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New Phytologist
Notes S2 Detailed description of the traits measured.
Canopy structure traits (height, crown area, stem diameter, and number of
leaves) were recorded immediately before plant harvest, and allometric traits
immediately after. Both groups of traits were recorded for 9 replicate plants per
treatment and species (plants were arranged in 3 blocks). For allometric traits, plants
were separated into leaves, stems and roots, oven-dried at 60ºC for 3 days and weighed
to calculate weight ratio of leaf (LWR), stem (SWR) and root (RWR) per total biomass.
Before oven-drying the material, leaf area of each whole individual was measured using
a Delta-T leaf area meter device (Delta-T devices, Cambridge), to calculate leaf area
ratio (LAR= leaf area/plant dry mass) and specific leaf area (SLA=leaf area/leaf dry
mass). Finally, we measured percentage of survival at the end of the experiment for
each species per treatment combination (n=36).
Leaf physiology traits were measured using a LI-6400 portable photosynthesis
system with a fluorescence chamber (LI-COR, Lincoln, NE) in one mid-height
undamaged fully expanded leaf (n=3 plants randomly selected per species and
treatment, except in Amax and WUE where n=6 plants). We constructed light response
curves at 10 light intensities of PAR following the order 0, 800, 1100, 1500, 1900, 500,
250, 150, 100, 50, 0 μmolm-2 s-1 and with the following constant conditions: CO2
concentration 400 ppm, flow 400 cm3min-1, air humidity 40-60% and block temperature
25ºC. First, we adapted the leaf to dark for 30 minutes to measure respiration (Rdark).
Then, the leaf was irradiated with saturating and non-inhibitory light (800 μmolm-2 s-1)
for 10 minutes to be sure that plants were photosynthetically active. We then changed
light intensity and recorded maximum photosynthetic rate (Amax) at each light level
when it was stable (i.e. every 3 minutes on average). At maximum light intensity (1900
μmolm-2 s-1), transpiration rate (T) was also recorded to subsequently calculate
Oscar Godoy, Fernando Valladares, Pilar Castro-Díez
New Phytologist
instantaneous water use efficiency (iWUE= Amax/T). From dark to maximum light
intensity, we measured a set of fluorescence parameters (Fv/Fm, ФPSII, qP, qN, NPQ
and ETR), as follows: First, we measured maximal efficiency of Photosystem II in
darkness (Fv/Fm) and at one saturating and potentially inhibitory and at another not
saturating light level (ФPSII at 1900 μmolm-2 s-1 and at 150 μmolm-2 s-1). Fv/Fm=(FmFo)/Fm), where Fo is the value of fluorescence in total darkness and Fm is the
fluorescence of a dark-adapted leaf during a saturating flash light and ФPSII=(Fm´Fo´)/Fm´, where Fo´ and Fm´ are respectively the minimal and the maximal fluorescence
of a light adapted leaf that has momentarily been darkened. Later, a suite of quenching
variables were measured at both light levels (150 and 1900 μmolm-2 s-1), to know the
capacity of leaf photo protection against a possible excess of light intensity.
Photochemical quenching (qP= (Fm´-Fs)/(Fm´-Fo)) involves photosynthesis and
photorespiration and tends to be higher under low light because of the higher light use
efficiency under low light conditions. Here, Fs is the basal fluorescence at a given light
and the rest of the parameters were mentioned above. The quenching nonphotochemical (qN=( Fm-Fm´)/( Fm- Fo´)) is associated to radiant energy dissipation (i.e.
heat dissipation), and reach the highest values at high light levels reflecting a plant
protection mechanism to avoid damages of the thylakoid membrane due to an excess of
energy. The non-photochemical quenching (NPQ=(Fm-Fm´)/ Fm´) accomplish the same
function but is associated to a non-radiant energy dissipation (i.e. photoprotective
pigments like xanthophylls). Finally, we measured the electron transport rate
(ETR=((Fm´-Fs)/ Fm´)fIαleaf ) which is the flux of electrons driving the photosystem II,
where f is the fraction of absorbed quanta that is used by the photosystem II, I is the
light intensity and αleaf is the leaf absorptance.
Oscar Godoy, Fernando Valladares, Pilar Castro-Díez
New Phytologist
We obtained photosynthetic parameters from light response curves using
Photosyn Assistant software version 1.1.1 (Richard Parsons, Dundee, U.K). This
software models the photosynthetic response of leaves to variation in light level by a
rectangular hyperbola following the quadratic equation presented by Chartier & Prioul
(1976), where the light compensation point (Γ) is estimated from intercept to x-axis, the
light saturation point (Ic) is the light level at which the leaf reaches its maximal
photosynthetic capacity and the convexity light curve factor (Θ) describes the
progressive rate of bending between the linear gradient and the maximum value.
Finally, we measured organic leaf nitrogen concentration per mass (Nmass) and
per area (Narea) at Nutrilab (University Rey Juan Carlos, Móstoles, Madrid, Spain) with
segmented flux autoanalyzer (S-F.A.S. Skalar San ++), after digestion with H2SO4 and
Cu-KSO4, which converts all organic nitrogen into ammonium (NH4+-N). Previously,
leaves of each species and treatment had been pooled within blocks and ground in a
Culatti mill to 1 mm particle size. After that, Narea was calculated by dividing N leaf
content by the leaf area mean and photosynthetic nitrogen use efficiency (PNUE) as the
division of Amax by Nmass.
References
Chartier P, Prioul JL. 1976. The effects of irradiance, carbon dioxide and oxygen on
the net photosynthetic rate of the leaf: a mechanistic model. Photosynthetica 10:
20-24.
Oscar Godoy, Fernando Valladares, Pilar Castro-Díez