Do initial S reserves and mineral S availability alter leaf S-N mobilization and leaf senescence in oilseed rape?

Winter oilseed rape is sensitive to S limitation, however few studies have clearly assessed the impact of initial S reserves on the remobilization of leaf N-S compounds and senescence dynamics within the leaves in S limited plants. As a consequence, the impacts of high or low initial S reserves on these parameters, further cross-combined with either high or low S availabilities, were examined using a ¹5N and ³4S double-labelling method associated with a study of gene expression of relevant tonoplastic sulphate transporters (BnSultr4;1 and BnSultr4;2) and a molecular indicator of leaf senescence (BnSAG12/BnCab). Plants with high initial S status and S limitation showed an optimal growth comparable to control plants. Moreover, in response to S limitation, leaf soluble protein content, total S, recently assimilated S (i.e., ³4S) and the sulphate content in the oldest leaves declined, and the expression of genes encoding tonoplastic sulphate transporters were up-regulated. However, compared to control plants, S limitation delayed leaf senescence. These data suggested that in response to S limitation, plants with high initial S were able to sustain optimized leaf growth by increasing endogenous N and S remobilization independently of the leaf senescence process. In contrast, if these low S plants had no initial S reserves, leaf N-S remobilization was not sufficient to allow optimal growth. As a conclusion, our study supports a model where oilseed rape is able to compensate transiently for S limitation through a fine management of leaf N-S remobilization and a delayed leaf senescence dynamics.

Effect of mineral sulphur availability on nitrogen and sulphur uptake and remobilization during the vegetative growth of Brassica napus L.

Because it has a high demand for sulphur (S), oilseed rape is particularly sensitive to S limitation. However, the physiological effects of S limitation remain unclear, especially during the rosette stage. For this reason a study was conducted to determine the effects of mineral S limitation on nitrogen (N) and S uptake and remobilization during vegetative growth of oilseed rape at both the whole-plant and leaf rank level for plants grown during 35 d with 300 microM (34)SO(4)(2-) (control plants; +S) or with 15 microM (34)SO(4)(2-) (S-limited plants; -S). The results highlight that S-limited plants showed no significant differences either in whole-plant and leaf biomass or in N uptake, when compared with control plants. However, total S and (34)S (i.e. deriving from S uptake) contents were greatly reduced for the whole plant and leaf after 35 d, and a greater redistribution of endogenous S from leaves to the benefit of roots was observed. The relative expression of tonoplast and plasmalemma sulphate transporters was also strongly induced in the roots. In conclusion, although S-limited plants had 20 times less mineral S than control plants, their development remained surprisingly unchanged. During S limitation, oilseed rape is able to recycle endogenous S compounds (mostly sulphate) from leaves to roots. However, this physiological adaptation may be effective only over a short time scale (i.e. vegetative growth).

Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability.

The impact of sulphur limitation on the remobilization of endogenous S compounds during the rosette stage of oilseed rape, and the interactions with N availability on these processes, were examined using a long-term (34)SO(4)(2-) labelling method combined with a study of leaf senescence progression (using SAG12/Cab as a molecular indicator) and gene expression of the transporters, BnSultr4;1 and BnSultr4;2, involved in vacuolar sulphate efflux. After 51 d on hydroponic culture at 0.3 mM (34)SO(4)(2-) (1 atom% excess), the labelling was stopped and plants were subject for 28 d to High S-High N (HS-HN, control), Low S-High N (LS-HN) or Low S-Low N (LS-LN) conditions. Compared with the control, LS-HN plants showed delayed leaf senescence and, whilst the shoot growth and the foliar soluble protein amounts were not affected, S, (34)S, and SO(4)(2-) amounts in the old leaves declined rapidly and were associated with the up-regulation of BnSultr4;1. In LS-LN plants, shoot growth was reduced, leaf senescence was accelerated, and the rapid S mobilization in old leaves was accompanied by decreased (34)S and SO(4)(2-), higher protein mobilization, and up-regulation of BnSultr4;2, but without any change of expression of BnSultr4;1. The data suggest that to sustain the S demand for growth under S restriction (i) vacuolar SO(4)(2-) is specifically remobilized in LS-HN conditions without any acceleration of leaf senescence, (ii) SO(4)(2-) mobilization is related to an up-regulation of BnSultr4;1 and/or BnSultr4;2 expression, and (iii) the relationship between sulphate mobilization and up-regulation of expression of BnSultr4 genes is specifically dependent on the N availability.

Dynamics of nitrogen uptake and mobilization in field-grown winter oilseed rape (Brassica napus) from stem extension to harvest. II. An 15N-labelling-based simulation model of N partitioning between vegetative and reproductive tissues.

BACKGROUND AND AIMS: Oilseed rape (Brassica napus) has often been used as a catch crop to deal with the issue of N leaching, but for this to be effective, prediction of the crop's N uptake capability and N partitioning is required. The aim of this work was to build a compartmental model of N dynamics in oilseed rape, based on the kinetic description of N uptake, partitioning and mobilization in each organ. MODEL: In this study, logistic and exponential equations were fitted to the N relations of each compartment, especially the leaf at each node. Data previously obtained from an 15N-labelling field experiment was used to quantify the partitioning of total N content, the allocation of N taken up and subsequent changes in the sink/source status for endogenous N in each tissue throughout the growth cycle. KEY RESULTS AND CONCLUSIONS: This modelling approach provides a unique tool for the quantitative estimation of cycling of endogenous N in relation to changes in N uptake at the whole-plant level. Furthermore, as oilseed rape is known to release large amounts of N to the soil during spring through leaf loss, this model was used to identify potential methods for improving the N harvest index of the crop. Simulations showed that N content or yield could be improved by 15% by optimizing N transfer from vegetative to reproductive tissues and by reducing the residual %N (DW) in abscised leaves.

Developmental changes in shoot N dynamics of lucerne (Medicago sativa L.) in relation to leaf growth dynamics as a function of plant density and hierarchical position within the canopy.

Shoot N concentration in plants decreases as they get bigger, due to the fact that N accumulates less rapidly than dry matter in plants during the plant growth process, leading to an allometric relationship between shoot N content (N(sh)) and shoot mass (W(sh)): N(sh)=a(W(sh))b. The results obtained on lucerne plants growing either under controlled low density conditions or in dense stands under field conditions show that the value of the allometric coefficient b that represents the ratio between the relative N accumulation rate in shoots [dN(sh)/(N(sh)dt)] and the relative growth rate [dW(sh)/(W(sh)dt)], decreases from 0.88 for a low plant density to 0.72 for a dense stand. Therefore, the fractional increase of shoot N per unit of shoot dry matter is lower when plants are in competition for light in dense canopies. This decrease can be entirely explained by the parallel decline in the leaf area per unit of shoot mass. Thus, a remarkably constant linear relationship can be established between N(sh) and leaf area (LA): N(sh)=1.7 g m(-2) LA, regardless of the conditions (low versus high density, controlled versus field conditions). Moreover, in a field dense stand, the comparison of plants with contrasting positions between the top and the bottom of the canopy (dominant, intermediate or suppressed plants), also shows that the difference in N(sh) at similar shoot mass is explained by the proportion of leaf mass to shoot mass. These data support the idea that leaf growth drives the dynamics of shoot N accumulation. These results also indicate that competition for light among individual plants within a dense canopy induces developmental changes in plant morphology (leaf:stem ratio) that explain the differences observed in shoot N concentration. This last observation could be extrapolated to multi-specific plant stands. Therefore, the sharing of N resources among plant species could partially be the result of the sharing of light within the canopy.

Dynamics of nitrogen uptake and mobilization in field-grown winter oilseed rape (Brassica napus) from stem extension to harvest: I. Global N flows between vegetative and reproductive tissues in relation to leaf fall and their residual N.

BACKGROUND AND AIMS: Despite its high capacity to take up nitrate from the soil, winter oilseed rape (Brassica napus) is characterized by a very low N recovery in the reproductive tissues under field conditions. A significant part of the N taken up is lost to the soil in dead leaves during the growth cycle. An accurate description of N dynamics at the whole plant level in each compartment under field conditions should lead to a better understanding of N allocation in B. napus and improvements in the nitrogen harvest index. METHODS: An experiment was conducted in field conditions using sequential weekly 15N labelling to follow N uptake, partitioning and mobilization. Nitrogen labelling (2.5 kg N ha(-1); 10 % excess) was analysed weekly (from stem extension to harvest) to distinguish between uptake of new N (labelled) and mobilized N (unlabelled) in the different plant components. KEY RESULTS AND CONCLUSIONS: N requirements for seed filling were satisfied mainly by N mobilized from vegetative parts (about 73 % of the total N in pods). Determination of the endogenous N flow showed that there was net transfer of N to the pods by leaves (36 %), stem (34 %), inflorescences (22 %) and taproot (8 %). Precise study of N flow from leaves at different nodes revealed the existence of two main groups of leaves in terms of their apparent capacity to mobilize N; 30-60 % and 70-80 % of peak N content occurring during flowering and pod filling, respectively. Moreover, the latter group was found to be the main source of endogenous N from leaves. The mobilization of endogenous N from these leaves was prolonged and concomitant with N accumulation in the pods. A complex pattern of N mobilization from the leaves, to vegetative or reproductive tissues, was revealed. These results will be used to model N partitioning during the growth cycle.

Influence of initial organic N reserves and residual leaf area on growth, N uptake, N partitioning and N storage in alfalfa (Medicago sativa) during post-cutting regrowth.

BACKGROUND AND AIMS: The influence of initial residual leaf area and initial N reserves on N uptake, final N distribution, and yield in alfalfa regrowing after cutting, were studied. METHODS: The effects of two levels of initial residual leaf area (plants cut to 15 cm, with (L+) or without (L-) their leaves) and two initial levels of N status [high N (HN) or low N (LN)] on growth, N uptake and N partitioning, allocation and storage after 29 d of post-cutting regrowth were analysed. KEY RESULTS: During most of the regrowth period (8-29 d after the initial harvest), HN and L+ plants had higher net N uptake rates than LN and L- plants, respectively, resulting in a greater final mineral N uptake for these treatments. However, the final partitioning of exogenous N to the regrowing shoots was the same for all treatments (67 % of total exogenous N on average). Final shoot growth, total plant N content, and N allocation to the different taproot N pools were significantly lower in plants with reduced initial leaf area and initial N reserve status. CONCLUSIONS: Although both initial residual leaf area and initial N reserves influenced alfalfa regrowth, the residual leaf area had a greater effect on final forage production and N composition in the taproot, whereas the N uptake rate and final total N content in plant were more affected by the initial N reserve status than by the residual leaf area. Moreover, N storage as proteins (especially as vegetative storage proteins, rather than nitrate or amino acids) in the taproot allowed nitrate uptake to occur at significant rates. This suggests that protein storage is not only a means of sequestering N in a tissue for further mobilization, utilization for growth or tissue maintenance, but may also indirectly influence both N acquisition and reduction capacities.

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: effects of methyl jasmonate on nitrate uptake, senescence, growth, and VSP accumulation.

The role of methyl jasmonate (MeJa) in promoting senescence has been described previously in many species, but it has been questioned in monocarpic species whether induced senescence is a result of a potential death hormone like MeJa, or a consequence of an increased metabolic drain resulting from the growth of reproductive tissue. In oilseed rape (Brassica napus L.), a polypeptide of 23 kDa has been recently identified as a putative vegetative storage protein (VSP). This polypeptide could be used as a storage buffer between N losses from senescing leaves putatively promoted by methyl jasmonate that might be produced by flowers, and grain filling which occurs later on, while N uptake is strongly reduced. In order to describe causal relationships during Brassica napus L. plant responses to MeJa treatment, a kinetic experiment was performed to determine the order and the amplitude with which general processes such as growth, photosynthesis, chlorophyll content, N uptake, and N storage under the form of the 23 kDa VSP are affected. One of the most immediate consequences of MeJa treatment was the strong reduction of nitrate uptake within 6 h, relative to control plants. However, this was not a specific effect as K(+) uptake was similarly affected. Photosynthesis was reduced later (after 24 h), while chlorophyll content as well as leaf growth also decreased in a similar way. Moreover, this was concomitant with a remobilization of endogenous unlabelled N from senescing leaves to roots. Accumulation of the 23 kDa VSP was induced in the taproot after 24 h of MeJa treatment and was increased 10-fold within 8 d. On the other hand, the reversible effect of a MeJa pretreatment was tested in the long term (i.e. along the growth cycle) using plants previously grown in field conditions induced for flowering. Results show that a MeJa pulse induced a reversible effect on N uptake inhibition. In parallel, protein immunologically related to the 23 kDa VSP was detected in stems with a similar molecular weight (23 kDa), and in flowers and leaves with a molecular weight of 24 kDa. This accumulation was concomitant with the remobilization of both subunits of Rubisco. During stem and pod development, this protein induced by MeJa is fully hydrolysed. The external and intermittent supply of MeJa mimic some of the plant physiological processes previously reported under natural conditions. This suggests that in oilseed rape, methyl jasmonate could be considered as a possible monocarpic senescence factor while accumulation/mobilization of the 23 kDa VSP in taproot could be a marker for the cessation of N uptake and the initiation of a massive leaf senescence.

Integrated physiological and agronomic modelling of N capture and use within the plant.

Today farmers have several constraints to take into account in managing their crops: (i) competitiveness: productivity must be maintained or increased whereas inputs must be decreased, (ii) the environmental consequences of cultural practices: pesticide and fertilizer use must be decreased, and (iii) product quality must be improved and nitrogen nutrition is an important factor in harvest quality. These new constraints sometimes conflict: maximum yield is often obtained with large amounts of N, increasing the risks of N leaching. The determination of rates and dates for nitrogen application must become more precise in this context. Tools are required for the forecasting of crop requirements, the diagnosis of N deficiencies during the crop cycle and breeding of new adapted varieties. Models and diagnosis indicators have been developed to meet these needs, but those relating to nitrogen are often based on empirical relationships. Moreover, the available models and indicators often fail to account for cultivar-specific responses. The improvement of agronomic tools and the breeding of new varieties adapted to new cropping systems should be based on a thorough understanding of the key metabolic processes involved, and the relative contributions of these processes to yield determination in conditions of fluctuating N supply. For both purposes, more information is required about plant and crop N economy. In this paper, the way in which N absorption and use within the plant and crop, plant responses to deficiencies and excesses of nitrogen are taken into account in major agronomic models is described first. The level of sophistication of the modules comprising these models depends on operational objectives. Secondly, the ways in which the most recent molecular plant physiology findings can, and indeed should, be integrated into models at the crop and crop cycle levels are described. The potential value of this approach for improving current agronomic models and diagnostic tools, and for breeding more efficient varieties is also discussed.

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: identification, characterization and immunolocalization of a putative taproot storage glycoprotein.

In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein patterns.

Oilseed rape (Brassica napus L.) is commonly grown for oil or bio-fuel production, while the seed residues can be used for animal feed. It can also be grown as a catch crop because of its efficiency in extracting mineral N from the soil profile. However, the N harvest index is usually low, due in part to a low ability to remobilize N from leaves and to the fall of N-rich leaves which allows a significant amount of N to return to the environment. In order to understand how N filling of pods occurs, experiments were undertaken to quantify N flows within the plant by (15)N labelling and to follow the changes in soluble protein profiles of tissues presumed to store and subsequently to remobilize N. Whereas N uptake increased as a function of growth, N uptake capacity decreased at flowering to a non-significant level during pod filling. However, large amounts of endogenous N were transferred from the leaves to the stems and to taproots which acted as a buffering storage compartment later used to supply the reproductive tissue. About 15% of the total N cycling through the plant were lost through leaf fall and 48%, nearly all of which had been remobilized from vegetative tissues, were finally recovered in the mature pods. SDS-PAGE analysis revealed that large amounts of a 23 kDa polypeptide accumulated in the taproots during flowering and was later fully hydrolysed. Its putative function of storage protein is further supported by the fact that when plants were grown at lower temperature, both flowering, its accumulation and further mobilization were delayed. The overall results are discussed in relation to plant strategies which optimize N cycling to reproductive sinks by means of buffering vegetative tissues such as stems and taproots.

A rapid and reliable method for NO quantification and 15NO/14NO determination using isotope ratio mass spectrometry: an application for the detection of NO synthesis in propionibacteria.

For the last decade, numerous studies have focused on the positive or toxic effects of nitric oxide (NO) in procaryotic and eucaryotic cells. This gas has fundamental roles in neurotransmission, vasodilatation, cytotoxicity, and intestinal motility. The ability to produce NO by intestinal microflora or probiotic bacteria is unknown. In this preliminary study, we present a rapid and reproducible procedure for NO quantification and 15NO/14NO determination (based on the reaction between nitrite and acidic potassium iodide) by isotope ratio mass spectrometry. Using this method, we have demonstrated for the first time in vitro production of NO by a dietary bacterium (Propionibacterium acidipropionici, Pa 1) under anaerobic culture conditions. Using different sources of nitrogen, we have clearly shown that propionibacteria can synthesize NO from reduction of nitrate or nitrite. In our experimental conditions, NO synthase was not involved in NO production by propionibacteria.

Nitrogen and Carbon Flows Estimated by 15N and 13C Pulse-Chase Labeling during Regrowth of Alfalfa.

The flow of 15N and 13C from storage compounds in organs remaining after defoliation (sources) to regrowing tissue (sinks), and 13C losses through root or shoot respiration were assessed by pulse-chase labeling during regrowth of alfalfa (Medicago sativa L.) following shoot removal. A total of 73% of labeled C and 34% of labeled N were mobilized in source organs within 30 d. Although all of the 15N from source organs was recovered in the regrowing tissue, much of the 13C was lost, mainly as CO2 respired from the root (61%) or shoot (8%), and was found to a lesser extent in sink tissue (5%). After 3, 10, or 30 d of regrowth, 87, 66, and 52% of shoot N, respectively, was derived from source tissue storage compounds; the rest resulted from translocation of fixed N2. Overall results suggest that most shoot C was linked to photosynthetic activity rather than being derived from mobilization of stored C in source organs. Furthermore, isotopic analysis of different chemical fractions of plant tissue suggests that between 14 and 58% of the shoot C derived from source tissues was linked to the mobilization of N compounds, not carbohydrates.

Nitrogen Reserve Mobilization during Regrowth of Medicago sativa L. (Relationships between Availability and Regrowth Yield).

An experiment was designed to study the role of N and C reserves on regrowth of the shoots following defoliation of forage species. Starch and N accumulation in root and crown tissue of nonnodulated Medicago sativa L. were modified during regrowth by applying different levels of N and different cutting heights. Plants were obtained with similar crown and root dry weights, but having either low starch and high tissue N or high starch and low tissue N. The plants were then submitted to a second defoliation and supplied with optimal N nutrition, and N flow from reserve was quantified using pulse-chase 15N labeling. Maximum yields following the second regrowth were obtained from those plants having a high tissue N, despite their low level of nonstructural carbohydrate. When N in the roots and crown exceeded 5 mg N plant-1 at the beginning of regrowth, about 68% was translocated to regrowing shoots. Highly significant correlations were also found between the amounts of N available in roots and crown at the beginning of regrowth and (a) the amount of N that was mobilized to new tissues, (b) the amount of N taken up during the regrowth period, and (c) the final shoot yield after 24 d of regrowth. No similar correlations were found for plants that varied in their initial starch content of roots and crown. It is suggested that N reserves were used mainly during the first 10 d after defoliation, and that the resulting aerial growth during this period should be sufficient to restore N2 fixation and/or N uptake to levels equal to those prior to defoliation. These data emphasize (a) the importance of root N reserves in initiating and sustaining new shoot growth, and (b) the need for a re-evaluation of the contribution of C reserves to shoot regrowth.

Effects of low temperature on nitrate uptake, and xylem and phloem flows of nitrogen, in Secale cereale L. and Brassica napus L.

The effect of low temperature on nitrate uptake and subsequent N translocation and cycling was investigated in Secale cereale L. and Brassica napus L. transferred to 7 °C for 9 d or kept at a warm temperature (20 °C). Some plants were grown with a split root system and 15 NO3 - , labelled to measure NO3 - uptake, root-to-shoot and shoot-to-root translocation of N from NO3 - , taken up. Other plants with single-root system were subjected to 15 N pulse-chase labelling to quantify endogenous N remobilization. Lowering growth temperature from 20 to 7 °C reduced nitrate uptake more strongly in rye (-59%) than in winter rape (-28%). A very large proportion of the nitrate taken up was further translocated to shoots in both species. However, lowering the temperature decreased, xylem N translocation by about 60 and 30% in S. cereale and B. napus, respectively. Most root N in the latter species came directly from root absorption, while in rye roots the proportions ascribe able to direct root allocation and phloem flow were well balanced. Cold treatment did not significantly modify the imbalance between the two origins. Mobilization of endogenous N from roots to shoot, estimated by pulse-chase labelling, was limited in B. napus and significant in S. cereale despite a depressive effect of low temperature. In general, low temperature led to an increase in root N concentration in both species. It is Suggested that low temperature may directly affect the nitrate uptake system, and also that N accumulation in the roots (resulting from greater inhibition of N xylem flow than of NO3 - uptake) might increase the inhibition of uptake by higher cellular NO3 , and/or amino acid content. The usefulness (for spring growth) of root N accumulation triggered by low temperature is discussed.