2.4. ACTITUDES LINGÜÍSTICAS
2.4.1. Actitudes y lengua
where IT is the amount of acidity required to alter the leaf homogenate by 1 pH unit, divided by the calculated change of H^
H ) concentration, per gram fresh weight (Wy).
2.8 Cation d eterm in ation
Extraction of the cations calcium (Ca^^), magnesium (Mg^3, and potassium (K^) took place by acid digest, following the routine of Allen (1974). 0.1 g of current year leaf material was added to 4.4 ml of acid digest mix (350 ml H2O2 (30% w/v), 420 ml 18 M H2SO4, 0.42 g selenium and 14.0 g L iSO J in acid washed, thick walled boiling tubes. Tubes were then heated to 360°C in 60°C steps for 10 min each. Once full temperature was achieved, the samples were left at 360°C for 2 hours until all plant material had been digested and the liquid became clear. Samples were left to cool to room temperature and ddH2 0 was added to achieve a total volume of 50 ml. Concentrations of Ca^^, Mg^^ and were determined from the 50 ml samples, using a Pye Unicam SP9 atomic absorption spectrophotometer.
2.9 Total nitrogen and phosphate d eterm in a tio n
Total N and phosphate (PO4) were measured from the 50 ml samples prepared as above. Total N was determined from 1.0 ml of neutralised sample by the ammonia method of McCullough (1967). PO4 was determined from 1 . 0 ml of sample by the molybdate - antimony method of Golterman (1978).
2 .1 0 stable isotope and total nitrogen analysis u sin g A N C A -M S
The technique of ANCA-MS used for stable isotope and total N analysis, is described by Barrie & Lemley (1989). Samples of plant material exposed to labelled nitrogen sources were firstly washed 3 times in ddHjO to remove surface adsorbed then dried in a microwave oven on low power for 10 min. Dried material was ground to talcum powder consistency (< 250 //m particle diameter) using a MM2 ball mill (Retsch GmbH and Co., W.Germany) and sealed in 6 mm X 4 mm methanol washed tin capsules (2 - 10 pig sample per capsule). Samples were then analysed for and total N using ANCA- MS, (Europa Scientific Ltd, Crewe, UK). Plant samples analysed for ô^^N natural abundance were prepared as above and analysed using ANCA-MS.
2.11 P hotosyn th etic m easu rem ents
2 .1 1 . 1 O2 ev o lu tio n
Measurements of PS using O2 evolution were adapted from the method of W alker (1987). Leaf portions excluding the midrib, whole leaflets or needles were detached from plants and immediately placed inside an air tight chamber (7 cm^ volume), form ing part of a gas phase leaf disc oxygen electrode (Hansatech, King’s Lynn, UK). The chamber allows for a 10 cm^ leaf to be placed inside. Leaves greater than 10 cm^ in area were cut with a 1 0 cm^ circular cutter to provide an accurately sized leaf portion. In the case o f needle shaped leaves, or leaflets that were smaller than 1 0 cm^ in total area; these were placed along side each other in the leaf chamber to avoid overlapping. A fter measurement of PS, the leaves were placed under a clear acetate sheet
and their combined area was scanned using a hand held scanner connected to a PC. This gave an exact area measurement, which could then be incorporated into the calculation o f PS. A mixture of 5% CO2 balanced with air was injected into the chamber via a gas port. This concentration of CO2 is required to allow continucj^ PS measurements, without the rapid depletion of CO2 (Walker,1987). A high concentration of CO2 also allows uptake o f CO2 directly via the cuticle, as stomata may close in excised leaves (Walker, 1987). The entire chamber and electrode assembly were maintained at a temperature of 20°C with water circulating inside an integral water jacket. A tungsten-halogen light source was connected directly to the top of the chamber and electrode assembly, providing a light intensity of 1800 //mol m ^ s'^ at the leaf surface. O2 evolution from the leaf surface was recorded polarographically using a platinum cathode and silver anode embedded in epoxy resin (Walker, 1987). The poles of the electrode were linked using an electrolyte consisting of 1 part 0.4 M H B O4 + 0.4 M KCl (final pH 9); 1 part saturated KCl; 2 parts 1.0 M NaHCOj (pH 9). The electrode assembly was separated from the leaf chamber using an O2 permeable polythene membrane. The electrode was connected via an analogue interface to an IBM computer allowing ‘real-time’ measurements of O2 evolution to be viewed on screen and recorded. The period of time between leaf detachment, placement in the chamber, the introduction of 5% CO2 and the establishment of steady state O2 evolution was approximately 2 minutes. O2 evolution was then recorded for a further 2 min to give control rates of PS. Prior to introduction of plant material, the chamber and electrode assembly were calibrated using zero grade N2 gas, to create optimal conditions for the measurement of O2 evolution.
2 .1 1 .2 COj fixation and w ater ex ch a n g e
In-situ leaf (6.25 cm^) was clamped inside an air tight portable leaf chamber (LCA 4, ADC Ltd, Hoddesdon, UK). Leaves were clamped routinely in the same position to standardise the leaf portion used. This consisted of a central portion of the leaf, excluding the midrib where possible. For species with a leaf area smaller than the leaf chamber, several non detached leaflets or needles were clamped inside the chamber, then scanned as described above after PS was measured to give a measurement of their combined leaf area. Measurements of PS using CO2 fixation were recorded using a portable LCA-4 Infra Red Gas Analyser (IRGA) (ADC Ltd, Hoddesdon, UK) connected to the portable leaf chamber. A reference gas of measured ambient CO2 balanced with air was allowed to pass over the leaf surface at 300 ml min'^ for 2 min for equilibration. The IRGA contrasted the reference ambient CO2 concentration to the CO; concentration within the chamber, to give a measurement of the rate of CO2 fixation. In addition, the IRGA was also used to determine stomatal conductance and transpiration rates, by comparisons o f the water vapour content the reference gas and the leaf chamber.
CHAPTER 3