4.1. Plant cultivation and Nitrogen Treatment

The experiment was carried out in a greenhouse at the Cotton Research Institute of the Chinese Academy of Agriculture Sciences, Anyang, China. Healthy seeds of two cotton genotypes (CCRI-69, N-efficient, and XLZ-30, N-inefficient) were placed in a mixture of sand and vermiculate for one week in a germinator. After the full opening of two cotyledons, seedlings with uniform height were selected and transplanted into 7 L containers in the growth chamber (16/8 h light/dark cycle, 28 °C temperature, 60% relative humidity). After transplanting, the seedlings were supplied with half-strength during the first week, followed by a full-strength, Hoagland solution as reported by Iqbal et al. [66]. At three-leaves stage, seedlings were exposed to nitrogen-free nutrients solution (N starvation) for five days. After five days, the seedlings were resupplied with 2 mM Ca(NO₃)₂ which contained 4 mM NO₃⁻ as normal nitrogen, and subsequently, the root and shoot samples were taken at 0 and 6 h after treatment.