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.