Physiological and molecular mechanisms of cytokinin involvement in nitrate-mediated adventitious root formation in apples

Potassium nitrate (KNO3) promotes adventitious root (AR) formation in apple stem cuttings. However, evidence for the possible involvement of cytokinin (CK) in KNO3-mediated AR formation in apples is still lacking. In this study, we cultured GL-3 apple microshoots in different treatment combinations. While the T1 (KNO3 9.4 mmol L-1+6- benzyl adenine (6-BA) 2.22 mu mol L-1) and T3 (6-BA 2.22 mu mol L-1) treatments completely inhibited AR formation, the control, T2 (KNO3 9.4 mmol L-1), and T4 (KNO3 9.4 mmol L-1+lovastatin (Lov) 1.24 mu mol L-1) treatments developed ARs. However, T4-treated microshoots developed fewer and shorter ARs, indicating that optimum CK synthesis is needed for normal AR growth. This also suggests that these fewer and shorter ARs developed because of the presence of KNO3 in the same medium. The anatomy of the stem basal part indicated that the inhibition of CK biosynthesis delayed AR primordia formation. The endogenous levels of indole-3-acetic acid (IAA) and zeatin riboside (ZR) were higher in T2-treated microshoots, while the abscisic acid (ABA), gibberellic acid 3 (GA3), and brassinosteroid (BR) levels were higher in T4-treated microshoots. The expression levels of MdNRT1.1 and MdNRT2.1 were higher in T2-treated microshoots at 3 and 8 days, while MdRR2 and MdCKX5 were higher at 8 and 16 days, respectively. Furthermore, higher IAA levels increased MdWOX11 expression, which in turn increased MdLBD16 and MdLBD29 expression in response to T2. The combined expression of these genes stimulated adventitious rooting by upregulating cell cycle-related genes ( MdCYCD1;1 and MdCYCD3;1) in response to T2 treatment. This study shows that specific genes and hormonal pathways contribute to KNO3-CK- mediated adventitious rooting in apples.