LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis
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Adventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood.
In this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia.
These results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.
KeywordsArabidopsis thaliana Adventitious root formation Auxin response factor Lateral organ boundaries domain LBD16 LBD18
Auxin response factor
Auxin/indole acetic acid protein
Lateral organ boundaries domain/asymmetric leaves2-like
Quantitative reverse transcription-PCR
Root architecture in higher plants, which is critical for anchorage in soil and the uptake of water and nutrients, is diverse at both the system and anatomical levels . In general, dicotyledonous plants, such as Arabidopsis thaliana, have a primary root that branches to form lateral roots (LRs). In both monocot and dicotyledonous plants, the primary root can develop adventitious roots (ARs) that arise naturally from the aerial organs as an adaptive response to environmental changes, such as flooding and dark-light transitions, or artificially by wounding [2, 3, 4, 5]. AR formation is critical for vegetative propagation of elite genotypes in agriculture and is also important for plant survival under a variety of biotic and abiotic stresses [4, 5]. Auxin plays a central role in both LR and AR formation . Although signaling and molecular mechanisms of auxin-regulated primary and LR development are relatively well characterized, our understanding of how auxin regulates AR formation is rudimentary [1, 7, 8, 9].
Plant root development is regulated by establishing auxin maxima at the primordium tip through auxin transport [10, 11, 12, 13]. During auxin transport, auxin travels acropetally or basipetally over long distances by the combined action of plasma membrane-localized auxin efflux and influx carriers and triggers various regulatory mechanisms along its path [14, 15, 16, 17]. Auxin efflux carriers, including two major transmembrane proteins, i.e., PIN-FORMED (PIN) and ATP-binding cassette subfamily B (ABCB), were shown to be involved in both LR and AR formation in different plant species [10, 18, 19, 20, 21, 22]. Studies with the polar auxin transport inhibitor, N-1-naphthylphthalamic acid, showed that PIN1-mediated auxin transport is necessary for AR emergence in rice plants . Auxin together with ethylene positively regulates AR initiation through DIAGEOTROPICA (DGT), which encodes a cyclophilin A-type protein (SlCYP) [24, 25]. SlCYP1 changes the abundance of PIN efflux carriers at the plasma membrane to modulate polar auxin transport during AR initiation [26, 27, 28, 29].
Auxin signaling is regulated by two large protein families: the AUXIN RESPONSE FACTOR (ARF) proteins, which act as the DNA-binding transcriptional regulators of auxin responses, and the Aux/IAA proteins, negative regulators of ARFs . Several ARFs have been identified as playing a role in AR formation in both Arabidopsis and rice. ARF6 and ARF8 have been shown to act as positive regulators of AR initiation in Arabidopsis hypocotyls, whereas ARF17 acts as a negative regulator [31, 32]. These ARFs regulate each other’s expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160, which targets ARF17 and miR167, subsequently targeting both ARF6 and ARF8 . This complex network of transcription factors regulates the expression of three auxin-inducible GRETCHEN HAGEN3 (GH3) genes, encoding acyl-acid-amido synthetases, which are required for fine-tuning AR initiation in the Arabidopsis hypocotyls by modulating jasmonic acid homeostasis . Some studies have indicated that auxin signaling modules involved in LR formation could play a role in AR formation as well. For instance, ARF7 and ARF19 control LR formation as well as AR formation in Arabidopsis [34, 35, 36, 37, 38]. OsARF16, which is a rice ortholog of ARF7 and ARF19, controls the initiation of adventitious crown root primordia in rice by activating the expression of CROWN ROOTLESS1/ADVENTITIOUS ROOTLESS1 (CRL1/ARL1), which encodes a LATERAL ORGAN BOUNDARIES DOMAIN (LBD) protein [39, 40, 41].
In Arabidopsis, several LBD genes, such as LBD16, − 18, − 29 and − 33, have been demonstrated to play critical and distinct roles in auxin-regulated LR development [37, 42, 43, 44, 45, 46, 47, 48, 49]. It has been shown that the auxin influx carriers AUXIN1 (AUX1) and LIKE-AUX1 3 (LAX3) are required for auxin-responsive expression of LBD16 and LBD18 to control various stages of LR development in Arabidopsis [45, 50, 51]. In the present study, we show that the AUX1/LAX3-ARF7/ARF19-LBD16/LBD18 signaling module is also important for AR formation in Arabidopsis, providing evidence of a common regulatory mechanism being utilized for LR and AR formation during auxin signaling.
Analysis of GUS expression patterns of Pro ARF7 :GUS, Pro ARF19 :GUS, Pro LAX3 :GUS, Pro LBD16 :GUS and Pro LBD18 :GUS during AR development
AUX1, LAX3, LBD16 and LBD18 are involved in AR formation in Arabidopsis hypocotyls
Both LBD16:SRDX and LBD18:SRDX suppress cell cycle gene promoter activities during induction of AR formation
LBD16 and LBD18 act downstream of ARF7 and ARF19 to regulate AR development
Previous studies have shown that ARF6 and ARF8 act as positive regulators of AR formation [31, 32]. Thus, we tested if ARF6 and ARF8 could control the expression of LBD16 and LBD18 for AR formation. The time-course response of LBD16 and LBD18 expression in arf6 and arf8 mutants after treatment with auxin indole-3-acetic acid was analyzed using RT-qPCR, but no alteration in the expression of LBD16 or LBD18 in arf6 and arf8 mutant backgrounds was observed compared with that of the wild type (Additional file 1: Figure S1), suggesting that ARF6 and ARF8 regulate AR formation through a distinct pathway, independent of LBD16 and LBD18, during auxin signaling. Taken together, these results indicated that LBD16 and LBD18 expression is regulated downstream of ARF7 and ARF19, but not of ARF6 and ARF8, for AR development.
Studies on genetic aspects and hormonal responses of LR and AR formation suggested that LRs and ARs share key elements of genetic and hormonal regulatory networks but with different regulatory mechanisms . While genetic components and molecular signaling pathways during LR development in Arabidopsis have been well characterized , those involved in AR development are largely unknown. In this work, we showed that the auxin-responsive ARF7/ARF19-LBD16/LBD18 transcriptional module, via AUX1/LAX3 transporters, plays an important role in AR formation in the Arabidopsis hypocotyl.
In Arabidopsis, the developmental processes of LRs are well defined, and consists of the priming of pericycle cells in the basal meristem of the primary root, the first anticlinal asymmetric cell division (initiation), ordered anticlinal and periclinal cell divisions to form a dome-shaped LR primordium, and the emergence of an LR primordium from the primary root [53, 54]. Although the developmental stages of AR formation are not well described in Arabidopsis, AR formation in apple cuttings have been similarly divided into four successive phases: cell dedifferentiation, induction as the beginning of cell division, the outgrowth of a dome-shaped primordium, and the emergence of the AR . In Arabidopsis, ARs initiate from the pericycle cells adjacent to the xylem pole in the hypocotyl, similar to how LRs initiate . ARF proteins, which are involved in LR initiation, were found to regulate AR initiation in Arabidopsis [32, 33], indicating that although AR and LR originate from different organs, similar molecular mechanisms may regulate AR initiation. In rice, CRL1/ARL1, which promotes crown root initiation, positively regulates 277 genes, and among those, it positively regulates many genes homologous to Arabidopsis genes involved in LR formation .
We noticed that the complementation lines of the lbd16 or lbd18 mutant generated by expressing LBD16 or LBD18 under the control of their own promoter exhibited much higher expression levels of each transgene compared with that of the wild-type LBD16 or LBD18, and yet the AR numbers in the complementation lines are comparable to that of the wild-type Col-0 [Fig. 3]. This result indicates that overexpression of a single LBD transcription factor gene regulated downstream of ARF7 is not sufficient to overproduce ARs in transgenic plants.
It has been previously reported that the apical part of argonaute1 (ago1) mutants displays a defect in AR formation, but not in LR development, in response to auxin . AGO1 is one of the components that plays a critical role in the regulation of posttranscriptional gene silencing . ARF17, which is upregulated in ago1 mutants, negatively regulates AR formation by repressing GH3 genes and thus perturbing auxin homeostasis in a light-dependent manner . Together, these results suggest that AR development has both unique and shared components with LR development in Arabidopsis. Identification of unique components, which play critical roles in AR development, aid in the discovery of the distinctive developmental processes between AR and LR development.
The ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in the Arabidopsis hypocotyl, suggesting that a common regulatory mechanism is utilized for LR and AR formation during auxin signaling.
Plant growth and tissue treatment
Arabidopsis thaliana seedlings were grown and treated as described previously . For treatment with auxin IAA, seedlings were grown in a 16-h photoperiod on a 3 MM Whatman filter paper on top of agar plates at 23°C. The filter paper with seedlings was then transferred to a plate containing IAA at 20 μM and incubated for a given period of time with gentle shaking in the light at 23°C. The light intensity was approximately 120 μmol m− 2 s− 1 and was provided by three daylight wavelength color fluorescent bulbs (Kumho Electric Co.).
The Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. We used the homozygous T-DNA insertion mutant lines lbd16, lbd18, lax3, aux1–21, lbd16 lbd18, aux1 lax3, lax3 lbd16, lax3 lbd18, lax3 lbd16 lbd18, aux1 lbd16, aux1 lbd18, aux1 lbd16 lbd18, aux1 lax3 lbd16, aux1 lax3 lbd18, and aux1 lax3 lbd16 lbd18, arf7, arf19, and arf7 arf19, which were developed in previous studies [37, 45, 51, 52]. We identified arf6–1 (CS24606) and arf8–2 (CS24608) knockout T-DNA insertion mutants from the Arabidopsis Biological Resource Center (ABRC). The ProLBD16:GUS and ProLBD18:GUS transgenic plants were obtained from a previous study . ProLAX3:GUS transgenic seeds were generously provided by Dr. Malcolm Bennett . ProARF7:GUS (CS24633) and ProARF19:GUS (CS24634) transgenic seeds were obtained from the ABRC. To generate ProLBD16:LBD16:4xMyc in the lbd16 mutant background, the promoter region of LBD16, encompassing − 1309 to − 21 bp relative to the AUG codon, was amplified by PCR using the pfu DNA polymerase with primers harboring the NotI (N-terminus) and AscI (C-terminus) sites, and was cloned into the pENTR™/SD/D-TOPO (Invitrogen) vector to yield pENTR™/SD/D-TOPO:ProLBD16. The LBD16 DNA fragment was inserted into the pENTR™/SD/D-TOPO:ProLBD16 plasmid with the AscI restriction site in both the N- and C-terminus to yield a pENTR™/SD/D-TOPO:ProLBD16:LBD16 plasmid. This construct was subcloned into the destination vector pGWB516 (Nakagawa, Shimane University, Japan) by an LR recombination reaction, and was then transformed into the lbd16 mutant by Agrobacterium-mediated transformation, generating ProLBD16:LBD16:4xMyc/lbd16 Arabidopsis. ProLBD18:LBD18:3xHA/lbd18 Arabidopsis generated from a previous study was used . Pro35S:LBD16:GR/arf7 arf19 and Pro35S:LBD18:GR/arf7 arf19 transgenic mutants were generated by crossing arf7–1 arf19–1 (female) with Pro35S:LBD16:GR (male) or Pro35S:LBD18:GR (male) . Homozygous lines were isolated according to genotype, the lack of lateral root phenotype for arf7–1 arf19–1 and by PCR detection of genomic DNA for the LBD16:GR or LBD18:GR transgenes. All mutants and transgenic plants were confirmed via genotyping prior to usage. The primer sequences used in this study are shown in Additional file 2: Table S1.
Induction of ARs in the hypocotyl was performed as previously described . After seed sterilization, the seeds were sown on plates, incubated at 4°C for 2 d for stratification, and transferred to the light for several hours to induce germination. Plates were then placed vertically in the dark for 3 d, until the hypocotyls reached approximately 6 mm length, and were then transferred to the light for 7 d before counting the emerged ARs.
RNA isolation and RT-qPCR analysis
Following treatment, Arabidopsis plants were immediately frozen in liquid nitrogen and stored at − 80°C. For the reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen), and real-time RT-PCR was carried out using a QuantiTect SYBR Green RT-PCR kit (Qiagen) in a CFX96™ Real-time system using a C1000™ Thermal cycler (Bio-Rad) as described previously . All RT-qPCR was conducted in triplicate biological replications and subjected to statistical analysis. Analysis of relative gene expression data for determining fold changes was conducted as described previously [60, 62]. Data analysis for determining the copy number of the transcripts and for determination of reaction specificities was performed as described previously . RT-qPCR conditions and primer sequences are shown in Additional file 2: Table S1. The experimental conditions used for RT-qPCR followed MIQE (minimum information for publication of quantitative real time PCR experiments) requirements as described in Additional file 3: Table S2.
Microscopy and histochemical GUS assays
Whole-mount visualization of the seedlings and histochemical assays for GUS activity were conducted as described previously .
Quantitative data were subjected to statistical analysis for every pair-wise comparison using software for Student’s t-Test (Predictive Analytics Software for Windows version 20.0).
This study was supported by grants from the Next-Generation BioGreen 21 Program (PJ013220), Rural Development Administration (RDA), Republic of Korea and the Mid-career Researcher Program (2016R1A2B4015201) and Basic Research Laboratory (2017R1A4A1015620) through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology of Korea to J. Kim. RDA and NRF did not participate in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.
Availability of data and materials
All the data supporting our findings is contained within the manuscript. Constructs and seeds are available upon request from JK.
HWL, CC, SKP, YP, and MJK designed and conducted the experiments and analyzed the data. SKP prepared the manuscript draft. JK conceived the project, designed the experiments, analyzed the data, and wrote the article. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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- 4.Geiss G, Gutierrez L, Bellini C. Adventitious root formation: new insights and perspectives. In: Annual plant reviews volume 37: root development; 2009.Google Scholar
- 25.Lombardi-Crestana S, da Silva Azevedo M, e Silva GF, Pino LE, Appezzato-da-Gloria B, Figueira A, Nogueira FT, Peres LE. The tomato (Solanum lycopersicum cv. Micro-tom) natural genetic variation Rg1 and the DELLA mutant procera control the competence necessary to form adventitious roots and shoots. J Exp Bot. 2012;63:5689–703.CrossRefGoogle Scholar
- 34.Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D, et al. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell. 2005;17:444–63.CrossRefGoogle Scholar
- 35.Vanneste S, De Rybel B, Beemster GT, Ljung K, De Smet I, Van Isterdael G, Naudts M, Iida R, Gruissem W, Tasaka M, et al. Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana. Plant Cell. 2005;17:3035–50.CrossRefGoogle Scholar
- 46.Berckmans B, Vassileva V, Schmid SP, Maes S, Parizot B, Naramoto S, Magyar Z, Alvim Kamei CL, Koncz C, Bogre L, et al. Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins. Plant Cell. 2011;23:3671–83.CrossRefGoogle Scholar
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