Leaf water content as a percentage of fresh mass was calculated according to the following equation: leaf water content (%) = 100 × (FM − DM)/FM, Endocrinology antagonist where DM and FM denote respectively dry matter and fresh matter of the flag leaves. Photosynthesis, chlorophyll and nitrogen content,

and activities of PEPC, Rubisco and carbonic anhydrase (CA) in flag leaves were measured at 14 DPA and 21 DPA. Photosynthesis was determined under the conditions of 28 °C, ambient CO2 concentration, and 65%–70% relative humidity using a LI-6400 portable photosynthesis system (LI-COR, Lincoln, Nebraska, USA). The photosynthetic photon flux density (PPFD) was controlled by a LED light source built into the portable photosynthesis system and was set to 1500 μmol m− 2 s− 1. Six leaves were measured for each treatment on each measurement date. Chlorophyll was extracted by shaking in methanol overnight and measured as described by Holden [22]. Leaf nitrogen content was determined by micro Kjeldahl digestion, distillation, and titration [23]. Activities of PEPC, Rubisco and CA were assayed according to the methods of Gonzalez et al. [24], Wei et al. [25] and Guo et al. learn more [26], respectively.

At 10, 17, 24 and 31 DPA, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of the flag leaves were determined according to the methods described by Giannopolitis and Ries [27] and by Zhao et al. [28], respectively. Root exudates and root oxidation activity (ROA) were determined at 14 and 28 DPA. Six hills of plants from each treatment were used for collection of root exudates. Each plant was cut at an internode about 12 cm above the soil surface at 18:00 h. An absorbent cotton ball was placed on the top of each decapitated stem and covered with a polyethylene sheet. The cotton ball with exudates was collected after 6 h. The volume of exudates was estimated from the increase in cotton weight with the assumption that the specific

gravity of the exudation sap was 1.0. For ROA measurement, a cube of soil (20 × 20 × 20 cm) around each individual hill was removed using a soil sampling corer. Plants of three hills from each plot formed a sample at each measurement. The roots of each hill were carefully rinsed with a hydropneumatic elutriation device (Gillison’s Variety Fabrications, Mannose-binding protein-associated serine protease Benzonia, MI, USA). The equipment employs a high-kinetic-energy first stage in which water jets erode the soil from the roots followed by a second low-kinetic-energy flotation stage that deposits the roots on a submerged sieve [29]. All the roots were detached manually from their nodal bases. A portion (10 g) of each root sample was used for measurement of ROA. The remaining roots were dried in an oven at 70 °C for 72 h and weighed. The method for measurement of ROA was according to Yang et al. [30]. Root activity was expressed as μg α-alpha-naphthylamine (α-NA) per gram dry weight (DW) per hour (μg α-NA g− 1 DW h− 1).