Plants were grown under these conditions for 8 days and then
transferred to different controlled-environment cabinets (Sanyo
Gallenkam Fitotron, Leicester, UK) fitted with an ADC 2000 CO2
gas monitor. The plants were kept under ambient CO2 levels
(400 -
L L−1) or elevated CO2 concentration (800 -
L L−1) under con-
stant conditions of photonic flux (400 -
mol m−2 s−1), temperature
(25/19 ◦C) and relative humidity (70/80%) for another 34 days.
High-purity CO2 was supplied from a compressed gas cylinder (Air
Liquid, Seville, Spain). Samples of primary leaves aged 16, 22, 32 or
42 days were collected 2 h after the start of the photoperiod. Whole
leaves were excised and pooled in two groups: one was used to
measure leaf area and specific leaf mass (SLM)–dry weight (DW),
and the other was immediately frozen in liquid nitrogen and stored
at −80 ◦C. The frozen plant material was ground in a mortar pre-
cooled with liquid N2 and the resulting powder distributed into
small vials that were stored at −80 ◦C until enzyme activity and
metabolite determinations.