Within a stand, size often varies considerably among individual plants. These size differences may be the result of genetic differences in growth rate and shoot elongation rate. Initial small differences can be amplified by competition for light. As a dense stand develops, the growth rate of the smallest individuals will decrease and they will eventually die due to the suppression by larger individuals. This self-thinning process is well documented on the basis of shoot mass and plant density. However, plants may change allometry and show physiological adaptations when they get into lower hierarchical positions within a stand. Yet, little is known about the physiological adaptations which occur as a function of the hierarchical position in a stand. Adaptations to the light environment are manifested in the internal vertical distribution of leaf nitrogen and in the gas exchange which reflects carbon (C) gains and losses (respiration) of the plants.

The objectives of the project were:

• to investigate effects of plant-plant interactions on self-thinning processes in herbaceous stands,

• to quantify C/N-budgets and -allocation to plant organs and substance classes in self-thinning stands and
• to develop techniques to quantify resource fluxes among and within individuals.

Plant material was Medicago sativa and Helianthus annuus grown in monoculture and M. sativa, Dactylis glomerata and Taraxacum officinale grown in mixture either with Lolium perenne, L. multiflorum or Poa pratensis. After germination, seedlings were transplanted in pots with sand (5 cm diameter, 35 cm long) and transferred to growth cabinets. Plant stands were formed by placing the pots in containers (76 cm long, 56 cm wide, 32 cm high). Each container held 165 pots resulting in a maximum density of 400 plants m^-2. Alternatively, isolated growing plants were artificially shaded to simulate light gradients typical for plant canopies. Root competition was simulated by supplying the individuals with nutrient solution containing a high (7.5 mM) or low (1.5 mM) concentration of nitrate-N.
The light profile within the stands was regularly measured with a photon flux meter (Fig. 1). Individuals were harvested weekly for growth analysis. Some of these plants were labelled with ¹³C/¹⁵N.
The growth cabinets were part of a steady-state ¹³CO₂/¹²CO₂ labeling system. In this system, atmospheric CO₂ was exchanged with CO₂ of known ¹³C composition (δ). Growth cabinets received CO₂ with δ -2.4 ‰ or δ -46.8 ‰. During a growth period of eight weeks, individuals were randomly selected and transferred between cabinets of different δ. Thus, all photosynthates an individual fixed during one photoperiod were labeled. At the same time plants were labeled with ¹³C, they were supplied with ¹⁵N-enriched nutrient solution.
At the end of the photoperiod, plants were enclosed in a respiration cuvette to measure shoot and root respiration for 8 h (Fig. 2). Air flows were recorded (mass flow meters, Tylan, CA, USA) and CO₂ was continuously analyzed for concentration (Infrared gas analyzer, LI-COR 6262, NE, USA) and C-isotopic composition (IRMS, Delta plus, Finnigan, Germany).
Afterwards, shoot height and leaf area were measured. Shoots were clipped in four to five layers beginning at the top of the plant. Plants were dissected into leaves, stems and roots. Plant material was weighed fresh and after oven drying at 70 °C for 72 h. Ground plant material was analyzed for total C/N and C/N-isotopic composition using an elemental analyzer (Carlo Erba NA1108, Italy) interfaced to the IRMS. Sub-samples were analyzed for soluble carbohydrates and their C-isotopic composition.
The amount of C fixed during the labeling photoperiod (C_new) was calculated from total C mass and isotopic composition in the plant material. Use of C_new in respiration (C_{new, R}) was assessed from the rate of respiration and the C-isotopic composition of respiratory CO₂.

Verical distribution of leaf nitrogen

Vertical gradients of leaf nitrogen are a common feature of plant canopies. The partitioning of leaf N per unit leaf area (N_LA) parallels the vertical light distribution within the canopy. N_LA profiles are viewed as plastic responses that optimise N utilization with respect to carbon assimilation. The objective was to investigate the N_LA distribution relative to the light gradient in grassland species. Grassland systems are characterized by diversity of species and individuals in different hierarchical positions exposed to different availabilities of light and nitrogen. In such systems, species composition and stand structure may affect the N_LA gradient of the individuals. It was hypothesized that the variability of the slope of the N_LA-I/I₀ regression is related to the N status of the plant. The N status was analyzed using the concept of the critical N concentration (N_crit) in which shoot N per unit dry mass (N_SM) decreases with shoot mass, and negative deviation of actual N_SM from N_crit indicates N shortage of the plant. The hypothesis was tested: i) with contrasting growth forms common in grassland systems (monocotyledonous versus dicotyledonous species, leaf rosette forming versus erect growing plants), and ii) by varying light and nitrogen availability, plant density and hierarchical positions of the individuals within stands (Lötscher et al. 2003).
Even when there is an ample supply of nitrogen N, the N content per unit dry mass (NDM) in shoots declines as plants grow. It has been found that, as the shoot mass increased, the nitrogen content per unit tissue water (NW) was rather constant and the decline in NDM was mainly explained by a decline in the tissue water content per unit dry mass (WDM).Individuals in a sward are exposed to different light regimes and N availabilities which may also affect the components of the NDM. We investigated effects of light and nitrogen supply on the contribution of NW and WDM to the variation in NDM in leaves of Medicago sativa (Lötscher et al. 2002).

C assimilation and respiration
The amount of recently fixed C respired in the dark (C_{new, R}) correlated positively with the net gain of C fixed during the prior photoperiod (C_new). Conversely, the amount of respired C that originated from root and shoot reserves correlated with the mass of the organs.Thus, the contribution of recently fixed C to respiration depended on the light availability an individual experienced in the stand and its actual mass. It was concluded that plants getting into low hierarchical positions had to invest increasingly more carbohydrates from reserves to sustain maintenance needs (Lötscher et al. 2001, 2004). Following this conclusion a new hypothesis was formulated for the proceeding project B6: self-thinning in plant stands is accelerated when stress defense causes high C costs.

Fig. 1. Stand of Helianthus annuus growing in the growth cabinet. High plant density and different sowing dates resulted in weak subordinate plants. Light sensors were installed to quantify the vertical light gradient within the stand.