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Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Adventitious root formation is a bottleneck for the mass propagation of microshoots, and nitrate is an essential nutrient regulating adventitious roots. WOX11 is involved in adventitious rooting. But the crosstalk between nitrate and WOX11 is completely unknown. In this study, MdWOX11 transgenic apple microshoots were grown on different nitrate treatments. Low nitrate promotes adventitious rooting in overexpressed microshoots more than wild type and RNA interference microshoots. In contrast, medium nitrate significantly inhibits it in overexpressed and RNA interference microshoots compared with wild type microshoots. Stem anatomy indicated that medium nitrate delays root primordia formation compared with low nitrate. Methyl jasmonate and zeatin riboside played positive and negative roles in adventitious rooting, respectively. Transcriptomic analysis was conducted to understand the molecular mechanisms behind the phenotypes better. Hormone signalling, sugar metabolism, nitrogen metabolism, cell cycle and root development pathway-related genes were selected for their potential involvement in adventitious rooting. Results suggest that nitrogen signaling and MdWOX11 expression affect cytokinin accumulation and response to cytokinin through regulating the expression of genes related to cytokinin synthesis and transduction pathways, which ultimately affect adventitious rooting. This study provided important insights into the complicated networks involved in adventitious rooting in transgenic microshoots under nitrate treatments.

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.

In the process of vegetative propagation of apple rootstocks, the development of adventitious roots (ARs) has crucial importance. Nitrate is an essential nutrient necessary for plant growth; however, the inhibitory effect of high nitrate on ARs formation has not been explored. The physiological and molecular mechanisms underlying ARs inhibition were examined in this study. Stem cuttings of B9 apple rootstock were cultured on two nitrate treatments (T1 = 18.7 mM L-1 and T2 = 37.5 mM L-1 ), where T2 was identified as ARs inhibiting treatment. Morphological and anatomical observations advocating that high availability of nitrate inhibited AR formation by delaying the ARs initiation and emergence stages, where the root number was 287%, and the length was 604.6% lower than the T1 cuttings. Moreover, the contents of endogenous hormones were also elevated in response to T2 at most of the time points, which may cause a hormonal imbalance within the plant body and drive toward ARs inhibition. Furthermore, 3686 genes were differentially expressed by high-throughput sequencing. Out of these, 1797 genes were upregulated, and 1889 genes were downregulated. Approximately 238 genes related to nitrate, hormones, root development, and cell-cycle induction pathways were selected according to their potential to be involved in ARs regulation. This is the first study providing information regarding the inhibitory effect of high nitrate on ARs formation in apple rootstock.

Malus hupehensis is an extensively used apple rootstock in China. In the current study, M. hupehensis seedlings were treated with exogenous 2.2 µm 6-benzyladenine (6-BA) so as to investigate the mechanism by which 6-BA affects lateral root development. The results indicate that 6-BA treatment promotes elongation and thickening of both root and shoot in M. hupehensis, but reduces the number of lateral roots, as well as reducing the auxin level after 6-BA treatment. Moreover, MhAHK4, MhRR1 and MhRR2 were also significantly up-regulated in response to 6-BA treatment. Expression levels of auxin synthesis- and transport-related genes, such as MhYUCCA6, MhYUCCA10, MhPIN1 and MhPIN2, were down-regulated, which corresponds with lower auxin levels in the 6-BA-treated seedlings. A negative regulator of auxin, MhIAA3, was induced by 6-BA treatment, leading to reduced expression of MhARF7 and MhARF19 in 6-BA-treated seedlings. As a result, expression of MhWOX11, MhWOX5, MhLBD16 and MhLBD29 was blocked, which in turn inhibited lateral root initiation. In addition, a lower auxin level decreased expression of MhRR7 and MhRR15, which repressed expression of key transcription factors associated with root development, thus inhibiting lateral root development. In contrast, 6-BA treatment promoted secondary growth (thickening) of the root by inducing expression of MhCYCD3;1 and MhCYCD3;2. Collectively, the changes in hormone levels and gene expression resulted in a reduced number of lateral roots and thicker roots in 6-BA-treated plants.

Adventitious root (AR) formation is a bottleneck for the mass propagation of apple rootstocks, and water stress severely restricts it. Different hormones and sugar signaling pathways in apple clones determine AR formation under water stress, but these are not entirely understood. To identify them, GL-3 stem cuttings were cultured on polyethylene glycol (PEG) treatment. The AR formation was dramatically decreased compared with the PEG-free control (CK) cuttings by increasing the endogenous contents of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (JA-me) and reducing the indole-3-acetic acid (IAA) and gibberellic acid 3 (GA3) contents. We performed a transcriptomic analysis to identify the responses behind the phenotype. A total of 3204 differentially expressed genes (DEGs) were identified between CK and PEG, with 1702 upregulated and 1502 downregulated genes. Investigation revealed that approximately 312 DEGs were strongly enriched in hormone signaling, sugar metabolism, root development, and cell cycle-related pathways. Thus, they were selected for their possible involvement in adventitious rooting. However, the higher accumulation of ABA, ZR, and JA-me contents and the upregulation of their related genes, as well as the downregulation of sugar metabolism-related genes, lead to the inhibition of ARs. These results indicate that AR formation is a complicated biological process chiefly influenced by multiple hormonal signaling pathways and sugar metabolism. This is the first study to demonstrate how PEG inhibits AR formation in apple plants.

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 (IAA), zeatin riboside (ZR), and jasmonic acid (JA) 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.

Apples are economically valuable and widely consumed fruits. The adventitious roots (ARs) formation is gridlock for apple trees mass propagation. The possible function of multiple hormones and sugar signaling pathways regulating ARs formation has not been completely understood in apple. In this study, B9 stem cuttings were treated with KCl treatment, where the highest root numbers (220) and maximum root length of 731.2 cm were noticed in KCl-treated cuttings, which were 98.2% and 215% higher than control cuttings. The content of endogenous hormones: IAA, ZR, JA, GA, and ABA were detected higher in response to KCl at most time-points. To figure out the molecular mechanisms underlying this effect, we investigated transcriptome analysis. In total, 4631 DEGs were determined, from which about 202 DEGs were considerably enriched in pathways associated with hormone signaling, sugar metabolism, root development, and cell cycle-related and were thereupon picked out on their potential involvements in ARs formation. Though, IAA accumulation and up-regulation of various genes contribute to induce AR formation. These results suggest that AR formation is a complex biological process in apple rootstocks, influenced mainly by the auxin signaling pathway and sugar metabolism.

Adventitious root (AR) formation is great significance for apple rootstock breeding. Transcriptome analyses were performed with cytokinins (CTKs) signal treatments to analyze the mechanism of AR formation. The results showed that 6-benzyadenine (6-BA) treatment inhibited AR formation. Histological analysis also observed that AR primordium cell formation was significantly suppressed by 6-BA treatment; the ratio of auxin/cytokinins exhibited the lowest values at 1 and 3 day (d) in the 6-BA treatment group. Furthermore, the differentially expressed genes were divided into five categories, including auxin, cytokinins, other hormones, cell cycle, and carbohydrate metabolism pathways. Due to the study of cytokinins signal treatment, it is important to understand the particular module mediated by the cytokinins pathway. The expression level of MdRR12 (a family member of B-type cytokinins-responsive factors) was significantly upregulated at 3 d by 6-BA treatment. Compared to the wild type, the 35S::MdRR12 transgenic tobaccos suppressed AR formation. The promoter sequence of MdCRF8 contains AGATT motif elements that respond to MdRR12. RNA-seq and RT-qPCR assays predicted cytokinins response factor (MdCRF8) to be a downstream gene regulated by MdRR12. The activity of the pro-MdCRF8-GUS promoter was obviously induced by 6-BA treatment and inhibited by lovastatin (Lov) treatment. Yeast one-hybrid, dual-luciferase reporter, and GUS coexpression assays revealed that MdRR12 could directly bind to the MdCRF8 promoter. Additionally, 35S::MdCRF8 transgenic tobaccos also blocked AR growth. Compared to the wild type, 35S::MdRR12 and 35S::MdCRF8 transgenic tobaccos enhanced sensitivity to cytokinins. Thus, we describe that MdRR12 and MdCRF8 function as integrators of cytokinins signals that affect cell cycle- and carbohydrate metabolism-related genes to regulate cell fate transition during AR formation. On the basis of these results, we concluded that the MdRR12-MdCRF8 module is involved in the negative regulation of AR formation in apple rootstock and can potentially be applied in agriculture using genetic approaches.

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 root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

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.

Our previous studies suggested that a direct hippocampo-cerebellar projection might exist in the chicken. To confirm such a presumption of hippocampo-cerebellar interactions, horseradish peroxidase (HRP) was used as a retrograde tracer to be injected into the white matter of the folia VI-VIII of the cerebellum in young and adult chickens. In another set of experiments, young chickens were subjected to electrolytic lesions of the hippocampal formation (HF), especially the ventromedial portion, and the cerebellum was observed with the electron microscope to find neuronal degeneration in the HF. Following injections of HRP into the cerebellum, a large number of labeled neurons were found in the area APHm-APHim of the HF in the young and adult chickens. As a result of the electrolytic lesions of the APHm-APHim in the HF, many large degenerated nerve fibers were found in the white matter in the vicinity of the lateral nucleus of the cerebellum, and some small degenerated fibers were found in the white matter of the folia VI-VIII. In the cerebellar cortex of folia VI-VIII, degenerated axonal terminals occurred in both the molecular and Purkinje layers, but not in the granular layer. In the lateral nucleus, some dark degenerating axonal terminals were recognized to connect with the perikarya of neurons of this nucleus. The present experiments demonstrate that the APHm-APHim of the HF directly projected to the cortex of folia VI-VIII and the lateral nucleus of the cerebellum in young chickens.

The performance management is a very important element of management for the modern enterprise management. Academic circles often study enterprise performance management based on the specific enterprise performance management, analyzing concretely based on concrete problems. This paper constructs the influencing factors of performance management effectiveness mainly based on the performance management of the relevant documents and materials, and does empirical analysis aimed at the high and new technology enterprises. So, it determines influencing factors according to above actions. They should not only improve enterprise performance evaluation index of the work, quantificating index, making clear duty, analyzing job, but also have to strengthen the enterprise leader’s attention and supervision, providing a reference on enterprise performance management, and guide the enterprises to improve and strengthen performance management effectiveness index factors in the performance management, realizing good effectiveness of enterprise performance management.