The soil column was placed in an 80 cm deep square

pit filled with soil both inside and outside the column and made soil compact by watering. The distance between soil columns was 11 cm, that is, the row width was 65 cm, and surrounded by the board rows (Fig. 1). Two plants MS-275 in vivo were grown in each soil column. Plants in one column were planted under normal spacing (NS, 27 cm), and the other under narrow spacing (CS, 6 cm). The columns were treated at two nitrogen levels, N0 (no N) and N1 (7.5 g N plant− 1), and for the N1 treatment, nitrogen fertilizer was applied by 20%, 50% and 30% at the seedling, male-tetrad and flowering stages, respectively. The experimental design included four treatments MAPK inhibitor (N0 × NS, N0 × CS, N1 × NS and N1 × CS) and 30 separate soil columns were planted in each treatment. Samples of the soil columns (top 40 cm) were mixed and screened with 20 mesh sieving. Then they were mixed

with clean river sand in a ratio of 3:1 by volume of topsoil to sand. The mixed soil nutrient contents were as follows: organic matter 7.1 g kg− 1, total nitrogen (N) 0.62 g kg− 1, mean available mineral phosphorous (P) 46 mg kg− 1, and exchangeable potassium (K) 59 mg kg− 1. All treatments were fertilized with P and K according to nutrient demand, and each unit of experimental treatment was fertilized with 2.5 g of phosphate (P2O5) and 6.25 g of potash (K2O), with both applied at the seedling stage. Required irrigation was also applied from the outlet of a pump by using plastic pipes. At the onset of pollination, three replicates of each treatment were sampled on the same day fortnightly. The above-ground plant parts were divided into leaves, grains and stems (remaining parts except for leaves and grains). Roots were separated from various layers of the soil profile, viz. 0–20, 20–40, 40–70 and

> 70 cm, and washed to remove all soil residues. Root layers were mixed well after removing impurities, and fine roots were selected and temporarily stored at 0 °C. 2,3,5-triphenyl tetrazolium chloride (TTC) reduction was applied to determine root reductive activity [19]; fresh root samples (0.5 g) were exposed mafosfamide to 0.4% TTC and 0.2 mol L− 1 tricine-HCl buffer (pH 8.4), then placed in a darkroom at 37 °C for 6 h to induce reduction of TTC to triphenyl formazan (TTF), following the method described by Duncan and Widholm [19]. To quantify the amount of TTC reduced, we extracted the tissues with 95% ethanol at room temperature for 48 h, and then performed spectrophotometric analysis at 485 nm. The results were expressed as μg TTF g− 1 root mass h− 1. At maturity, the remaining aboveground parts were completely harvested to calculate average dry matter weight per plant and weight distribution. The remaining roots and above-ground samples were fixed at 105 °C for 30 min. Samples were subsequently baked at 75 °C until a constant weight was reached and recorded.