Spad 502 dl plus

Complete functional leaves of similar shape and size were selected, and a SPAD 502 DL Plus instrument (Konica Minolta, Tokyo, Japan) was used to measure the leaves at the center of maize plants under different PGR treatments at V6, V10 and V14.

Infrared Gas Analyzer (CID-340, Photosynthesis System, Bio-Science, USA) was used for the measurements of gas-exchange parameters, i.e., net photosynthesis (Pn), stomatal conductance (gs), and intercellular CO₂ concentration (Ci). All gas-exchange measurements were performed in fully expanded uppermost leaves, at atmospheric CO₂, 380 ± 5 µmol mol⁻¹, relative humidity, ~70%, photosynthetically active radiation (PAR), 800 µmol m⁻² s⁻¹, and atmospheric temperature, 32 ± 1 °C, in between 10:00 to 11:30 am.
Chlorophyll content or greenness was measured in intact leaves with the help of SPAD chlorophyll meter (SPAD 502 DL PLUS, produced by Konica Minolta, Japan) in the early morning hours and represented in SPAD values.
Rubisco activity was determined following the method of Usuda [25 ] by monitoring NADH oxidation at 30 °C at 340 nm during the conversion of 3-phosphoglycerate to glycerol 3-phosphate after the addition of enzyme extract to the assay medium. The details of the method is provided in the Supplementary file S1.

The fully expanded third leaf of each treatment was taken for gas exchange measurements. An infrared gas analyzer (CID-340, Photosynthesis System, Bio-science, Washington, DC, USA) was used for measurements of net photosynthesis (P_N), stomatal conductance (gs), and intercellular CO₂ concentration (Ci). The details of the method are given in Supplementary File S1. Chlorophyll content was measured with a SPAD chlorophyll meter (SPAD 502 DL PLUS, Konica Minolta, Japan).
The maximal efficiency of photosystem II (PSII), as given by Fv/Fm, was determined with a chlorophyll fluorometer (Junior-PAM, Heinz Walz, GmbH, Effeltrich, Germany). The process is detailed in Supplementary File S1.
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity was spectrophotometrically determined by adopting the method of Usuda [73 (link)]. The details are given in Supplementary File S1.