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Adventitious root (AR) formation is an essential step in the vegetative propagation of apple rootstocks. Nitrate serves as an essential signaling molecule for regulating root architecture by inducing the expression of auxin-related genes. However, the underlying mechanisms of nitrate-mediated ARs remain to be explored in apple. In this study, stem cuttings of B9 apple rootstocks were treated with different nitrate treatments: T1 (9.4 mM/L), T2 (28.1 mM/L), and T3 (46.9 mM/L). The root morphological parameters indicated that T2 was the optimum nitrate level for AR formation and development in B9 apple rootstocks. Therefore, to identify the underlying molecular mechanism by which nitrate promotes AR formation, stem cuttings of B9 were grown on T2 and T3. Furthermore, morphological and anatomical observations of stem cuttings also revealed that the nitrate treatment (T2) promoted AR formation. The results indicated that nitrate perceptibly upregulated the relative expression of genes related to nitrate (MdNRT1.1, MdNRT2.1, MdNIA1, and MdANR1) and auxin biosynthesis (MdIAA14 and MdIAA23) in T2 cuttings compared with T3 cuttings. This resulted in enhanced expression of AR development-related genes (MdWOX11, MdARRO1, and MdSHR), collectively resulting in elevated expression of the cell cycle-related genes (MdCYCD1;1, MdCYCD3;1, and MdCYCP4;1). Overall, this study established a foundation for applied research work and shed light on nitrate-mediated AR formation in B9 apple rootstock and other fruit rootstock cuttings.

Adventitious root (AR) formation plays a critical role for the clonal propagation of horticultural crops. In this study, Malus prunifolia var. ringo micro-cuttings were treated with IBA and darkness treatments to investigate the mechanism of darkness effects AR formation. Morphological and anatomical observation revealed that darkness promotes more ARs formation. Root morphology, and hormones level were evaluated during AR formation. Results indicated that darkness promotes AR formation by increasing auxin level at stages of 1 d and 3 d, moreover, ZR, ABA, GA3, BR and JA also changed. The number of ARs in darkness-treated group was significantly higher than that of control treatment; Additionally, genes related to light signal pathway-, hormones-, carbohydrate metabolism-, cell cycle-, and root development were determined by RT-qPCR. Auxin- and cytokinin-related genes, such as MdARF1, MdGH3;1, MdPAT1-1; MdPIN1, MdCRF2, MdCRF4, and MdRR10 were significantly induced by darkness treatment, which corresponded with higher hormones levels. Furthermore, light-signal-related genes (MdASP3, MdATPC1, MdBGLU13, MdCBA1, MdLHCB6, MdLHCA1, MdNPQ4, MdPTE1 and MdPSAL) were also induced by darkness-treatment, resulting changes in the expression profiles of cell cycle-related genes (MdCYCB2;1, MdCYCB2;3–1 and MdCYCB2;3–2) and also in the expression of root development-related genes (MdLRP1, MdRHS19, MdSGR1 and MdSHI). Collectively, darkness treatment mediated auxin-, cytokines-, light signal-, cell cycle-, carbohydrate metabolism-, and root development-related pathways to regulating AR formation. This work laid the foundation for applied research and could be useful in future crop improvement programs. This study revealed that darkness treatment during AR induction phase promoted more AR formation in plant tissue culture, and suggest that natural conditions of darkness should be simulated to improve rooting system in future research.

Lateral roots (LRs) are critical for plant stress tolerance and productivity. Understanding how hormones and genes interact in a fluctuating environment to coordinate LR development is a major challenge. Abscisic acid (ABA) is the primary stress-responsive hormone and mediates LR development in various plant species. However, the effect of exogenous ABA on LR development has not been elucidated in apple. In this study, ‘Qingzhen 1’ was treated with exogenous 5 µM ABA for 20 days to investigate the regulation mechanism of ABA on LR development. Morphological observations advocated that ABA inhibited both LR and shoot development in ‘Qingzhen 1’ apple plants, where the root number was 16.94%, the root length was 30.32%, the plant height was 10.88%, and the stem thickness was 8.08% lower than those in the control plants. Meanwhile, the endogenous ABA concentration was significantly increased, but the indole-3-acetic acid, zeatin riboside, and jasmonic acid concentrations were significantly decreased with ABA treatment. Furthermore, the expression levels of ABA-related genes (MdCYP707A2, MdABI1, MdAREB2, and MdABF3) were significantly upregulated, while the expression levels of auxin-related genes (MdYUCCA3, MdYUCCA8, MdPIN1, MdPIN2, MdPIN3, and MdARF19), root development-related genes (MdWOX5 and MdWOX11), and cell cycle-related genes (MdCYCD1;1 and MdCYCD3;1) were significantly downregulated at the early stage of ABA treatment, which act together on the inhibition of LR development. Taken together, the changes in hormone levels and gene expression resulted in inhibited LR development of apple plants in response to ABA. This study revealed the mechanism that exogenous ABA application inhibits the LR development of ‘Qinzhen 1’ apple rootstock by affecting the auxin signaling and the expression of growth-related genes.

Adventitious root (AR) formation is required for the vegetative propagation of economically important horticultural crops, such as apples. Asexual propagation is commonly utilized for breeding programs because of its short life cycle, true-to-typeness, and high efficiency. The lack of AR formation from stem segments is a barrier to segment survival. Therefore, understanding the AR regulatory mechanisms is vital for the prolonged and effective use of biological resources. Several studies have been undertaken to comprehend the molecular and physiological control of AR, which has greatly extended our knowledge regarding AR formation in apples and other crops. Auxin, a master controller of AR formation, is widely used for inducing AR formation in stem cutting. At the same time, cytokinins (CKs) are important for cell division and molecular reprograming, and other hormones, sugars, and nutrients interact with auxin to control excision-induced AR formation. In this review, we discuss the present understandings of ARs’ formation from physiological and molecular aspects and highlight the immediate advancements made in identifying underlying mechanisms involved in the regulation of ARs. Despite the progress made in the previous decades, many concerns about excision-induced AR formation remain unanswered. These focus on the specific functions and interactions of numerous hormonal, molecular, and metabolic components and the overall framework of the entire shoot cutting in a demanding environment.

The adventitious roots (ARs) formation is a major setback for the mass proliferation of apple rootstocks. Nitrate is a vital signaling molecule for the regulation of root architecture. However, the inhibitory effect of nitrate at a high dose is missing. In this study, the physiological and molecular mechanism underlying ARs inhibition was tested. The B9 stem cuttings were grown on low and high nitrate concentrations, namely T1 and T2, respectively, and the T2 appeared as ARs inhibiting dose, where the rooting percentage was only 28% that was 130% less than T1 cuttings. Moreover, the average root numbers of T2 were only 31, which was 78% lower than T1 cuttings. At most time points, the endogenous hormones: JA, BR, ABA, CTK, GA, and AUX were exhibited at higher levels in response to T2, leading to hormonal imbalance in the plant and led to ARs inhibition. In addition, 205 known miRNAs belonging to 42 families and 438 novel miRNA were observed differentially expressed by the nitrate treatments. Where miR160a, miR390a, miR167, miR394, and miR169a were found to be related to auxin and ABA signaling, and miR171, miR166, miR156, miR319, and miR396 were related with cell fate transformation, proliferation, and enlargement. Our results propose that ARs formation is a complicated biological process, which was influenced by multiple hormone signaling pathways. This work is a pioneer giving knowledge on the inhibitory effects of high nitrate concentration on AR formation for future studies in woody plants.

Adventitious roots (ARs) formation is crucial for the vegetative propagation of horticultural crops. Nitrate availability is the main factor determining plant growth and development. Nitrate uptake and distribution is carried out by nitrate transporters (NRTs). In present work, we identified 84 MdNRT genes in the apple genome and analyzed them in terms of phylogenetic analysis, syntenic relationships, domain structure, cis-elements, and motif analysis. We further analyzed their amino acid composition, physical and chemical characteristics. Our results revealed that apple NRT family can be divided into three subfamilies; MdNRT1/PTR, MdNRT2, and MdNRT3 having 77, 5, and 2 members, respectively. The syntenic relationships indicated that almost 48.8 % of the total NRT genes appeared from genome duplication. The composition of cis-elements suggests that most of the MdNRT genes might be involved in hormones, light, and stress responses. Moreover, different nitrate treatments (9.4 mM/L, 18.8 mM/L, 28.1 mM/L, 46.9 mM/L, and 84.5 mM/L) were applied, and 28.1 mM/L was identified as an optimum level of nitrate for ARs formation and development in B9 apple rootstock. Additionally, RNA sequencing results revealed that several MdNRT genes showed an organ-specific expression pattern suggesting their role in nitrate distribution. These MdNRT genes indicated diverse expression profiles in response to nitrate treatments (28.1 mM/Land 46.9 mM/L) in the stem basal parts of B9 apple rootstock during AR formation. Interestingly, MdNRT2.1 gene has the strongest response to nitrate availability, advocating that MdNRT2.1 may have a significant role in the apple ARs formation and development. Furthermore, over-expression of 35 s MdNRT2.1 in tobacco increased root number (53.6 %), length (72.5 %), volume (54.6 %), and surface area (58.4 %) compared with wild type. Therefore, we concluded that MdNRT2.1 has a direct role in AR formation and development. Our research provides new genetic resources for functional characterization of NRT genes.