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Eunkyeong Jeon, Na Young Kang, Chuloh Cho, Pil Joon Seo, Mi Chung Suh, Jungmook Kim, LBD14/ASL17 Positively Regulates Lateral Root Formation and is Involved in ABA Response for Root Architecture in Arabidopsis, Plant and Cell Physiology, Volume 58, Issue 12, December 2017, Pages 2190–2201, https://doi.org/10.1093/pcp/pcx153
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Abstract
The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family members play key roles in diverse aspects of plant development. Previous studies have shown that LBD16, 18, 29 and 33 are critical for integrating the plant hormone auxin to control lateral root development in Arabidopsis thaliana. In the present study, we show that LBD14 is expressed exclusively in the root where it promotes lateral root (LR) emergence. Repression of LBD14 expression by ABA correlates with the inhibitory effects of ABA on LR emergence. Transient gene expression assays with Arabidopsis protoplasts demonstrated that LBD14 is a nuclear-localized transcriptional activator. The knock-down of LBD14 expression by RNA interference (RNAi) resulted in reduced LR formation by delaying both LR primordium development and LR emergence, whereas overexpression of LBD14 in Arabidopsis enhances LR formation. We show that ABA (but not other plant hormones such as auxin, brassinosteroids and cytokinin) specifically down-regulated β-glucuronidase (GUS) expression under the control of the LBD14 promoter in transgenic Arabidopsis during LR development from initiation to emergence and endogenous LBD14 transcript levels in the root. Moreover, RNAi of LBD14 enhanced the LR suppression in response to ABA, whereas LBD14 overexpression did not alter the ABA-mediated suppression of LR formation. Taken together, these results suggest that LBD14 promoting LR formation is one of the critical factors regulated by ABA to inhibit LR growth, contributing to the regulation of the Arabidopsis root system architecture in response to ABA.
Introduction
The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes plant-specific transcription factors, which harbor a conserved LOB domain in the N-terminus, and this gene family plays an important role in regulating diverse aspects of plant development (Majer and Hochholdinger 2011, Xu et al. 2016). There are 42 LBD genes in Arabidopsis (Iwakawa et al. 2002), 35 LBD genes in rice (Yang et al. 2006) and 43 LBD genes in maize (Majer and Hochholdinger 2011), and LBD homologs have been further identified in various other plant species (Coudert et al. 2013). After the LBD/ASL gene family was first identified in Arabidopsis (Iwakawa et al. 2002, Shuai et al. 2002), a number of subsequent studies revealed the roles of certain LBD genes in Arabidopsis in the establishment of adaxial–abaxial polarity in the leaf (Lin et al. 2003, Xu et al. 2003), the auxin-dependent embryonic apical–basal polarity and patterning processes in the embryo (Borghi et al. 2007, Bureau et al. 2010), tracheary element development (Soyano et al. 2008), male gametophyte development (Oh et al. 2010, Kim et al. 2015) and lateral root (LR) development (Okushima et al. 2007, H.W. Lee et al. 2009, Berckmans et al. 2011, Feng et al. 2012). Recent studies have further identified the roles of some LBD genes in brassinosteroid regulation of organ boundary formation (Bell et al. 2012), directing callus formation for plant regeneration (Fan et al. 2012), plant defense (Thatcher et al. 2012) and root-knot nematode pathogenesis (Cabrera et al. 2014). The functions of some of the LBD homologs in other plant species have also been identified in crown root initiation (Inukai et al. 2005, Liu et al. 2005, Coudert et al. 2015), developmental regulation of floral organs and megagametophytes (Zhang et al. 2015), and leaf rolling in rice (Li et al. 2016) as well as flower development (Bortiri et al. 2006, Evans 2007) and shoot-borne root initiation (Taramino et al. 2007, Xu et al. 2015) in maize.
The plant root system consists of a primary root derived during embryogenesis and LRs and secondary roots that form post-embryonically (Tian et al. 2014). LRs are a major determinant of the root system architecture, which is important for root anchoring, nutrient and water uptake, and storage, and it is vital for the growth and survival of plants (Tian et al. 2014). In Arabidopsis, LR formation is a complex developmental process that consists of pre-initiation events in the basal meristem, initiation, primordium development, emergence and meristem activation (Dastidar et al. 2012). Arabidopsis LRs originate from founder cells formed from xylem pole pericycle cells. These founder cells undergo anticlinal and asymmetric divisions, resulting in two shorter daughter cells at the center and two longer cells at the flanks (Malamy and Benfey 1997). These cells undergo further anticlinal and periclinal divisions to generate a dome-shaped LR primordium (LRP) that emerges from the primary root via cell separation (Parizot et al. 2008, Péret et al. 2009a, Péret et al. 2009b, Lavenus et al. 2013). Auxin critically regulates every step of the process of Arabidopsis LR development via several AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA)–AUXIN RESPONSE FACTOR (ARF) modules, including SOLITARY-ROOT/IAA14–ARF7–ARF19, BODENLOS/IAA12–ARF5, IAA28–ARF5, 6, 7, 8, 19 and IAA3–ARF7, as well as auxin transporters, such as AUXIN1 LIKE-AUXIN1 3 and PINs (Fukaki et al. 2002, Benková et al. 2003, Fukaki et al. 2005, Okushima et al. 2005, Vanneste et al. 2005, Laskowski et al. 2008, Swarup et al. 2008, De Rybel et al. 2010, De Smet et al. 2010, Goh et al. 2012a, Goh et al. 2012b, Lavenus et al. 2013, Marhavý et al. 2013, Péret et al. 2013). Several transcription factors, including LBD proteins such as LBD16, LBD18, LBD29 and LBD33, have been identified to act downstream of Aux/IAA–ARF modules in response to auxin to control LR development (Okushima et al. 2007, D.J. Lee et al., 2009, H.W. Lee et al. 2009, Berckmans et al. 2011, Goh et al. 2012a, Kang et al. 2013, Lee and Kim 2013, Lee et al. 2013, Lee et al. 2015).
ABA is known as an antistress plant hormone that plays a role in stress response and is also important in many plant developmental processes, such as stomatal closing, root growth, seed maturation and dormancy, germination and seedling establishment (Leung and Giraudat 1998, Finkelstein et al. 2002, Raghavendra et al. 2010, Umezawa et al. 2010). Accumulating evidence suggests that ABA plays an important role in LR formation (De Smet et al. 2006, Ding and De Smet 2013). ABA reversibly inhibits LR development at a specific developmental stage, immediately after the emergence of the LRP and prior to the activation of the LR meristem (De Smet et al. 2003).
In this study, we showed that LBD14 is a transcriptional activator promoting LR formation and is involved in the ABA response to suppress LR formation in Arabidopsis. Our RNA interference (RNAi) and gain-of-function studies suggested that LBD14 promotes LR formation by enhancing developmental kinetics of both LR primordium and LR emergence. Analysis of β-glucuronidase (GUS) expression showed that LBD14 is expressed in the root stele and the primordium during LR development and that exogenous treatment with ABA down-regulates LBD14 expression in those root tissues. ABA further suppresses the LR formation in dexamethasone (DEX)-inducible LBD14RNAi transgenic Arabidopsis lines upon DEX treatment, relative to that observed with mock treatment, indicating that LBD14 is involved in the ABA response to suppress LR formation.
Results
LBD14 is expressed in root stele and in LRPs, and encodes a transcriptional activator
Reverse transcription–PCR (RT–PCR) analysis of the expression profiles of 24 different LBD genes in Arabidopsis has shown that the transcripts from LBD14, LBD16, LBD29 and LBD33 were detected only in roots (Shuai et al. 2002). LBD16, LBD18, LBD29 and LBD33 have been previously reported to play roles in LR development (Okushima et al. 2007, H.W. Lee et al. 2009, Berckmans et al. 2011, Lee and Kim 2013, Lee et al. 2013). However, the role of LBD14 in root development has not been reported yet. We first analyzed the tissue-specific expression patterns of LBD14 to investigate the function of LBD14 in plant development. We generated transgenic Arabidopsis harboring the approximately 1.7 kb promoter region of LBD14 fused to a green fluorescent protein (GFP):GUS reporter gene (ProLBD14:GFP:GUS) and conducted histochemical GUS assays for this transgenic plant. ProLBD14:GFP:GUS plants showed GUS expression in roots, in particular root stele, but undetectable GUS expression in other tissues (Fig. 1A, B), which is consistent with a previous analysis reporting on RT–PCR data (Shuai et al. 2002). We then determined GUS expression of ProLBD14:GFP:GUS plants during LR development at given developmental stages based on the classification by Malamy and Benfey (1997), showing that GUS expression was detected throughout LR development from stage I to stage VII (Fig. 1C). After emergence of the LR, GUS expression was weak, and GUS expression eventually disappeared in the fully grown LR (Fig. 1B, C). These GUS expression patterns indicated the potential role of LBD14 during LR development. A subcellular localization assay of LBD14:enhanced green fluorescent protein (EGFP) in Arabidopsis protoplasts showed that LBD14:EGFP is co-localized with IAA1:monomeric red fluorescent proten (mRFP) used as a marker for nuclear localization (Abel et al. 1994, Kim et al. 2015), demonstrating that LBD14 is a nuclear-localized protein (Fig. 2A). Transient gene expression assay of LBD14 fused with the Gal4 DNA-binding domain as an effector plasmid and the Gal4 DNA-binding site fused to the luciferase (LUC) reporter gene as a reporter plasmid with Arabidopsis protoplasts showed that LBD14 displays transcription-activating activity (Fig. 2B). These results suggested that LBD14 acts as a transcriptional activator in plant cells.
LBD14 knock-down by RNAi results in reduced LR formation by delaying both primordium development and LR emergence
To evaluate the role of LBD14 in LR development, we generated an RNAi construct to knock down transcript levels of LBD14 in Arabidopsis [Pro35S:LhGR:LBD14RNAi (LBD14RNAi)], since LBD14 null mutants are not available. This construct allows the formation of a hairpin RNA containing 235 bp of the LBD14 coding region under the control of the 35S:LhGR that encodes the DEX-responsive LhGR transcription factor (Wielopolska et al. 2005). Three LBD14RNAi transgenic lines exhibiting the most significantly reduced LBD14 transcript levels by DEX (55.1, 44.5 and 27.6% for #3-2, #12-1 and #20-1 compared with those of –DEX, respectively) were selected for the phenotypic analysis (Fig. 3A). As shown in Fig. 3A, DEX treatment of these transgenic lines did not result in a decrease in the levels of the transcripts of other LBD family genes, such as LBD16, LBD18 and LBD29 which are highly homologous to LBD14, and LBD13 and LBD15, that are relatively distant from LBD14, in a phylogenetic tree (Supplementary Fig. S1). These results indicated the specific down-regulation of LBD14 expression by DEX (Fig. 3A). DEX treatment significantly reduced LR density (emerged LRs per primary root length, #/cm) up to 50% (#3-2) [1.78 ± 0.56 (mean ± SD)], 49.7% (#12-1) (1.78 ± 0.44) and 39.2% (#20-1) (1.47 ± 0.37), respectively, compared with that of –DEX (#3-2, 3.56 ± 0.35; #12-1, 3.58 ± 0.46; #20-1, 3.75 ± 0.57), without affecting primary root length (Fig. 3B–E). The processes of LR development can be divided into eight stages according to specific anatomical characteristics and cell divisions (Malamy and Benfey 1997). We determined the LRP number at the seven given developmental stages and found that DEX treatment of these three LBD14RNAi transgenic lines did not display statistically significant differences in the LRP number for every given stage (Supplementary Fig. S2A). The total numbers of LRPs of DEX-treated LBD14RNAi lines were also similar to those of mock-treated lines (Supplementary Fig. S2A).
Previous studies have shown that quantitative analysis of LR developmental kinetics is a more sensitive assay for determining the mutation effects on LR development than the analysis of numbers of LRPs and LRs at a given developmental stage of plants (Lee and Kim 2013, Lee et al. 2015). Thus to analyze further the impact of LBD14RNAi on LR development, we conducted an LR induction experiment that allows for a more direct and quantitative analysis of LR developmental kinetics (Péret et al. 2012). We used gravitropic stimuli to induce LR initiation (Ditengou et al. 2008). LBD14RNAi and wild-type plants grown vertically for 3 d were exposed to a gravitropic stimulus by rotating the agar plate 90°, and the number of LRPs newly developed on the convex side of the curves was measured after 18 or 42 h of gravitropic induction. The LRPs were grouped according to their developmental stages (Malamy and Befey 1997), and stage VIII indicates an emerged LRP. Wild-type plants showed accumulation of stages I and II LRPs at 18 h post-gravitropic induction (p.g.i.) and accumulation of stages VII and VIII 42 h p.g.i. in both the absence and presence of DEX (Fig. 4A). Two LBD14RNAi lines treated without or with DEX showed similar stages of LR formation at 18 h p.g.i. compared with the wild type, indicating that early stages of LR development were not affected by LBD14RNAi (Fig. 4). However, the LBD14RNAi plants treated with DEX at 42 h p.g.i. exhibited accumulation of stages V and VI but a decreased profile at stages VII and VIII compared witgh the absence of DEX. The LRP distribution of mock-treated LBD14RNAi lines in response to a gravitropic stimulus is similar to that of the wild type. These result indicated that LRP development at later stages and LR emergence are delayed in the LBD14RNAi line, which is consistent with the decreased density of emerged LRs, as shown in Fig. 3E.
Overexpression of LBD14 in transgenic Arabidopsis enhances LR formation and primordium development
To evaluate the gain-of-function phenotype of LBD14 during LR formation, we constructed transgenic Arabidopsis that overexpresses LBD14 under the control of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (Pro35S:LBD14). Three independent transgenic lines expressing high levels of LBD14 expression were chosen for the phenotypic analysis (Fig. 5A). The densities of emerged LRs in three lines of Pro35S:LBD14 transgenic plants increased to some extent without affecting the primary root growth relative to that of the wild type (Fig. 5B–E). As equal densities of LRPs were found between these transgenic lines and the wild type (Supplementary Fig. S2B), we conducted LR induction experiments at 18, 32 and 42 h p.g.i. for Pro35S:LBD14 plants and the wild type, and found that LBD14 overexpression slightly enhanced LRP developmental kinetics at 32 h p.g.i. relative to that of the wild type (Fig. 5F).
LBD14RNAi enhances the suppression of LR formation in response to ABA
Thus far, the roles of LBD genes during LR development have been linked to the auxin signaling pathway. To investigate further the LBD14 gene function in connection with phytohormones, we examined GUS expression of ProLBD14:GFP:GUS plants in response to the auxin IAA, brassinolide, the cytokinin kinetin and ABA. Histochemical GUS assays showed that ABA decreases GUS staining in the root, but other phytohormones did not affect the GUS expression patterns except for a slight enhancement of GUS expression around the root tip by IAA (Fig. 6A; Supplementary Fig. S3). We next determined the effect of ABA on GUS expression of ProLBD14:GFP:GUS plants during LR development, and found that treatment with ABA at 50 µM for 1 d greatly decreases GUS expression at all LR developmental stages as well as emerged LRs (Fig. 6B). We also quantified the response of LBD14 to 50 µM ABA by using quantitative RT–PCR (qRT–PCR) analysis, and showed that the LBD14 transcript levels gradually decrease with increasing incubation time from 4 to 24 h compared with mock treatments, while the expression of RD29A, a marker gene used for ABA response (Yamaguchi-Shinozaki and Shinozaki 1994), greatly increases after 4 h treatment with ABA and then gradually declines until 24 h (Fig. 6C). To understand the role of LBD14 during ABA response for LR formation, we incubated 3-day-old LBD14RNAi, Pro35S:LBD14 and wild-type plants with ABA at 0, 250 and 500 nM concentrations for 4 d and then measured the primary root length and LR number. While LR densities of the wild type incubated at 250 and 500 nM ABA were reduced to 91.5% and 81.7%, respectively, compared with that of wild type incubated without ABA, LR densities of LBD14RNAi line #12-1 incubated at 250 and 500 nM ABA were reduced to 52.5% and 52.1%, respectively, compared with that of LBD14RNAi plants incubated without ABA (Fig. 7A). Similar results were obtained with the other line (#20-1) of LBD14RNAi plants. These results suggested that LBD14RNAi enhances the ABA-induced suppression of LR density compared with the wild type (Fig. 7A). In contrast, lbd16 single mutant and possibly the lbd16 lbd18 double mutant display slightly increased ABA-induced suppression of LR density, which is consistent with the unresponsiveness of LBD16 and LBD18 expression to ABA (Supplementary Fig. S4). It is noted that ABA at 250 or 500 nM greatly reduced GUS expression of ProLBD14:GFP:GUS plants during LR development under the same treatment conditions used for LBD14RNAi plants (Supplementary Fig. S5A). The qRT–PCR analysis also showed that the endogenous LBD14 transcript level was decreased by almost 50% by treatment with ABA at 500 nM for 1 d compared with mock treatment (Supplementary Fig. S5B). These results showed that LBD14RNAi specifically enhances the suppression of LR formation in response to ABA. However, LBD14 overexpression failed to produce the opposite phenotype as compared with the knock-down mutants, i.e. LBD14 overexpression failed to reduce the sensitivity of LR density to ABA (Fig. 7B). The LBD14 transcript levels in transgenic Arabidopsis overexpressing LBD14 were not significantly altered by ABA treatment at both 250 and 500 nM, while RD29A expression was increased by the same treatments, as expected (Supplementary Fig. S6).
Discussion
A number of LBD transcription factors are involved in a plethora of plant developmental processes, but the manner in which LBD proteins integrate phytohormones to modulate developmental programs is largely unknown, except for the auxin-responsive LR development in Arabidopsis. Recent studies have indicated that ABA plays a role in regulating the root architecture by preventing LR growth toward a highly saline environment (Duan et al. 2013, Geng et al. 2013). In this work, we showed that LBD14 promotes LR formation and is a critical component down-regulated by ABA to inhibit LR growth without affecting primary root growth, contributing to the regulation of root architecture in response to ABA in Arabidopsis.
We initially tried to isolate a homozygous line of the LBD14 knock-out T-DNA insertion mutant (SALK_093993), but only heterozygous lines were produced after self-pollination of the initial LBD14 T-DNA insertion line. So we used an RNAi approach to investigate the role of LBD14 in LR formation. Although the CaMV 35S promoter was used to express a hairpin RNA to knock down the LBD14 transcript levels, the transcript levels of other LBD family members, such as LBD13, LBD15, LBD16, LBD18 and LBD29, which are homologous to LBD14, were not altered in the LBD14RNAi lines with or without DEX treatment, indicating that LBD14 is specifically down-regulated by DEX treatment (Fig. 4B). Moreover, besides LR phenotypes, we did not detect any apparent phenotypes, including primary root length, other than LR phenotypes in the DEX-treated LBD14RNAi lines. Although the LRP densities from stage I to stage VII of the DEX-treated LBD14RNAi lines and LBD14 overexpression lines were similar to those of the wild type (Supplementary Fig. S2), the densities of the emerged LRs are respectively reduced or enhanced by LBD14RNAi or LBD14 overexpression, relative to that of the wild type (Figs. 3E, 5E). Analysis of the LR developmental kinetics showed that LBD14RNAi or LBD14 overexpression did not affect LR initiation but delayed or enhanced LRP development, respectively (Figs. 4, 5F). These results demonstrate that LBD14 plays a positive role in LR formation, primarily by promoting LRP developmental processes and LR emergence, thus providing additional evidence for the prominent role of several LBD genes in Arabidopsis LR development given that LBD16, 18, 29 and 33 genes function in LR formation (Okushima et al. 2007, H.W. Lee et al. 2009, Berckmans et al. 2011, Feng et al. 2012, Goh et al. 2012a).
LR branching displays plasticity in response to changing environmental conditions, and several genetic components and signaling pathways have been identified to regulate LR branching in response to nutrients (including phosphate, nitrate, iron and sulfur), gravity and abiotic stresses such as salt and cold (Dastidar et al. 2012, Ding and De Smet 2013, Tian et al. 2014, Jeon et al. 2016). Recent studies indicated that ABA signaling in the endodermis suppresses LR growth during salt stress by regulating tissue-specific transcriptional programs, whereas ABA signaling plays a minor role in salt-regulated primary root growth repression (Duan et al. 2013, Geng et al. 2013). This change in the architecture of the root system prevents further root growth into highly saline environments.
The down-regulation of LBD14 expression by ABA (Fig. 6; Supplementary Fig. S5) may contribute to ABA-mediated inhibition of LR growth. Consistent with this, ABA treatment of LBD14RNAi lines enhances the suppression of LR formation compared with control plants (Fig. 7A). This effect was marginal in single and double lbd16 and lbd18 mutants, which is in agreement with the microarray data showing that these genes are not responsive to ABA (Supplementary Fig. S4), indicating that LBD16 and LBD18 may play minor roles in LR development in response to ABA. The overexpression of LBD14 failed to prevent ABA-mediated inhibition of LR growth (Fig. 7B). Thus, LBD14 may be a critical factor that is negatively regulated by ABA to limit LR growth, providing a mechanism for an adaptive response of the root system architecture under adverse environmental conditions, such as high salinity or drought stress. Alternatively, it is possible that LBD14 and ABA may act in parallel pathways or converge on common downstream targets to control LR formation. In addition, as reduced expression of LBD14 and ABA synergistically decrease not only LR density but also primary root length (Fig. 7A), the decrease in LR density could be due to an overall inhibition of root growth, causing delayed emergence of LRs and thus a lower LR density.
The gene regulatory network which is crucial for an adaptive response of LR development in Medicago truncatula under salt stress has been revealed (Ariel et al. 2010a, Ariel et al. 2010b). Constitutive expression of HB1, an HD-Zip I transcription factor gene, in M. truncatula roots alters their root architecture such as a longer primary root and a significantly lower root dry weight compared with control plants, whereas hb1 mutants showed increased LR emergence (Ariel et al. 2010b). The root biomass of the HB1-overexpressing plants was not significantly affected by salt stress, in contrast to control plants (Ariel et al. 2010b). Moreover, the increase in LR emergence in the hb1 mutants was greater under salt stress compared with control plants. HB1 directly recognizes a specific cis-acting element in the promoter of LBD1, repressing LBD1 expression (Ariel et al. 2010b). The expression of HB1 is induced by both salt stress and ABA, whereas LBD1 expression is induced by auxin (Ariel et al. 2010b). These results together showed that HB1 directly suppresses the expression of LBD1, modulating root architecture in response to high salt or ABA in M. truncatula. It will be of interest to investigate if an HB1-like transcription factor is involved in the repression of LBD14 in response to ABA, suppressing LR growth in Arabidopsis.
Materials and Methods
Plant growth and tissue treatment
Arabidopsis thaliana (Col-0) seedlings were grown and treated as previously described (Park et al. 2002). For the phenotypic measurement of the seedlings, the plants were grown vertically for 3 d under a 16 h photoperiod on a half-strength Murashige and Skoog (MS) agar plate, transferred to a plate containing 10 µM DEX and/or phytohormone at a given concentration and incubated vertically for an additional 4 d. LR development analysis was conducted by applying a gravitropic stimulus to the plants grown vertically for 3 d by rotating the agar plate through 90° and measuring the number of newly developed LRPs on the convex side of the curves after 18 or 42 h gravitropic induction. LRPs were classified according to developmental stages as defined previously (Malamy and Benfey 1997), but stage VIII was added to indicate the emerged LRPs (Peret et al., 2012). To determine GUS expression of seedlings in response to phytohormone, the seedlings were grown for 5 d on a half-strength MS agar plate, then transferred to a half-strength MS agar plate containing a given concentration of hormone, and incubated vertically for an additional 2 d. The light intensity was approximately 120 µmol m–2 s–1 and was provided by three wavelength daylight color fluorescent bulbs (Kumho Electric Co.).
Plasmid construction and Arabidopsis transformation
To construct ProLBD14:GFP:GUS, the promoter region of LBD14, which encompasses base pair –1,752 to base pair –1 relative to the AUG initiation codon was isolated from the genomic DNA of Arabidopsis Col-0 by PCR with primers harboring NotI (N-terminus) and AscI (C-terminus) sites using the Pfu DNA polymerase (Stratagene). The PCR product was inserted into the pGEM-T Easy vector (Promega). The insert DNA fragment was cut with the corresponding restriction enzymes, subcloned into pENTR™/SD/D-TOPO® (Invitrogen), and then transferred to pK7WGF2 (VIB) through an LR recombination reaction using Gateway® LR Clonase™ II Enzyme Mix (Invitrogen), yielding the ProLBD14:GFP:GUS plasmid. To construct the DEX-inducible LBD14RNAi, the LBD14 coding region of base pairs 324–558 was isolated from the genomic DNA of Arabidopsis Col-0 by PCR with primers harboring NotI (N-terminus) and AscI (C-terminus) sites using the Pfu DNA polymerase (Stratagene). The PCR product was inserted into pGEM-T Easy vector (Promega), and the insert of the DNA fragment was cut with the corresponding restriction enzymes and subcloned into pENTR™/SD/D-TOPO® (Invitrogen) and then transferred to pOpOff2 (hyg) (VIB) via LR recombination reaction using Gateway® LR Clonase™ II Enzyme Mix (Invitrogen), yielding the DEX-inducible LBD14RNAi construct. All these constructs were introduced into Arabidopsis Col-0 by Argobacterium-mediated transformation, and T3 homozygous transformants were generated. All constructs were confirmed by DNA sequencing prior to plant transformation. To generate transgenic Arabidopsis overexpressing LBD14, the full-length LBD14 coding region was isolated by PCR from the genomic DNA of Arabidopsis Col-0, and subcloned into pDONR™221 (Invitrogen) using the Gateway® BP recombination reaction to yield pDONR-LBD14. This construct was subcloned into the pB7WG2,0 vector (destination vector, VIB) using the Gateway® LR recombination reaction to yield the Pro35S:LBD14 plasmid. To construct Pro35S:EGFP:LBD14, the LBD14 full-length coding region was isolated from the existing Pro35S:EGFP:LBD14 (pK7WG2,0) plasmid via PCR using the primers with XmaI (N-terminus) and BamHI (C-terminus) sites and the Pfu DNA polymerase (Stratagene). The PCR product was inserted into pGEM-T easy vector (Promega), and the insert DNA fragment was cut with the corresponding restriction enzymes and cloned into pBI221:EGFP vector, yielding the Pro35S:EGFP:LBD14 plasmid. The primer sequences used in this study are shown in Supplementary Table S1.
RNA isolation, RT–PCR and qRT–PCR analysis
Following treatment, the Arabidopsis plants were immediately frozen in liquid nitrogen and stored at –80°C. The total RNA was isolated from frozen Arabidopsis using TRI reagent® (Molecular Research Center, Inc.), and the total RNA was isolated using an RNeasy Plant Mini kit (Qiagen) and subjected to RT–PCR analysis with the Access RT–PCR system (Promega) according to the manufacturer’s instructions. Real-time RT–PCR was carried out using a QuantiTect SYBR RT-PCR kit (Qiagen) in the CFX96™ Real-time system (BIO-RAD). Data analysis and determination of reaction specificities were performed as described previously (Jeon et al. 2010). All real-time RT–PCR assays were conducted in duplicate for the same RNA isolated from each biological replicate. qRT–PCR analysis was carried out for three different biological replicates and subjected to statistical analysis.
Microscopy and histochemical GUS assay
For whole-mount visualization, the seedlings were cleared in 80% (v/v) ethanol for 24 h, mounted in 90% glycerol and observed under a Leica DM2500 microscope with differential interference contrast (DIC) according to the method described by Malamy and Benfey (1997). Histochemical assays for GUS activity were performed with 5-bromo-4-chloro-3-indolyl glucuronide (Duchefa) as previously described (Jefferson and Wilson 1991). The samples were observed under a Leica DM2500 microscope at × 200 magnification with DIC.
Reporter and effector plasmids
For the transcriptional activity assays, the LBD14 full-length DNA amplified by PCR using the Pfu DNA polymerase was inserted into the Pro35S:GD:ARF1M vector (Tiwari et al. 2003) at SalI (N-terminus) and NotI (C-terminus) sites for translational fusion after removing the ARF1M DNA fragment, yielding the Gal4BD:LBD14 construct. Gal4(3X):LUC was used as a reporter plasmid (Lee et al. 2013). The Pro35S:GUS vector was used as a transfection control (Lee et al. 2008), and the PCR-amplified DNA sequences were used for subcloning after verification by DNA sequencing. The PCR conditions and primer sequences are shown in Supplementary Table S1.
Transient expression assays with Arabidopsis protoplasts
The plasmids were purified using a Qiagen Plasmid Midi kit prior to protoplast transfection, and the protoplasts from Arabidopsis plants were prepared as previously described (Lee et al. 2008). The protoplasts were isolated from the rosette leaves of 2- to 3-week-old Arabidopsis plants or from root tissues of 8-day-old seedlings grown vertically on an MS plate under a 16 h photoperiod. The mesophyll protoplasts isolated were transfected with plasmid DNA by polyethylene glycol (PEG)-mediated protoplast transfection and incubated for 18 h in the dark at room temperature. The samples were collected in liquid nitrogen, and the total proteins were extracted using 1 × Passive Lysis buffer (Promega) according to the manufacturer’s protocol. The LUC activity was then determined using the Dual-Luciferase Reporter Assay System (Promega) with GloMax™ Luminometer (Promega). GUS activity was assayed with 1 mM 4-methylumberlliferyl-β-d-glucuronide (MUG) in GUS extraction buffer, as previously described (Lee et al. 2008). After terminating the reaction with 0.2 M Na2CO3, the appearance of the GUS reaction product MU was measured with a fluorescence FLx800 microplate fluorescence reader (BIO-TEK Instruments). LUC activity was normalized to the GUS activity. Transfection was performed in triplicate, and duplicate LUC and GUS assays were performed for each transfection.
Statistical analysis
The t-tests of quantitative data for statistical analysis were conducted for every pair-wise comparison, using the software for Student’s t-test (Predictive Analysis Software for Windows version 23.0) under the assumption of equal variances. Analysis of variance (ANOVA) was also conducted using Predictive Analysis Software for Windows version 23.0 under the assumption of equal variances.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Rural Development Administration, Republic of Korea [Next-Generation BioGreen 21 Program grant No. PJ01104701] and the Ministry of Education, Science, and Technology of Korea [Mid-career Researcher Program (2016R1A2B4015201) and Basic Research Laboratory (2017R1A4A1015620) through the National Research Foundation of Korea to J.K.].
Disclosures
The authors have no conflicts of interest to declare.
Abbreviations
- ARF
auxin response factor
- Aux/IAA
auxin/indole aceticacid protein
- CaMV
Cauliflower mosaic virus
- DEX
dexamethasone
- EGFP
enhanced green fluorescent protein
- GFP
green fluorescent protein
- GUS
β-glucuronidase
- LBD/ASL
lateral organ boundaries domain/asymmetric leaves2-like
- LR
lateral root
- LRP
lateral root primordium
- LUC
luciferase
- MS
Murashige and Skoog
- p.g.i.
post-gravitropic induction
- qRT–PCR
quantitative reverse transcription–PCR
- RFP
red fluorescent protein
- RNAi
RNA interference
- RT–PCR
reverse transcription–PCR