Canopy conductance and hormone levels
To measure midday whole plant transpiration pots were enclosed in a
black plastic bag and covered in aluminium foil secured around the stem
with a reversible cable tie to eliminate evaporation from the soil
medium. During drought pots were weighted (Mettler Toledo, OH, USA) 30
minutes before and after solar midday. Drought was initiated by
withholding water. Samples for Ψl determination and
foliage hormone analysis were collected 30 minutes after solar midday
after final masses were taken. Samples were wrapped in damp paper towel,
then aluminium foil, placed inside a ziplock bag and then an insulated
box, for transportation to the lab. Ψl was measured
using a Scholander pressure chamber (PMS Instrument Company, OR, USA)
and microscope for accurate determination of balance pressure, by slowly
pressurizing and depressurizing the chamber. After measuring
Ψl, a subsample of tissue was then taken for hormone
analysis (see below). Water was withheld from plants until they reached
-6 MPa (at this Ψl there was not signs of leaf death or
damage). Once this threshold Ψl was reached (at least 10
d after a peak in ABA levels was measured), plants were rewatered to
soil field capacity. Measurements were made on rehydrated plants until
whole plant transpiration approached the levels measured prior to
drought (4 d). At the end of the experiment total plant foliage area was
determined. In U. californica leaf area was calculated by
scanning leaves (Epson Perfection V39 Scanner, Epson America, Inc., CA,
US) and quantifying leaf area using ImageJ (NIH). A mean leaf area for
an individual leaf was determined from these images (12.68
cm2) which was used to adjust total plant leaf area
during the experiment to account for leaves periodically harvested for
Ψl and foliage hormone analysis. In C. rhomboidealeaf area was calculated from the ratio of leaf dry weight to leaf area.
Whole plant leafy branch area was harvested and dried to completeness at
70°C for 48h, after which dry mass was taken. Total leaf area was
calculated from the ratio of dry mass to leaf area (70.80
cm2 g-1) determined from sub-samples
prepared in the same way. In C. rhomboidea a mean area of sample
collected for Ψl and hormone analysis was determined
from 10 random samples (2.86 cm2). This mean sample
area was used to correct whole plant leaf area for declines caused by
sampling. Temperature and relative humidity measurements were recorded
every 10 min using a HOBO MX2301A Data Logger (Onset Computer
Corporation, MA USA), suspended at plant height in the glasshouse. The
gravimetric determination of whole plant water loss was then used to
calculate canopy conductance conductance (gc ) by
calculating whole plant transpiration (E, mol
m-2 s-1) using equation 1 and the
leaf area determined above.
E = \(\frac{\text{moles\ of\ water\ lost}}{area\ \ s}\) (1)
Mean vapor pressure deficit of the atmosphere (VPD) for the hour during
which E was measured using equation 2.
VPD = \((610.7*10^{\frac{7.5*T}{237.3+T}})*\frac{(100-RH)}{100}\)(2)
Where T is mean air temperature and RH is relative humidity for the
duration of the transpiration measurements.
gc was then calculated from E and VPD using equation 3:
gc = \(\frac{E}{VPD\ \times\ P_{\text{atm}}}\) (3)
Where Patm is atmospheric pressure. We assumed
negligible boundary layer conductance due to the constant air
circulation in the glasshouse, and that leaf temperature approximated
air temperature.