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Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

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 shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants. We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.

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.

Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation.

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.

Adventitious roots (ARs) induced are essential for apple rootstock vegetative propagation, however, a transcriptomic analysis of long non-coding RNAs (lncRNAs) that mediate AR formation under exogenous indole-3-butyric acid (IBA) treated in the apple dwarf rootstock ‘M26’ (Malus pumila Mill.) rarely been explored. To explore this, we examined ‘M26’ stem cuttings treated with IBA (1 mg L−1) and control (without IBA treatment), where anatomical analysis showed IBA-treated stems began to develop AR primordia within third day and ARs occurred after 16 days, while the control had no ARs developed. An assessment of hormone content levels in basal stem cuttings showed that, after IBA treatment, indole-3-acetic acid, zeatin riboside, and jasmonic acid were increased at third day but subsequently decreased later, however gibberellin-3 was decreased at third day. The control and IBA-treated third day basal stem cuttings were sent for whole-genome, high-throughput RNA sequencing. The results showed a total of 855 reliable lncRNAs were identified as novel lncRNAs and 63 novel lncRNAs were defined as IBA-responsive lncRNAs. Fifteen IBA-responsive lncRNAs were predicted to be putative targets for 94 miRNAs and three IBA-responsive lncRNA (CUFF.16781, CUFF.2143 and CUFF.41325) was predicted to be the target mimic of mdm-miR156 and mdm-miR396. Functional annotations of these potential target genes suggest that they are involved in many different biological processes, including plant hormone and sucrose signaling, which suggests that IBA-responsive lncRNAs may participate in AR formation in apple rootstocks. This study sheds light on IBA-mediated AR formation in apple rootstock cuttings and provides a foundation for further research.

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.

Background Melatonin (MT) plays a key role in the plant growth and development, however, whether MT involved in apple adventitious root (AR) development is not entirely understood. In present study, we set up four MT-treated groups at different times according to the stages of AR development and one control group in Malus domestica (MP), endogenous hormones levels of MT, auxin (IAA), zeatin-riboside (ZR), gibberellins (GA1 + 3), abscisic acid (ABA) were analyzed in five groups, then the expression of MT signal, IAA synthesis, transport, and signal transduction, cell cycle, and root development related genes were measured by RT-qPCR, the function of MdWOX11 was analyzes by technology of apple transgenic. Results Advance of AR development by MT is depended on its promotion of stage of AR induction at 0–2 d in the apple rootstocks. MT-treated increased IAA levels, there were crosstalk between MT and IAA during inducing AR formation. Expression analysis exposed that the expression of MdWOX11 induced by MT-treatment, and positively regulated AR formation in apple. Furthermore, MdWOX11 over-expressed lines produced more ARs than ‘GL3’, phenotypic analysis indicated that MdWOX11 over-expressed lines were more sensitive to exogenous MT-treatment than ‘GL3’, it was also indicated that MdWOX11 regulated AR formation in response to MT in apple rootstock. Conclusions MT promotes AR formation mainly during AR induction stage by inducing IAA levels as well as by up-regulation of MdWOX11.

Additional file 1: Figure S1. Morphological observations of AR formation in tissue culture plantlets of Malus prunifolia at 20 d. IBA group were continuously cultivated in 3.45 μM IBA and 1.29 μM MT, NPA group were continuously cultivated in 10 μM NPA and 1.29 μM MT, TIBA group were continuously cultivated in 10 μM TIBA and 1.29 μM MT. Figure S2. Identification of DNA level of in overexpression MdWOX11 transgenic lines, marker was 2000 bp, wild type is named as WT, there are three lines in overexpression MdWOX11 transgenic lines, they are MdWOX11OE-15#, MdWOX11OE-16#, MdWOX11OE-20#, H2O was set as negative control. Table S1. Composition of medium. Table S2. The gene name (the abbreviation and full name) and the apple MDP number, as well as the species and protein of the homologue on which the apple protein was based on. Table S3. Sequence of primers used for expression analysis, F for the former primer, R for the rear primer, MDP number of gene and length of primers.

Cytokinin (CK) inhibits adventitious root (AR) formation in stem cuttings. Little is known, however, about the mechanism underlying the inhibitory effect. In this study, 2 mg l-1 of exogenous 6-benzyl adenine (6-BA) was administered to 3 and 7-day-old apple rootstocks 'M.26' cuttings (3 and 7 days 6-BA) by transferring them from a rooting medium containing indole-3-butanoic acid to the medium containing 6-BA. Anatomical and morphological observations revealed that the exogenous application of 6-BA inhibited primordia formation in the 3 days 6-BA but not the 7 days 6-BA group. The concentration of auxin (IAA), the ratios of IAA/CK and IAA/abscisic acid were lower in 3 days 6-BA than in 7 days 6-BA. Expression analysis of genes known to be associated with AR formation was also analyzed. In the 3 days 6-BA group, high level of CK inhibited the synthesis and transport of auxin, as a result, low endogenous auxin level suppressed the auxin signaling pathway genes, as were other AR development and cell cycle related genes; all of which had an inhibitory impact on AR primordium formation. On the contrary, low CK level in the 7 days 6-BA, reduced the inhibitory impact on auxin levels, leading to an upregulated expression of genes known to promote AR primordia formation. Collectively, our data indicated that 3-7 days is the time period in which AR primordia formation occurs in cuttings of 'M.26' and that the inhibition of AR development by CK is due to the suppression of AR primordia development over 3-7 days period after culturing in rooting medium.

Malus hupehensis seedlings were treated with exogenous applications of IBA (0.1 mg L−1 and 0.5 mg L−1) in order to investigate the mechanism by which IBA promotes and effects lateral root (LR) development. Root morphology, plant height, plant crown diameter, and endogenous levels of hormone in LRs were evaluated. Additionally, patterns of differential gene expression related to root development were examined by RT-qPCR in IBA-treated and untreated roots during LR development. Results indicated that IBA treatment promoted both root and shoot growth, and that 0.5 mg L−1 IBA had a more obvious effect on growth promotion than 0.1 mg L−1 IBA. IBA treatment also induced an increase in the levels of endogenous auxin (IAA), zeatin-riboside (ZR), and the ratios of IAA/ABA and ABA/GA1+3, however, reduced gibberellin (GA1+3) and abscisic acid (ABA) levels. In addition, there was also a significant upregulation in the expression of MdYUCCA4, MdPIN1, MdPIN2 and MdAUX1 in response to the IBA treatment; thus leading to an increase in auxin levels. Although MdIAA23 was induced, the expression of MdIAA5 and MdIAA14, a negative regulator of MdARF7 and MdARF19, were downregulated in IBA-treated apple seedlings. This resulted in elevated expression of MdARRO1, MdGATA1 and MdSCR1, which was associated with an increase in lateral root development. Additionally, higher levels of endogenous auxin elevated the expression of MdWOX11, and thus the expression of MdWOX5, MdLBD16 and MdLBD29; collectively resulting in enhanced expression of the cell cycle related gene: MdCYCD3;1. This pattern of expression was again associated with a promotion in lateral root development. In summary, changes in gene expression and hormone levels resulted in an increased number of LRs and other root growth parameters in IBA-treated plants.

Malus hupehensis is regarded as a superior rootstock in China. In this study, in order to investigate the mechanism by which MeJA promotes and affects lateral root (LR) development, M. hupehensis seedlings were treated with exogenous applications of 1 μM methyl-jasmonate (MeJA). Results indicated that MeJA treatment promoted LR development, but inhibited shoot development. MeJA treatment increased the levels of both endogenous auxin (IAA) and zeatin-riboside (ZR), and reduced gibberellin (GA1+3). Additionally, a significant upregulation in the expression of MdCOI1, MdMYC2 and MdJDL1 were also observed in response to the MeJA treatment. Although transcript levels of key IAA synthesis related genes increased, but those genes related to IAA transport were declined, which corresponded with higher IAA levels in the MeJA-treated samples. The expression of MdIAA5, which is a negative regulator of MdARF7, was dramatically downregulated in MeJA-treated apple seedlings, resulted in elevated expression of MdLBD16 and MdLBD29. Additionally, the expression of MdCYCD3;1 and MdCYCP4;1 were induced, which in turn increased LR development. Collectively, MeJA treatment increased the number of LRs and other growth characteristics.

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.

The plant hormone auxin is essential for plant growth and development. YUCCA proteins catalyse the rate-limiting step for endogenous auxin biosynthesis. In this study, we isolated 20 MdYUCCA genes from apple genome. MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a were expressed in most organs and could support whole plant basal auxin synthesis. MdYUCCA4a, MdYUCCA10b, and MdYUCCA11a expression indicated roles for these genes in auxin biosynthesis in vegetative organs. MdYUCCA2b, MdYUCCA11b, and MdYUCCA11d were mainly expressed in flower organs. High temperature induced the expression of MdYUCCA4a, MdYUCCA6a, MdYUCCA8a, and MdYUCCA10a, and down-regulated the expression of MdYUCCA2b and MdYUCCA6b. Dual-luciferase assay indicated that MdPIF4 could trans-activate the MdYUCCA8a promoter. Overexpression of MdYUCCA8a increased IAA content, increased stem height, enhanced apical dominance, and led to silique malformation. These results provide a foundation for further investigation of the biological functions of apple MdYUCCAs.

Adventitious root (AR) formation is an essential process for the vegetative propagation of apple rootstocks. However, the mechanisms of miR160 mediate AR formation in apple remains to be seen. In this study, five mdm-miR160 family members were identified in apple rootstocks and two AUXIN RESPONSIVE FACTOR (MdARF16 and MdARF17) were found as the targets of mdm-miR160. Furthermore, the sequences of mdm-miR160 family members’ precursors were cloned from four different AR rooting ability apple rootstocks, whereas, mdm-miR160a, mdm-miR160c and mdm-miR160e were highly conserved than mdm-miR160b and mdm-miR160d. Mdm-miR160 and their targets expression patterns showed obviously negative relationship in different stages during AR formation. In addition, the transcript level of mdm-miR160 was higher in ‘SH6’ and lower in ‘MP’, where it responds to exogenous indole-3-butyric acid (IBA). Mdm-miR160a was cloned from ‘M26’ and used for further experiments. The result of over-expressed mdm-miR160a in tobacco (Nicotiana tabacum L.cv.SR1) inhibited AR formation in 35S:MdMIR160a transgenic lines as compared with WT, whereas, exogenous IBA treatment could promote AR formation in 35S:MdMIR160a transgenic lines. Expression of five endogenous miRNAs: miR167c, miR393a, miR396a, miR396b, and miR156 in transgenic lines were lower than in WT. Moreover, the targets of endogenous miRNAs in transgenic lines showed different expression patterns with WT. In consistent, mdm-miR160 was an auxin responsive factor and participated in AR formation in apple rootstock, however, over-expressed mdm-miR160a has inhibitory effect on AR formation while exogenous IBA treatment could raise the rooting ability of transgenic lines.

Adventitious root (AR) formation is a critical process for the clonal propagation of horticultural crops. Polyamines are involved in several processes associated with root growth such as cell division and cell development. In this study, Malus prunifolia var. ringo stem cuttings were treated with exogenous spermidine (Spd) 20 μmol/L in order to investigate the physiological and molecular mechanism of Spd to promote ARs formation. The results of the present study indicated an involvement of Spd in AR formation in apple rootstock by interacting with IAA. Morphological and anatomical observation revealed that the Spd application promotes AR formation. According to our results exogenous Spd application promoted AR formation by improving the concentration of endogenous IAA and GA3 at 1, 3 and 1, 8 days respectively as well as by decreasing the ABA and JA contents at all time points except JA at 16 day in response to Spd treatment. Furthermore no significant difference was observed in BR and ZR levels at all time points except at 8 day in both groups. Spd application obviously up-regulated the expression of Spd related genes (MdSAMDC1, MdPAO and MdSPDS6) and auxin biosynthesis related genes (MdIAA7, MdIAA14 and MdIAA23) in the basal parts of apple rootstock. On the other hand MdWOX11 up-regulated the expression of MdLBD16 and MdLBD29, which induced the expression of cell cycle related genes MdCYCD1;1 and MdCYCP4;1. Collectively, our work laid the foundation for applied research and could be useful in future crop improvement programs.

Background Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. Results Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein–protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of ‘Nagafu 2’ (CF)/CF than in dwarf trees of CF/‘Malling 9’ (M.9), and in CF than in the spur-type bud mutation “Yanfu 6” (YF). Conclusion This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth. Electronic supplementary material The online version of this article (10.1186/s12864-018-5342-1) contains supplementary material, which is available to authorized users.

Adventitious root (AR) formation is essential for the vegetative propagation of apple rootstocks. miRNAs play a significant role in regulating AR development, however, large-scale transcriptomic data on miRNA mediated AR formation in apple rootstocks is lacking. Therefore, in order to identify the molecular mechanisms underlying AR formation in ‘M9-T337’ apple rootstocks, transcriptomic changes occurring during key time points of AR formation (0, 3, and 16 days) were analyzed using high-throughput sequencing with a focus on miRNAs. A total of 84 known miRNAs and 56 novel miRNAs have differentially expressed were identified. Additionally, a total of 88 target genes of known miRNAs and 76 target genes of novel miRNAs were identified by degradome sequencing. The expression levels of the miRNAs and target genes were quantified by RT-qPCR. Results indicate that miRNAs and their target genes are associated with auxin signal-related (miR160 and miR390), stress response-related (miR398, miR395 and miR408), cell fate transformation-, proliferation- and enlargement-related (miR171, miR156, miR166, miR319 and miR396). These all involve pathways that participate in AR formation in ‘M9-T337’ apple rootstock. In addition, hormones (AUX, CTK, GA3, BR, JA, and ABA) are also involved in regulating AR formation. The candidate genes belonging to pathways associated with AR formation exhibited significantly higher expression levels, providing evidence that they may be involved in the regulation of AR development. The collective results of the present study indicate that the developmental process associated with AR formation in apple rootstock is extremely complex. The known and novel miRNAs and target genes that were identified by high-throughput and degradome sequencing, respectively, provide a framework for the future analysis of miRNAs associated with AR development in apple rootstocks, and provide new information that can be used to better understand AR development in woody plants.

Growth-regulating factor (GRF) proteins comprise a family of plant-specific transcription factors that play crucial roles in regulating plant architecture. However, information pertaining to the GRF gene family in apple (Malus × domestica) is limited. In the present study, 16 GRF family members were identified in the apple genome, and nine were predicted to be putative targets of apple microRNA396 (miR396). Phylogenetic and synteny analyses of MdGRF genes indicated that gene duplication events have likely contributed to the expansion and evolution of MdGRFs. Moreover, several pairs of MdGRF/AtGRF homologs were identified. The tissue-specific expression patterns of MdmiR396–MdGRF genes in different tissues and in the ArrayExpress database were subsequently characterized. The analyses indicated that they had opposing functions. Exogenous brassinolide, gibberellic acid, 1-naphthylacetic acid, and 6-benzylaminopurine increased primary shoot lengths and diameters, and elevated the expression levels of several MdGRF genes. The expression levels of MdmiR396 and MdGRF genes were reduced in the standard cultivar “Nagafu 2” compared with its short branch spur-type bud mutation “Yanfu 6,” and in the standard graft combination Nagafu 2/Nagafu 2 compared with the dwarfing graft combination Nagafu 2/“Malling 9.” Thus, MdmiR396-MdGRF may be involved in the regulation of apple tree size in response to hormone signals and in different apple varieties and grafting combinations. This work provides a basis for further functional research on MdmiR396-MdGRFs and their potential involvement in the regulation of apple tree architecture.

In plants, brassinosteroid biosynthesis and metabolism genes affect endogenous brassinosteroid metabolic processes as well as stem and root growth. However, there is little information available regarding these genes in apple. In this study, 22 brassinosteroid biosynthesis and metabolism genes were identified in apple (Malus domestica). These genes were named according to their chromosomal locations and the Arabidopsis thaliana homologs. Their conserved characteristic domains, evolutionary relationships, syntenic relationships, chemical characteristics, gene/protein structures, interactions among the encoded proteins, promoter sequences, and functions were investigated. These 22 genes were clustered with their A. thaliana homologs based on bioinformatics analyses, which suggested they are functionally similar in apple and A. thaliana. Tissue-specific expression levels revealed that most of these genes are important for stem growth and development, while several of these genes affect lateral root formation. The transcription patterns of these genes in shoot tips were investigated following diverse treatments [brassinosteroid (shoot tips and roots), brassinazole, auxin, and temperature]. Gene expression levels were also examined in different grafting combinations ('Nagafu No. 2'/Malling 9 and 'Nagafu No. 2'/'Nagafu No. 2') and shoot varieties ('Yanfu No. 6' and 'Nagafu No. 2'). The results indicated that these genes may be involved in apple stem and root growth. The comprehensive genome-wide analysis of brassinosteroid biosynthesis and metabolism genes presented herein may be useful for breeding new apple cultivars with increased vigor. The data also represent a rich genetic resource for future apple gene functional investigations that may have implications for the genetic improvement of apple tree species.

Adventitious root (AR) formation, which is controlled by endogenous and environmental factors, is indispensable for vegetative asexual propagation. However, comprehensive proteomic data on AR formation are still lacking. The aim of this work was to study indole-3-butyric acid (IBA)-induced AR formation in the dwarf apple rootstock ‘T337’. In this study, the effect of IBA on AR formation was analysed. Subsequent to treatment with IBA, both the rooting rate and root length of ‘T337’ increased significantly. An assessment of hormone levels in basal stem cuttings suggested that auxin, abscisic acid, and brassinolide were higher in basal stem cuttings that received the exogenous IBA application; while zeatin riboside, gibberellins, and jasmonic acid were lower than non-treated basal stem cuttings. To explore the underlying molecular mechanism, an isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic technique was employed to identify the expression profiles of proteins at a key period of adventitious root induction (three days after IBA treatment). In total, 3355 differentially expressed proteins (DEPs) were identified. Many DEPs were closely related to carbohydrate metabolism and energy production, protein homeostasis, reactive oxygen and nitric oxide signaling, and cell wall remodeling biological processes; as well as the phytohormone signaling, which was the most critical process in response to IBA treatment. Further, RT-qPCR analysis was used to evaluate the expression level of nine genes that are involved in phytohormone signaling and their transcriptional levels were mostly in accordance with the protein patterns. Finally, a putative work model was proposed. Our study establishes a foundation for further research and sheds light on IBA-mediated AR formation in apple as well as other fruit rootstock cuttings.

Table S1. Primers and their sequences used for qRT-PCR analyses. a Internal control. Table S2. Best hits for putative apple SERK proteins in BLAST searches against apple ESTa assemblies. a Downloaded from NCBI database. Table S3. Sequences of MdSERK proteins. Table S4. Amino acid composition, physical and chemical characteristics, and subcellular localization of MdSERK proteins. aGrand average of hydropathicity bThree main amino acids in each protein (A, Ala; S, Ser; G, Gly; L, Leu; V, Val). Table S5. Motif sequences identified using MEME tools. Motif numbers correspond to motifs in Additional file 2: Figure S1. Table S6. Secondary structures of MdSERK proteins. Table S7. Synteny analysis of MdSERK genes. Table S8. Synteny analysis of MdSERK and AtSERK genes. (DOCX 37 kb)

Background The root architecture of grafted apple (Malus spp.) is affected by various characteristics of the scions. To provide information on the molecular mechanisms underlying this influence, we examined root transcriptomes of M. robusta rootstock grafted with scions of wild-type (WT) apple (M. spectabilis) and a more-branching (MB) mutant at the branching stage. Results The growth rate of rootstock grafted MB was repressed significantly, especially the primary root length and diameter, and root weight. Biological function categories of differentially expressed genes were significantly enriched in processes associated with hormone signal transduction and intracellular activity, with processes related to the cell cycle especially down-regulated. Roots of rootstock grafted with MB scions displayed elevated auxin and cytokinin contents and reduced expression of MrPIN1, MrARF, MrAHP, most MrCRE1 genes, and cell growth-related genes MrGH3, MrSAUR and MrTCH4. Although auxin accumulation and transcription of MrPIN3, MrALF1 and MrALF4 tended to induce lateral root formation in MB-grafted rootstock, the number of lateral roots was not significantly changed. Sucrose, fructose and glucose contents were not decreased in MB-grafted roots compared with those bearing WT scions, but glycolysis and tricarboxylic acid cycle metabolic activities were repressed. Root resistance and nitrogen metabolism were reduced in MB-grafted roots as well. Conclusions Our findings suggest that root growth and development of rootstock are mainly influenced by sugar metabolism and auxin and cytokinin signaling pathways. This study provides a basis that the characteristics of scions are related to root growth and development, resistance and activity of rootstocks. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2484-x) contains supplementary material, which is available to authorized users.

Gene-specific primers used for quantitative real-time PCR. (DOC 37Â kb)

Clusters of annotated GO terms in the biological process category enriched in Up-regulated (A) and Down-regulated (B) between roots of grafted WT and MB apple. (DOC 2423Â kb)

Photosynthetic parameters of WT and MB grafted apple leaves. Comparison of photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci), in WT and MB leaves. Values are means ± SE (n = 10). Significant differences (*P

Selected genes related to sugar metabolism. (DOC 93Â kb)

Differentially expressed genes related to hormone signaling. (DOC 194Â kb)

Selected differentially expressed genes related to cell division, differentiation and growth. (DOC 39Â kb)

Selected differentially expressed genes related to salicylic and jasmonic acid signaling and nitrogen metabolism. (DOC 48Â kb)

Adventitious roots (AR) play an important role in the vegetative propagation of apple rootstocks. The potential role of hormone, wounding, and sugar signalling pathways in mediating AR formation has not been adequately explored and the whole co-expression network in AR formation has not been well established in apple. In order to identify the molecular mechanisms underlying AR formation in &lsquo, T337&rsquo, apple rootstocks, transcriptomic changes that occur during four stages of AR formation (0, 3, 9 and 16 days) were analyzed using high-throughput sequencing. A total of 4294 differentially expressed genes were identified. Approximately 446 genes related to hormones, wounding, sugar signaling, root development, and cell cycle induction pathways were subsequently selected based on their potential to be involved in AR formation. RT-qPCR validation of 47 genes with known functions exhibited a strong positive correlation with the RNA-seq data. Interestingly, most of the candidate genes involved in AR formation that were identified by transcriptomic sequencing showed auxin-responsive expression patterns in an exogenous Indole-3-butyric acid (IBA)-treatment assay: Indicating that endogenous and exogenous auxin plays key roles in regulating AR formation via similar signalling pathways to some extent. In general, AR formation in apple rootstocks is a complex biological process which is mainly influenced by the auxin signaling pathway. In addition, multiple hormones-, wounding- and sugar-signaling pathways interact with the auxin signaling pathway and mediate AR formation in apple rootstocks.