New leaves appeared approximately 15 days after the beginning
of the experiment and from then on the total Chl concentration was
estimated non destructively in mature and the youngest fully
expanded apical leaves using a portable SPAD-502 m (Minolta,
Osaka, Japan). Five readings per leaf were recorded for at least three
leaves per plant. SPAD readings were converted to total Chl using
the equation:Y ¼ 0.4453x2 1.1114x þ 32.562 (r2 ¼ 0.98; n ¼ 31; P < 0.001)
where Y is the Chl content (mmolm1) and x is the SPAD reading [9].
This calibration curve was established by analysing leaf disks that
showed different degrees of Fe deficiency with the SPAD-502, extracting
the pigments from the same leaf area with 100% acetone in
the presence of Na ascorbate [28], and measuring Chl spectrophotometrically
according to [29].