Root system growth and nodule establishment on pea (Pisum sativum L.)

Transcription

1 Journal of Experimental Botany, Vol. 48, No. 316, pp , November 1997 Journal of Experimental Botany Root system growth and nodule establishment on pea (Pisum sativum L.) Frederique Tricot 1, Yves Crozat 2 and Sylvain Pellerin 3 ' 4 1 ESA, Laboratoire de Biotechnologie des Sols, 55 rue Rabelais, F Angers, France 2 CRAD-CA, Avenue du Val de Montferrand, F Montpellier, France 3 NRA, Laboratoire d'agronomie, 71 avenue Edouard-Bourleaux, BP81, F Villenave d'ornon, France Received 10 June 1997; Accepted 8 July 1997 Abstract Development of the root system, appearance of nodules, and relationships between these two processes were studied on pea {Pisum sativum L., cv. Solara). Plants were grown in growth cabinets for 4 weeks on a nitrogen-free nutrient solution inoculated with Rhizobium leguminosarum. Plant stages, primary root length, distance from the primary root base to the most distal first-order lateral root, and distance from the root base to the most distal nodule, were recorded daily. Distribution of nodules along the primary root and distribution of laterals were recorded by sampling root systems at two plant stages. Primary root elongation rate was variable, and declined roughly in conjunction with the exhaustion of seed reserves. First-order laterals appeared acropetally on the primary root. A linear relationship was found between the length of the apical unbranched zone and root elongation rate, supporting the hypothesis of a constant time lag between the differentiation of first-order lateral's primordia and their emergence. Decline of the primary root elongation rate was preceded by a reduction in density and length of first-order laterals. Nodules appeared not strictly but roughly acropetally on the primary root. A linear relationship was found between the length of the apical zone without nodule and root elongation rate, supporting the hypothesis of a constant time lag between infection and appearance of a visible nodule. A relationship was found between the presence/absence of nodules on a root segment and the root elongation rate between infection and appearance of nodules on the considered root segment. Regulation of both processes by carbohydrate availability, as a causal mechanism, is proposed. Key words: Pisum sativum L, root system, nodules. ntroduction Pea (Pisum sativum L.), as most legumes, establishes in root nodules a symbiotic association with Rluzobium leguminosarum bacteria. Atmospheric N 2 is reduced to NH^ within nodule cells and then incorporated into amino acids before being released into the phloem, thus providing assimilated nitrogen to the whole plant. At the root segment level, much has been achieved in recent years in understanding the steps which are essential for the formation of a functional nodule (see review articles from Sprent, 1989; Hirsch, 1992; Mylona et ai, 1995). nfections by Rhizobium bacteria leading to nodule formation are restricted to only a narrow band of cells above the zone of root elongation and just below the smallest emergent root hairs (Bhuvaneswari, 1981; Bhuvaneswari et ai, 1980, 1981; Pueppke, 1986). The interaction of Rhizobia and legumes involves signal exchange and recognition of the symbiotic partners, followed by attachment of the Rhizobia to the plant root hairs. The root hair deforms, and the bacteria invade the plant by a newly formed infection thread growing through it. Simultaneously, cortical cells are mitotically activated, giving rise to the nodule primordium. Nodule morphogenesis is therefore elicited by at least two distinct signals: one from Rhizobium, a product of the nod genes (Nod factor), and a second signal, which is generated within plant tissues after treatment with Nod factor (Hirsch, 1992). At the whole-root system level, the number of nodules depends on both internal and environmental factors (see review article from Caetano-Anolles and Gresshoff, 1991; * To whom correspondence should be addressed. Fax: O Oxford University Press 1997

2 1936 Tricot et al. Mengel, 1994). Regulation by internal factors has often been termed autoregulation. t is manifested as inhibition of nodule formation on one part of the root by prior initiation elsewhere on the root. Environmental factors known to affect the number of nodules are nitrate concentration in the growing medium (Macduff et al., 1996), soil compaction (Katoch et al., 1983), air and soil temperature (Munevar and Wollum, 1981; Rawsthorne et al., 1985), air CO 2 concentration (Phillips et al., 1976; Murphy, 1986), and light intensity (Kosslak and Bohlool, 1984). The mechanism by which these environmental factors affect nodulation and interact with the process of autoregulation is not understood. nvestigations at this whole-plant level require a good preliminary knowledge of the dynamics of nodule establishment, which is in strong interaction with root system formation. Unexpectedly, relationships between these two processes apparently have been rarely investigated. The objective of this work was to study the development of the root system, the appearance of nodules, and relationships between these two processes for pea plants grown in hydroponic conditions. Materials and methods Growth of seedlings Seeds of pea (cultivar Solara) were weighed and only those of similar weight (between and g) were used in order to reduce plant-to-plant variability. Healthy, viable seeds were surface-sterilized with a 0.2% mercuric chloride solution for 5 min, and then rinsed well in sterile distilled water to remove traces of the sterilizing chemicals. They were then transferred aseptically into Petri dishes on blotting paper moistened with sterile distilled water for germination in darkness at a constant temperature (20 C). During germination, seeds were also inoculated by scattering a suspension of Rhizobium leguminosarum P221 at cells ml" 1. After 5 d, 20 seedlings were transferred in aseptic conditions on a wine mesh support at the top of sterilized flasks (0.18 m depth, volume). The flasks were kept filled with a nitrogen-free nutrient solution so that the whole root system was under submerged conditions. This solution was inoculated with a suspension of Rhizobium leguminosarum to provide a concentration of 10 7 Rhizobium ml" 1. t was completely replaced twice during the experiment and reinoculated. Each flask was covered with a nontransparent plastic sheet to keep roots in darkness. Air was bubbled through each flask to provide aeration and mixing of the nutrient solution. Plants were grown for 6 weeks in a growth chamber with the following conditions: 16/8 h day/ night; 18 C day/18 C night; 90% air relative humidity. Light was provided by cool-white fluorescent tubes (58 W, Sylvania, Germany). The photosynthetically active radiation at the top of the plants was 14.1 J m" 2 s" 1. Measurements on shoots, roots and nodules Developmental plant stages were recorded daily using the measurement scale proposed by Maurer et al. (1966). Length of the primary root, length of the basal unbranched zone, and length of the apical unbranched zone were measured daily by very carefully removing each plant and its root system from the flask. The number of nodules carried by the primary root, and distance between the most distal nodule and the root apex, were also recorded daily. Nodules were recorded as soon as they became visible (>0.5 mm). All plant manipulations were performed under a laminar flow hood to avoid contamination. At the 6 leaf stage and at the beginning of flowering, 10 plants were harvested, and the positions of first-order laterals and nodules along the primary root were recorded. Assumptions tested and calculations Differentiation of lateral roots occurs within the pericycle of the mother root, a few millimetres behind the root tip, whereas emergence of laterals occurs several centimetres behind, thus leading to the existence of an apical unbranched zone (KJepper, 1990). Data were used to test the assumption of a constant time lag between differentiation and emergence of first-order laterals. Figure 1 shows the theoretical relationship between the length of the apical unbranched zone, the root elongation rate, and the time required from primordia formation to appearance of laterals on a root segment. f the duration required from primordia formation to appearance of laterals is constant, then the relationship between the length of the apical unbranched zone (LAUZ) and the root elongation rate (RER) is expected to be linear. A similar assumption can be tested for the appearance of nodules. f the time lag between Rhizobium infection and nodule appearance is constant, then the relationship between length of the apical zone without nodule (LAZWN) and root elongation rate is expected to be linear. Results Elongation of the primary root Figure 2 shows the average length of the primary root versus time after the beginning of seed imbibition. The elongation curve was almost linear during the first 18 d, with an average root elongation rate of 22 mmd" 1. LAUZ, i \ \± 1 LAUZ Fig. 1. Theoretical relationship between length of the apical unbranched zone, parent root elongation rate and the time required from pnmordia formation to appearance of laterals on a root segment. /,: date at which lateral primordia differentiated in the considered root segment; l 2 : date at which laterals emerged on this root segment. Length of the apical unbranched zone (LA UZ) is the sum of the distance from the root tip to the root segment when lateral primordia differentiated (LAUZ 0 ) and the root length increase between t l and t 2. Thus, LAUZ = LAUZ 0 + RERy.(t 1 t l ), where RER is the average root elongation rate between t, and t 2 (adapted from Pellerin and Tabourel, 1995).

3 Root growth and nodulation on pea T 4.07 X r" = E E S Primary root length Lateral front Days after seed mbibition RER (mm per day) 40 Fig. 2. Length of the primary root, and position of the front of firstorder lateral emergence, versus the number of days after the beginning of seed imbibition. Vertical bars represent ± one standard deviation. Arrows indicate plant stages: ll: one leaf; 4L: four leaves; 6L: six leaves; BF. beginning of flowering. Elongation rate drastically decreased between 18 and 25 d, and started again thereafter. t definitely stopped after 35 d. Such an elongation curve was observed on all individual primary roots. Emergence of first-order laterals First-order laterals appeared acropetally on the primary root. Basal and apical unbranched zones were clearly visible on all primary roots, and at all sampling dates. Since laterals appeared acropetally, the length of the basal unbranched zone did not vary during the experiment (data not shown). ts average length was 3 mm. By contrast, length of the apical unbranched zone varied widely between sampling dates. Values ranged between 5 and 185 mm. Figure 3 shows the relationship between the length of the apical unbranched zone and the root elongation rate calculated as proposed in Fig. 1. A rough linear relationship was observed, thus supporting the hypothesis of a constant time lag required from primordia formation to Fig. 3. Length of the apical unbranched zone (LAUZ) versus primary root elongation rate (RER). appearance of laterals. According to Fig., this delay is given by the slope of the regression line (about 4 d under our experimental conditions). The intercept of the regression line was statistically different from zero (T=3.22, p> 7 = ). t measures the distance between the most apical lateral and the root tip when primary root elongation rate is zero. t also shows the distance between the root tip and the root segment where lateral primordia differentiate. n our set of data it was about 11 mm. Density and length of first-order laterals Figure 4 shows the density of first-order laterals (number of laterals mm" 1 of primary root) and their average length versus the distance from the root base. Only plants harvested at the beginning of flowering were used for these calculations. Both curves have a similar shape, with the density of laterals and their average length being minimal between 160 and 200 mm from the root base. Considering the position of the front of emergence of laterals, it appears that laterals on this less densely branched root segment were produced just before the decline of the primary root elongation rate (Fig. 2).

4 1938 Tricot eta\. Density of first-order laterals Distance from the root base (mm) ,1 500 Fig. 4. Number of first-order laterals per millimetre of primary root and average length of first-order laterals versus distance from the pnmary root base Counting of first-order laterals was performed on root segments of 20 mm. Vertical bars represent!one standard deviation Appearance of nodules Nodules appeared on the root systems of all plants studied, thus confirming that these conditions were suitable for nodulation. Nodules were found mainly on the primary root, but some first-order laterals also were nodulated (Fig. 5). Nodules appeared roughly but not strictly acropetally. As for root branching, basal and apical non-nodulated zones were visible. Average length of the basal zone without nodules was constant throughout the experiment (data not shown). By contrast, length of the apical zone without nodules varied between observation dates. Figure 6 shows the relationship between the length of the apical zone without nodules and root elongation rate, calculated as indicated in Fig. 1. Only roots whose nodulated zone was elongating were considered for this calculation. A linear relationship exists between the length of the apical zone without nodules and the root elongation rate. This supports the hypothesis of a constant lag time required from Rhizobium infection to the appearance of a macroscopic nodule. This delay is given by the slope of CM i: m Number of laterals per root segment of 20 mm a Number of laterals carrying nodules e z L li..ll 1 1.ill. 1 p in CM Distance from the root base (mm) Fig. 5. Total number of first-order laterals per segment of 20 mm, and number of them carrying nodules, versus distance from the primary root base.

5 Roof growth and nodulation on pea > 200 dott«d linei y-7.71jc r'« W / «70 c O) 50 D / D S Root elongation rate (mm per day) Class of root elongation rate (mm/day) >40 Fig. 6. Length of the apical zone without nodules {LAZWN) versus primary root elongation rate. The dotted line is the regression line obtained when the intercept is imposed to zero (y = 7.7l.v; ^ = 047). the regression line. The distance between the root tip and the root segment where infections occur is theoretically given by the intercept. However, because experimental points were missing at low elongation rates, the intercept could not be estimated with accuracy. According to the literature, infections mainly occur at a short distance behind the root tip (about 10 mm) (Bhuvaneswari, 1981; Bhuvaneswari et ai, 1980, 1981; Pueppke, 1986). The delay between infection and the appearance of a macroscopic nodule can, therefore, be approximated by the slope of the regression line passing through the origin, which is 7.7 d for our experimental conditions. Density of nodules At the beginning of flowering, the average density of nodules within the nodulated segment of the primary root (i.e. the root segment between the most proximal and the most distal nodule) ranged between 0.04 and 0.25 nodules mm" 1, depending on the plant. Nodules, however, were unevenly distributed within this nodulated segment. Some Fig. 7. Percentage of root segments carrying nodules per class of root elongation rate. non-nodulated root segments were found within the nodulated zone. The question thus arises as to the reason for this. n Fig. 7, the assumption of a link between the presence or absence of nodules and primary root elongation rate was tested. Each root segment was characterized by (i) presence or absence of nodules at the beginning of flowering and (ii) root elongation rate between infection and actual (or theoretical) emergence of nodules on the root segment under consideration. Calculations were performed using the delay between infection and emergence described above. Apical root segments, on which nodules could not have appeared because the time elapsed since infection was lower than the time required for nodule appearance, were discarded. The percentage of root segments carrying nodules was plotted for each root elongation rate class (Fig. 7). Percentage of root segments carrying nodules was found to increase with root elongation rate. Discussion Strong relationships were found between nodule establishment and root system growth, thus emphasizing the

6 1940 Tricot et al. importance of considering the latter in nodulation studies. An unexpected decline in primary root elongation rate was observed between 18 and 25 d after seed imbibition which could not be attributed to an artefact. Growing conditions were constant during the experiment, and this decline did not occur when primary roots reached the bottom of the flasks. Bourdu and Gregory (1983), Deleens et al. (1984) and Derieux et al. (1989) have observed a decline of maize root elongation rate at the beginning of the translocation of photosynthates from leaves to roots. A possible explanation is that the reduction of primary root elongation rate observed in this experiment corresponded to the exhaustion of seed reserves. Actually weighing seeds has shown that, at this moment, the percentage of utilization of seed reserves was 86%. Carbon shortage due to the transition from the heterotrophic to the autotrophic phase is very likely to occur in growth chambers because of the low radiation, compared to field conditions. The decline of primary root elongation rate was preceded by a reduction in branching rate and elongation of first-order laterals, suggesting a link between these processes. Carbon shortage at the time of transition from the heterotrophic to the autotrophic phase, which may be involved in the decline of primary root elongation rate, may also explain a lower branching rate and elongation of laterals. n this regard, Bingham and Stevenson (1993) observed that the number of lateral wheat root primordia was affected by carbohydrate supply. Reduction of branching rate and elongation of laterals occurred just before the decline of primary root elongation rate, suggesting that the primary root has priority for carbon allocation. This is consistent with the findings of Aguirrezabal et al. (1993), who observed with sunflower that the elongation rate of the taproot was less reduced at low irradiance than the elongation rate of first-order laterals. Consistent with results obtained for other species (MacLeod, 1990; Pellerin and Tabourel, 1995), these results suggest that a constant time lag exists between the differentiation of first-order lateral roots and their emergence (4 d under this study's conditions). The estimated distance between the root tip and the root segment where lateral primordia differentiate was 10.6 mm. These data are in agreement with those of MacLeod and Thompson (1979), who observed with pea the most apical root primordium to be located between 11.8 and 18.8 mm behind the root tip. These findings suggest that the most apical laterals which arise on roots whose elongation stopped correspond to the last primordium which differentiated behind the root tip. This observation is consistent with that of Abadia-Fenoll et al. (1986), who showed that lateral primordia can differentiate only after root tissues have reached a certain level of maturity. Nodules appeared roughly but not strictly acropetally on the primary roots. A linear relationship was found between length of the apical part of the root without nodules and root elongation rate. This strengthens the assumption of a constant time lag between infection and appearance of nodules (about 7.7 d under our experimental conditions). The not strictly acropetal sequence of appearance of nodules may be due either to a not strictly acropetal sequence of infections or to slight variations in the delay between infections and appearance of macroscopic nodules. A relationship was found between the presence/absence of nodules on a root segment and root elongation rate during the period between infection and appearance of nodules on the considered root segment. The proposed interpretation, which should be tested by further studies, involves the regulation of both processes by carbohydrate availability. Calvert et al. (1984) observed that many infections formed on soybean roots, but relatively few developed into nodules. Kosslak and Bohlool (1984) have shown that the number of successful infections may be affected by photosynthetic capacity of the host plant. Kasperbauer et al. (1984) and Kasperbauer and Hunt (1994) showed for soybean and southern pea that greater photoassimilate allocation to roots was associated with formation of more nodules. Moreover, Murphy (1986) showed for pea and other legumes that increasing CO 2 atmospheric concentrations from 330 to 0 [A " 1 was associated with the formation of more nodules. On the other hand, root elongation was shown to be dependent on the short-term on carbohydrate availability (Aguirrezabal^al., 1994; Bingham and Stevenson, 1993). Under these experimental conditions, variations in primary root elongation rate probably reflected carbon allocation to the root system. Therefore, the link between elongation rate of the primary root and presence/absence of nodules may reflect the limitation of both processes by carbohydrate availability. References Abadia-Fenoll F, Casero PJ, Lloret PG, Vidal MR Development of lateral primordia in decapitated adventitious roots of A lium cepa. Annals of Botany 58, Aguirrezabal LA, Pellerin S, Tardleu F Carbon nutrition, root branching and elongation: can the present state of knowledge allow a predictive approach at a whole-plant level? Environmental and Experimental Botany 33, Aguirrezabal LA, Deleens E, Tardieu F Root elongation rate is accounted for by intercepted PPFD and source-sink relations in field and laboratory-grown sunflower. Plant, Cell and Environment 17, Bhuvaneswari TV Recognition mechanisms and infection process in legumes. 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