Root and Nodule Growth in Pisum sativum L. in Relation to Photosynthesis: Analysis Using ¹³C‐labelling

Abstract

The effect of the nitrogen source (gaseous nitrogen, N₂, or nitrate ions, NO₃^–) on the use of carbon (C) for root and nodule growth of pea (Pisum sativum L.) was investigated using ¹³C‐labelling of assimilated CO₂ at various stages of growth. Nitrate supply and growing conditions (sowing dates, air CO₂ concentration) were varied to alter photosynthetic rates. Nodules are the sink with the highest demand for C in both the vegetative and flowering stages, growing at the expense of shoot and root in the vegetative stage, but only at the expense of roots at flowering. Until flowering, the addition of C into root and nodule biomass was linearly related to pre‐existing biomass, thus determining net sink strengths which decreased with root and nodule age. Nodule growth patterns did not depend on the N source, whereas root growth was increased by nitrate when nodule biomass was low. At seed filling, the increase in C of biomass of the root system was no longer related to pre‐existing biomass and C was preferentially diverted to roots of plants assimilating nitrate, or to nodules for plants fixing N₂.

INTRODUCTION

Symbiotic gaseous nitrogen (N₂) fixation is inhibited by nitrate ions (NO₃^–) (Streeter, 1988), but it has been difficult to determine which aspect of the process is affected. It may be nitrogenase activity, or growth, or maintenance of nodules. Biomass partitioning between roots and nodules is also affected by nitrate, as frequently reported (Pate et al., 1979; Ryle et al., 1979b; Atkins et al., 1980; Jensen, 1986; Lodeiro et al., 2000). Relationships between root and nodule growth and the N source (N₂, NO₃^–) are still unclear: plants relying exclusively on symbiotic N₂ fixation have less root biomass, but more nodule biomass, than plants supplied with nitrate (Voisin et al., 2002a), which have impaired symbiotic N₂ fixation (Streeter, 1988; Voisin et al., 2002b). Nitrate application can stimulate root production in the surface soil at the early growth stage, as reported for non‐leguminous crops (Oikeh et al., 1999) but increased root growth in the presence of nitrate could also result from the inhibition of symbiotic N₂ fixation (Voisin et al., 2002a, b).

The metabolic relationships and material fluxes between root and nodule growth throughout the growth cycle and how they are linked are poorly understood and quantified. Root growth of legumes may cease at flowering (Salter and Drew, 1965) but other evidence shows that it also continues during reproduction (Andersen and Aremu, 1991; Armstrong et al., 1994; Voisin et al., 2002a). Regulation of the number of nodules per plant could be systemic (Francisco and Harper, 1995; Harper et al., 1997) regulated by hormones, e.g. ethylene (Markwei and Larue, 1997), or controlled by availability of assimilates (Atkins et al., 1989; Tricot et al., 1997). Nodule growth may be linked to total plant growth (Mengel, 1994) and depend upon environmental factors (e.g. nitrate supply, salt stress, drought stress) directly or through global effects on plant growth (Sprent et al., 1988; Streeter, 1988). There are several hypotheses concerning the decrease of both nodule activity and/or senescence towards the end of the growth cycle. They could arise either from competition between nodulated roots and filling seeds for assimilates from leaves (Lawrie and Wheeler, 1974), or be regulated by signal molecules arising from the shoot (Malik, 1983; Silsbury et al., 1986) or by internal factors associated with nodule ageing (Klucas, 1974; Minchin et al., 1986).

Nodule functioning depends directly on current photosynthesis (Kouchi et al., 1986). The proportion of assimilates allocated to nodulated roots does not depend upon the N source but is related to the photosynthetic rate (Voisin et al., 2003). However, the amount of carbon (C) used in respiration by below‐ground organs (roots and nodules) varies according to the N source, being greater for nodulated than for non‐nodulated roots (Pate et al., 1979; Ryle et al., 1979a, b; Atkins et al., 1980; Schulze et al., 1999; Voisin et al., 2003). Root and nodule growth may thus depend upon assimilate use within nodulated roots, in relation to the respiratory C costs induced by roots and nodules. Thus, C seems to be a good candidate for explaining (a) interactions between root and nodule growth and (b) root and nodule growth patterns during the growth cycle. The objective of this study was therefore to examine and quantify the effect of the N source on C use for root and nodule growth of pea during the growth cycle. Also, the aim was to determine priority for assimilate allocation between roots and nodules at each stage of growth.

MATERIALS AND METHODS

Plant material and growing conditions in the glasshouse

Peas (Pisum sativum ‘Baccara’) were sown on 15 Sep. 1999, on 2 and 16 Mar. 2000 and on 26 May 2000 and grown in a naturally lit glasshouse (Dijon, France). The aim was to vary conditions using natural variations in temperature, radiation and photoperiod during growth. The genotype Baccara requires long‐days for flower initiation and has a facultative vernalization requirement. Supplementary artificial light was therefore provided to give a 16‐h day in autumn 1999 to induce flowering. Temperatures in the glasshouse were maintained between 10 and 30 °C during the day and between 10 and 25 °C at night.

Eight seeds were sown in each of 180 5‐L PVC pots filled with a 1 : 1 v/v mixture of sterilized attapulgite and clay balls (diameter 2–6 mm). The seeds were inoculated with Rhizobium leguminosarum bv vicieae. At emergence, plants were thinned to four per pot. All plants were regularly watered with a similar volume of nutrient solution to maintain the soil water near pot capacity. Nutrient solution was supplied automatically, using two balances which automatically triggered watering of all pots of a treatment when the cumulated transpiration since the last watering exceeded one‐tenth of the soil water reserve. This system was checked three times per week to ensure that soil water retention was maintained approximately equal to field capacity, as measured in plots at an INRA experimental station near Dijon (France).

For each sowing date, two treatments were applied in the glasshouse: half of the plants (90 pots) were provided with N‐free nutrient solution (N₀ treatment) and the other half (90 pots) with 5 mol m^–3 KNO₃ in the nutrient solution (N₅ treatment). In a preliminary experiment this concentration of nitrate limited symbiotic N₂ fixation by approx. 50 % and reduced nodule biomass by 60–70 % compared with plants only fixing N₂. Nutrient solutions adjusted to neutral pH, containing 0·8 mol m^–3 K₂HPO₄, 1·0 mol m^–3 MgSO₄, 0·2 mol m^–3 NaCl, 0·05 mol m^–3 iron chelate and oligo‐elements. In addition the nutrient solutions for the N₀ and N₅ treatments, respectively, contained 0 and 1·25 KNO₃ mol m^–3, 0 and 1·875 mol m^–3 Ca(NO₃)₂, 0·7 and 0·075 mol m^–3 K₂SO₄, 2·5 and 0·625 mol m^–3 CaCl₂.

¹³C‐labelling

For each of the four sowing dates, plants were exposed to ¹³CO₂ at three stages of growth: ‘vegetative’ (10 nodes on main stem), ‘flowering’ (flowers on the 6th node of the main stem, before the beginning of seed filling), and ‘seed filling’ (between final stage in seed abortion and start of physiological maturity). The early flowering stage (two reproductive nodes) was also studied for plants sown in autumn 1999. Three pots of each treatment were measured. The equipment (Warembourg et al., 1982; Voisin et al., 2000, 2003) allowed ¹³CO₂ labelling of plant parts and simultaneous measurement of below‐ground respiration. The pots of plants grown in the glasshouse were inserted in individual air‐tight PVC containers (cylinder 0·022 m diameter and 0·024 m height) to separate root and shoot atmospheres. Air tightness was ensured using physiological moulding material (Qubitac; Qubit Systems Inc., Kingston, Canada) and silicone rubber (RTV; Zundel Kohler, France). Oxygen concentration of the root atmosphere was maintained at 20 ± 1 % during labelling to compensate for O₂ consumption due to respiration. Expansion bags regulated pressure resulting from samplings or injections. The pots (three pots per each treatment) with separate root atmospheres were placed in a transparent air‐tight labelling chamber (0·95 × 0·95 × 1·5 m) made of Plexiglas.

Shoots were exposed to a ¹³CO₂‐enriched atmosphere for 10 h with a constant ¹³C : ¹²C ratio (10 atom % ¹³C). An air‐conditioning unit (Voilot, Dijon, France) was used for circulation of the air at a flow rate of 80 L s^–1 within the air‐tight labelling chamber. In 2000, in order to vary the photosynthetic rates at the three growth stages (phenological stages) and three sowing dates, plant were exposed to three different concentrations of CO₂ in air (150, 360 and 750 µL L^–1). During ¹³C‐labelling, the air in the chamber was regularly sampled to assess its ¹³C enrichment by mass spectrometry; this showed that the ¹³C air enrichment remained within ±5 % of the set value during labelling. Labelling was followed by a non‐labelling ‘chase’ period of 4 d in 1999 and 2 d in 2000, with the plants maintained at 360 µL L^–1 CO₂ in air. The chase period was to allow transport of labelled C to the metabolic pools in the plant (Montange et al., 1981; Kouchi et al., 1986). A chase period of 4 d was used in 1999 to study the components of nodulated root respiration (data not shown). Moreover, such a duration of the chase period allowed us to verify that, for peas, isotope partitioning among plant parts was similar after 4 d to that observed after 2 d of chase.

Within the chamber, temperature and humidity of the air around the shoot were maintained using an air‐conditioning unit (Voilot, Dijon, France). Day and night temperature and humidity in the chamber during each labelling experiment were chosen to match the mean temperature and humidity observed the previous day in the glasshouse. Light was provided by four 400 W sodium lamps on each side of the enclosure and two 1000 W mercury lamps above it. The photosynthetically active radiation was 600–900 µmol m^–2 from the bottom to the top of the canopy, respectively. Soil water was measured gravimetrically and adjusted daily. The whole system was computer monitored and controlled using special software (Dasylab, Newport Omega, Amherst, NH, USA).

Harvesting and measurements

Plants were sampled at the beginning of the labelling experiment (control plants) and at the end of the chase period (labelled plants). All plants in each pot were harvested together and separated into shoots, roots and nodules. Dry matter was determined after oven drying at 80 °C for 48 h. The C and N concentrations were determined by the Dumas procedure on ground samples. Their ¹³C enrichment (atom % ¹³C) was measured using a dual inlet mass spectrometer (Fison Isochron; Micromass, Villeur banne, France) and an internal standard calibrated using Pee Dee Bellemnite (1·1111 % ¹³C).

Carbon dioxide produced by below‐ground respiration (i.e. arising from roots plus nodules and rhizosphere organisms) was measured by trapping the CO₂ evolved from the root compartment in NaOH and titration with hydrochloric acid. The traps were calibrated for daily measurements (50 ml 2 m NaOH, two pots per treatment). The ¹³C enrichment of the respired CO₂ was measured spectrophotometrically on the CO₂ produced by acidification of the trap solutions.

Calculations

Isotopic composition was measured according to the isotope dilution principle. The percentage (I) of carbon (% ¹³C) derived from the labelled source was calculated from:

I = 100 (L – C)/(A – C)

where A is atom % ¹³C of labelled atmosphere, L is atom % ¹³C of labelled plants and C is atom % ¹³C of unlabelled plants (control) plant material (shoot, roots, nodules) and respired C (Deléens et al., 1994). In order to take into account the small variations of A (see above), its average value was calculated using the values of ¹³CO₂ enrichment measured during the labelling.

For each component (plant sample, CO₂), the quantity of C derived from photosynthesis during the labelling period (Q_C) was calculated using %¹³C (I), dry matter (M) and carbon (C) determinations:

Q_c = M(C/100)(I/100)

Statistics

Analysis of variance was performed using ANOVA (SAS Institute, 1987). Means were compared using the least significant difference (LSD) test at 0·05 probability. For each treatment, each value is the average of three measurements (three pots).

RESULTS

The amount of photosynthetic C, determined from ¹³C‐labelling, incorporated into roots and nodules of pea plants was related to their biomass at the start of labelling (Fig. 1) at each phenological stage. Nodule biomass was greater for plants fixing N₂ (N₀ treatment) than for those assimilating nitrate (N₅ treament) (Fig. 1B, D and F) but root biomass was greater for the plants supplied with nitrate (Fig. 1A, C and E).

Fig. 1. Daily increase in carbon in roots (A, C and E) or nodules (B, D and F) of a plant calculated from ¹³C‐labelled carbon as a function of root biomass (A, C and E) or nodule biomass (B, D and F) at the start of the labelling period, during growth of Pisum sativum L. A and B, Vegetative stage; C and D, flowering stage; E and F, seed filling stage. The influence of the N source is indicated for plants grown in 1999 (circles) and 2000 (diamonds): N₂‐fixing plants (N₀ treatment, open symbols), nitrate‐assimilating plants (N₅ treatment, filled symbols).

At the vegetative stage, whatever the N treatment and for both roots (Fig. 1A) and nodules (Fig. 1B), there was a single, linear relationship between C addition into biomass and pre‐existing biomass: the slope of the regression was similar for roots and nodules. At flowering, another single, linear relationship was obtained for the roots (Fig. 1C), but the slope was smaller than at the previous stage. However, two significantly different linear relationships were obtained for the nodules at this stage (Fig. 1D), with one for plants fixing N_{2 (}N₀ treatment) in 1999 and plants assimilating nitrate (N₅ treatment) in both years, and another for plants fixing N₂ (N₀ treatment) in 2000. At seed filling, the amount of C entering the biomass was no longer related to pre‐existing biomass for either roots (Fig. 1E) or nodules (Fig. 1F). For a given initial biomass, more C was incorporated into biomass of roots in the N₅ treatment than in the N₀ treatment (Fig. 1E), but less C was incorporated into nodules in the N₅ treatment compared with the N₀ treatment (Fig. 1F).

For both roots (Fig. 2A, C and E) and nodules (Fig. 2B, D and F), C addition to biomass per unit of pre‐existing biomass at the start of labelling decreased with age of the plants and stage of the growth cycle. This held regardless of the N treatment. Until early flowering, C addition to biomass per unit of pre‐existing biomass nodules (Fig. 2B) was far greater for nodules (Fig. 2B) than for roots (Fig. 2A). Carbon addition to root or nodule biomass per unit of their pre‐existing biomass at the start of labelling was plotted as a function of net photosynthesis (Fig. 2), measured from ¹³C‐labelling as the sum of the carbon integrated into plant biomass plus carbon respired by the root system, including nodules (Voisin et al., 2003). Net photosynthesis ranged from 51 to 124 mg plant^–1 d^–1 at the vegetative stage, from 110 to 346 mg plant^–1 d^–1 at flowering, and 112 to 378 mg plant^–1 d^–1 at seed filling (Fig. 2). For both roots and nodules, C addition to biomass per unit of pre‐existing biomass increased with net photosynthesis at the vegetative stage (Fig. 2A and B) but was not significantly affected by the amount of photosynthetic C either at flowering (Fig. 2C and D) or seed filling (Fig. 2E and F). There was no difference related to the N source, except at flowering when average C addition to root biomass per unit of pre‐existing root biomass was higher for the N₅ treatment than the N₀ treatment (Fig. 2C).

Fig. 2. Influence of the N source (with N₂ fixing plants: N₀ treatment, open symbols; nitrate assimilating plants, N₅ treatment, filled symbols) on the changes in root (A, C and E) and nodule (B, D and F) net sink strength (daily carbon increment in root, or nodule, per unit of root, or nodule, biomass at the start of the labelling period) as a function of daily net photosynthesis, during growth of Pisum sativum. A and B, Vegetative stage; C and D, flowering stage; E and F, seed filling stage for 1999 (circles) and 2000 (diamonds) experiments.

DISCUSSION

The effects of the source of N, either N₂ fixation or nitrate assimilation, and photosynthetic rate on C use for growth of nodulated roots of peas were investigated using ¹³CO₂ labelling under different concentrations of CO₂ in air. Net photosynthesis was varied, both by changing the growing conditions (different sowing dates) and, by altering air CO₂ concentration used during ¹³C‐labelling at each stage of growth for the different sowing dates (see Materials and Methods). This experimental design allowed the daily photosynthetic rate to be directly altered, and thus tested the hypothesis that the availability of photoassimilates is a main determinant of root and nodule growth.

Carbon allocation to root and nodule growth

Vegetative stage.

In this stage of development of pea plants, C addition to roots and nodules was linearly related to their initial biomass (Fig. 1) whatever the N treatment, with similar slopes. This result is similar to that found earlier for pea seeds (Jeuffroy and Warembourg, 1991). According to Ho et al. (1989), the net accumulation rate of dry matter is a measure of ‘net sink strength’ while the net gain of dry matter plus respiratory loss of dry matter is a measure of gross sink strength. The percentage of C in dry matter of different shoot and root parts (including nodules) of the plant was identical at the start and at the end of the labelling period (data not shown). Hence the slope of the linear regression relating C addition into roots (or nodules) of a plant and the pre‐existing biomass of roots (Fig. 1A) or nodules (Fig. 1B) suggests that they have similar net sink strengths at this stage. Net sink strength (i.e. C addition in biomass during the labelling per unit of pre‐existing biomass at the start of labelling) was plotted for roots (Fig. 2A) and nodules (Fig. 2B) vs. net photosynthesis. When net photosynthesis rate per plant increased, net sink strength also increased, suggesting that the C supply from photosynthesis was limiting. It has been shown (Voisin et al., 2003a) that the percentage of C respired by below‐ground parts decreased when net photosynthesis rate increased. This suggests that the amount of C respired (for synthesis, maintenance of structures and/or symbiotic N₂ fixation or root mineral N assimilation activities) was decreasing the C supply available for investment in biomass of the root system (Warembourg, 1983). Net sink strength of roots ranged between 8 and 27 mg C per pre‐existing gram of root biomass and per day (Fig. 2A). Net sink strength of nodules ranged between 18 and 58 mg C per pre‐existing gram of nodule biomass and per day (Fig. 2B). Net sink strength of nodules were generally higher for the plants supplied with nitrate (N₅ treatment; Fig. 2B) than for the plants fixing N₂ (N₀ treatment, Fig. 2B). Differences between net sink strength of nodules of the N₅ and N₀ treatments may arise from differences in pre‐existing nodule biomass (Fig. 1B) and were associated with nodule age, as a result of delayed nodulation due to nitrate in the N₅ treatment (Streeter, 1988). As the net sink strength of nodules decreased during growth (Fig. 2B, D and F), young nodules in the N₅ treatment may have had a higher net sink strength than nodules initiated earlier (and therefore bigger) for the plants of the N₀ treatment, consistent with earlier reports using ¹⁴C‐labelling (Hacin et al., 1997).

Thus, at the vegetative stage, it can be hypothesized that roots are the secondary sink for C assimilates from photosynthesis and that their growth is limited by that of nodules. Indeed, nodules were a far more competitive sink for assimilates than roots, as shown by their respective net sink strength at the vegetative stage (Fig. 2A and B). The results presented here are consistent with those of Hacin et al. (1997), who reported that developing nodules create a strong sink for assimilates at the expense of roots and of nodules initiated later. This contradicts the suggestion of Tricot et al. (1994, 1997) that root elongation would have priority over nodule formation and development when assimilates are limiting.

Flowering stage.

There was a linear relationship between C increment into roots or nodules and their pre‐existing biomass (Fig. 1C and D) as at the vegetative stage. A single linear relationship was obtained for roots, regardless of N treatment (Fig. 1C), showing that net sink strength of roots did not depend upon the N source. However, the net sink strength of roots of plants fixing N_{2 (}N₀ treatment; Fig. 2C) was lower than the net sink strength of roots of plants supplied with nitrate (N₅ treatment; Fig. 2C). At flowering, net photosynthesis no longer affected the net sink strength of either roots or nodules (Fig. 2C and D). Thus, photosynthesis was not the main factor limiting growth of the root system and could not account for variability in growth of roots and nodules. In contrast, the net sink strength of nodules did not differ with the N treatment (Fig. 2D). This suggests that root growth is limited by nodules, which presumably decrease C availability for root growth. The results presented here show that nodules are still the main sink within the root system at flowering. They do not agree with those of Tricot et al. (1994, 1997) which showed that nodule growth was more inhibited than root growth under C‐limiting conditions. However, as nodule biomass is always far smaller than root biomass, any effect of C supply on relative growth should affect nodule biomass more than roots.

Two different linear relationships between C increment into nodules and pre‐existing biomass were obtained for nodules at flowering (Fig. 1D) in the two years, with the second relationship observed in the plants fixing N₂ (N₀ treatment) in 2000. Even if the developmental stage of the shoots was identical between treatments, plants fixing N₂ in 2000 probably had the oldest nodules among all treatments (N source and growing conditions), as shown by their biomass (Fig. 1D), because nodule establishment in other treatments was presumably delayed. This delayed nodule establishment may be attributed either to the presence of nitrate, for plants of the N₅ treatment sown either in 1999 or in 2000, or to limiting growing conditions for plants of N₀ treatment, sown in autumn 1999 (Voisin et al., 2003).

Seed filling.

At seed filling, C allocation to roots and nodules (Fig. 1E and F) was no longer related to their pre‐existing biomass. This suggests that C was mainly used for maintenance of the structures at this late stage. Net sink strength of roots and nodules (Fig. 2E and F) was low and independent of net photosynthesis, whatever the N treatment. This could be related to the low sink strength of the root system at the end of the growth cycle (Voisin et al., 2003) due to the high demand for assimilates of the filling seeds (Jeuffroy and Warembourg, 1991). However, for roots (Fig. 1E), C addition in biomass was higher for plants supplied with nitrate (N₅ treatment) than for plants fixing N₂ (N₀ treatment) while the opposite was observed for nodules (Fig. 1F). This suggested that organs linked to the main N uptake pathway would have priority for C within the root system. Therefore nodule senescence may not be directly linked to nodule age (Klucas, 1974; Minchin et al., 1986) and/or C availability (Lawrie and Wheeler, 1974; Bethlenfalvay and Phillips, 1977) but may also depend on the N source. In the presence of nitrate, the higher photosynthetic ability of the plant at seed filling (Voisin, 2002), compared with plants only fixing N₂, should benefit root survival through higher C allocation to the root system (Fig. 1E).

Explanations of differences between shoot and root biomass induced by the N source

Assuming that C uptake by photosynthesis does not depend upon the N source (Ryle et al., 1979b), and because C partitioning between shoot and roots is not affected by the N source (Voisin et al., 2003), photosynthetic C supply cannot account for differences in biomass partitioning between plants fixing N₂ or assimilating nitrate (Mahon and Child, 1979; Pate et al., 1979; Ryle et al., 1979b; Atkins et al., 1980; Butler and Ladd, 1985; Jensen, 1986; Lodeiro et al., 2000). Therefore, those differences can be attributed to differences in C‐utilization within the root system, related to the presence of nodules. At the vegetative stage, nodule growth presumably occurred at the expense of root growth since net sink strength of nodules was always higher than net sink strength of roots. In the case of limited C availability, nodule growth also occurred at the expense of shoot growth, since nodulated roots have priority for C over the shoot at the early stages of growth (Voisin et al., 2003). This could explain the smaller shoot biomass of plants fixing N₂ and hyper‐nodulating plants compared with plants assimilating nitrate (Salon et al., 2001). At flowering, the root system was more limited by C supply, presumably because of the appearance of reproductive organs (Voisin et al., 2003). Nodule growth and the associated maintenance costs presumably occurred at the expense of root growth. At seed filling, despite low sink strength of the root system, both roots and nodules were limited by C supply, because of the large demand for assimilates for seed filling. Within the root system, C was mainly used for maintenance processes at this late stage of the growth cycle and C was preferentially diverted to below‐ground organs (i.e. roots or nodules) linked to the main N uptake pathway (assimilation of nitrate or symbiotic fixation of N₂, respectively).

The results presented here demonstrate that, once initiated, nodule growth is not affected by the N source, whereas root growth depends on the presence of nodules. The negative effect of nitrate on nodule biomass, which has often been reported (Streeter, 1988), may therefore only result from the delayed nodule establishment due to the presence of nitrate. Differences of biomass partitioning in shoot, roots and nodules due to the N source are explained by changes in competition for C during the growth cycle. These results demonstrate that C availability is the key variable determining the structure and functioning of legume root systems.

ACKNOWLEDGEMENTS

We thank V. Durey, J. Gonthier and P. Mathey for their excellent technical assistance, B. Mary and O. Delfosse (ENSAIA, Nancy) for ¹³C isotopic analysis of plant material and J. N. Thibault and P. Garnier (INRA, Rennes) for gaseous ¹³C isotopic analysis, and two anonymous referees for constructive critiscism. This work was funded by INRA, UNIP and Conseil Régional de Bourgogne.

Received: 13 December 2002; Revised: 3 April 2003. Accepted: 23 June 2003