CASE STUDY 5.1  Partitioning of carbon and nitrogen in a legume

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J.S. Pate

Plant composition in respect of carbon and nitrogen
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Figure 1 (A) Transmission electron micrograph of a root nodule; (B) flowering specimen of white lupin (Lupinus albus); (C) nodules on the root system of a white lupin plant. Some of the nodules are cut open to reveal the pink haemoglobin pigmentation typical of actively N2-fixing nodules; (D) fruits of lupin with their ends cut off to induce bleeding from the phloem; (E) 'cryopuncturing' of the fruit of cowpea (Vigna unguiculata) with a needle cooled in liquid nitrogen; (F) phloem bleeding from a cryopunctured fruit

Dry matter of a typical green plant contains a large and relatively constant proportion of carbon (35–50%), virtually all of which is derived from photosynthetic fixation of CO2 by leaves or other green parts. Comparable values for nitrogen in dry matter tend to be more variable, with lowest levels in roots and stems (0.5–1.5%) and considerably higher levels in leaves (3–5%) where much nitrogen is associated with enzymes involved in photosynthesis. Higher levels are en-countered in seeds (6–7%) and other storage organs in which large quantities of protein are being stored.

Most plants rely on nitrate (NO3) or ammonium (NH4+) in the soil as their prime sources of nitrogen but certain leguminous plants gain nitrogen directly from the atmosphere by engaging in symbiosis with N2-fixing bacteria in nodules on their roots (Figure 1A and B). White lupin is one such leguminous plant. Accompanying photographs show a flowering shoot (Figure 1B), nodules (Figure 1C) and pods (Figure 1D) of white lupin (Lupinus albus L.). Subcellular details of a nodule, with bacterial symbionts clearly visible, are seen in a transmission micrograph (Figure 1A).

Requirements of plant parts for carbon and nitrogen

Consider lupin plants which are effectively nodulated and growing in a rooting medium to which all nutrients except nitrogen have been supplied. Allocation of carbon and nitrogen within the plant can then be examined in terms of the interrelationships of two mutually dependent, self-supporting (autotrophic) processes — photosynthesis and N2 fixation. Within such a system, nodules supply further fixed nitrogen only so long as shoots export photosynthetically produced sugars. Shoots provide energy for nitrogen fixation, as well as carbon skeletons for synthesis of amino acids and other nitrogenous solutes which carry fixed nitrogen from nodules to other plant parts. Conversely, increasing demands for photosynthetic products as plants progress through their life cycles can be met only if nitrogen from nodules continues to be available for expansion and functioning of new photosynthetic surfaces.

One complication which pervades experimental study on a plant such as lupin is that the carbon and nitrogen status of differently aged parts of the plant change continuously over a growth cycle. This is principally because new, actively growing tissues continuously come on stream as users of carbon and nitrogen whereas older plant parts progressively lose dry matter and especially nitrogen as they approach senescence. Because of their high protein content and relatively unthickened walls, young tissues require nitrogen rather than carbon. At the same time, nitrogen can be mobilised most effectively back into the plant from older organs prior to their senescence, since this nitrogen is attached mostly to degradable molecules such as protein. Conversely, carbon in dry matter is associated mostly with the non-degradable fabric of cell walls and is accordingly retrieved to a very limited extent prior to organ and tissue death. Thus, large proportions of the already accumulated resources of nitrogen within a plant are continuously recycled from old stem and leaf tissue to new leaves and eventually to fruits. Such recycling occurs to a much smaller extent with respect to carbon.

Before one can understand how carbon and nitrogen are allocated to different organs over a prescribed interval of growth of a plant such as lupin, quantitatively accurate information is required on the following:

1. changes in carbon and nitrogen contents of dry matter in each plant part;

2. assessment of net gaseous exchanges of carbon as CO2 occurring in photosynthesis or respiration of each plant part;

3. estimates of total nitrogen gain of plants by N2 fixation, as determined by summation of net increments or losses of nitrogen in plant parts over the study period;

4. estimates of net daytime photosynthetic gain of carbon by whole plants, as determined from measurements of increments or losses of carbon in organ dry matter, respiratory losses of carbon by root and nodules and night-time respiratory losses of carbon by the shoot system.

It then becomes possible to construct simple models illustrating patterns of consumption of carbon and nitrogen by various plant parts. These are shown in Figure 2 for a specific week in the life of our lupin plant. Each model visualises the proportional amounts of carbon or nitrogen being processed, using appropriately shaped ‘cartoons’ for each class of organ drawn to an area proportional to the respective amounts they gain (positive values, shapes in solid lines) or lose (negative values, shapes in broken lines) during the interval under examination. The model for carbon (Figure 2a), depicting proportional allocation of the 1061 mg carbon produced as net photosynthate, includes a stippled halo around each plant part to represent the respiratory losses accompanying a specified gain or loss of carbon by that part. Overall, we find that the nodulated root (NR) consumes a very large proportion (43%) of carbon generated by the plant in net photosynthesis, and most of the carbon so used is lost as respired CO2 rather than incorporated into dry matter. The two upper strata of leaflets (L3 and L4, Figure 2) consume the next greatest proportion (24%) followed by the various levels of stem axis plus petioles (SP1–SP4) (22%), shoot apex (A) (8%) and older leaves (L1 and L2) (4%).

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Figure 2 Representation of the consumption of (a) carbon and (b) nitrogen by plant parts of white lupin (Lupinus albus) during mid-vegetative growth (51-58 d after sowing). L1-L4, four strata of leaflets; SP1-SP4 corresponding strata of stem segments and associated petioles; NR, nodulated root; A, shoot apex. Each profile for utilisation depicts net gain or loss of carbon or nitrogen. The profile for carbon includes losses of carbon as respired CO2. Absolute amounts entering the plant as carbon of net photosynthate or fixed N2 are indicated (Based on Pate and Layzell 1981)

The equivalent allocation ‘cartoon’ for the 34.8mg nitrogen fixed in the study period (Figure 2b) is shaped very differently. L4 is the dominant sink for nitrogen (38%) followed by the nodulated root (NR) (29%), shoot apex (A) (19%) and L1 (9%); extremely modest gains by stem and petiole tissue (SP) and net losses of nitrogen from the lower strata of leaves (L1 and L2) are observed.

Exchanges of carbon and nitrogen in phloem and xylem

So far we have not considered the role of long-distance transport conduits of xylem and phloem when carbon and nitrogen are partitioned to fulfil the requirements of plant parts (Figure 2). White lupin bleeds xylem fluid from the top of its root after the shoot has been cut off, thus enabling an inventory to be made of organic solutes passing up from roots and nodules to shoots through the xylem. Importantly, white lupin also bleeds from the sieve tubes of its phloem, if shallow cuts are made into stem, petiole or fruit (Figure 1D shows phloem sap bleeding from pods of lupin). Data from these sap samples are used to assess what carbon and nitrogen solutes are moving in the various streams of translocate being transported from any of the age groups of leaves to each of a number of possible consuming (sink) organs within the system. Another legume, cowpea (Vigna unguiculata), also bleeds from phloem and has been used for studies similar to those described here for lupin. Cowpea, however, bleeds only from its fruits and only if these are cryopunctured (Figure 1E and F). To determine which source leaves serve which sink regions within plants, 14C-feeding studies need to be conducted in single source leaves which are fed 14CO2 and the fate of 14C-labelled sucrose and other assimilates which they export are subsequently traced within the plant.

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Figure 3   Carbon to nitrogen weight ratios in the xylem and various phloem streams of plants of white lupin during mid-vegetative growth (51-58 d after sowing). Plants were entirely dependent on N2-fixing nodules for nitrogen. Coding of plant parts as in Figure 2  (From Pate and Layzell 1981; reproduced with permission of Kluwer Academic Publishers)

Armed with the above information, a transport profile can be gained, a picture of where resources move in a lupin plant (Figure 3). Directions of flow between source leaves and consuming parts of the plant, as determined by 14C feeding, are indicated by arrowed pathways in the figure. Local ratios (by weight) of carbon to nitrogen within different plant parts are shown; they reflect outcomes of carbon and nitrogen transport. For example, the xylem (drawn in black) delivers fluid to transpiring parts with solutes of very low C : N ratio, because sugars are virtually absent and the major solute is the amino acid asparagine (C:N of 2:1). Conversely, the youngest leaves (L4), which are most active in exporting sugars, are not yet losing much nitrogen, so they produce phloem translocate (dotted pathways) of relatively high C:N ratio (59:1). Downward-moving translocate from the shoot shows a C:N ratio which could be predicted from a mixture of the streams of translocate originating in the three strata of leaves which supply the root. One surprising finding is that the apical part of a shoot receives phloem translocate with a relatively low C:N ratio of 20:1, despite being fed from the upper leaves (L3 and L4) which are generating much less nitrogen rich solute streams (C:N ratios of 57:1 and 59:1 respectively). Clearly phloem sap must be modified en route from source leaves to shoot apex. How is this differential partitioning of nitrogen towards the apex achieved?

Combining the information described so far the model for partitioning of 1000 units of carbon of net photosynthate is dominated by phloem-mediated transfer of photosynthate from leaves to root and shoot apex. Corresponding xylem flow of carbon is proportionately much less and reflects the extent to which carbon supplied to nodules is returned to the shoot in the form of xylem-exported amino compounds formed in N2 fixation. Approximately 8% of the total carbon exported from photosynthesising leaves cycles through roots and nodules. This substantial demand by nodules for photosynthate is most striking. During mid-vegetative growth, 24% of the carbon of net photosynthate is translocated to nodules, over half of which is respired, and the remainder incorporated into nodule dry matter or returned to the plant attached to fixation products.

The complementary model for partitioning 1000 units of fixed nitrogen revolves around bulk export of newly fixed nitrogen from nodules. As expected, transpiring leaves act as principal initial destinations for such nitrogen. However, a proportion of nitrogen is transferred from xylem to phloem within veins of lower leaves, becoming available to roots. Similar xylem to phloem transfer in upper leaves provides nitrogen for shoot apices.

Note that the three lower strata of leaves (L1–L3), are, in effect, short changed for nitrogen through withdrawal of nitrogen from xylem traces supplying these lower leaves. After withdrawal, this nitrogen passes back into xylem traces, moving further up the stem.

As a result, xylem sap in the body of the stem becomes progressively enriched with nitrogen, and upper leaves thus receive considerably more nitrogen per unit of transpirational activity than nitrogen-deprived older leaves further down the canopy. This subtle shuttle system within a stem results in over 40% of currently fixed nitrogen being targeted towards upper, still expanding leaves — the very site of greatest demand for nitrogen at this stage in plant growth.

Two other features of this model for nitrogen deserve mention:

1. In a legume totally dependent on its nodules for nitrogen, parts of the root system extending below or laterally outwards beyond the nodules cannot receive nitrogen directly from xylem since the sap in this tissue moves upwards. Instead they gain nitrogen indirectly as phloem translocate supplied from the shoot (Section 3.6). Consequently, roots growing in soils deficient in nitrogen will be tightly controlled by nitrogen from shoots.

2. Substantial amounts of nitrogen moving through stem xylem into uppermost parts of the shoot are shuttled laterally across to the phloem stream moving photosynthate from upper leaves to the shoot apex. The apex accordingly acquires much more nitrogen than one would ever expect from its weak transpirational activity or from the phloem streams generated in the upper nurse leaves. Such stem-located processes of xylem to phloem transfer, together with xylem to xylem transfers in lower stems, comprise extremely important elements in differential partitioning of nitrogen and, indeed, of a number of other nutrient elements in herbaceous plants such as barley, castor bean, pea and lupin (Pate and Jeschke 1995). Demonstrations of such transfer activity between xylem and phloem clearly call into dispute previous suppositions that vascular tissues in stems play merely passive roles in straight throughput of solutes. Transport can be regulated at all points along the pathway.

Predictive use of partitioning models

Although our model is ‘empirically’ based on actual observations and measurements constructed for a particular set of growth circumstances, it can still be used to predict what might happen if the system were to be perturbed in any of a number of ways. For instance, were N2 fixation suddenly to cease, the model would suggest that xylem export of nitrogen from the nodulated root would cease almost immediately and that those organs first to suffer would be the shoot apex and any young leaves still dependent on xylem to meet the demands of their growing photosynthetic tissues for nitrogen. Then, if nitrogen starvation continued, senescence of lower leaves would commence and, according to the model, nitrogen released would become available mostly to root growth as opposed to rescuing upper shoots from nitrogen deficiency. Indeed, from observation, nitrogen starvation reduces new shoot growth, followed in turn by yellowing of leaves, and increased rather than decreased root growth.

Further study

Empirical models of the kind described in this essay may describe adequately events involved in partitioning of carbon and nitrogen, but they still tell us virtually nothing of the nature of the underlying cellular and molecular processes which modulate and regulate resource distribution in whole plants. To achieve such a picture, much more has to be learned about regulation of solute loading and unloading into both xylem and phloem elements. Particularly interesting in this connection would be a detailed study of how xylem to xylem or xylem to phloem transfers are coordinated along the stem throughout the life of a plant and how these activities change in relation to supply and demand of various donor and receptor regions of a plant during a cycle of growth and development.

References and further reading

Pate, J.S. (1996). ‘Photoassimilate partitioning and consumption in nitrogen-fixing crop legumes’, in Photoassimilate Partitioning in Plants and Crops: Source : Sink Relationships, eds E. Zamski and A.A. Schaffer, 467–477, Marcel Dekker: New York.

Pate, J.S.and Jeschke, W.D. (1995). ‘Role of stems in transport, storage and circulation of ions and metabolites by the whole plant’, in Stems and Trunks: Their Roles in Plant Form and Function, ed. Barbara L. Gartner, 177–204, Academic Press: New York.

Pate, J.S. and Layzell, D.B. (1981). ‘Carbon and nitrogen partitioning in the whole plant — a thesis based on empirical modelling’, in Nitrogen and Carbon Metabolism, ed. J.D. Bewley, 94–134, Martinus Nijhoff/Dr. W. Junk Publishers: The Hague

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