plant) 71 ±4 86 + 4 26 ± 1 23 ± 1 (a) Whole plant 13±2 10± 1 15 ± 1 9.9 ± 0.5 (b) Root (reduced-N) 14± 1 5.9 ± 0.5 -0.4 ± 0.1 -2.8 ± 0.3 (c) Root (NO3-N) 11 ± 0.8 55±4 50±3 17 ± 0.5 (d) Shoot (reduced-N) 3.2 ± 0.3 5 ± 0.4 -0.6 ± 0.1 -1.2 ± 0.1 (e) Shoot (NO3-N) 2. C Utilization (mg C/plant) 1104 ± 62 1725 ± 110 1221 ±46 1358 ± 63 (a) Net photosynthesis 321 ±40 189 ± 32 269 ± 46 302 ± 25 (b) Root C increment 315 ± 14 379 ± 17 453 ± 18 304± 15 (c) Root respiration 521 ± 51 918 ± 99 548 ± 35 573 ± 46 (d) Shoot C increment 79 ±4 67 ± 3 85 ± 5 86± 13 (e) Shoot respiration 314 ± 26 304± 19 387 ± 17 534 ± 35 3. Transpiration (g H20/plant) 4. Xylem composition 275 1267 107 1198 (a) Total N (jig N/ml) 1114 232 99 1055 (b) Reduced-N (Mg N/ml) 43 ± 3 153± 11 8±3 143 ± 8 (c) N03-N (Mg N/ml) 171 404 1939 1821 (d) Organic C (Mg C/ml)a 1.47 1.53 1.6 1.52 (e) C:N ratio (g C/g N) 5. Phloem composition 19±3 26 ±4 9.9± 1.4 10.7± 1.6 (a) C:N ratio (g C/g N)b 2.31 6.36 5.89 3.32 (b) Total N (mg N/ml)C 6. Xylem transport to shoot' 222 75 191 64 (a) C (mg C/plant) 40 51 146 125 (b) N (total) (mg N/plant) 43 37 129 110 (c) Reduced N (mg N/plant) 17 15 8 3 (d) N03-N (mg N/plant) 7. Phloem transport to root' 682 694 764 943 (a) C (mg C/plant) 35 29 70 88 (b) N (mg N/plant) 69 73 60 56 (c) Phloem N/Xylem N (%)' 8. N cycling through root 14 75 25 60 (a) N (mg N/plant) 14% 28% 52% 15% (b) Phloem N used by root' 9. NO3- reduction 23 18 50 55 (a) Root (mg N/plant)' 9 3 10 14 (b) Shoot (mg N/plant)" 66% 88% 83% 79% (c) Percent in root 10. Xylem N concentration 168 398 (a) Total N (Mgg N/ml)' 103 273 141 (b) Reduced N (Mg N/mly 96 242 350 11. Phloem N concentration:Xylem N concentration 24.3 25.3 18.2 (a) Reduced N 24.5 a Calculated from the xylem sap N content (item 4a) and the xylem sap C content of xylem in a comparable study with soybean by Layzell and LaRue (15). b Calculated from total sugars and ninhydrin-N analysis of EDTA phloem exudates. CAssumes 15% sucrose (63 mg C/ml) in the phloem and C:N ratio as in item 5a. d Calculated from the equations in Pate et al. (22). ' Calculated as (item 7b/item 6b x f Calculated as (item lb/item 7b x 100). ' Calculated as ([item Ia - item lc] - [item 4c/item 4a 100). h Calculated as (item la - [item lc + item le] - 9a). x item 6b]). 'Calculated as (item 6b/item 3 x 1000). i Calculated as (item 6c/item 3 x 1000).

ofthis extra C was cycled through the root, returning to the shoot in the xylem (Table II, item 6a). From the xylem flow of N (Table II, item 6b) and the transpiration rate (Table II, item 3) it was possible to determine the average in situ xylem N concentration over each of the 4 d study periods. The values obtained (Table II, item 10) were similar to the measured N concentrations in the root bleeding exudates of the 2 mm NO3- fed plants (Table II, items 4a, b). However, in the plants that were fed 10 mM NO3-, the root bleeding exudates

were 3.1 to 4.4 times the N concentration predicted from the model. Root bleeding xylem exudates generally have higher solute concentrations than xylem sap which is vacuum extracted from stem segments (18). This observation is consistent with the differing rates of water flux in detopped and whole plants. However, since it is very difficult to collect vacuum extracts of stem segments in the absence of contamination from cut and burst cells, xylem bleeding is generally considered as a more accurate reflection of the relative, in situ composition, if not the

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Plant Physiol. Vol. 83, 1987

2e

FIG. 6. Diagrammatic representation of flow and accumulation of nitrogen (A) and carbon (B) for the transport models (Table II). Large boxes represent shoot and root components of the plants. Number and letter labels correspond to the items of the model from Table II. In the N flow diagram (A) the shaded lines represent the flow of N03-N and blank lines represent the flow of reduced N.

concentration, of the xylem sap transported in the intact plant (18). Since the model is based on the C:N ratio of the xylem exudate, its predictions for xylem N concentration (Table II, item 1Oa) were considered to be the more accurate estimates, and indicated that the xylem sap in the 10 mm plants was 2.4 to 2.7 times more concentrated in N than that of the xylem sap of the 2 mm NO3- fed plants. The N concentration of the phloem sap was estimated from its relative composition of sucrose and amino N, assuming sucrose to be present at a concentration of 15% (w/v) (22). Since C transport to roots was only marginally affected by N supply over the study period, it seems reasonable that the concentration of sucrose would have been consistent between treatments. Given this assumption, phloem sap N concentration in the 10 mM treatment was estimated to be 1.9 to 2.5 times that in the 2 mm treatment (Table II, item Sb). When xylem and phloem sap concentrations were compared, it was noted that the reduced N concentrations in the phloem were consistently 18 to 25 times higher than the reduced N in the xylem, regardless of the nitrate treatment (Table II, item 1 1). This indicated that the concentration and amount of N being transported in xylem to shoots is positively correlated with the concentration and amount of N that is subsequently transported back to the roots via the phloem. While the mechanism which regulates phloem loading of N is not known (27), it is thought to occur from the apoplast (10). It is possible that in stems, petioles and minor veins of leaves, the apoplast from which phloem is loaded may be in equilibrium with the fluids moving through the xylem. Whatever the mechanism, it is possible that the regulation of N transport to roots in the phloem may be simply a function of the rate of C transport to the roots and the reduced N concentration in the xylem fluid. This proposal would be consistent with the observation that in general within a species the organic N composition of xylem and phloem fluids are similar (18). Of the N returning to the roots in the phloem, the roots of the 2 mm NO3 fed plants retained a much higher proportion (2852%) than did the roots of the plants fed 10 mm NO3- (14-15%) (Table II, item 8b). Consequently, the increment of N within the root tissue was similar among treatments (Table II, item b) and N cycling through the root was greater in the 10 mm treatment than in the 2 mm treatment (Table II, item 8a).

Cycling of N through roots has been indicated in other studies (6, 21, 22). Israel et al. (13) reported the cycling of nonureide, reduced-N from phloem to xylem in the roots of male sterile soybeans and associated this occurrence with a lack of sinks for nitrogen utilization. Similarly, in the present study, the cycling of N was greatest in the 10 mm treated plants (Table II, item 8a) which had the largest pools of stored NO3- in the tissues (Fig. 4b). Concerning the cycling of N through the root, it is important to consider that over each 4 d study period, the same N may be cycling through the root several times, and that a large N cycling component does not necessarily mean that a large percentage of whole plant N is soluble and moving within the plant. To demonstrate this point, an attempt was made to calculate (a) the soluble pool of N within the xylem and phloem at a given period of time, and (b) the average time required for N to cycle through the plant. The calculations (Table III) were based on the observations by Pate et al. (20) that xylem and phloem tissues accounted for 30 and 7 %, respectively, of tissue in lupin, a legume similar to soybean in that secondary growth of vascular tissue occurs in the stems and roots. When these values for xylem and phloem volume were applied to the plants in the present study, the xylem N pool was estimated to contain 0.65 to 1.6 mg N and the phloem N pool from 3 to 8 mg N (Table III, item 2). As expected, the larger N pools were associated with the 10 mm NO3- treatment, but in both treatments transport fluid N accounted for a similar proportion (3.6-4.5%) of whole plant N (Table III, item 4c). The turnover time of N in the xylem streams was estimated to be between 37 and 55 min and in the phloem stream was estimated to be between 7.5 and 11.2 h for all treatments examined (Table III, item 5). Consequently, the high NO3- treatment altered only the absolute concentration of NO3in the transport fluids and shoot tissues, not the proportion or rate of N cycling through the plant. Root Nitrate Reduction and Respiratory Cost. The amount of reduced N in xylem sap as a proportion of the total N content

has frequently been used as an index of the relative importance of root versus shoot NO3- reduction (2). However, Rufty et al. (24) have noted that errors in the estimate may be significant if the plant is cycling reduced N through the root. The transport model outlined in Table II allows one to correct for the N cycling

REGULATION OF ASSIMILATE PARTITIONING IN SOYBEANS Table III. Characterization of Transport Fluid N Pools Calculation of amount and turnover time of N in xylem and phloem for 2 and 10 mm NO3- fed plants over 26 to 29 and 30 to 33 DAP time periods. Treatment Item

2 mM

26-29 d 1. Volume (ml) (a) xylem (b) phloem (c) Total 2. N pool (mg N/ plant) (a) xylemb (b) phloemc (c) Total 3. Whole plant N Content (mg N/ plantp 4. Percent of whole plant N in transport fluids

10 mM

30-33 d

26-29 d

30-33 d

3.9 0.91 4.8

6.4 1.48 7.9

3.6 0.85 4.5

5.8 1.36 7.16

0.65 3.0 3.7

0.66 3.6 4.3

1.4 5.4 6.8

1.6 8.0 9.6

95

119

151

230

(%)C

0.55 0.93 0.70 0.68 (a) xylem 3.0 3.6 3.2 3.5 (b) phloem 3.9 3.6 4.5 4.2 (c) Total 5. Turnover time of N in transport fluids (h) 0.72 0.92 0.65 0.61 (a) xylem' 8.2 11.2 7.5 8.3 (b) phloem" a Assumes xylem and phloem account for 30 and 7% of the volume b Calculated as (item la * [Table II, item of stem and root tissue (20). lOa/1000]). c Calculated as (item lb x [Table II, item Sb]). dAverage for growth interval, Figure lc. ICalculated as ([item 2/ item 3)] x 100). f Assumes 14 h/d period of transpiration, or 56 h/ g Assumes no diurnal and interval: (item 2a/[Table II, item 6b/56]). 96 h/interval: (item 2b/[Table II, item 7b/96]).

component and therefore obtain a more accurate estimate of root versus shoot nitrate reduction. For example, direct analysis of xylem sap composition (Table II, items 4a, b) revealed that 84 to 93% of the xylem N was reduced, suggesting a major role for root nitrate reduction. By comparison, the transport model indicated that from 66 to 88% of the whole plant NO3- reduction occurred in roots and that the proportion of root and shoot nitrate reduction was not affected by the N treatment (Table II, item 9c). A previous study using only xylem sap composition has estimated that soybean roots are responsible for 53 to 66% of whole plant NO3 reduction (7) while the study of Rufty et al. (24) estimated root NO3- reduction at less than 20% of whole plant assimilation. The lower estimates in the study by Rufty et al. (24) may be attributed to differences from the present study in soybean cultivars or light irradiance used in plant growth. Alternatively, the '5N approach used in the Rufty et al. study may have underestimated root NO3- reduction as a result of compartmentization of NO3- and reduced N pools leading to a temporal separation of uptake and transport. The observation that the proportion of whole plant nitrate reduction which occurred in roots was not affected by the N treatment (Table II, item 9c), was in agreement with the investigations of Andrews et al. (1) for legumes of tropical origin such as field bean, soybean, and pigeon pea. However, they found that the proportion of nitrate reduction occurring in roots declined with increasing nitrate supply in legumes of temperate

347

origin (2). While the NO3- treatment did not affect the proportion of NO3- reduction localized in roots, the 10 mM plants reduced 2.4 to 2.8 times as much NO3- as that which was reduced in the plants fed 2 mm NO3- (Table II, item 9a). As mentioned previously, the roots of the plants fed 10 mm NO3- also displayed dark respiration rates which were 6 to 20% greater than those of the plants fed 2 mM NO3- (Table I). Assuming an energetic cost of 4 electron pairs per NO3- reduced, a P:O ratio of 3.0, and glucose as a substrate, the theoretical respiratory cost of reducing this additional NO3- is able to account for 120% ofthe measured difference in root respiration. This is not to imply that the respiratory cost associated with root NO3- reduction either fully or solely accounted for the differences in root respiration rates among the N treatments, but that root NO3- reduction significantly contributed to these differences. Regulation of the Shoot:Root Ratio. The transport models presented in Table II, and the data on the patterns of C and N partitioning in all 12 treatments were used to develop a hypothetical mechanism to account for the way in which changes in the rate of NO3- supply regulates the shoot:root ratio of soybeans before N limitation causes a decline in specific net photosynthetic rates. This hypothetical mechanism, shown diagramatically in Figure 7, is illustrated as a series of cause and effect relationships. It is proposed that if NO3- supply to roots is decreased or curtailed, the resultant decline in NO3- uptake (A) would decrease the absolute amount of root NO3- assimilation (B) and therefore the root respiration associated with this process (C). The concentration of N in the xylem would decline (D) and would lead to a decrease in the shoot N pool (E), which in this study resulted in a slight decrease in shoot growth (F). Due to an apparent relationship between xylem and phloem N concentrations (Table II, items 7c and 1 1), the N content of the phloem sap leaving the shoot would decrease (G) but at a proportion similar to that of plants fed higher concentrations of nitrate. Of the N sent to the roots in the phloem a much higher proportion, but similar amount, is unloaded and used by the roots (H) thus maintaining the reduced N content of the root tissue (I), and decreasing the amount of N left to cycle through the root (J). This decrease in N cycling would result in a further decline in the N concentration in the xylem (K). The maintenance of the root reduced N content (I), and the decrease in root respiration (C) would promote root dry matter accumulation (L, M) as would any C diverted to roots (N) as a result of shoot growth being limited by N supply (F). Ultimately therefore, root growth would be promoted, shoot growth retarded, and the shoot:root ratio would decline. Eventually, of course, this would result in a decrease in leaf area expansion and therefore a decrease in net photosynthesis and whole plant growth. It is important to note that through the simple series of cause and effect events outlined above and in Figure 7, it is possible to account for the effect of nitrate supply on growth and dry matter partitioning in soybeans and there is no need to invoke secondary factors such as plant growth regulators (25) to account for these changes in assimilate partitioning. In fact, the maintenance of reduced N levels in the roots of the lowest NO3- treated plants (Fig. 4c) contradicts the hypothesis that assimilate partitioning could have been influenced by a reduced level of cytokinin production in roots due to N limitation of this tissue (29). Two features of long distance transport in plants that are essential to the 'chain-reaction' hypothesis proposed here are (a) that phloem N concentration may be regulated by xylem N concentration, possibly through phloem loading from an apoplast which is in equilibrium with xylar fluid, and (b) that roots remove from the phloem sufficient N to satisfy their own requirements for growth and the excess is cycled back to the shoot in

348

VESSEY AND LAYZELL N METABOLISM

Plant Physiol. Vol. 83, 1987

C METABOLISM NO3 SUPPLY iTO ROOTS

I

LEG

tINCRE s DECRE 1

SE1I FIG. 7. Model of the effect of decreasing N03- supply on C and N assimilation and partitioning. Vertical arrows within boxes indicate an increase (T) or decrease (l) in the component. Arrows between boxes indicate an influence of one component on another. Letters accompany the expla-

nation in the text.

the xylem. It would be of interest to determine whether these features characterize N transport under a wide variety of environmental conditions, and whether a simple chain-reaction of cause and effect events can explain other environmentally induced changes in assimilate and dry matter partitioning. Acknowledgments-The technical assistance of K. Walsh, B. King, G. Weagle, and M. L. Vessey is gratefully acknowledged. LITERATURE CITED 1. ANDREWS M, JM SUTHERLAND, RJ THOMAS, JI SPRENT 1984 Distribution of nitrate reductase activity in six legumes: the importance of the stem. New Phytol 98: 301-3 10 2. ATKINS CA, JS PATE, DB LAYZELL 1979 Assimilation and transport of nitrogen in nonnodulated (N03-grown) Lupinus albus L. Plant Physiol 64: 10781082 3. BEEVERS L, RH HAGEMAN 1980 Nitrate and nitrite reduction. In BJ Miflin, ed, The Biochemistry of Plants-A Comprehensive Treatise. Academic Press, Toronto 4. BROUWER R 1962 Nutritive influences on the distribution of dry matter in the plant. Neth J Agric Sci 10: 399-4084 5. BRUGGE R, JHM THORNLEY 1984 Shoot-root-nodule partitioning in a vegetative legume-a model. Ann Bot 54: 653-671 6. COOPER HD, DT CLARKSON, MG JOHNSTON, JN WHITEWAY, BC LOUGHMAN 1986 Cycling of amino-nitrogen between shoots and roots in wheat seedlings. Plant Soil 91: 319-322 7. CRAFTS-BRANDNER SJ, JE HARPER 1982 Nitrate reduction by roots of soybean (Glycine max [L.] Merr.) seedlings. Plant Physiol 69: 1298-1303 8. ELRIFI I, DB LAYZELL, BJ KING, GE WEAGLE, DH TURPIN 1986 Inexpensive, computer-automated HPLC for ion exchange separation and quantification of aminoacids in physiological fluids. J Liq Chromatogr. In press 9. FALES 1 1951 The assimilation and degradation of carbohydrates by yeast cells. J Biol Chem 193: 113-124 10. GIAQUINTA RT 1983 Phloem loading of sucrose. Annu Rev Plant Physiol 34: 347-387 11. GREENWOOD EAN 1976 Nitrogen stress in plants. Adv Agron 28: 1-35 12. HERRIDGE DF 1984 Effects ofnitrate and plant development on the abundance of nitrogenous solutes in root-bleeding and vacuum-extracted exudates of soybean. Crop Sci 25: 173-179 13. ISRAEL DW, JW BURTON, RF WILSON 1985 Studies on genetic male-sterile soybeans. IV. Effect of male sterility and source of nitrogen nutrition on

14.

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22. 23. 24.

25.

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28. 29.

30.

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