Adventitious Root Formation In Apple Research Articles

Strigolactone regulates adventitious root formation via the MdSMXL7-MdWRKY6-MdBRC1 signaling cascade in apple.

Propagation through stem cuttings is a popular method worldwide for species such as fruit tree rootstocks and forest trees. Adventitious root (AR) formation from stem cuttings is crucial for effective and successful clonal propagation of apple rootstocks. Strigolactones (SLs) are newly identified hormones involved in AR formation. However, the regulatory mechanisms underpinning this process remain elusive. In the present study, weighted gene co-expression network analysis, as well as rooting assays using stable transgenic apple materials, revealed that MdBRC1 served as a key gene in the inhibition of AR formation by SLs. We have demonstrated that MdSMXL7 and MdWRKY6 synergistically regulated MdBRC1 expression, depending on the interactions of MdSMXL7 and MdWRKY6 at the protein level downstream of SLs as well as the direct promoter binding on MdBRC1 by MdWRKY6. Furthermore, biochemical studies and genetic analysis revealed that MdBRC1 inhibited AR formation by triggering the expression of MdGH3.1 in a transcriptional activation pathway. Finally, the present study not only proposes a component, MdWRKY6, that enables MdSMXL7 to regulate MdBRC1 during the process of SL-controlled AR formation in apple, but also provides prospective target genes to enhance AR formation capacity using CRISPR (i.e. clustered regularly interspaced short palindromic repeats) technology, particularly in woody plants.

Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

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.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone-mediated inhibition of adventitious root formation in apple.

Apple is one of the most important fruit crops in temperate regions and largely relies on cutting propagation. Adventitious root formation is crucial for the success of cutting propagation. Strigolactones have been reported to function in rooting of woody plants. In this study, we determined that strigolactones have inhibitory effects on adventitious root formation in apple. Transcriptome analysis identified 12 051 differentially expressed genes over the course of adventitious root initiation, with functions related to organogenesis, cell wall biogenesis or plant development. Further analysis indicated that strigolactones might inhibit adventitious root formation through repressing two core hub genes, MdLAC3 and MdORE1. Combining small RNA and degradome sequencing, as well as dual-luciferase sensor assays, we identified and validated three negatively correlated miRNA-mRNA pairs, including mdm-miR397-MdLAC3 and mdm-miR164a/b-MdORE1. Overexpression of mdm-miR164b and silencing MdORE1 exhibited enhanced adventitious root formation in tobacco and apple, respectively. Finally, we verified the role of mdm-miR164b-MdORE1 in strigolactone-mediated repression of rooting ability. Overall, the identified comprehensive regulatory network in apple not only provides insight into strigolactone-mediated adventitious root formation in other woody plants, but also points to a potential strategy for genetic improvement of rooting capacity in woody plants.

Different miRNAs and hormones are involved in PEG-induced inhibition of adventitious root formation in apple

• PEG inhibited AR formation in GL-3 apple cuttings. • Lower IAA and higher ABA, JA-me, and ZR content lead to the inhibition of ARs. • 32 known miRNAs belonging to 22 families and 60 novel miRNAs were found to be differentially expressed. • A total of 81 target genes of known miRNAs and 62 targets of novel miRNAs were identified using degradome sequencing. In apples, adventitious root (AR) formation plays a crucial role in the life cycle, but drought stress limits this process. Multiple hormones and miRNAs play key roles in regulating ARs under drought conditions, but they are lacking in GL-3 apple clones. Therefore, to identify physiological and molecular changes behind the phenotype, GL-3 apple cuttings were treated with 6% polyethylene glycol (PEG) during the activation, induction, initiation, and emergence stages of AR formation and compared with a PEG-free control. Anatomical analysis indicates that 6% PEG treatment delays AR primordia formation and produces fewer thin ARs than control plants by decreasing the indole-3-acetic acid (IAA) endogenous content and promoting the abscisic acid (ABA), methyl jasmonate (JA-me), and zeatin riboside (ZR) content at all time points. Furthermore, 32 known miRNAs belonging to 22 families and 60 novel miRNAs were found to be differentially expressed. A total of 81 target genes of known miRNAs and 62 targets of novel miRNAs were identified using degradome sequencing. The known miRNA targets were classified into 50 GO terms. In biological processes, various terms related to the response to water deprivation; salt, heat, and cold stress; abscisic acid-activated signaling pathways; and so on, were observed. The KEGG analysis indicates 20 pathways from which plant hormone signal transduction, ribosomes, etc., were the most enriched. Further analyses indicated that miRNAs (miR167b, miR393g, and miR477a) related to IAA, ABA, and JA were induced; various stress-responsive miRNAs (miR171c, miR169o, miR159a, miR10979, and miR167a) were also promoted; and cell division and development-related miRNAs (miR396a and miR482a) were repressed by 6% PEG treatment. These results indicate that AR formation is an intricate biological process in apples under stressed conditions regulated by various hormones, related miRNAs, and target genes. This study delivers new information about the underlying regulatory mechanisms of AR formation in GL-3 and other plants.

The BTB protein MdBT2 recruits auxin signaling components to regulate adventitious root formation in apple.

Ubiquitination is an important post-translational protein modification. Although BROAD-COMPLEX, TRAMTRACK AND BRIC A BRAC and TRANSCRIPTION ADAPTOR PUTATIVE ZINC FINGER domain protein 2 (BT2) is involved in many biological processes, its role in apple (Malus domestic) root formation remains unclear. Here, we revealed that MdBT2 inhibits adventitious root (AR) formation through interacting with AUXIN RESPONSE FACTOR8 (MdARF8) and INDOLE-3-ACETIC ACID INDUCIBLE3 (MdIAA3). MdBT2 facilitated MdARF8 ubiquitination and degradation through the 26S proteasome pathway and negatively regulated GRETCHEN HAGEN 3.1 (MdGH3.1) and MdGH3.6 expression. MdARF8 regulates AR formation through inducing transcription of MdGH3s (MdGH3.1, MdGH3.2, MdGH3.5, and MdGH3.6). In addition, MdBT2 facilitated MdIAA3 stability and slightly promoted its interaction with MdARF8. MdIAA3 inhibited AR formation by forming heterodimers with MdARF8 as well as other MdARFs (MdARF5, MdARF6, MdARF7, and MdARF19). Our findings reveal that MdBT2 acts as a negative regulator of AR formation in apple.

Transcriptome Analysis Reveals Multiple Genes and Complex Hormonal-Mediated Interactions with PEG during Adventitious Root Formation 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.

Melatonin promotes adventitious root formation in apple by promoting the function of MdWOX11

BackgroundMelatonin (MT) is important for plant growth and development; however, it is not known whether MT is involved in apple adventitious root (AR) development. In this study, we treated Malus prunifolia (MP) at four different stages of AR development, and analyzed the level of the endogenous hormones MT, auxin (IAA), zeatin-riboside (ZR), abscisic acid (ABA), and gibberellins (GA1 + 3) in all four treatment groups and the untreated control group. The expression of MT, IAA biosynthesis, transport and signal transduction, the cell cycle, and root development related genes were quantified by RT-qPCR. The function of MdWOX11 was analyzed in transgenic apple plants.ResultsThe promotion of AR development by MT was dependent on the stage of AR induction between 0 and 2 d in apple rootstocks. MT-treatment increased the level of IAA and crosstalk existed between MT and IAA during AR formation. The expression of MdWOX11 was induced by MT treatment and positively regulated AR formation in apple. Furthermore, transgenic lines that overexpressed MdWOX11 lines produced more ARs than ‘GL3’. Phenotypic analysis indicated that MdWOX11 overexpression lines were more sensitive to exogenous MT treatment than ‘GL3’, suggesting that MdWOX11 regulates AR formation in response to MT in apple rootstock.ConclusionsMT promotes AR formation mainly during the AR induction stage by inducing IAA levels and upregulating MdWOX11.

Auxin and Its Interaction With Ethylene Control Adventitious Root Formation and Development in Apple Rootstock.

Adventitious root (AR) formation is indispensable for vegetative asexual propagation. Indole-3-butyric acid (IBA) functioned indirectly as precursor of IAA in regulating AR formation. Ethylene affects auxin synthesis, transport, and/or signaling processes. However, the interactions between auxin and ethylene that control AR formation in apple have not been elucidated. In this study, we investigated the effects of IBA and its interaction with ethylene on AR development in apple. The results revealed that IBA stimulated the formation of root primordia, increased the number of ARs, and upregulated expression of genes (MdWOX11, MdLBD16, and MdLBD29) involved in AR formation. Comparison of different periods of IBA application indicated that IBA was necessary for root primordium formation, while long time IBA treatment obviously inhibited root elongation. RNA-seq analysis revealed that many plant hormone metabolism and signal transduction related genes were differentially expressed. IBA stimulated the production of ethylene during AR formation. Auxin inhibiting ARs elongation depended on ethylene. Together, our results suggest that the inhibitory role of auxin on AR elongation in apples is partially mediated by stimulated ethylene production.

Mdm-miR160 Participates in Auxin-Induced Adventitious Root formation of apple rootstock

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.

ITRAQ-Based Proteomic Analysis Reveals Potential Regulation Networks of IBA-Induced Adventitious Root Formation in Apple.

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.

Involvement of the ARRO-1 gene in adventitious root formation in apple

Adventitious root formation is a limiting factor for vegetative propagation in apple. The molecular mechanisms underlying the rooting process are still largely unknown and need to be extensively investigated. The Adventitious Rooting Related Oxygenase (ARRO-1) has previously been isolated from apple and has been shown to be upregulated during adventitious root induction. However, the function of this gene is still unclear. The aim of this study was to downregulate the ARRO-1 gene expression, using RNAi technique, to study the function of ARRO-1 in adventitious root formation in apple. The apple rootstock M26 was transformed with the RNAi- ARRO-1 construct. The transgenic clones, confirmed by PCR and Southern blot analysis, showed significantly reduced adventitious root formation both with microcuttings and stem discs, indicating the involvement of ARRO-1 in adventitious root formation. The transgenic clones also appeared to be more sensitive to exogenous hormones compared to the untransformed control plants, suggesting that ARRO-1 may be involved in regulating hormone homeostasis. Relative quantification of the ARRO-1 mRNA showed no obvious difference in transcript levels between untransformed control and the transformants. A possible explanation for this is that the downregulation of the ARRO-1 gene might be translational rather than transcriptional.

Isolation and characterization of a cDNA encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during adventitious root formation in apple (Malus domestica 'Jork 9') stem discs

ODDs typically catalyse such reactions as hydroxylation A cDNA clone encoding a novel 2-oxoacid-dependent di- and epoxidation and are involved in a variety of diVerent oxygenase which is up-regulated during the early phases of biosynthetic pathways (Prescott and John, 1996). The isol- adventitious root formation in Malus domestica 'Jork 9' has ated cDNA has subsequently been designated ARRO-1 been isolated. The corresponding gene has been designated (Adventitious Rooting Related Oxygenase). Sequence com- ARRO-1 (Adventitious Rooting Related Oxygenase) and is parison with other plant dioxygenases revealed that the represented in the apple genome by a number of distinct ARRO-1 cDNA was missing approximately 200 bp from its gene copies. N-terminus. A strategy based on the polymerase chain reac- tion (PCR) was used to amplify the remaining ARRO-1

Gene note. Isolation and characterization of a cDNA encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during adventitious root formation in apple (Malus domestica 'Jork 9') stem discs

Gene note. Isolation and characterization of a cDNA encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during adventitious root formation in apple (Malus domestica 'Jork 9') stem discs