MxMPK6-2-mediated phosphorylation enhances the response of apple rootstocks to Fe deficiency by activating PM H+ -ATPase MxHA2.

Iron (Fe) deficiency significantly affects the growth and development, fruit yield and quality of apples. Apple roots respond to Fe deficiency stress by promoting H+ secretion, which acidifies the soil. In this study, the plasma membrane (PM) H+ -ATPase MxHA2 promoted H+ secretion and root acidification of apple rootstocks under Fe deficiency stress. H+ -ATPase MxHA2 is upregulated in Fe-efficient apple rootstock of Malus xiaojinensis at the transcription level. Fe deficiency also induced kinase MxMPK6-2, a positive regulator in Fe absorption that can interact with MxHA2. However, the mechanism involving these two factors under Fe deficiency stress is unclear. MxMPK6-2 overexpression in apple roots positively regulated PM H+ -ATPase activity, thus enhancing root acidification under Fe deficiency stress. Moreover, co-expression of MxMPK6-2 and MxHA2 in apple rootstocks further enhanced PM H+ -ATPase activity under Fe deficiency. MxMPK6-2 phosphorylated MxHA2 at the Ser909 site of C terminus, Thr320 and Thr412 sites of the Central loop region. Phosphorylation at the Ser909 and Thr320 promoted PM H+ -ATPase activity, while phosphorylation at Thr412 inhibited PM H+ -ATPase activity. MxMPK6-2 also phosphorylated the Fe deficiency-induced transcription factor MxbHLH104 at the Ser169 site, which then could bind to the promoter of MxHA2, thus enhancing MxHA2 upregulation. In conclusion, the MAP kinase MxMPK6-2-mediated phosphorylation directly and indirectly regulates PM H+ -ATPase MxHA2 activity at the protein post-translation and transcription levels, thus synergistically enhancing root acidification under Fe deficiency stress.

The Root-Lesion Nematode, Pratylenchus penetrans, Affects Early Growth and Physiology of Grafted M.9, G.41, and G.935 Apple Rootstocks Similarly Under Field Microplot Conditions.

The root-lesion nematode, Pratylenchus penetrans, is a ubiquitous parasite of roots of temperate fruit trees. It affects early growth of trees replanted into former orchard sites where populations have built up and may contribute to decline complexes of older trees. Most British Columbia, Canada, apple acreage is planted with M.9 rootstock, but growers are increasingly considering Geneva-series rootstocks such as G.41 and G.935. Among these rootstocks, responses to P. penetrans, specifically, are poorly known. To compare the resistance and tolerance to P. penetrans of G.41, G.935, and M.9 rootstocks ('Ambrosia' scion), a field microplot experiment was established in spring of 2020 at the Summerland Research and Development Centre. The experimental design was a two by three factorial combination of: P. penetrans inoculation (+/-) and rootstock (G.41, G.935, and M.9), with 20 replicate microplots of each of the six treatment combinations arranged in a randomized complete block design. The P. penetrans inoculum was 5,400 nematodes per microplot (54 P. penetrans liter-1 soil), which is below commonly accepted damage thresholds. Though P. penetrans population densities were lower for the G.41 rootstock by the end of the 2021 growing season, the effects of P. penetrans were similar among rootstocks. In the establishment year (2020), P. penetrans caused significant reductions in aboveground growth. In 2021, shoot growth and root weight were reduced by P. penetrans. The nematode also reduced rates of leaf gas exchange and stem water potential. These data suggest that while G.41 and G.935 may have other horticultural benefits over M.9, they are equally susceptible to P. penetrans at the early stages of tree growth.

Nitrate transporter MdNRT2.4 interacts with rhizosphere bacteria to enhance nitrate uptake in apple rootstocks.

Plants have developed complex mechanisms to adapt to changing nitrate (NO3-) concentrations and can recruit microbes to boost nitrogen absorption. However, little is known about the relationship between functional genes and the rhizosphere microbiome in NO3- uptake of apple rootstocks. Here, we found that variation in Malus domestica NO3- transporter (MdNRT2.4) expression contributes to nitrate uptake divergence between two apple rootstocks. Overexpression of MdNRT2.4 in apple seedlings significantly improved tolerance to low nitrogen via increasing net NO3- influx at the root surface. However, inhibiting the root plasma membrane H+-ATPase activity abolished NO3- uptake and led to NO3- release, suggesting that MdNRT2.4 encodes an H+-coupled nitrate transporter. Surprisingly, the nitrogen concentration of MdNRT2.4-overexpressing apple seedlings in unsterilized nitrogen-poor soil was higher than that in sterilized nitrogen-poor soil. Using 16S ribosomal RNA gene profiling to characterize the rhizosphere microbiota, we found that MdNRT2.4-overexpressing apple seedlings recruited more bacterial taxa with nitrogen metabolic functions, especially Rhizobiaceae. We isolated a bacterial isolate ARR11 from the apple rhizosphere soil and identified it as Rhizobium. Inoculation with ARR11 improved apple seedling growth in nitrogen-poor soils, compared with uninoculated seedlings. Together, our results highlight the interaction of host plant genes with the rhizosphere microbiota for host plant nutrient uptake.

Siderophore production in pseudomonas SP. strain SP3 enhances iron acquisition in apple rootstock.

AIMS: The purpose of this study was to analyse the effects of siderophore-producing bacteria and bacterial siderophore on the iron nutrition of apple rootstocks under iron-deficient conditions. METHODS AND RESULTS: We isolated three Pseudomonas strains, SP1, SP2 and SP3 from the rhizosphere of the Fe-efficient apple rootstocks using the chrome azurol S agar plate assay. We found that all three strains had the ability to secrete indole acetic acid-like compounds and siderophores, especially SP3. When Fe-inefficient rootstocks treated with SP3 were grown in alkaline soil, an increase in the biomass, root development, and Fe concentration was observed in the plants. In addition, SP3 secreted pyoverdine, a siderophore that can chelate Fe3+ to enhance the bioavailability of Fe for plants. We purified the pyoverdine from the SP3 culture supernatant. Hydroponic experiments were conducted with a Fe-deficient solution supplemented with pyoverdine, resulting in a reduction in the chlorosis caused by Fe deficiency and marked improvement in Fe uptake. CONCLUSIONS: Under iron-deficient conditions, Pseudomonas sp. strain SP3 can effectively promote apple rootstock growth and improve plant iron nutrition by secreting siderophores that enhance Fe availability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that plant growth-promoting rhizobacteria from Fe-efficient plants have the potential to improve iron nutrition in Fe-inefficient plants, and Fe-siderophore chelates can be used as an effective source of iron for apple plants. Based on these findings, it may be possible to develop biological agents such as siderophore-producing bacteria for sustainable agricultural and horticultural production.

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.

Ethylene Response Factors MbERF4 and MbERF72 Suppress Iron Uptake in Woody Apple Plants by Modulating Rhizosphere pH.

Iron (Fe) deficiency limits the yield of fruit trees. When subjected to Fe deficiency, H+ secretion increases in the rhizosphere of dicotyledonous plants and pH decreases. This leads to the acidification of the soil and promotes Fe3+ to Fe2+ conversion, which plants can better uptake. This study investigated the relationship between two inhibitory transcription factors (ethylene response factors MbERF4 and MbERF72) and the H+-ATPase gene MbHA2. Two species of apple woody plants were studied: the Fe-inefficient Malus baccata and the Fe-efficient Malus xiaojinensis. Yeast one-hybrid and electrophoretic mobility shift assays showed that both MbERF4 and MbERF72 bind to the GCC cassette (AGCCGCC) of the MbHA2 promoter. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that MbERF4 interacts with MbERF72. Furthermore, ß-glucuronidase and luciferase reporter assays showed that the MbERF4- and MbERF72-induced repression of MbHA2 expression is synergistic. Virus-induced gene silencing of MbERF4 or MbERF72 increased MbHA2 expression, and thus lowered the rhizosphere pH in M. baccata. Consequently, the high expressions of MbERF4 and MbERF72 induced by Fe deficiency contributed to the Fe sensitivity of M. baccata. Moreover, the low expressions of MxERF4 and MxERF72 contributed to the Fe-deficiency tolerance of M. xiaojinensis via different binding conditions to the HA2 promoter. In summary, this study identified the relationship of two inhibitory transcription factors with the H+-ATPase gene and proposed a model in which ERF4 and ERF72 affect the rhizosphere pH in response to Fe deficiency.

Apple rootstocks with different phosphorus efficiency exhibit alterations in rhizosphere bacterial structure.

AIMS: The purpose of this study was to select phosphorus-efficient apple rootstocks under phosphorus deficiency and to reveal the effects of different apple rootstocks on the rhizosphere bacterial community. METHODS AND RESULTS: We used 83 hybrid lines of Malus robusta Rehd. × Malling 9 (M.9) to investigate their physiological traits and the phosphorus deficiency phenotypes of leaves in response to phosphorus deficiency (0·1 mmol l-1 PO4 3- ). All the plants were cultivated in pots in the greenhouse and watered using drip irrigation. In accordance with the results of investigation, we selected the phosphorus-efficient hybrid lines (PE) and the phosphorus-inefficient hybrid lines (PI) to research their root morphology and root hairs (RH). In addition, we used Illumina MiSeq sequencing to determine the bacterial community of the rhizosphere from different rootstocks. The results showed that the PE plants had better growth characteristics and stronger root plasticity than that of the PI plants, and phosphorus deficiency can stimulate the RH growth of PE plants. There was no significant difference in the rhizosphere bacterial diversity, but we found that the bacterial community structure was significantly different at the genus levels; in addition, 89 genera were found to have significant differences between PE and PI plants, especially Bacillus. The PE rhizosphere had more abundant Bacillus compared to the PI. High positive Pearson correlations with the phosphorus concentration in the plantlets of apple rootstocks were detected for the bacterial genera Bacillus (r: 0·776). CONCLUSIONS: The phosphorus-efficient apple rootstocks adapted to phosphorus deficiency by shaping the root morphology. Notably, different apple rootstocks showed alteration of the microbes in rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study can provide the materials for exploring the mechanism of apple rootstock phosphorus absorption. In accordance with the different bacterial community compositions, we can develop the inoculants to promote nutrient uptake.

Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks.

Plants experiencing salt-induced stress often reduce cytokinin levels during the early phases of stress-response. Interestingly, we found that the cytokinin content in the apple rootstock "robusta" was maintained at a high level under salt stress. Through screening genes involved in cytokinin biosynthesis and catabolism, we found that the high expression levels of IPT5b in robusta roots were involved in maintaining the high cytokinin content. We identified a 42 bp deletion in the promoter region of IPT5b, which elevated IPT5b expression levels, and this deletion was linked to salt tolerance in robusta×M.9 segregating population. The 42 bp deletion resulted in the deletion of a Proline Response Element (ProRE), and our results suggest that ProRE negatively regulates IPT5b expression in response to proline. Under salt stress, the robusta cultivar maintains high cytokinin levels as IPT5b expression cannot be inhibited by proline due to the deletion of ProRE, leading to improve salt tolerance.

Transcriptome Analysis Reveals Multiple Hormones, Wounding and Sugar Signaling Pathways Mediate Adventitious Root Formation in Apple Rootstock.

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 'T337' 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.

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.

Methylation effect on IPT5b gene expression determines cytokinin biosynthesis in apple rootstock.

The molecular mechanism of dwarfing in the apple rootstock is poorly understood, and has been attributed to low root cytokinin biosynthesis levels. Here we identified differences in root trans-zeatin content and expression of cytokinin metabolic pathway genes between dwarfing and vigorous rootstocks. Specifically, a stable low expression of IPT5b was identified in the dwarfing rootstocks. Bisulfite sequencing showed that two CpG islands (CpG2 and CpG4) in the IPT5b promoter region showed higher methylation levels in the M9 rootstock (dwarfing) compared to the Mr (vigorous) rootstock. Furthermore, IPT5b expression increased when M9 rootstocks were treated with 5-azaC, a methylation blocker, indicating that methylation levels influence IPT5b expression. In conclusion, we found low IPT5b expression with high level methylations in promoter region, leading to poor root trans-zeatin biosynthesis in the M9 rootstock, which may induce dwarfing.

Significant and unique changes in phosphorylation levels of four phosphoproteins in two apple rootstock genotypes under drought stress.

Drought stress is a major problem around the world and there is still little molecular mechanism about how fruit crops deal with moderate drought stress. Here, the physiological and phosphoproteomic responses of drought-sensitive genotype (M26) and drought-tolerant genotype (MBB) under moderate drought stress were investigated. Our results of the physiology analysis indicated that the MBB genotype could produce more osmosis-regulating substances. Furthermore, phosphoproteins from leaves of both genotypes under moderate drought stress were analyzed using the isobaric tags for relative and absolute quantification technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites, and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Five and thirty-five phosphoproteins with the phosphorylation levels significantly changed (PLSC) were identified in M26 and MBB, respectively. Among these, four PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlap of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins represent a unique combination of metabolism, transcription, translation, and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new and unique homeostasis of major cellular processes. The basic trend was an increase in protein and organic molecules abundance related to drought. These increases were higher in MBB than in M26. Our study is the first to address the phosphoproteome of apple rootstocks in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.

Effect of solution pH on root architecture of four apple rootstocks grown in an aeroponics nutrient misting system.

The pH of the solution in the rhizosphere is an important factor that determines the availability and mobility of nutrients for plant uptake. Solution pH may also affect the root distribution and architecture of apple rootstocks. In this study, we evaluated the effect of solution pH on root system development of apple rootstocks using an aeroponics system designed and developed at Cornell AgriTech Geneva, USA. Four Geneva® apple rootstocks (G.210, G.214, G.41, and G.890) were grown in an aeroponic system under nutrient solution misting featuring continuously adjusted pH levels to three pH treatments (5.5, 6.5, and 8.0). Root development was monitored for 30 days and evaluated regularly for distribution and root mass. Images of the developed roots grown in the aeroponic system were collected at the end of the experiment using a high-resolution camera and analyzed using GiA Roots® software, which generates root architecture parameter values in a semi-automated fashion. The resulting root architecture analysis showed that the Geneva® rootstocks were significantly different for two architecture parameters. The length-to-width ratio analysis represented by two GiA Roots parameters (minor-to-major ellipse ratio and network width-to-depth ratio) showed that G.210 was flatter than G.890, which had a greater tendency to grow downward. Rootstocks G.214 and G.41 displayed similar growth values. The solution pH affected most root architecture parameter measurements where overall root growth was higher at pH 8 than at pH 5.5 and 6.5, which showed similar growth. In general, the average root width tended to decrease at higher pH values. While there were no significant differences in the leaf nutrient concentrations of P, K, Ca, Mg, S, B, Zn, Cu, and Fe within the four rootstocks, the pH level of the solution had a significant effect on P, Ca, and Mn. This study is the first of its kind to investigate the effect of pH on root architecture in a soil-free (aeroponic) environment and may have implications for apple root behavior under field conditions where pH levels are different.

Genome-wide analysis of the MdZR gene family revealed MdZR2.2-induced salt and drought stress tolerance in apple rootstock.

The DNL-type zinc finger protein constitutes a zinc ribbon protein (ZR) family, which belongs to a branch of zinc finger protein and plays an essential role in response to abiotic stress. Here, we identified six apple (Malus domestica) MdZR genes. Based on their phylogenetic relationship and gene structure, the MdZR genes were divided into three categories, including MdZR1, MdZR2, and MdZR3. Subcellular results showed that the MdZRs are located on the nuclear and membrane. The transcriptome data showed that MdZR2.2 is expressed in various tissues. The expression analysis results showed that MdZR2.2 was significantly upregulated under salt and drought treatments. Thus, we selected MdZR2.2 for further research. Overexpression of MdZR2.2 in apple callus improved their tolerance to drought and salt stress and ability to scavenge reactive oxygen species (ROS). In contrast, transgenic apple roots with silenced MdZR2.2 grew more poorly than the wild type when subjected to salt and drought stress, which reduced their ability to scavenge ROS. To our knowledge, this is the first study to analyze the MdZR protein family. This study identified a gene that responds to drought and salt stress. Our findings lay a foundation for a comprehensive analysis of the MdZR family members.

Appropriate and Constant Potassium Supply Promotes the Growth of M9T337 Apple Rootstocks by Regulating Endogenous Hormones and Carbon and Nitrogen Metabolism.

Potassium (K) is an indispensable nutrient element in the development of fruit trees in terms of yield and quality. It is unclear how a stable or unstable supply of K affects plant growth. We studied the root morphology and physiological and molecular changes in the carbon and nitrogen metabolism of M9T337 apple rootstock under different K levels and supply methods using hydroponics. Five K supply treatments were implemented: continuous low K (KL), initial low and then high K (KLH), appropriate and constant K (KAC), initial high and then low K (KHL), and continuous high K (KH). The results showed that the biomass, root activity, photosynthesis, and carbon and nitrogen metabolism of the M9T337 rootstocks were inhibited under KL, KH, KLH and KHL conditions. The KAC treatment promoted root growth by optimizing endogenous hormone content, enhancing carbon and nitrogen metabolism enzyme activities, improving photosynthesis, optimizing the distribution of carbon and nitrogen, and upregulating the transcription levels of nitrogen assimilation-related genes (nitrate reductase, glutamine synthetase, glutamate synthase, MdNRT1.1, MdNRT1.2, MdNRT1.5, MdNRT2.4). These results suggest that an appropriate and constant K supply ensures the efficient assimilation and utilization of nitrogen and carbon.

Genome-wide identification of apple PPI genes and a functional analysis of the response of MxPPI1 to Fe deficiency stress.

Iron (Fe) deficiency affects plant growth and development. The proton pump interactor (PPI) in plants responds to multiple abiotic stresses, although it has not been well characterized under Fe deficiency stress. In this study, we systematically identified and analyzed the PPI gene family in apple. Three PPI candidate genes were found, and they contained 318-1349 amino acids and 3-7 introns. Under Fe deficiency stress, we analyzed the expression of all the PPI genes in roots of apple rootstock Malus xiaojinensis. Expression of the gene MD11G1247800, designated PPI1, is obviously induced by Fe deficiency treatment in M. xiaojinensis. We first cloned MxPPI1 from M. xiaojinensis and determined its subcellular localization, which indicated that it is localized in the cell membrane and nucleus in tobacco. We found that the level of expression of the MxPPI1 protein increased significantly under Fe deficiency stress in apple calli. Moreover, overexpressing MxPPI1 in apple calli enhanced the activities of ferric chelate reductase and H+-ATPase, H+ secretion, MxHA2 gene expression and total Fe content when compared with the wild type calli. We further found that MxPPI1 interacted with MxHA2 using bimolecular fluorescence complementation and luciferase complementation assays. Overall, we demonstrated that MxPPI1 interacts with MxHA2 to enhance the activity of H+-ATPase to regulate Fe absorption in M. xiaojinensis.

Transcriptome analysis reveals that cytokinins inhibit adventitious root formation through the MdRR12-MdCRF8 module in apple rootstock.

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.

Transcriptome analysis reveals the promotive effect of potassium by hormones and sugar signaling pathways during adventitious roots formation in the apple rootstock.

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.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock.

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.