JXB Advance Access originally published online on September 25, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 54, No. 392, pp. 2457-2465,
November 1, 2003
© 2003 Oxford University Press
Can sucrose content in the phloem sap reaching field pea seeds (Pisum sativum L.) be an accurate indicator of seed growth potential?
Received 12 May 2003; Accepted 17 July 2003
INRA, Unité de Génétique et d’Ecophysiologie des Légumineuses, BP 86510, F-21065 Dijon Cedex, France
* To whom correspondence should be addressed. Fax: +33 (0)3 80 69 32 62. E-mail: munierjo{at}dijon.inra.fr
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The composition of the translocates reaching the seeds of pea plants having various nitrogen (N) nutrition regimes was investigated under field situations. Sucrose flow in the phloem sap increased with the node number, but was not significantly different between N nutrition levels. Because N deficiency reduced the number of flowering nodes and the number of seeds per pod, the sucrose flow bleeding from cut peduncles was divided by the number of seeds to give the amount of assimilates available per seed. The sucrose concentration in phloem sap supplied to seeds at the upper nodes was higher than that at the lower nodes. The flow of sucrose delivered to the seeds during the cell division period was correlated with seed growth potential. Seeds from the more N-stressed plants had both the highest seed growth rate and received a higher sucrose flux per seed during the cell division period. As seed growth rate is highly correlated with the number of cotyledonary cells produced during the cell division period, sucrose flow in phloem sap is proposed to be an important determinant of mitotic activity in seed embryos. The carbon (C)/N ratio of the flow of translocates towards seeds was higher under conditions of N-deficiency than with optimal N nutrition, indicating that N flux towards seeds, in itself, is not the main determinant of seed growth potential.
Key words: C/N ratio, mitotic activity, nitrogen nutrition, pea, phloem, Pisum sativum, seed growth rate, sucrose.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Field pea is the most cultivated legume species in Europe. However, pea yields are still low and highly variable. Pea yield is highly correlated with numbers of seeds obtained per square metre but, for a given genotype, the effect of the environment on the mean seed weight explains the remaining variability (Munier-Jolain and Ney, 1995): amongst abiotic stresses, impaired nitrogen (N) nutrition appears to be the most detrimental for pea seed yield (Salon et al., 2001).
Seed development is characterized by three developmental stages: fertilization, the end of seed abortion, which corresponds to both the end of cell division and the beginning of storage reserve accumulation, and physiological maturity (Ney et al., 1993). Consequently, seed development comprises two successive phases: during the first phase, which starts at flowering and ends at the beginning of seed filling (Ney and Turc, 1993), little biomass accumulates, but there is high mitotic activity in the seed embryo (Ney et al., 1993) and seeds can abort during this cell division period. The second phase begins when cell divisions cease in the embryo and thereafter most of the seed biomass is accumulated up to physiological maturity. As seeds beginning to fill can no longer abort, seed number is already set at the start of seed filling (Duthion and Pigeaire, 1991). The three developmental stages (fertilization, the end of seed abortion and physiological maturity) progress linearly along the stem versus cumulative degree-days (Ney and Turc, 1993). Progression rates differ between stages, the later the stage, the higher the progression rate. However, progression rates do not depend upon environmental conditions.
Seed size is the product of seed growth rate by seed growth duration. Seed growth rate, in contrast to duration of seed filling, is rather insensitive to the availability of assimilates during seed filling (Munier-Jolain et al., 1998). Seed growth rate is highly correlated with the number of cotyledonary cells that have been produced during the cell division period (Munier-Jolain and Ney, 1998). As the progression rate of flowering is lower than the progression of the beginning of seed filling, the duration of the cell division period of upper seeds is shorter than that of seeds produced earlier. At a given node, even if the duration of the cell division period varies slightly for a given genotype under optimal growth conditions (Ney and Turc, 1993), this duration might also differ according to environmental constraints such as N nutrition (Sagan et al., 1993). Hence, although the duration of the cell division period is shorter for upper seeds, these are larger as compared with seeds of the first reproductive nodes (Munier-Jolain and Ney, 1998). This implies that mitotic activity increases with the position of the reproductive node. To date, several physiological explanations of the variations of mitotic activity in seeds as a function of the nodal position have been cited: (i) either ABA (Myers et al., 1992), gibberellins and auxin (Swain et al., 1997; Ozga et al., 2002) or cytokinins (Binns, 1994) have been proposed to control seed mitotic activity; (ii) environmental factors such as temperature (Jones et al., 1985); (iii) partially, the carbon (C) and N supply (Singh and Jenner, 1984; Egli et al., 1989; Munier-Jolain and Ney, 1998) provided by the plant to the embryos during cell division. Concerning this latter proposal, it has been demonstrated that the composition of the assimilates reaching seeds depends upon the mode of N nutrition (Rochat and Boutin, 1991), and may be affected by the efficiency of N remobilization and environmental factors such as temperature.
In order to study the determinism of seed growth potential, the objectives of this study were (i) to determine the composition (quantity and quality) of the flow of assimilates reaching seeds during the cell division period, (ii) to detect any modification of this flow by either the N nutrition regime or dependence upon nodal position, and (iii) to relate such variations, if any, to seed growth potential. This study was carried out in the field in order to compare both the feasibility of the sap sampling method employed and the correspondence of the data with those obtained under controlled conditions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Site
Experiments were carried out in 1997 in Dijon (France, 47°20’ N lat.) on a clayey calcic brown soil (clayey eutric Cambisol). Soil clay content and CaCO3 content were, respectively, 41.6% and 0.1
in the ploughed layer (0–35 cm). At sowing, the soil contained 2.7 g N m–2 inorganic N and soil organic N content was less than 1.5. Adequate P, K and Mg fertilization was supplied (P2O5, K2O and MgO supplied as 10.4, 10.4 and 4.9 g m–2, respectively) during the autumn that preceded the experiment. The field was well irrigated in order to avoid moisture stress. Rhizobium leguminosarum was naturally present in the soil, precluding any inoculation.
Treatment and experimental design
Both a nodulated pea (Pisum sativum L.) ‘Frisson’ and its non-nodulating P2 mutant (Duc and Messager, 1989) were used in order to vary the mode and/or the level of N nutrition: ‘Frisson’ either relied exclusively on symbiotic fixation (F-0 treatment) or was provided with 25 g N m–2 at sowing (F-25 treatment). The P2 mutant was provided with mineral N applied as NH4NO3: at sowing, 5 g N m–2 were mechanically spread for the P2-5 treatment, while the P2-25 and P2-50 treatments received 25 g N m–2. Subsequently the P2-50 treatment received an additional amount of 25 g N m–2 at the flowering stage.
On 15 March, 1997, peas were sown at a density of 80 m–2, in a randomized split-plot design with crops as main plots, N treatments as sub-plots and with four replications. Each plot had six rows of plants spaced 0.2 m apart. Border plots of peas sown at the same density surrounded the experimental plots. A low-level-15N enrichment of the soil was used to estimate symbiotic N fixation: 0.5 g m–2 of 15N labelled ammonium nitrate was dissolved in water and sprayed at a rate of 30 ml m–2 (Duc et al., 1988), the 15N isotope dilution technique allowed the contribution of symbiotic fixation to overall N acquisition by the crops to be calculated.
Sampling procedures and measurements
Harvest and development: Three times a week, from the vegetative stage until the end of seed filling, aerials parts of pea plants were collected from the four 1 m long central rows. Ten plants were randomly selected for observations of development that were performed on the main stem since branches have similar development (Jeuffroy and Sébillotte, 1997): according to the development stage, the number of flowering nodes and the number of nodes that had reached the stage of early seed filling were recorded. Progress through the growth cycle was expressed as cumulative degree-days from emergence, using 0 °C as the base temperature (Etévé and Derieux, 1982; Ney and Turc, 1993). Seed water concentration was measured to assess the duration of both reproductive phases, duration of cell division and duration of seed filling (Ney et al., 1993). The beginning of seed filling and physiological maturity corresponded with seed water concentrations of 0.80 and 0.55 g g–1, respectively (Le Deunff and Rachidian, 1988; Dumoulin et al., 1994). These dates were determined for each of the nodes studied (see below), by using linear regressions of seed water content decrease.
Sap harvesting: After the beginning of flowering, during the cell division period according to Ney et al. (1993), phloem sap was collected on 3 d a week from peduncles of various reproductive nodes, located at the bottom, in the middle and at the top of the main stem: for all the treatments the ‘bottom node’ was the first reproductive node. Because of the variations among treatments of crop growth due to the various levels of N nutrition, for P2-5 and P2-25 the medium and top nodes were, respectively, the third and fifth ones, while the fifth and seventh reproductive nodes were selected for P2-50, F-0 and F-25. Eppendorf tubes (1.5 ml total volume) containing 0.5 ml of an EDTA/HEPES buffer solution (5 mM/10 mM; pH 7.5) were used in order to harvest phloem sap exuding from the freshly cut peduncles (King and Zeevaart, 1974): three plants for each replicate (i.e. 12 plants in total) were randomly selected and a razor blade was used to cut the peduncles, the cut surface was quickly rinsed with the EDTA/HEPES buffer solution. Peduncles were then immersed in buffer in the Eppendorf tubes. Supple masking rubber was used to fix the tubes onto the peduncles, carefully but tightly, so that they could not fall during 6 h of sap harvesting. After that time, the Eppendorf tubes were recovered, placed on ice and subsequently frozen at –18 °C for further analysis.
Sap analysis: The major organic compounds in the sap were determined by enzymatic analysis: soluble sugars (sucrose, glucose and fructose) were assayed by using ß-fructosidase, hexokinase, glucose-6-P dehydrogenase and phosphoglucose isomerase (Roche Diagnostic, Meylan, France) as previously described (Bergmeyer and Bernt, 1974). Amino acid concentrations in the phloem sap were measured by derivatization (Moore and Stein, 1968). C/N ratio of the saps were calculated using the molar concentration of C and N atoms in the sugars and amino acid fractions.
Dry matter and C and N content: Total dry matter was determined after oven-drying at 80 °C for 48 h. Samples were ground and N content was determined by the Dumas procedure using a CHN analyser (Carlo Erba), 15N enrichment of samples was measured with a single inlet double collector mass spectrometer (Fison Isochrom, Micromass, Lyon, France) operating in line with a CHN analyser (Carlo Erba).
For each reproductive node studied, seed number was counted and seed dry matter was determined after oven drying at 80 °C for 48 h. Individual seed growth rates were assessed as the slopes determined by linear regression of individual seed weight versus thermal time during the filling period defined by water concentration.
Calculation of symbiotic N fixation
Using the isotope dilution technique (Amarger et al., 1977; Mariotti et al., 1983), the percentage of fixed N (%Ndfa) was calculated using as control plants the non-nodulating mutants of Frisson (P2) which were provided with similar amounts of N-fertilizers:
%Ndfa=100(
15Nlegume–15Ncontrol plant)/(
fix–15Ncontrol plant)
where 15Nlegume is the isotopic composition of the legume-fixing plant expressed as a percentage; 15Ncontrol plant is the isotopic composition of the non-fixing plant P2 expressed as a percentage; fix (–1 for Pisum sativum) is the isotopic enrichment factor associated with symbiotic N fixation. (Mariotti et al., 1980, 1983).
Calculations and statistical analysis
Analysis of variance was performed with the GLM and NLIN procedures of SAS and means were classified using the least significant difference test (LSD) at the 0.05 probability level (SAS Institute, 1987).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Variation of the N nutrition level
In order to relate variations of the sap composition reaching seeds from the various plant nodes to the level of N nutrition, the pea genotype Frisson and its non-nodulating isogenic mutant P2 were used. Frisson, relying on atmospheric and soil N resources (F-0), acquired across the growth cycle 89±3% of its total N through symbiotic N fixation. When provided with a supplemental amount of N-fertilizer (25 g N m–2; treatment F-25) the contribution of symbiotic N fixation to overall N accumulation by the crop dropped to about 44%. At sowing, P2 was provided with either 50 g N m–2 mineral N supply (treatment P2-50), a dose sufficient to sustain all N needs (Sagan et al., 1993) or with limiting amounts such as 25 g N m–2 (treatment P2-25) and 5 g N m–2 (treatment P2-5). Throughout its growth cycle, P2 relied exclusively on soil mineral N.
As already reported (Greenwood et al., 1991) N was progressively diluted in the biomass as it accumulated in plants during the growth cycle (Fig. 1). The N nutrition level of the peas was estimated using the critical N dilution curve (Ney et al., 1997). The dilution curve (Fig. 1) indicates the optimal N nutrition level, defined as the lowest N concentration leading to the maximum biomass production. When P2 received only 5 g N m–2, its growth was greatly impaired and its N content in the shoot was lower than the one expected from the critical dilution curve (Fig. 1). When P2 received 25 g N m–2, its growth rate was optimal to approximately 800 g m–2, which occurred during the cell division period. According to the dilution curve, after this date the crop growth rate was impaired, which indicates that the crop was N-deficient during the seed-filling period. For all the other treatments, experimental values were located close to the dilution curve, demonstrating that plants were never N-stressed during the growth cycle (Fig. 1).
|
Phloem sap reaching the pods
Throughout the growth cycle, in order to analyse variation in assimilate delivery as a function of the seed position on the plant, phloem sap was collected from the pod peduncles of different reproductive nodes. Because plants were not subjected to similar N-nutrition levels, their development and the number of reproductive nodes were different. As a consequence, different nodes were chosen as the medium and higher reproductive nodes in different plants (see Materials and methods). Phloem sap sugars consisted mostly of sucrose, accounting for the bulk of total sugars, with hexoses representing a minor fraction of C weight at every date and for all of the treatments (data not shown). Because flowering progressed linearly along the main stem, phloem sap reaching seeds during the cell division period was harvested later for the upper nodes (Fig. 2). As the beginning of seed filling progressed quicker than flowering did along the main stem, the cell division period was shorter as the number of reproductive nodes increased: as a consequence, the number of phloem sap harvests was reduced for higher nodes (Fig. 2). Between 40 and 160 nmol sucrose were delivered h–1 to the pods of the various nodes and the flow of this carbohydrate was rather constant throughout the duration of the cell division period, whichever treatment or reproductive node is considered (Fig. 2).
|
Whatever the mode (i.e. P2 versus ‘Frisson’ treatments) or the level of N nutrition (i.e. comparison of different P2 treatments), although for some dates the sucrose flux in phloem sap was significantly different between the lower and upper nodes, most of the points were not significantly different (Fig. 2). However, sucrose flux in phloem sap tended to show a positive gradient from the bottom to the top of the stem (Fig. 2). Because of a large frequency of seed abortion affecting the lowest reproductive nodes of F-25, sucrose flux reaching these pods was not considered as representative of the translocate flow and has therefore been disregarded.
Phloem sap reaching seeds
The phloem sap harvested from cut peduncles does not reflect adequately the flow of assimilates reaching individual seeds, as the various N-nutrition regimes resulted in different numbers of seeds per node. Hence, sucrose flux was divided according to the number of seeds borne by the pods of the reproductive nodes at each sucrose sampling date, in order to get an accurate estimate of sucrose flow towards seeds during the cell division period (Table 1). Summarizing all treatments, sucrose flux per seed rose with the position of the reproductive node that was harvested (Table 1). For a given reproductive node, sucrose flux per seed of the phloem sap was significantly higher for the N-stressed treatment (P2-5) as compared with the other treatments, and the F-0 treatment always displayed the lower sucrose flux per seed (Table 1).
|
Seed growth rate as a function of nodal position and N-nutrition regime
Seed growth rate varied both intra-plant and between treatments (Fig. 3): as an example, the growth rate of seeds borne by the lowest reproductive node of the P2-5 treatment (0.393 mg degree-days–1) was much higher than that of the same node on F-0 (0.275 mg degree-days–1). As the number of cells being formed prior to seed filling is correlated with seed growth rate (Munier-Jolain and Ney, 1998), an attempt was made to relate the seed growth rate to the flux of sucrose into seeds during the cell division period (Fig. 3). A hyperbolic relationship was established between the seed growth rate of both different nodes and the various treatments, and the sucrose flux per seed during the cell division period (Fig. 3). When the inverse of seed growth rate was plotted against the reciprocal of sucrose flux per seed, a linear relationship was established, suggesting Michaelis–Menten kinetics (Fig. 3 insert).
|
C/N ratio of phloem sap and seed
Sucrose and amino acid concentrations of phloem sap were used to calculate the C/N ratio reaching the seeds of the medium reproductive node during the seed cell-division period. Because they are the more contrasted concerning both sucrose flux in the phloem sap per seed and seed growth rate (Fig. 3; Table 1) only the phloem sap C/N ratios supplying the reproductive nodes of P2-5 and F-0 treatments are presented (Fig. 4A). The C/N ratio remained relatively low, less than 20, during the cell division period for the F-0 treatment (Fig. 4A). By contrast, for the low N treatment, (P2-5), the C/N ratio of phloem sap was larger throughout the cell division period (Fig. 4A).
|
The C/N ratio of the seeds borne by the medium reproductive node whose phloem sap was harvested, was determined (Fig. 4B). Seed C/N ratio was always lower than that of the phloem sap: the C/N ratio of seeds from the pea genotype whose N-nutrition was deficient (P2-5) increased throughout the study while the C/N ratio of the seeds from F-0 remained much lower (Fig. 4B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Variation of N nutrition level
Treatments F-0 and F-25 and P2-50 had similar biomass and N accumulation throughout the growth cycle. It has already been shown (Duc et al., 1988) that when providing a fertilization of 50 g m–2 to the non-nodulating pea mutant, similar seed yields to the nodulating WT line were obtained. For these treatments, a diagnosis of the N nutrition level was made with the dilution curve, which demonstrated that there never was any N stress during the growth cycle (Fig. 1). This result is supported by earlier studies with the same (Sagan et al., 1993) or other pea genotypes (Voisin et al., 2002) indicating that a similar level of N nutrition may result whatever the mode of N acquisition by the crop. Supplying P2 with a limited amount of N fertilizer (5 g N m–2) resulted in reduced biomass and N accumulation, and the plants displayed N stress symptoms early during their growth cycle, whereas supplying P2 with 25 g N m–2 delayed N-deficiency after flowering. It has previously been shown that seed N concentration at maturity is significantly lower for crops suffering from N-deficiency (either throughout the growth cycle or only during the reproductive period) than for crops with an optimal N-nutrition level (Lhuillier-Soundele et al., 1999). Those results demonstrate that assimilate fluxes towards the seed are affected by N-nutrition levels.
Intra-plant variations in N-nutrition levels of phloem sap reaching pods
At every date and for all of the treatments, a very low amount of hexoses was recovered in phloem sap and sucrose predominated. Although recent studies demonstrated that invertase is only present significantly in wounded stem tissues (Zhang et al., 1996) the possibility of solute leakage (monosaccharides and sucrose) from either cells surrounding phloem or broken cells cannot be excluded (Atkins, 1999) and compounds collected using the EDTA technique cannot be considered unambiguously as representing those present in a pure phloem sap (King and Zeevaart, 1974; Pate et al., 1974). By contrast with some other legumes (Pate et al., 1974) pea does not show prolonged exudation. Thus, only aphids (Kennedy and Mittler, 1953; Atkins, 1999) or isotopic labelling of the neighbouring leaf (using 14CO2 and comparing the specific activity of the source 14CO2 with that of sucrose recovered in the EDTA solution) could prove that phloem sap was pure. These techniques, however, could not be used in this study carried out in the field. The concentration of sucrose and amino compounds found in phloem sap in that study were close to that found in lupin (Lupinus albus L; Pate et al., 1979a), and soybean (Glycine max L. Merr.; Layzell and LaRue, 1982) at the same stage. However, because the sink has been removed during sap collection, the measured assimilate flow is likely to represent qualitatively, but not quantitatively, the actual translocates reaching seeds, and thus allows comparisons between treatments or nodal positions within the plant.
These field data confirm that the sucrose concentration of phloem sap increases with the node number (Fig. 2), supporting findings obtained earlier on isolated plants under controlled conditions (Pate, 1980). Such an increase may be the consequence of xylem-to-phloem transfer (Pate et al., 1975; Layzell et al., 1981) and/or enhanced contribution of the neighbouring leaves to the C economy of the closest fruits (Pate, 1980). Conversely, no significant differences of sucrose concentration of phloem sap were observed when comparing plants grown under different N-nutrition levels. As concluded by Sagan et al. (1993), a major effect of N starvation was to reduce the number of flowering nodes. Consequently, even if the reduction in dry matter and N accumulation in the vegetative parts due to N starvation reduces photosynthesis (Grima-Pettenati et al., 1987), the sucrose concentration of phloem sap reaching each pod is maintained among treatments because of the reduced number of reproductive sinks (nodes and pods) on N-deficient treatments.
Phloem sap reaching seeds
As observed by Sagan et al. (1993), the lower the N-nutrition level, the smaller the number of seeds per pods. This was indeed the case in this study and, consequently, as the number of seeds aborted differed between both N treatments and genotypes, it was necessary to divide the sucrose flow released from cut peduncles by the number of seeds in order to get a useful comparison of the amount of assimilates available per seed (Table 1). Significant differences concerning sucrose flux per seed at the various nodes of the main stem and among N-nutrition treatments were then demonstrated: phloem sap theoretically feeding seeds from the upper nodes contained more sucrose than that supplying the lower nodes. N-stressed plants (Fig. 1) such as P2-5 translocated more sucrose per unit of time and per seed than well-fed plants such as those of the F-0 treatment.
Sucrose flux in phloem sap and seed growth potential
These data show that the seeds from the more N-stressed plants both had the highest seed growth rates (Fig. 3) and received the higher sucrose flux per seed during the cell division period (Table 1). By contrast, seeds from plants having an optimal N-nutrition (F-0) which had the lowest seed growth rate, fed their seeds with a low sucrose flux (Table 1). Munier-Jolain and Ney (1998) showed a tight relationship between individual seed growth rate and cotyledon cell number. Consequently, individual seed growth rate might be considered as a convenient indicator of cell division between the beginning of flowering and the beginning of seed filling. The relationship between seed growth rate and sucrose flux per seed conformed to Michaelis–Menten kinetics (Fig. 3). Using reciprocal plots (insert of Fig. 3), a maximum seed growth rate of 0.85 mg degree-day–1 and a Km of 23 nmol h–1 seed–1 were calculated. A linear relationship may, however, be employed, instead of the Michaelis–Menten kinetics, for the range of values that were encountered in this study (Fig. 3) and which covered all N-nutrition regimes and nodal positions on the main stem (Table 1).
Beyond any mathematical consideration these results already suggest that the flow of sucrose delivered to the seeds during the cell division period is an important determinant of seed growth potential. This explanation is likely to be valid because, according to the literature, the seed growth rate observed during seed filling is determined during the preceding period of cell division in the embryo and the variability of seed growth rate among treatments and intraplant position is strongly correlated with cotyledon cell number (Munier-Jolain and Ney, 1998). Hence it is tempting to speculate that sucrose may be instrumental in the modulation of seed embryo mitotic activity. Recent knowledge of the plant cell cycle regulation has been gathered and provides a mechanistic explanation. Cyclin-dependent kinase a (CDK-a) forms heterodimers with cyclin D-type proteins (CycD2 and CycD3) and is involved in the progression from the G1 to the S phase during cell division (Den Boer and Murray, 2000). In tomato fruits CDK-a proteins accumulate during the early period of cell division (Joubes et al., 1999). Both phytohormones and sucrose modulate CycD expression. In Arabidopsis, cytokinin activation of the cell cycle is mediated by transcriptional regulation of CycD3 in both cultured cells and intact plants (Soni et al., 1995; Riou-Khamlichi et al., 1999) and in suspension-cultured cells the effect is more marked when sucrose is also added (Soni et al., 1995). Auxin increases CycD2 mRNA levels in pea root tissues (John et al., 1993) and is supposed to be responsible for the induction of the expression of the kinase subunit of the CDK–CycD complex (Soni et al., 1995). In Arabidopsis, CycD2 transcripts are reduced in sucrose-depleted cell suspensions (Hemerly et al., 1993), while CycD2 is induced by C source availability (Soni et al., 1995). CycD3 is also induced by sucrose in both cell-suspension cultures and young seedlings of Arabidopsis, but this appears in late G1 compared with the effect of sucrose on CycD2 (Riou-Khamlichi et al., 2000). Although in this study sucrose level, but not auxin and cytokinin levels, were measured in phloem exudates, this does not preclude that these phytohormones might be involved in the regulation of seed embryo mitotic activity.
Apart from sucrose, high N flux has also been suggested as an important determinant of cell division in meristems. The relative composition of C and N (C/N ratio) in the phloem sap was analysed for the treatments having highly contrasting N-nutrition levels, F-0 and P2-5 (Fig. 4). The C/N ratio of the flow of translocates towards the seeds was much larger for the N-deficient treatment which displayed the higher seed growth potential (Fig. 3) compared with F-0 (Fig. 4). These results indicate that N flux towards the seeds, in itself, is not the main determinant of seed growth potential. The values found for pea genotypes having contrasting N nutrition regimes agree well with earlier published data concerning the C/N ratio of phloem translocates serving fruits as a function of phenology: for nodulated white lupin with an optimal N nutrition (Pate et al., 1979b), the C/N ratio of phloem sap was about 20, a value similar (Fig. 4A) to that found for the pea genotype Frisson (F-0) under similar growth conditions; for nodulated white lupin transiently exposed to argon in order to inhibit symbiotic N fixation (Pate et al., 1984), the C/N ratio of the petiole phloem sap peaked to high values (higher than 200) soon after argon exposure, and returned to values found in the air 4 d later; lastly for non-nodulated white lupin supplied with increasing amount of nitrate (Atkins et al., 1979), the C/N ratio of fruit phloem sap decreased from 50 with a very low concentration of 1 mM nitrate in nutrient solution, to about 20 when the nitrate supply (15 mM and up to 30 mM nitrate in nutrient solution) produced optimal growth, values close to those encountered in this study for P2-5 and F-0, respectively.
The level of N supply had a marked effect on phloem composition and, consequently, on nutrition of the corresponding organs receiving the translocates. Seed C/N ratio during the cell division period (Fig. 4B) was always lower than C/N ratio of phloem sap (Fig. 4A). Although pea pod photosynthesis can improve the economy of C usage in a fruit by as much as 20% (Atkins et al., 1977), pea pods generally have low rates of gas exchange, whether CO2 and water, but are still able to acquire some N from the xylem stream (Flinn et al., 1977). While N carried in the phloem sap is incorporated in seed dry matter with virtually no loss, C can, at the same time, either be incorporated in seed biomass or be lost in the respiration processes. Carbon dioxide derived from seed respiration is, in part, reassimilated by the photosynthetic machinery present in the pods (Flinn et al., 1977; Atkins et al., 1977), which produces a substantial C recovery in the fruit C economy. Despite this recovery, fruits and seeds still accumulate dry matter with a C/N ratio lower than that of phloem sap (Pate et al., 1977; Pate and Layzell, 1981). However, as pointed out above, the use of EDTA could have led to solute leakage from the cells around the phloem. As such, the possibility of an overestimation of the C/N ratio of phloem sap compared with that of the seeds cannot be excluded.
Although tedious, harvesting of phloem exudates was successfully used in a field experiment and contributed to the determination of assimilate distribution among sinks. This technique provided a qualitative view of the assimilates reaching the seeds during the cell division period, which was related to the determinism of seed growth potential. As far as is known, this study is the first one demonstrating, in the field and for a range of contrasted N-nutrition regimes, possible links between sucrose flux in the phloem sap and the establishment of seed growth rate. To increase this understanding, both mitotic activity and the duration of cell divisions in the embryo should be more thoroughly investigated in relation to C and N assimilates and possibly phytohormones (auxin and cytokinins) reaching seeds during the cell division period. For this, isotopic labelling and in vitro studies might prove to be useful tools to vary both the flow of translocates (in quantity and quality, taking into account the C/N ratio) and the environment (temperature) to which the embryos are subjected.
|
Acknowledgements |
|---|
Our grateful thanks are due to V Durey, J Gonthier, C Jeudy, and P Mathey for their excellent technical assistance.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Amarger N, Mariotti A, Mariotti F. 1977. Essai d’estimation du taux d’azote fixé symbiotiquement chez le lupin par le traçage isotopique naturel (15N). Comptes Rendus de l’Académie des Sciences 284, 2179–2182.
Atkins GA. 1999. Spontaneous phloem exudation accompanying abscission in Lupinus mutabilis (Sweet). Journal of Experimental Botany 50, 805–812.
Atkins GA, Kuo J, Pate JS, Flinn AM, Steele TW. 1977. Photosynthetic pod wall of pea (Pisum sativum L.). Plant Physiology 60, 779–786.
Atkins GA, Pate JS, Layzell DB. 1979. Assimilation and transport of nitrogen in nonnodulated (NO3-grown) Lupinus albus L. Plant Physiology 64, 1078–1082
Bergmeyer HU, Bernt E. 1974. In: Methods of enzymatic analysis, 2nd edn. Vol. III. New York and London: Academic Press, Inc., 1176–1179.
Binns AN. 1994. Cytokinin accumulation and action: biochemical, genetic, and molecular approaches. Annual Review of Plant Physiology and Plant Molecular Biology 45, 173–196.[CrossRef][ISI]
Den Boer BGW, Murray JAH. 2000. Triggering the cell cycle in plants. Trends in Cell Biology 10, 245–250.[CrossRef][ISI][Medline]
Duc G, Messager A. 1989. Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation. Plant Science 60, 207–213.[CrossRef]
Duc G, Mariotti A, Amarger N. 1988. Measurements of genetic variability for symbiotic dinitrogen fixation in field grown fababean (Vicia faba L.) using a low level 15N tracer technique. Plant and Soil 106, 262–276.
Dumoulin V, Ney B, Etévé G. 1994. Variability of pea seed and plant development in pea. Crop Science 34, 992–998.
Duthion C, Pigeaire A. 1991. Seed length corresponding to final stage in seed abortion of three grain legumes. Crop Science 31, 1579–1583.
Egli DB, Ramseur EL, Zhen-Wen Y, Sullivan CH. 1989. Source–sink alterations affect the number of cells in soybean cotyledons. Crop Science 29, 732–735.
Etévé G, Derieux M. 1982. Variabilité de la durée de la phase végétative chez le pois (Pisum sativum L.). Application à la sélection de types résistants à l’hiver et à la détermination de la date de semis. Agronomie 2, 813–817.
Flinn AM, Atkins CA, Pate JS. 1977. Significance of photosynthetic and respiratory exchanges in the carbon economy of the developing pea fruit. Plant Physiology 60, 412–418.
Greenwood DJ, Gastal F, Lemaire G, Draycott A, Millard P, Neeteson JJ. 1991. Growth rate and%N of field grown crops: theory and experiments. Annals of Botany 67, 181–190.
Grima-Pettenati J, Bailly-Fenech G, Latché JC. 1987. Etude comparative des migrations d’assimilats chez deux variétés de soja, de type déterminé ou indéterminé. Influence d’une carence en azote. Agronomie 6, 447–456.
Hemerly AS, Ferreira P, de Almeida Engler J, Van Montagu M, Engler G, Inzé D. 1993. cdc2a expression in Arabidopsis is linked with competence for cell division. The Plant Cell 5, 1711–1723.[Abstract]
Jeuffroy MH, Sébillotte M. 1997. The end of flowering in pea: influence of plant nitrogen nutrition. European Journal of Agronomy 6, 15–24.
John PCL, Zhang K, Dong C, Diedreich L, Wightman F. 1993. p34cdc2 related proteins in control of cell cycle progression, the switch between division and differentiation in tissue development, and stimulation of division by auxin and cytokinin. Australian Journal of Plant Physiology 20, 503–526.
Jones RJ, Roessler J, Ouattar S. 1985. Thermal environment during cell division in maize: effects on number of endosperm cells and starch granules. Crop Science 25, 830–834.
Joubes J, Phan TH, Just D, Rothan C, Bergounioux C, Raymond P, Chevalier C. 1999. Molecular and biochemical characterization of the involvement of cyclin-dependent kinase A during the early development of tomato fruit. Plant Physiology 121, 857–869.
Kennedy JS, Mittler TE. 1953. A method of obtaining phloem sap via the mouth-parts of aphids. Nature (London) 171, 528.
King RW, Zeevaart JA. 1974. Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiology 53, 96–103.
Layzell DB, LaRue TA. 1982. Modelling C and N transport to developing soybean fruits. Plant Physiology 70, 1290–1298.
Layzell DB, Pate JS, Atkins CA, Canvin DT. 1981. Partitioning of carbon and nitrogen and the nutrition of root and shoot apex in a nodulated legume. Plant Physiology 67, 30–36.
Le Deunff Y, Rachidian Z. 1988. Interruption of water delivery at physiological maturity is essential for seed development, germination and seedling growth in pea (Pisum sativum L.). Journal of Experimental Botany 39, 1221–1230.
Lhuillier-Soundele A, Munier-Jolain NG, Ney B. 1999. Dependence of seed nitrogen concentration on plant nitrogen availability during the seed filling in pea. European Journal of Agronomy 11, 157–166.[CrossRef]
Moore S, Stein WH. 1968. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. Journal of Biological Chemistry 21, 907–913.
Mariotti A, Mariotti F, Amarger N. 1983. Utilization du traçage isotopique naturel par 15N pour la mesure du taux d’azote fixé symbiotiquement par les légumineuses. Physiologie Végétale 21, 279–291.[ISI]
Mariotti A, Mariotti F, Amarger N, Pizelle E, Ngambi JM, Champigny ML, Moyse A. 1980. Fractionnements isotopiques de l’azote lors des processus d’absorption des nitrates et de fixation de l’azote atmosphérique par les plantes. Physiologie Végétale 18, 163–181.
Munier-Jolain NG, Munier-Jolain NM, Roche R, Ney B, Duthion C. 1998. Seed growth rate in grain legumes. I. Effect of photoassimilate availability on seed growth rate. Journal of Experimental Botany 49, 1963–1969.
Munier-Jolain NG, Ney B. 1995. Variability of seed growth rate for various pea cultivars (Pisum sativum L.) in relation to their seed cell number. In: AEP, ed. Improving production and utilization of grain legumes, Proceedings of the 2nd European Conference on grain legumes, Copenhagen, Denmark, 32–33.
Munier-Jolain NG, Ney B. 1998. Seed growth rate in grain legumes. II. Seed growth rate depends on cotyledon cell number. Journal of Experimental Botany 49, 1971–1976.
Myers PN, Setter TL, Madison JT, Thompson JF. 1992. Endosperm cell division in maize kernels cultured at three levels of water potential. Plant Physiology 99, 1051–1056.
Ney B, Doré T, Sagan M. 1997. The nitrogen requirement of major agricultural crops: grain legumes. In: Lemaire G, ed. Diagnosis of the nitrogen status in crops. Heidelberg: Springer-Verlag, 107–118.
Ney B, Duthion C, Fontaine E. 1993. Timing of reproductive abortions in relation to cell division, water content, and growth of pea seeds. Crop Science 33, 267–70.
Ney B, Turc O. 1993. A heat unit description of the reproductive development of pea (Pisum sativum L.). Crop Science 33, 267–70.
Ozga JA, van Huizen R, Reinecke DM. 2002. Hormones and seed-specific regulation of pea fruit growth. Plant Physiology 128, 1379–1389.
Pate JS. 1980. Transport and partitioning of nitrogenous solutes. Annual Review of Plant Physiology 31, 313–340.[ISI]
Pate JS, Atkins CA, Hamel K, McNeil DL, Layzell DB. 1979a. Transport of organic solutes in phloem and xylem of a nodulated legume. Plant Physiology 63, 1082–1088.
Pate JS, Atkins CA, Layzell DB, Shelp BJ. 1984. Effects of N2 deficiency on transport and partitioning of C and N in a nodulated legume. Plant Physiology 76, 59–64.
Pate JS, Layzell DB. 1981. Carbon and nitrogen partitioning in the whole plant. A thesis based on empirical modeling. In: Bedley JD, ed. Nitrogen and carbon metabolism. The Hague, Boston, London: Martinus Nijhoff/Dr W Junk Publishers, 95–134.
Pate JS, Layzell DB, McNeil DL. 1979b. Modelling the transport and utilization of carbon and nitrogen in a nodulated legume. Plant Physiology 63, 730–737.
Pate JS, Sharkey PJ, Atkins CA. 1977. Nutrition of a developing legume fruit. Functional economy in terms of carbon and nitrogen in a nodulated legume. Plant Physiology 59, 506–510.
Pate JS, Sharkey PJ, Lewis OAM. 1974. Phloem bleeding from legume fruits. A technique for study of fruit nutrition. Planta 120, 229–243.[CrossRef][ISI]
Pate JS, Sharkey PJ, Lewis OAM. 1975. Xylem to phloem transfer of solutes in fruiting shoots of legumes, studied by a phloem bleeding technique. Planta 122, 11–26.[CrossRef]
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JAH. 1999. Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283, 1541–1544.
Riou-Khamlichi C, Menges M, Sandra Healy JM, Murray JAH. 2000. Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Molecular and Cellular Biology 20, 4513–4521.
Rochat C, Boutin JP. 1991. Metabolism of phloem-borne amino acids in maternal tissues of fruit of nodulated or nitrate fed pea plants (Pisum sativum L.). Journal of Experimental Botany 42, 270–214.
Sagan M, Ney B, Duc G. 1993. Plant symbiotic mutants as a tool to analyse nitrogen and yield relationship in field grown peas (Pisum sativum L.). Plant and Soil 153, 33–45.[CrossRef][ISI]
Salon C, Munier-Jolain NG, Duc G, Voisin AS, Grandgirard D, Larmure A, Emery RJN, Ney B. 2001. Grain legume seed filling in relation to nitrogen acquisition: a review and prospects with particular reference to pea. Agronomie 21, 539–552.[CrossRef][ISI]
SAS Institute. 1987. SAS/STAT guide for personal computers, 6th edn. Cary, NC: SAS Institute.
Singh BK, Jenner CF. 1984. Factors controlling endosperm cell number and grain weight in wheat: effects of shading on intact plants and of variation in nutritional supply to detached cultured ears. Australian Journal of Plant Physiology 11, 151–163.
Soni R, Carmichael JP, Shah ZH, Murray JAH. 1995. A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. The Plant Cell 7, 85–103.[Abstract]
Swain SM, Reid JB, Kamiya Y. 1997. Gibberellins are required for embryo growth and seed development in pea. The Plant Journal 12, 1329–1338.[CrossRef][ISI]
Voisin AS, Salon C, Munier-Jolain NG, Ney B. 2002. Effect of mineral nitrogen on nitrogen nutrition and biomass partitioning between the shoot and roots of pea (Pisum sativum L.). Plant and Soil 242, 251–262.[CrossRef][ISI]
Zhang L, Cohn NS, Mitchell JP. 1996. Induction of a pea cell-wall invertase gene by wounding and its localized expression in phloem. Plant Physiology 112, 1111–1117.[Abstract]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. M. Scarpari, L. W. Meinhardt, P. Mazzafera, A. W. V. Pomella, M. A. Schiavinato, J. C. M. Cascardo, and G. A. G. Pereira Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa J. Exp. Bot., March 1, 2005; 56(413): 865 - 877. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||