Effects of GR24 and Tis108 on root growth and development of Malus baccata Borkh. seedlings under low nitrogen stress

【Objective】Low nitrogen stress is one of the major abiotic constraints limiting fruit tree growth, development, and nutrient utilization. Insufficient nitrogen supply inhibits cell division and differentiation, reduces the synthesis of proteins and carbohydrates, and consequently suppresses plant growth, while also markedly affecting root morphological development and nutrient absorption. As the primary organ responsible for nitrogen uptake, the adaptive physiological and morphological plasticity of the root system constitutes a fundamental basis for improving nitrogen use efficiency in plants. Plant hormones play crucial roles in regulating root responses to low nitrogen stress. Among them, strigolactones, a class of recently identified plant hormones, regulate root growth and development and participate in plant stress responses through interactions with auxin signaling pathways. Given the widespread low organic matter content in orchard soils in China and the resulting decline in nitrogen use efficiency and increased environmental risks caused by excessive nitrogen fertilizer application, this study used Malus baccata Borkh., a commonly employed apple rootstock, as the experimental material. By applying strigolactone analog, GR24, and its inhibitor under low nitrogen conditions, we systematically investigated their effects on root respiratory metabolism, root activity, root architecture, endogenous hor-mone levels, and the expression of related genes. The objective of this study was to elucidate the physiological roles of strigolactone in regulating root adaptation of M. baccata Borkh. to low nitrogen stress, thereby providing a theoretical basis for improving nitrogen use efficiency and promoting sustainable orchard production.【Methods】The experiment was conducted at the research station of Shenyang Agricultural University, using one- year- old seedlings of M. baccata Borkh., a commonly used apple rootstock, as the experimental material. The seedlings were grown in sand culture in 18 cm × 16 cm plastic pots. Full- strength Hoagland nutrient solution was used as the normal nitrogen treatment (MN: NO3 - concentration of 868 mg·L-1 ). The low-nitrogen treatment (LN: NO3 - concentration of 310 mg·L-1 ) was prepared by reducing KNO3 (607 mg ·L-1 ) and Ca(NO3)2 · 4H2O (944 mg ·L-1 ) in Hoagland solution proportionally to one-third of their original concentrations, while KCl and CaCl2 were added to compensate for the corresponding deficiencies in K+ and Ca2+ . GR24 was dissolved in acetone and diluted with water to prepare solutions of different concentrations. Foliar application was performed once daily for three consecutive days, followed by a five-day interval and another three consecutive days of application. Each treatment consisted of three biological replicates, with five seedlings per replicate. After treatment, root samples were collected, and the optimal GR24 concentration was determined based on measurements of root respiration and sugar contents, which was identified as 10 μmol·L-1 . Based on this optimal concentration, experiments were conducted in the following year under both normal and low nitrogen conditions, with three treatments established: water control (CK), GR24 treatment, and treatment with the strigolactone biosynthesis inhibitor Tis108. The application concentrations for both GR24 and Tis108 were 10 μmol·L- 1 . The application method and treatment schedule were identical to those used in the GR24 concentration-screening experiment. Root samples were collected after treatment for subsequent analyses.【Results】Low nitrogen stress significantly increased the total root respiration rate of M. baccata Borkh. seedlings as well as the contributions of the EMP, TCA, and AOX respiratory pathways, and markedly promoted the accumulation of glucose, fructose, soluble sugars, and TCA cycle intermediates, including malic acid, citric acid, and α-ketoglutaric acid, in the roots. Under normal nitrogen conditions, GR24 had no significant effect on root respiration rate but increased the contents of sugars and some organic acids. Under low nitrogen conditions, increasing GR24 concentration increasingly and significantly enhanced the total respiration rate, the contributions of the three respiratory pathways, and the accumulation of sugars and organic acids, with the 10 μmol · L- 1 GR24 treatment showing the most pronounced effects. Low nitrogen stress significantly reduced root activity and increased total root length but significantly decreased total surface area, total volume, average diameter, number of root tips, number of links, and external link length. Under normal nitrogen conditions, GR24 significantly increased root activity and promoted root growth. Under low nitrogen conditions, GR24 markedly enhanced root activity, and increased total root length, total surface area, total volume, average diameter and the number of root tips and links, whereas Tis108 significantly suppressed root activity and root growth under low nitrogen conditions. Under low nitrogen stress, the contents of auxin and strigolactones in roots were both significantly increased. Under normal nitrogen conditions, GR24 decreased auxin content and increased strigolactone content, whereas Tis108 showed opposite effects. Under low nitrogen conditions, GR24 showed greater effect in reducing auxin and strigolactone contents, while Tis108 significantly decreased them. Gene expression analysis showed that low nitrogen stress significantly upregulated the expression of strigolactone biosynthesis- related genes CCD7, CCD8b, and MAX1a, and altered the expression of auxin biosynthesis and transport-related genes TAR2, TAA1, and PIN1. Under low nitrogen conditions, GR24 and Tis108 exerted opposite regulatory effects on these genes as well as on the strigolactone signaling-related genes MAX2 and D14.【Conclusion】This study provides new insights into the physiological roles of strigolactones in regulating the responses of M. baccata Borkh. roots to low nitrogen stress. Under low nitrogen conditions, treatment of GR24 increased the contents of sugars and organic acids in roots, enhanced root respiration rates, and improved root activity and functional capacity. The treatment increased endogenous strigolactone levels by regulating the expression of strigolactone biosynthesis-related genes, including CCD7, CCD8b, and MAX1a, but reduced auxin levels by downregulating the expression of auxin biosynthesis and transport- related genes TAR2, TAA1, and PIN1. These coordinated regulatory effects alleviated the inhibitory effects of low nitrogen stress on root growth and development, promoted the formation of a herringbone-type root architecture, and enhanced the adaptability of M. baccata Borkh. seedlings to nitrogen-deficient conditions.