Auxin-induced Adventitious Root Formation Research Articles

Temporal profiling of physiological, histological, and transcriptomic dissection during auxin-induced adventitious root formation in tetraploid Robinia pseudoacacia micro-cuttings.

Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.

Transcriptome Profiling Provides Molecular Insights into Auxin-Induced Adventitious Root Formation in Sugarcane (Saccharum spp. Interspecific Hybrids) Microshoots

Adventitious root (AR) formation was enhanced following the treatment of sugarcane microshoots with indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) combined, suggesting that auxin is a positive regulator of sugarcane microshoot AR formation. The transcriptome profile identified 1737 and 1268 differentially expressed genes (DEGs) in the basal tissues (5 mm) of sugarcane microshoots treated with IBA+NAA compared to nontreated control on the 3rd and 7th days post-auxin or water treatment (days post-treatment—dpt), respectively. To understand the molecular changes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. This analysis showed that DEGs associated with the pathways were associated with plant hormone signaling, flavonoid and phenylpropanoid biosyntheses, cell cycle, and cell wall modification, and transcription factors could be involved in sugarcane microshoot AR formation. Furthermore, qRT–PCR analysis was used to validate the expression patterns of nine genes associated with root formation and growth, and the results were consistent with the RNA-seq results. Finally, a hypothetical hormonal regulatory working model of sugarcane microshoot AR formation is proposed. Our results provide valuable insights into the molecular processes associated with auxin-induced AR formation in sugarcane.

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.

Transcriptomic profiling and discovery of key genes involved in adventitious root formation from green cuttings of highbush blueberry (Vaccinium corymbosum L.)

BackgroundPropagation of cuttings is frequently used in various plant species, including blueberry, which shows special root characteristics that may hinder adventitious root (AR) formation. AR formation is influenced by various factors, and auxin is considered to play a central role; however, little is known of the related regulatory mechanisms. In this study, a comparative transcriptome analysis of green cuttings treated with or without indole-butyric acid (IBA) was performed via RNA_seq to identify candidate genes associated with IBA-induced AR formation.ResultsRooting phenotypes, especially the rooting rate, were significantly promoted by exogenous auxin in the IBA application. Blueberry AR formation was an auxin-induced process, during which adventitious root primordium initiation (rpi) began at 14 days after cutting (DAC), root primordium (rp) was developed at 21 DAC, mature AR was observed at 28 DAC and finally outgrowth from the stem occurred at 35 DAC. Higher IAA levels and lower ABA and zeatin contents might facilitate AR formation and development. A time series transcriptome analysis identified 14,970 differentially expressed genes (DEGs) during AR formation, of which there were 7467 upregulated and 7503 downregulated genes. Of these, approximately 35 candidate DEGs involved in the auxin-induced pathway and AR formation were further identified, including 10 auxin respective genes (ARFs and SAURs), 13 transcription factors (LOB domain-containing protein (LBDs)), 6 auxin transporters (AUX22, LAX3/5 and PIN-like 6 (PIL6s)) and 6 rooting-associated genes (root meristem growth factor 9 (RGF9), lateral root primordium 1 (LRP1s), and dormancy-associated protein homologue 3 (DRMH3)). All these identified DEGs were highly upregulated in certain stages during AR formation, indicating their potential roles in blueberry AR formation.ConclusionsThe transcriptome profiling results indicated candidate genes or major regulatory factors that influence adventitious root formation in blueberry and provided a comprehensive understanding of the rooting mechanism underlying the auxin-induced AR formation from blueberry green cuttings.

Stimulation of adventitious root formation by the oligosaccharin OSRG at the transcriptome level

ABSTRACTOligosaccharins, which are biologically active oligosaccharide fragments of cell wall polysaccharides, may regulate the processes of growth and development as well as the response to stress factors. We characterized the effect of the oligosaccharin that stimulates rhizogenesis (OSRG) on the gene expression profile in the course of IAA-induced formation of adventitious roots in hypocotyl explants of buckwheat (Fagopyrum esculentum Moench.). The transcriptomes at two stages of IAA-induced root primordium formation (6 h and 24 h after induction) were compared after either treatment with auxin alone or joint treatment with auxin and OSRG. The set of differentially expressed genes indicated the special importance of oligosaccharin at the early stage of auxin-induced adventitious root formation. The list of genes with altered mRNA abundance in the presence of oligosaccharin included those, which Arabidopsis homologs encode proteins directly involved in the response to auxin as well as proteins that contribute to redox regulation, detoxification of various compounds, vesicle trafficking, and cell wall modification. The obtained results contribute to understanding the mechanism of adventitious root formation and demonstrate that OSRG is involved in fine-tuning of ROS and auxin regulatory modes involved in root development.

Auxin-Induced Adventitious Root Formation in Nodal Cuttings of Camellia sinensis.

Adventitious root (AR) formation is essential for the successful propagation of Camellia sinensis and auxins play promotive effects on this process. Nowadays, the mechanism of auxin-induced AR formation in tea cuttings is widely studied. However, a lack of global view of the underlying mechanism has largely inhibited further studies. In this paper, recent advances including endogenous hormone changes, nitric oxide (NO) and hydrogen peroxide (H2O2) signals, secondary metabolism, cell wall reconstruction, and mechanisms involved in auxin signaling are reviewed. A further time course analysis of transcriptome changes in tea cuttings during AR formation is also suggested to deepen our understanding. The purpose of this paper is to offer an overview on the most recent developments especially on those key aspects affected by auxins and that play important roles in AR formation in tea plants.

High miR156 Expression Is Required for Auxin-Induced Adventitious Root Formation via MxSPL26 Independent of PINs and ARFs in Malus xiaojinensis

Adventitious root formation is essential for the vegetative propagation of perennial woody plants. During the juvenile-to-adult phase change mediated by the microRNA156 (miR156), the adventitious rooting ability decreases dramatically in many species, including apple rootstocks. However, the mechanism underlying how miR156 affects adventitious root formation is unclear. In the present study, we showed that in the presence of the synthetic auxin indole-3-butyric acid (IBA), semi-lignified leafy cuttings from juvenile phase (Mx-J) and rejuvenated (Mx-R) Malus xiaojinensis trees exhibited significantly higher expression of miR156, PIN-FORMED1 (PIN1), PIN10, and rootless concerning crown and seminal roots-like (RTCS-like) genes, thus resulting in higher adventitious rooting ability than those from adult phase (Mx-A) trees. However, the expression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE26 (SPL26) and some auxin response factor (ARF) gene family members were substantially higher in Mx-A than in Mx-R cuttings. The expression of NbRTCS-like but not NbPINs and NbARFs varied with miR156 expression in tobacco (Nicotiana benthamiana) plants transformed with 35S:MdMIR156a6 or 35S:MIM156 constructs. Overexpressing the miR156-resistant MxrSPL genes in tobacco confirmed the involvement of MxSPL20, MxSPL21&22, and MxSPL26 in adventitious root formation. Together, high expression of miR156 was necessary for auxin-induced adventitious root formation via MxSPL26, but independent of MxPINs and MxARFs expression in M. xiaojinensis leafy cuttings.

Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development

The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCF(TIR1) ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation.

ZmHO-1, a maize haem oxygenase-1 gene, plays a role in determining lateral root development

Previous results revealed that haem oxygenase-1 (HO-1)/carbon monoxide (CO) system is involved in auxin-induced adventitious root formation. In this report, a cDNA for the gene ZmHO-1, encoding an HO-1 protein, was cloned from Zea mays seedlings. ZmHO-1 has a conserved HO signature sequence and shares highest homology with rice SE5 (OsHO-1) protein. We further discovered that N-1-naphthylacetic acid (NAA), haemin, and CO aqueous solution, led to the induction of ZmHO-1 expression as well as the thereafter promotion of lateral root development. These effects were specific for ZmHO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) differentially blocked the above actions. The addition of haemin and CO were able to reverse the auxin depletion-triggered inhibition of lateral root formation as well as the decreased ZmHO-1 transcripts. Molecular evidence showed that the haemin- or CO-mediated the modulation of target genes responsible for lateral root formation, including ZmCDK and ZmCKI2, could be blocked by ZnPPIX. Overexpression of ZmHO-1 in transgenic Arabidopsis plants resulted in promotion of lateral root development as well as the modulation of cell cycle regulatory gene expressions. Overall, our results suggested that a maize HO-1 gene is required for the lateral root formation.

N-3-Oxo-Decanoyl-l-Homoserine-Lactone Activates Auxin-Induced Adventitious Root Formation via Hydrogen Peroxide- and Nitric Oxide-Dependent Cyclic GMP Signaling in Mung Bean

N-Acyl-homoserine-lactones (AHLs) are bacterial quorum-sensing signaling molecules that regulate population density. Recent evidence demonstrates their roles in plant defense responses and root development. Hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and cyclic GMP (cGMP) are essential messengers that participate in various plant physiological processes, but how these messengers modulate the plant response to N-acyl-homoserine-lactone signals remains poorly understood. Here, we show that the N-3-oxo-decanoyl-homoserine-lactone (3-O-C10-HL), in contrast to its analog with an unsubstituted branch chain at the C3 position, efficiently stimulated the formation of adventitious roots and the expression of auxin-response genes in explants of mung bean (Vigna radiata) seedlings. This response was mimicked by the exogenous application of auxin, H(2)O(2), NO, or cGMP homologs but suppressed by treatment with scavengers or inhibitors of H(2)O(2), NO, or cGMP metabolism. The 3-O-C10-HL treatment enhanced auxin basipetal transport; this effect could be reversed by treatment with H(2)O(2) or NO scavengers but not by inhibitors of cGMP synthesis. Inhibiting 3-O-C10-HL-induced H(2)O(2) or NO accumulation impaired auxin- or 3-O-C10-HL-induced cGMP synthesis; however, blocking cGMP synthesis did not affect auxin- or 3-O-C10-HL-induced H(2)O(2) or NO generation. Additionally, cGMP partially rescued the inhibitory effect of H(2)O(2) or NO scavengers on 3-O-C10-HL-induced adventitious root development and auxin-response gene expression. These results suggest that 3-O-C10-HL, unlike its analog with an unmodified branch chain at the C3 position, can accelerate auxin-dependent adventitious root formation, possibly via H(2)O(2)- and NO-dependent cGMP signaling in mung bean seedlings.

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H 2O 2), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H 2O 2 was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H 2O 2 for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H 2O 2 scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H 2O 2. H 2O 2 may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H 2O 2. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H 2O 2 formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H 2O 2 or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H 2O 2 through NADPH oxidase was inhibited, and H 2O 2 mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H 2O 2 was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H 2O 2 was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H 2O 2 in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H 2O 2. Results obtained suggest that H 2O 2 may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.

Nitric Oxide Triggers Phosphatidic Acid Accumulation via Phospholipase D during Auxin-Induced Adventitious Root Formation in Cucumber

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transduction leading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR) formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- and NO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with 32P-inorganic phosphate and treated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in the presence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs were separated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Both auxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or 30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation via phospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR number compared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally, exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA is an early signaling event during AR formation induced by auxin and NO in cucumber explants.

Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber

A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca2+ and the regulation of Ca2+-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca2+ channel inhibitors and Ca2+ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca2+ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.

Nitric oxide and cyclic GMP are messengers in the indole acetic acid-induced adventitious rooting process.

This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.

Genetic analysis of variation for auxin-induced adventitious root formation among eighteen ecotypes of Arabidopsis thaliana L. Heynh.

Eighteen ecotypes and two inbred lines of Arabidopsis thaliana L. Heynh. were analyzed for variation in the number of adventitious roots formed (hereafter referred to as rooting) on seedling hypocotyls in response to auxin treatment. Mean root counts varied from 1.7 to 23.1. Stable high (HA) and low (LA) rooting lines selected from ecotype Columbia, a low rooting ecotype (Mt-0), and unselected Columbia populations were evaluated for vegetative and reproductive growth parameters to determine correlated phenotypic effects of selection for rooting response. High rooting in HA correlated with compact, highly branched shoot growth. Genetic analysis of HA, Mt-0, and their F1, F2, and reciprocal backcross generations indicated that high and low rooting responses in this population may be controlled by several genes acting independently in additive-dominance fashion. Genetic variance partitioned into principally additive effects, with dominance favoring low rooting.