1. Introduction
In plants, small regulatory RNAs (sRNAs) of primarily 21 to 24 nucleotides (nt) in length play a key role in controlling gene expression [1,2,3,4,5]. In the genetic model species Arabidopsis thaliana (Arabidopsis), multiple classes of sRNA are produced with each class generated from a structurally distinct double-stranded RNA (dsRNA) precursor transcript [1,2,3,4,5]. Members of two protein families, including the DICER-LIKE (DCL) and dsRNA BINDING (DRB) protein families, form core functional partnerships to recognize, bind and process each structurally distinct dsRNA precursor transcript to produce each class of sRNA [6,7,8,9]. Upon functional maturity, the sRNA is loaded by a specific protein effector complex, termed the RNA-induced silencing complex (RISC), which harbors a member of the ARGONAUTE (AGO) protein family at its functional core [10,11]. RISC uses the loaded sRNA as a sequence specificity guide to direct gene expression regulation of cognate target genes at either the transcriptional or posttranscriptional level [12,13].
The microRNA (miRNA) class of sRNA is the most thoroughly characterized class of sRNA in Arabidopsis, with many of the miRNAs, which accumulate in Arabidopsis cells, regulating the expression of developmentally important genes [6,8,12]. Each Arabidopsis miRNA originates from an individual genomic locus, termed a MICRORNA (MIR) gene [14,15,16], which, like protein-coding genes, are transcribed by RNA polymerase II (Pol II) [17,18]. The Pol II transcription product from a MIR gene, the primary miRNA (pri-miRNA), contains a region of partial self-complementarity which forms a stem-loop structure of imperfectly paired dsRNA. In the Arabidopsis cell nucleus, pri-miRNAs are recognized and bound by SERRATE1 (SE1), a zinc finger protein with the capacity to bind stem-loop-structured RNAs [19]. SE1 transports the bound pri-miRNA to specialized nuclear bodies, called nuclear Dicing bodies (D-bodies), where the pri-miRNA is further bound by DRB1, the functional partner of DCL1 [20,21,22]. DRB1 correctly positions DCL1 on the dsRNA stem-loop region of the pri-miRNA to direct accurate and efficient DCL1-catalyzed pri-miRNA processing to produce the processing intermediate, the precursor-miRNA (pre-miRNA) [6,8,23]. Together with DRB1, DCL1 further processes the pre-miRNA to liberate the miRNA/miRNA* duplex [6,8,23]. The 2′ hydroxyl group at the 3′ terminal nt of each duplex strand is then methylated by the sRNA-specific methyltransferase, HUA ENHANCER1 (HEN1), to stabilize the miRNA guide strand post its separation from the miRNA* passenger strand [24,25]. The miRNA guide strand is preferentially selected over the miRNA* passenger strand by DRB1 for loading into RISC to form the miRISC (miRNA-induced silencing complex) [26]. AGO1 forms the catalytic core of miRISC in Arabidopsis and uses the loaded miRNA as a sequence specificity determinant to direct either mRNA cleavage or translational repression to control target-gene expression [12,13,27].
In addition to DRB1, two other DRB protein family members, DRB2 and DRB4, are localized to the nucleus of the Arabidopsis cell, a cellular localization which indicates potential roles for DRB2 and DRB4 in miRNA biogenesis which occurs exclusively in the nucleus [9,27,28,29,30,31]. Accordingly, DRB4, together with DCL4, are required to produce the newly evolved subclass of miRNAs from highly structured stem-loop precursors, which form due to the extensive base pairing between the two stem arms, which are separated by a minimal loop region [26,29,32]. Unlike DRB1 and DRB4, which exclusively interact with DCL1 and DCL4, respectively [9,23,29,32], DRB2 is likely able to present miRNA precursor transcripts to both DCLs, forming a functional interaction with DCL1 to produce a specific cohort of conserved miRNAs, or with DCL4 to produce newly evolved miRNAs [29,30,31,32]. These DRB2/DCL interactions appear to interrupt the functionality of the DRB1/DCL1 and DRB4/DCL4 partnerships in the developmentally important tissues of wild-type Arabidopsis plants where DRB2 is expressed [28,29,30,33,34].
Arabidopsis, like all other plant species, synthesizes auxin (IAA; indole-3-acetic acid), primarily in the region which surrounds the shoot apical meristem (SAM) [35,36,37]. Auxin is a crucial phytohormone for plant development [37], playing a role in regulating gravitropism [38], phototropism [39], and organ patterning [40], as well as acting as a key regulator of floral development [41] and seed dormancy and/or germination [42,43]. Auxin also forms a key signaling molecule to play a pivotal role in root structure formation, directing cell identity in the root apical meristem (RAM) for the subsequent promotion of primary root elongation and lateral root initiation [44,45]. Auxin continues to accumulate at the RAM of the primary root throughout Arabidopsis development to maintain stem cell proliferation in the RAM to ensure the continued elongation of the primary root [45]. In addition, the continued accumulation of auxin throughout root system development directs the formation of ‘local auxin maxima’, which form the sites for lateral root initiation via the formation of localized populations of undifferentiated cells [44,46]. After lateral root initiation, the level of auxin remains constant at these sites to ensure continued growth and elongation of lateral roots [44,46,47].
At the molecular level, when auxin is perceived in the nucleus, members of the AUXIN RESPONSE FACTOR (ARF) transcription factor family are activated, and once activated, the ARF regulates the expression of sets of AUXIN RESPONSE GENES (ARGs) via binding the specific motifs in ARG promoter regions, termed AUXIN RESPONSE ELEMENTS (AREs), to elicit an appropriate response to auxin [48]. In Arabidopsis, ARF10, ARF16 and ARF17 form a subclade within the larger multimember ARF gene family, with the expression of these three ARFs regulated at the posttranscriptional level by miR160 [49,50]. Our previous research [26] on the drb2345 quadruple mutant showed that miR160 levels were increased considerably in this mutant background where of the five members of the Arabidopsis DRB protein family, only DRB1 is functional. This finding strongly suggested that in addition to DRB1 functioning with DCL1, another DRB family member is involved in miR160 production in Arabidopsis. In a subsequent study characterizing the drb235 triple mutant, we identified that DRB2 most likely occupies this additional role in controlling the level of the miR160 sRNA in Arabidopsis vegetative tissues [30].
Here, we used a molecular modification approach to further explore the degree to which the interplay between DRB1 and DRB2 is required for miR160 production and miR160-directed target gene expression regulation in the Arabidopsis root system. Towards this goal, wild-type Arabidopsis plants (ecotype Columbia-0; Col-0) and the drb1 and drb2 single mutants were transformed with the miR160 overexpression transgene, MIR160B, and miR160-resistant transgene versions of ARF10 and ARF16, mARF10 and mARF16. Molecular profiling of the roots of the Arabidopsis lines Col-0, Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16, revealed that DRB2 acts both synergistically and antagonistically to DRB1 function to tightly control the rate of miR160 production from its three precursor transcripts, PRE-MIR160A, PRE-MIR160B and PRE-MIR160C. Comparison of the molecular profiles obtained for the transformant series to those constructed for unmodified Col-0, drb1 and drb2 plants, further revealed that promotion of primary root elongation was the result of enhanced miR160-directed repression of ARF17 expression. This profiling exercise also showed that the promotion of lateral root development in individual plant lines of the generated transformant series was the result of altered miR160-directed expression regulation of ARF17, and to a lesser degree, ARF10 and ARF16 expression regulation. Similarly, altered expression regulation of ARF10, ARF16 and ARF17 by miR160 was likely responsible for the promotion of adventitious root development observed in individual transformant lines expressing either the MIR160B, mARF10 or mARF16 transgene. Taken together, we demonstrate the requirement of the interplay between DRB1 and DRB2 to tightly control the rate of miR160 production, to in turn ensure the correct degree of miR160-directed ARF10, ARF16 and ARF17 gene expression regulation as part of normal root system development in Arabidopsis.
2. Materials and Methods
2.1. Plant Lines, Growth Conditions, and Phenotypic Assessments
For all experimental analyses performed in this study, the Arabidopsis Col-0 ecotype was used as the wild-type control. The single mutants drb1-1 (SALK_064863) and drb2-1 (GABI_348A09), which harbor T-DNA insertion mutations in the DRB1 (AT1G09700) and DRB2 (AT2G28380) locus, respectively, are also in the Col-0 background and have been described in detail previously [28,29,30]. Col-0, drb1 and drb2 plants were also used to introduce the three plant expression vectors generated for use in this study, including the MIR160B, mARF10 and mARF16 transgenes. A standard Agrobacterium-mediated transformation approach was used to introduce the three transgenes into the Col-0, drb1 and drb2 backgrounds to produce nine additional Arabidopsis plant lines, including the Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines.
The seeds sourced from Col-0, drb1 and drb2 plant lines and the plant lines selected to represent the Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant populations were surface sterilized via incubation at room temperature for 90 min (min) in a sealed chamber containing chlorine gas. The sterilized seeds were transferred to petri dishes that contained full strength Murashige and Skoog (MS) plant growth medium supplemented with 1.0% (w/v) sucrose and 0.8% (w/v) agar. The dishes were sealed with permeable tape and the seeds stratified via incubation for 48 h (h), in the dark, at 4 °C. Following stratification, dishes were transferred to a temperature-controlled growth cabinet (A1000 Growth Chamber, Conviron®, Melbourne, Australia) and cultivated for 10 days under standard Arabidopsis growth conditions of 16 h of light (100–150 µmol m−2 s−1)/8 h of dark and a day/night temperature of 22/18 °C. At day 10, six healthy seedlings per Arabidopsis line were transferred to a large petri dish that contained fresh plant growth medium, and post sealing with permeable tape, the plates were orientated vertically and cultivated for a further 11-day period in the temperature-controlled growth cabinet under the growth conditions stated above. For each of the twelve Arabidopsis lines analyzed in this study, three plates were prepared via this method, with each plate forming a biological replicate for use in subsequent phenotypic and molecular analyses.
At day 21, images of each of the 12 Arabidopsis lines under assessment were captured using a Canon IXUS 801S digital camera with a 4.0 cm2 red calibration square included in each captured image. Images with the calibration square were loaded into the software, Easy Leaf Area (
2.2. Construction of the Transgene Vectors MIR160B, mARF10 and mARF16
The miR160-resistant versions of the ARF10 and ARF16 transcripts (termed mARF10 and mARF16, respectively) were commercially synthesized by GenScript (
2.3. Introduction of the MIR160B, mARF10 and mARF16 Transgenes into Col-0, drb1 and drb2 Plant Lines via Agrobacterium-Mediated Transformation
Post their introduction and sequence confirmation in Escherichia coli (E. coli; DH5α strain), the plasmids which harbored the MIR160B, mARF10, and mARF16 transgenes were extracted from E. coli and introduced into Agrobacterium tumefaciens (Agrobacterium; GV3101 strain) and the resulting Agrobacterium cultures were used to transform Col-0, drb1 and drb2 plants according to the protocol of [53]. The seeds were harvested from ‘floral dipped’ plants and in the T1 generation putative transformants were selected on plant growth medium supplemented with the in planta selective agent glufosinate ammonium (Sigma Aldrich, Sydney, Australia) at a concentration of 5.0 μg/mL, and 150 μg/mL of Timentin (PhytoTech Labs, Lenexa, KS, USA), which was also included in the growth medium to remove any unwanted residual Agrobacterium growth. The transgene copy number and the zygosity of each putative transformant line was determined via a standard PCR-based genotyping approach of resistant seedlings that were germinated and cultivated on Arabidopsis growth medium supplemented with 5.0 μg/mL glufosinate ammonium. Also in the T2 generation, the ‘best performing’ transformant line determined to be homozygous for a single chromosome insertion event was selected to represent the Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant populations for subsequent phenotypic and molecular analyses, with all experimentation reported here conducted on the T3 generation of plants. All DNA oligonucleotides used for PCR-based genotyping are listed in Table S2.
2.4. RNA Extraction, Complementary DNA Synthesis, and Gene Expression Analysis
For the reported molecular assessments, total RNA was extracted from three biological replicates of pooled root material sampled from six 3-week-old vertically grown seedlings of plant lines Col-0, Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16, using TRIzolTM Reagent according to the manufacturer’s protocol (ThermoFisher Scientific, Sydney, Australia). The quality of the extracted total RNA was assessed via standard electrophoretic separation of the nucleic acid on an ethidium bromide-stained 1.2% (w/v) agarose gel. For each high-quality total RNA preparation, a NanoDrop spectrophotometer (NanoDrop® ND-1000, Thermo Scientific, Sydney, Australia) was subsequently employed to determine total RNA concentration in micrograms per microliter (µg/µL).
For the generation of a global high molecular weight complementary DNA (cDNA) library for gene expression quantification, 5.0 µg of total RNA was digested with 5.0 units (U) of DNase I according to the manufacturer’s instructions (New England Biolabs, Melbourne, Australia). The DNase I-treated total RNA was next purified using a RNeasy Mini Kit (Qiagen, Melbourne, Australia) according to the manufacturer’s protocol, and 1.0 µg of this purified preparation was then used as the template to synthesize cDNA via the use of 1.0 U of the ProtoScript® II Reverse Transcriptase and 2.5 mM of oligo dT(18) according to the manufacturer’s instructions (New England Biolabs, Melbourne, Australia).
A miR160-specific cDNA was synthesized via the treatment of 500 nanograms (ng) of total RNA with 0.2 U of DNase I (New England Biolabs, Melbourne, Australia). Each DNase I-treated total RNA sample was directly used as a template for miR160-specific cDNA synthesis using miRNA-specific stem-loop DNA oligonucleotides (Table S2) and 1.0 U of ProtoScript® II Reverse Transcriptase (New England Biolabs, Melbourne, Australia). The cycling conditions of (1) 1 cycle of 16 °C for 30 min, (2) 60 cycles of 30 °C for 30 s, 42 °C for 30 s and 50 °C for 2 s, and (3) 1 cycle of 85 °C for 5 min were used for miR160-specific cDNA synthesis. All generated single-stranded cDNAs were subsequently diluted to a working concentration of 50 ng/µL in RNase-free water prior to their use as a template for the quantification of the abundance of either a gene transcript or miR160.
Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assessment of gene expression or miR160 abundance used the same cycling conditions of (1) 1 cycle of 95 °C for 10 min, and (2) 45 cycles of 95 °C for 10 s and 60 °C for 15 s. The GoTaq® qPCR Master Mix (Promega, Sydney, Australia) was used as the fluorescent reagent for all performed RT-qPCR experiments. miR160 abundance and gene transcript expression were quantified using the 2−∆∆CT method with the small nucleolar RNA, snoR101, and ELONGATION FACTOR-1α (EF-1α; AT5G60390) used as the respective internal controls to normalize the relative abundance of miR160 or of each assessed transcript. For all RT-qPCR experiments reported here, three biological replicates were used per sample, and three technical replicates were performed per biological replicate. The sequence of each DNA oligonucleotide used in this study either for the synthesis of a miR160-specific cDNA, or to quantify gene transcript abundance via RT-qPCR is provided in Table S2.
2.5. Statistical Analysis
Analytical data from this study were obtained from three biological replicates with each biological replicate consisting of a pooled sample of six individual plants. Statistical analysis was performed using a standard two-tailed t-test. Different degrees of statistical significance are represented in Figures 1–4 via the use of asterisks, where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
3. Results
3.1. Molecular Profiling of the miR160 Expression Module in Wild-Type Arabidopsis Plants and the drb1 and drb2 Mutants
Phenotypic assessment of the root architecture of 3-week-old wild-type Arabidopsis plants revealed Col-0 plants (Figure 1A) to have an average primary root length of 38 mm (mm) (Figure 1B) and to develop an average of 12 lateral roots (Figure 1C) and 1.5 adventitious roots (Figure 1D). The severe phenotypic alteration of the drb1 mutant throughout all aspects of its vegetative and reproductive development has been reported previously [6]. Consistently, global alteration of root system architecture was observed in 3-week-old drb1 mutants (Figure 1A), characterized by a 34% reduction to primary root length (25 mm) (Figure 1B), a 66% reduction to lateral root number (Figure 1C), and a 167% increase in adventitious root number (Figure 1D). While mild in comparison to drb1, drb2 plants also displayed a pattern of vegetative and reproductive development distinct to that of wild-type Arabidopsis [28,29,30]. Figure 1A clearly shows the enhanced development of the root system of the drb2 mutant compared to the root system of Col-0 plants. More specifically, 3-week-old drb2 mutants showed an average primary root length of 81 mm, and developed an average of 21 lateral roots and 2.3 adventitious roots (Figure 1B–D), representing increases of 113%, 75% and 53%, respectively over Col-0. Impaired vegetative development was also observed in the aerial tissues of drb1 plants (Figure S1A), with rosette leaf area reduced by 41% to 1.6 cm2 from 2.7 cm2 of 3-week-old Col-0 plants (Figure S1B). Similar to the root tissue, an enhanced growth status was also observed for the aerial tissues of 3-week-old drb2 plants, when compared to Col-0 plants, with rosette leaf surface area increased by 52% to 4.1 cm2 (Figure S1A,B).
Our previous analyses of 15-day-old Col-0, drb1 and drb2 seedlings following a 7-day cadmium stress period [54], strongly suggested that in addition to DRB1, DRB2 also plays a role in the multitiered regulation of the miR160/ARF10/ARF16/ARF17 expression module, most likely via its antagonism of the DRB1/DCL1 functional partnership [30,31]. Therefore, we next assessed the expression of the three miR160 target genes in the inflorescence tissues of Col-0, drb1 and drb2 plants grown under standard conditions using semi-quantitative RT-PCR. As shown in Figure S1C, all three assessed miR160 target genes were upregulated in the inflorescence tissues of the drb1 mutant (Figure S1C). Importantly, the abundance of the ARF10 and ARF16 transcripts was elevated to a similar degree in drb2 inflorescences as in drb1 inflorescences, whereas the level of the ARF17 transcript remained unchanged in the drb2 mutant compared to Col-0 inflorescences (Figure S1C). To investigate the antagonistic effect of DRB2 on the DRB1/DCL1 and DRB4/DCL4 functional partnerships [29,30,31], we next quantified the expression level of DRB1, DRB2, and DRB4 in the inflorescence tissues of Col-0, drb1 and drb2 plants. As shown in Figure S1D–F, the expression of DRB1 and DRB4 was elevated by 1.7- and 1.8-fold in the drb2 mutant background, supporting the potential antagonistic effect of DRB2 function in the correct regulation of all Arabidopsis miRNA expression modules.
The miR160/ARF10/ARF16/ARF17 expression module was next profiled by RT-qPCR in the roots of 3-week-old Col-0, drb1 and drb2 plants. In Arabidopsis, the miR160 sRNA is processed from three structurally distinct precursor transcripts, including PRE-MIR160A, PRE-MIR160B and PRE-MIR160C [55]. As shown in Figure 1E–G, the abundance of the three miR160 precursors was elevated by 30.0-, 2.9- and 14.8-fold, respectively, in drb1 roots. In drb2 roots, a similar trend of increased transcript abundance was observed for PRE-MIR160A and PRE-MIR160C, showing 3.0- and 2.0-fold upregulation, respectively, when compared to the level of expression of these two miR160 precursor transcripts in Col-0 plants (Figure 1E,G). In contrast, the abundance of the PRE-MIR160B was reduced by 1.8-fold in drb2 roots compared to Col-0 (Figure 1F). Using stem-loop RT-qPCR, SL-RT-qPCR, miR160 abundance was revealed to be reduced by 5.0-fold in drb1 roots (Figure 1H), which is likely the direct result of defective precursor transcript processing in the absence of DRB1 function (Figure 1E–G). The loss of DRB2 function in drb2 roots had a mild effect on miR160 accumulation, and unlike drb1, miR160 abundance was increased by 1.5-fold compared to Col-0 roots (Figure 1H). This moderate elevation in miR160 accumulation in drb2 roots is likely accounted for by the enhanced processing of the PRE-MIR160B precursor in the absence of DRB2 antagonism of the DRB1/DCL1 functional partnership.
The large change in miR160 precursor transcript abundance compared to the relatively mild alteration to mature miR160 abundance (Figure 1E–H) raised the possibility that other regulatory factors are involved in regulating the miR160 expression module. Previous studies have implicated endogenous target mimics (eTM160-1 and eTM160-2) in adding to the regulatory complexity of the miR160 expression module in Arabidopsis [56]. These two eTMs are expressed in a spatiotemporal manner to finetune miR160 abundance [56]. We therefore assessed the expression of eTM160-1 and eTM160-2 in the roots of 3-week-old drb1, drb2 and Col-0 plants. Compared to their abundance in Col-0 roots, the expression of eTM160-1 and eTM160-2 was reduced by 1.8- and 1.5-fold, respectively, in drb1 roots (Figure 1I,J). A shared trend in the abundance of miR160 and its eTMs suggests that eTM transcript abundance was ‘scaling’ with that of its regulated miRNA, miR160 (Figure 1H–J). In drb2 roots, the expression of eTM160-1 was elevated 2.0-fold (Figure 1I), while the abundance of the eTM160-2 transcript was mildly reduced by 1.4-fold (Figure 1J). The elevated abundance of both miR160 and eTM160-1 in drb2 roots again suggested that the endogenous target mimic scaled in abundance with its regulated miRNA, miR160.
In drb1 roots, the 5.0-fold reduction in miR160 abundance correlated with increased expression of the ARF10 (2.3-fold), ARF16 (1.5-fold) and ARF17 (2.5-fold) target genes (Figure 1K–M). In drb2 roots, the level of expression of ARF10, ARF16 and ARF17 was elevated by 1.3-, 2.1- and 1.1-fold, respectively (Figure 1K–M), in response to the 1.5-fold increase in miR160 abundance (Figure 1H). Taken together, the molecular analyses presented in Figure 1 indicated that both the canonical DRB1-dependent and non-canonical DRB2-dependent miR160-directed mode of target gene expression regulation was required for the absolute control of ARF10, ARF16 and ARF17 transcript abundance in Arabidopsis roots. These results also indicated that DRB1 is the primary DRB partner of DCL1 for miR160 precursor transcript processing, playing a principal role in the regulation of ARF10, ARF16 and ARF17 expression in the roots of 3-week-old Arabidopsis plants (Figure 1K–M). In addition, the altered abundance of both the precursor and mature forms of miR160 in drb2 roots suggested that DRB2 played a distinct role in the processing of miR160 from its precursors, which would result in potential additional layers of regulation complexity, including a translational repression mode of RNA silencing, operating in Arabidopsis roots to control the expression of ARF10 and ARF16.
3.2. Phenotypic and Molecular Characterization of the Root Systems of Col-0 Plants and the Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 Transformant Lines
To further understand the role of DRB2 in adding to the regulatory complexity of the miR160 expression module, three plant expression vectors were constructed and introduced into Col-0, drb1 and drb2 plants. These constructs included the (1) PRE-MIR160B overexpression vector (MIR160B), (2) miR160-resistant transgene version of ARF10 (mARF10), and (3) miR160-resistant transgene version of ARF16 (mARF16). Typical root phenotypes of the three transformant populations, and unmodified Col-0 plants are shown in Figure 2A–D. The average length of the primary root of Col-0/MIR160B transformants was 84 mm, representing a 121% increase to the primary root length of unmodified Col-0 plants (38 mm) (Figure 2B). A similar degree of promotion to primary root length was also observed for the Col-0/mARF10 and Col-0/mARF16 transformant lines, with increases of 123% to 85 mm, and 111% to 80 mm documented for these two transformant lines, respectively (Figure 2B). The introduction of the MIR160B transgene into Col-0 plants also significantly promoted lateral and adventitious root development, with Col-0/MIR160B plants developing an average of 34 lateral and 4 adventitious roots at 3 weeks of age compared to 12 and 1.5 lateral and adventitious roots in unmodified Col-0 plants, which represented increases of 183% and 167%, respectively (Figure 2C,D). The Col-0/mARF10 and Col-0/mARF16 transformant lines displayed much milder changes in lateral and adventitious root development than did the Col-0/MIR160B transformant line (Figure 2C,D). That is, 3-week-old Col-0/mARF10 plants developed an average of 15 lateral roots and 2.1 adventitious roots, representing increases of 25% and 40% compared to untransformed Col-0 plants. Similarly, 3-week-old Col-0/mARF16 transformants showed mild increases in lateral root (15.5 lateral roots or a 29% increase) and adventitious root (2.8 adventitious roots or a 87% increase) development in comparison to unmodified Col-0 plants of the same age (Figure 2C,D).
Consistent with the 17.2-fold increase in miR160 abundance in Col-0/MIR160B roots, the expression of the two target genes, ARF10 and ARF16, was repressed by 2.5- and 3.3-fold, respectively (Figure 2I,J). This moderate decrease in target gene expression compared to the strong 17.2-fold increase in miR160 abundance in Col-0/MIR160B roots could be accounted for by the increased abundance of the target mimics eTM160-1 and eTM160-2 which were potentially ‘buffering’ miR160-directed repression of target-gene expression. The Col-0/mARF10 transformant showed a 4.3-fold increase in ARF10 transcript abundance in its root tissues (Figure 2I), with ARF16 expression being largely unaffected (Figure 2J). Similarly, in Col-0/mARF16 roots, the level of ARF16 transcript was elevated by 4.7-fold with ARF10 expression levels being similar to Col-0 roots (Figure 2I,J). Although miR160 levels were revealed by SL-RT-qPCR to be elevated by 2.8- and 1.8-fold in Col-0/mARF10 and Col-0/mARF16 roots, respectively (Figure 2F), the highly increased abundance of the two miR160 target transcripts in the respective transformant lines was consistent with the modified transcripts being resistant to miR160 binding, and therefore, miR160-directed expression repression. This molecular alteration to the miR160 expression module also likely accounted for the phenotypic changes in the root systems of these transformant lines.
It is important to note here that in the roots of the Col-0/mARF10 transformant line, the 2.8-fold elevation in miR160 abundance correlated with a 2.0-fold reduction in ARF17 expression (Figure S2G), indicating that the miR160-directed target transcript cleavage mode of RNA silencing of the ARF17 transcript was enhanced. Interestingly, however, in Col-0/mARF16 roots, the 1.8-fold increase in miR160 abundance (Figure 2E) failed to alter the expression of the ARF17 target gene (Figure S2G). In addition, in all three transformant populations, the expression of DRB1, DRB2 or DRB4 was not altered in the roots compared to their respective levels of expression in unmodified Col-0 plants (Figure S2H–J). When considered together, RT-qPCR analysis suggested that alteration to the abundance of either the targeting miRNA (miR160), or its target genes (ARF10 and ARF16), failed to induce a feedback mechanism to promote DRB1, DRB2 or DRB4 gene expression in Col-0 roots to compensate for the molecular alterations introduced into the miR160 expression module in Col-0 plants.
3.3. Phenotypic and Molecular Characterization of the Root System of the drb1 Single Mutant and Transformant Lines drb1/MIR160B, drb1/mARF10 and drb1/mARF16
Compared to the unmodified drb1 mutant, the introduction of the MIR160B, mARF10 and mARF16 transgenes caused considerable alteration of the root architecture (Figure 3A). More specifically, the average primary root length of the drb1/MIR160B transformant was increased by 44% to 36 mm from 25 mm for drb1 plants (Figure 3B), and the average number of lateral roots was increased by 200% to 12 lateral roots from 4 lateral roots for drb1 plants (Figure 3C). However, the number of adventitious roots per drb1/MIR160B plant remained unchanged from that of drb1 plants (Figure 3D). The drb1/mARF10 transformant showed enhanced root development in all three assessed root phenotypic metrics when compared to 3-week-old drb1 plants (Figure 3A–D), with average primary root length of 33 mm (33% increase), an average of 10 lateral roots (150% increase) and 6.7 adventitious roots (67.5% increase). Similarly, the development of all three assessed root phenotypic metrics was promoted in 3-week-old drb1/mARF16 plants with 33 mm average primary root length (33% increase), 9.5 lateral roots (138% increase) and 5.4 adventitious roots (35% increase) per drb1/mARF16 plant (Figure 3B–D).
In drb1/MIR160B roots, the overexpression of PRE-MIR160B resulted in a 79-fold increase in miR160 precursor transcript levels (Figure 3E). However, surprisingly, the abundance of mature miR160 remained unchanged in drb1/MIR160B roots (Figure 3F). This result indicates that DRB1 function is absolutely required for efficient processing of the PRE-MIR160B precursor transcript by DCL1 in the roots of 3-week-old Arabidopsis plants. The transcript abundance of the miR160 target mimics eTM160-1 was significantly elevated by 10.4-fold, whereas that of eTM160-2 was reduced by 2.0 fold, in the roots of drb1/MIR160B plants (Figure 3G,H). The strongly increased abundance of eTM160-1 could partly account for the unchanged miR160 level in drb1/MIR160B plants due to increased sequestering or buffering of the miRNA. Consistent with the stable miR160 level, the expression of the two ARF genes assessed, ARF10 and ARF16, remained unchanged in the roots of the drb1/MIR160B transformant line compared to unmodified drb1 plants (Figure 3I,J).
In the roots of the drb1/mARF10 transformant, the level of both PRE-MIR160B and its processed sRNA, miR160, was significantly elevated by 3.2- and 4.0-fold, respectively, compared to the drb1 mutant (Figure 3E,F). The level of eTM160-1 expression was also significantly elevated by 5.9-fold in drb1/mARF10 roots (Figure 3G), whereas the eTM160-2 transcript level remained largely unchanged (up by 1.1-fold) (Figure 3H). As expected, a significant 2.4-fold increase in ARF10 expression was detected in drb1/mARF10 roots (Figure 3I). Interestingly, a mild 1.8-fold increase in ARF16 expression was observed in the root system of drb1/mARF10 plants. Like the drb1/mARF10 plants, the expression of the mARF16 transgene in the roots of 3-week-old drb1/mARF16 transformant induced a 4.0-fold increase in PRE-MIR160B expression (Figure 3E), which in turn led to the 4.4-fold increase in miR160 abundance compared to untransformed drb1 plants (Figure 3F). Also similar to the drb1/mARF10 plants, increased miR160 abundance in drb1/mARF16 roots was associated with a 5.7-fold increase in eTM160-1 transcript abundance, and a 1.4-fold decrease in eTM160-2 abundance (Figure 3G,H). In addition, the over-expression of the mARF16 transgene in drb1/mARF16 roots led to a significant 3.2-fold increase in ARF16 expression and a moderate 1.6-fold elevation in ARF10 expression (Figure 3I,J). Taken together, the expression profiles of the individual components of the miR160 expression module in the roots of the drb1/mARF10 and drb1/mARF16 transformants suggested that over-expression of the miR160-resistant ARF genes induced a positive feedback response at all levels of the targeted miRNA expression module to accommodate the introduced molecular modifications in these plant lines.
In contrast to the considerable alterations to root architecture, the drb1/MIR160B, drb1/mARF10 and drb1/mARF16 transformants only displayed mild changes in aerial tissue development compared to drb1 plants. Specifically, rosette leaf area was increased by 19% to 1.9 cm2 in drb1/MIR160B plants, moderately decreased by 31% to 1.1 cm2 in drb1/mARF10 plants, and remained unchanged at 1.6 cm2 in drb1/mARF16 plants (Figure S3B). Notably, all three miR160 precursor transcripts (PRE-MIR160A, PRE-MIR160B and PRE-MIR160C) showed increased accumulation in the roots of drb1/MIR160B, drb1/mARF10 and drb1/mARF16 transformants when compared to the 3-week-old drb1 plants (Figure S3C,D). However, the level of mature miR160 was unchanged in the drb1/MIR160B roots, whereas the drb1/mARF10 and drb1/mARF16 plants showed 4.0- and 4.4-fold increase in miR160 abundance, respectively, compared to the untransformed drb1 plant (Figure 3F). Consistent with the unchanged miR160 abundance, the expression level of ARF17 remained unchanged in drb1/MIR160B roots (Figure S3E). Surprisingly, the level of ARF17 expression also remained largely unchanged in the roots of the drb1/mARF10 and drb1/mARF16 plants despite the greater than 4-fold increase in miR160 levels in these two transformant lines. This result raised the possibility that DRB1 activity is required for efficient miR160-directed ARF17 target transcript cleavage, and in the absence of DRB1 function, enhanced ARF17 transcript cleavage due to increased miR160 abundance could not be achieved. This finding also suggests that in Arabidopsis roots, ARF17 transcript abundance is solely regulated by a DRB1-dependent miR160-directed transcript cleavage mode of RNA silencing.
DRB2 and DRB4 expression was only mildly affected in drb1/mARF10 and drb1/mARF16 roots, with respective 1.2- and 1.4-fold increases for DRB2 expression (Figure S3F) and 1.1- and 1.4-fold increases for DRB4 expression (Figure S3G). RNA secondary structure prediction showed that the PRE-MIR160A and PRE-MIR160C stem-loop structures possess a highly compacted loop, with only 3 and 4 unpaired nucleotides, respectively (Figure S3H,J). By comparison, the loop structure in the PRE-MIR160B transcript is less compact, composed of 7 unpaired nucleotides. The relatively compact loop region structure of PRE-MIR160A and PRE-MIR160C could have potentially enabled more efficient processing of these two precursors by DRB2/DCL1 or DRB2/DCL4 partnership in the absence of a canonical DRB1/DCL1 partnership [7,9,29,30], which in turn could account for the 4.0- and 4.4-fold increase to miR160 abundance in the drb1/mARF10 and drb1/mARF16 transformants (Figure 3F).
3.4. Phenotypic and Molecular Characterization of the Root System of the drb2 Single Mutant and the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 Transformant Lines
The expression changes of components of the miR160 expression module, including PRE-MIR160B, miR160, ARF10 and ARF16 in the roots of drb2 plants (Figure 1), prompted us to investigate the consequence of introducing the MIR160B, mARF10 and mARF16 transgenes into the drb2 background. As shown in Figure 4A, the roots of the drb2/MIR160B transformant line showed no significant change in overall root architecture when compared to the untransformed drb2 mutant. In contrast, the drb2/mARF10 transformant displayed dramatically altered root development (Figure 4A), showing primary root length of 97 mm (19.8% increase) (Figure 4B), and an average of 38.5 lateral (131% increase) and 3.9 adventitious (69.6% increase) roots (Figure 4C,D). All three quantified root phenotype metrics were also altered in the drb2/mARF16 transformant line, however, the degree of alteration was mild compared to drb2/mARF10 plants, with an average primary root length of 87 mm (7.4% increase), an average lateral root number of 28.5 (35.7% increase), and adventitious root number decreased by 17.4% from 2.3 in drb2 plants to 1.9 in drb2/mARF16 plants (Figure 4B–D).
Molecular profiling of drb2/MIR160B roots revealed that PRE-MIR160B transcript abundance was reduced by 1.8-fold, and that the accumulation of the miR160 sRNA was significantly elevated by 17.0-fold in comparison to the untransformed drb2 plants (Figure 4E,F). The expression level of eTM160-1 and eTM160-2 was moderately, yet significantly reduced by 2.2- and 1.9-fold, respectively (Figure 4G,H). This formed an unexpected result as elevated miR160 abundance in drb2/MIR160B roots was expected to lead to elevated eTM transcript abundance, and not decreased eTM abundance as determined by RT-qPCR., The 17.0-fold increase in miR160 abundance also correlated with enhanced repression of ARF10 and ARF16 expression, with the abundance of these two miR160 target transcripts reduced by 2.0- and 4.0-fold in the roots of drb2/MIR160B plants, respectively (Figure 4I,J). In summary, the data presented in Figure 4E–J shows that PRE-MIR160B transcript abundance was decreased in the drb2/MIR160B root system (Figure 4E), indicating that in the absence of DRB2 antagonism of DRB1 function, PRE-MIR160B is more efficiently processed by the DRB1/DCL1 functional partnership. This in turn led to elevated miR160 accumulation, and therefore, enhanced degrees of miR160-directed repression of its target genes, ARF10 and ARF16 (Figure 4G–J). Furthermore, the expression data presented in Figure 4E–J clearly reveals that DRB2-mediated mechanisms add an additional layer of regulatory complexity as part of the absolute control of ARF10 and ARF16 expression by miR160 in Arabidopsis roots.
In the roots of the drb2/mARF10 transformant, PRE-MIR160B transcript abundance was only marginally elevated by 1.1-fold (Figure 4E), which was associated with a 2.6-fold increase in miR160 abundance compared to the roots of 3-week-old drb2 plants (Figure 4F). The increased miR160 abundance caused a 2.0-fold reduction in eTM160-1 expression (Figure 4G). Surprisingly, the level of eTM160-2 expression was increased by 1.9-fold which was likely to be in response to the increased miR160 accumulation in drb2/mARF10 roots (Figure 4H). As expected, the expression of ARF10 was elevated in drb2/mARF10 roots by 3.4-fold (Figure 4I). In contrast to elevated ARF10 abundance, the expression level of ARF16 was reduced by 2.7-fold (Figure 4J), which was likely due to the higher level of miR160 directing increased repression of the expression of its target gene. In drb2/mARF16 roots, PRE-MIR160B expression was moderately reduced by 1.8-fold, and the abundance of miR160 was significantly increased by 4.6-fold, compared to drb2 roots (Figure 4E,F). This result suggested that the PRE-MIR160B precursor transcript is more readily processed in drb2/mARF16 than drb2 root tissues, leading to the enhanced accumulation of the miR160 sRNA. Consistent with the elevated miR160 abundance, the expression of eTM160-1 was significantly reduced by 5.0-fold (Figure 4G), and eTM160-2 transcript abundance slightly decreased by 1.1-fold (Figure 4H) in drb2/ARF16 roots. ARF10 expression was also decreased by 4.0-fold (Figure 4I), and ARF16 transcript abundance was elevated by 2.7-fold in the root system of the drb2/mARF16 transformant line.
Compared to unmodified 3-week-old drb2 plants, the introduction of all three transgenes repressed aerial tissue growth (Figure S4A). Total rosette leaf area was decreased by 43%, 12% and 51% to 2.3 cm2, 3.6 cm2 and 2.0 cm2 in the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines, respectively, compared to 4.1 cm2 for the unmodified drb2 mutant (Figure S4B). The abundance of the PRE-MIR160A precursor transcript was reduced by 2.3-, 2.9- and 2.5-fold in the roots of the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines (Figure S4C), which correlated with significantly increased miR160 accumulation (Figure 4F), suggesting an increased efficiency of miR160 precursor processing in the roots of these transformants. Interestingly, the abundance of PRE-MIR160C transcript was similar in the roots of the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines compared to the unmodified drb2 mutant (Figure S4D). This suggested that DRB2 is not involved in regulating the biogenesis of miR160 from the PRE-MIR160C precursor transcript in the roots of 3-week-old Arabidopsis plants. Consistent with the increased miR160 abundance, ARF17 expression was considerably reduced by 2.1-, 3.3- and 2.9-fold, respectively, in drb2/MIR160B, drb2/mARF10 and drb2/mARF16 roots (Figure S4E). In addition, DRB1 transcript abundance was increased by 3.2-, 1.8- and 1.9-fold, respectively, in these three transformant lines (Figure S4F). The highest degree of promotion of DRB1 expression in the roots of the drb2/MIR160B transformant line once again suggested that in the absence of DRB2 antagonism, the ability of DRB1 to form a functional partnership with DCL1 for efficient precursor transcript processing is enhanced. Interestingly, DRB4 expression was increased by a similar degree (approximately 1.5-fold) in the roots of the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines (Figure S4G). Elevated DRB4 expression in all three transformant lines of the drb2 mutant background again identifies the importance of DRB2 antagonism on DRB4 function to ensure the correct level of regulatory complexity for the miR160 expression module in the root system of 3-week-old Arabidopsis plants.
4. Discussion
4.1. Interplay between DRB1 and DRB2 Is Required for the Appropriate Regulatory Control of the miR160 Expression Module in Arabidopsis Roots
Of the five members of the Arabidopsis DRB gene family, DRB1 is firmly established as the primary functional partner of DCL1, the miRNA pathway-specific DCL endonuclease [6,8,9]. In Arabidopsis, as with all plant species, miRNAs are master regulators of gene expression throughout development, including coordinating the transition from juvenile to adult vegetative development, and the subsequent transition from vegetative growth to reproductive development [57,58,59]. The retarded development of the drb1 root system displayed by 3-week-old drb1 plants (Figure 1) therefore formed an expected result, and confirmed the abnormalities associated with all aspects of Arabidopsis development in the absence of DRB1 function [6,26,28]. At three weeks of age, defective root development of the drb1 single mutant was characterized by reductions to primary root length and lateral root number in combination with promotion of adventitious root development (Figure 1B–D). Considering that molecular alteration to the miR160 expression module, including altered abundance of miR160 or of its ARF10, ARF16 and ARF17 target genes has been previously associated with changes in Arabidopsis root development [49,50,55], we applied here a standard RT-qPCR approach to molecularly assess this expression module. Elevated abundance of PRE-MIR160A, PRE-MIR160B and PRE-MIR160C, together with reduced miR160 accumulation in the roots of 3-week-old drb1 plants (Figure 1E–H), readily confirmed the absolute requirement of DRB1 function for efficient miRNA precursor processing by DCL1 for miR160 production [6,8,9,20,21,22,23,26]. Furthermore, an elevated degree of ARF10, ARF16 and ARF17 expression (Figure 1K–M) in response to the reduced miR160 abundance (Figure 1H), showed that the canonical DRB1-dependent mRNA cleavage mode of RNA silencing [12,13,26,34] formed the primary mechanism of miR160-directed gene expression regulation in the root system of 3-week-old wild-type Arabidopsis plants.
Previous research has shown that developmental defects in the drb2 mutant are relatively mild in comparison to those displayed by the drb1 mutant [28,30,31]. However, the data presented in Figure 1 provides further solid evidence of the requirement of DRB2 function for normal Arabidopsis development. Specifically, primary root elongation, and the formation of lateral and adventitious roots were all promoted in 3-week-old drb2 plants. Our previous research has repeatedly demonstrated the requirement of DRB2 for producing specific miRNA cohorts in the tissues where DRB2 is expressed in Col-0 plants [30,31,33,34]. Here we provide further evidence to indicate that DRB2 functions both synergistically and antagonistically on the DRB1/DCL1 functional partnership for miR160 production. More specifically, the increased abundance of PRE-MIR160A and PRE-MIR160C in drb2 roots (Figure 1E,G) indicates that these miR160 precursor transcripts are processed at a reduced level of efficiency by DCL1 in the absence of DRB2 activity. This demonstrates that DRB2 acts in a synergistic manner with DRB1 to produce miR160 from these two precursor transcripts in Arabidopsis roots. In contrast, the reduced level of PRE-MIR160B abundance in drb2 roots (Figure 1F) suggests that DRB2 acts antagonistically on the DRB1/DCL1 partnership for miR160 processing from this precursor transcript, that is; in the absence of competitive binding of PRE-MIR160B by DRB2, the PRE-MIR160B precursor is more readily available for processing by the DRB1/DCL1 functional partnership.
Pélissier et al. [29] have shown that in addition to DRB4, together with its functional partner protein DCL4, DRB2 is required for the production of the newly-evolved class of Arabidopsis miRNAs from their highly complementary stem-loop structured precursors. Comparison of the folding structures of the three miR160 precursors (Figure S3H–J) shows that PRE-MIR160A and PRE-MIR160C form stem-loop structures that more closely resemble those adopted by precursors of the newly-evolved miRNAs [32] than does the PRE-MIR160B precursor. This suggests that the folding structure formed by an individual miRNA precursor transcript likely forms the primary determinant of the degree of involvement, and therefore, the mechanism of action by DRB2 (i.e., DRB2 antagonism or synergism), to fine tune the rate of production of a DRB2-dependent miRNA in Arabidopsis. RT-qPCR also showed both elevated abundance of miR160 and increased expression of ARF10 and ARF16 in the roots of 3-week-old drb2 plants (Figure 1). This shared trend in both miR160 abundance and its target gene expression raised the possibility that translational repression is operating as the mode in miR160-mediated gene repression [27,31,33,34]. Thus, in Arabidopsis roots, miR160 may direct both the canonical mode of RNA silencing, mRNA cleavage, and the noncanonical mode of RNA silencing, translational repression, to regulate ARF10 and ARF16 expression in this tissue.
4.2. Development of the Arabidopsis Root System Is Altered in Plant Lines That Harbor Molecular Modifications to the miR160 Expression Module
The initial phenotypic and molecular analyses performed on the roots of 3-week-old Col-0, drb1 and drb2 plants strongly inferred that the involvement of DRB2 on the miR160 expression module was limited to specific components of the module, including regulation of the rate of miR160 production from the PRE-MIR160B precursor transcript, and by potentially mediating translational repression as a mode of miR160-directed RNA silencing to add an additional layer of regulation to afford a tighter degree of control over the level of ARF10 and ARF16 gene expression. To gain further insight into the role(s) of DRB2 in the miR160 expression module as part of root development, the MIR160B, mARF10 and mARF16 transgenes were constructed and introduced in Col-0, drb1 and drb2 plants.
Compared to unmodified 3-week-old Col-0 plants, of the three root system metrics assessed in the Col-0/MIR160, Col-0/mARF10 and Col-0/mARF16 transformant lines (Figure 2B–D), altered primary root morphology formed the most pronounced phenotypic change (Figure 2B). The similar phenotypic alterations between the miR160 overexpression and miRNA-resistant target gene transformants was surprising, as decoupling ARF10 and ARF16 posttranscriptional regulation by miR160 in Col-0/mARF10 and Col-0/mARF16 plants was expected to have the opposite phenotypic effect to the enhancement of miR160-directed ARF gene expression repression in Col-0/MIR160B plants. For example, at the molecular level it was also surprising that elevated miR160 abundance in Col-0/mARF10 and Col-0/mARF16 roots failed to influence the expression of the unmodified ARF target gene (Figure 2F,I,J). That is, although miR160 abundance was significantly elevated by 2.8-fold in Col-0/mARF10 roots, ARF16 expression remained largely unchanged (Figure 2F,I), and in Col-0/mARF16 roots, ARF10 expression remained at a similar level to that observed in unmodified Col-0 roots, even though miR160 abundance was elevated by 1.8-fold (Figure 2F,J). However, in contrast to ARF10 and ARF16 transcript abundance in these transformant lines, the extent to which the expression of the ARF17 was decreased in Col-0/mARF10, Col-0/mARF16 and Col-0/MIR160B roots (Figure S2G) did correlate with elevated miR160 abundance (Figure 2F). Via a similar approach to that adopted here, Mallory and colleagues [60] showed that in Arabidopsis transformants which highly expressed the introduced mARF17 transgene (i.e., transformant lines with a high abundance of the miR160-resistant ARF17 transcript), primary root length was reduced. This previous report [60] provides strong support that enhancement of miR160-directed gene expression repression of ARF17 was the likely cause of the promotion of primary root development in the Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 transformant lines (Figure 2A,B and Figure S2E).
Among the transformant lines in the Col-0 background, promotion to lateral root development was observed only in Col-0/MIR160B plants (Figure 2C). This phenotypic change appeared to be directly related to highly elevated miR160 abundance (Figure 2F) and reduced ARF10, ARF16 and ARF17 expression (Figure 2I,J and Figure S2E). ARF17 is a well-known negative regulator of lateral root development [57,60,61], and therefore, enhanced miR160-directed repression of ARF17 expression was the likely initiator of promoted lateral root development in Col-0/MIR160B plants. All three transformant lines generated in the drb1 background developed an increased number of lateral roots (Figure 3C). This finding is significant considering that the unmodified drb1 mutant has impeded lateral root development compared to Col-0 plants (Figure 1). While ARF10 and ARF16 play opposing roles in lateral root initiation [57], both ARFs promote the growth of lateral roots once lateral root initials have emerged through the root epidermis [62]. It is therefore likely that the increased expression of ARF10 and ARF16 in the drb1/mARF10 and drb1/mARF16 transformant lines (Figure 3I,J) can rescue the deleterious effects of disrupted DRB1 function on miR160-controlled lateral root development. Lateral root number was also increased in the drb2/mARF10 and drb2/mARF16 transformant lines (Figure 4C). In these two plant lines, ARF16 and ARF10 expression was reduced (Figure 4I,J) as a result of elevated miR160 abundance (Figure 4F) and therefore, enhancement of the miR160-directed target transcript cleavage (Figure S4H). These results suggest that ARF10 and ARF16 can promote lateral root development independently of each other.
As observed with lateral root development for the transformant lines in the Col-0 background, promotion of adventitious root development appeared to be related to elevated miR160 accumulation (Figure 2D). Among the Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 transformant lines, adventitious root development was promoted to the greatest extent in Col-0/MIR160B plants where miR160 accumulation increased to its highest level (Figure 2F). Accordingly, the expression of ARF10, ARF16 and ARF17 showed the most pronounced reduction in Col-0/MIR160B roots (Figure 2I,J and Figure S2E). In adventitious roots, a previous report has shown that ARF6 and ARF8 expression is negatively regulated by ARF17 activity [63], thereby identifying ARF17 as a repressor of adventitious root development in Arabidopsis [60,61,63]. Therefore, reduced ARF17 expression in Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 roots due to increased miR160-directed repression likely accounts for the observed promotion of adventitious root development in these three transformant lines. Adventitious root development was also promoted in the drb1/mARF10 and drb1/mARF16 transformant lines (Figure 3D). Interestingly, in the roots of all three transformant lines generated in the drb1 background, ARF17 expression remained largely unchanged from its level in drb1 roots (Figure S3E). However, ARF10 and ARF16 expression was increased in both transformant lines which could indirectly promote adventitious root development via their reported transcriptional regulation of ARF6 and ARF8 gene expression, with both ARF6 and ARF8 previously identified as positive regulators of adventitious root development [63]. Altered adventitious root development in the transformant lines in the drb2 mutant background was primarily restricted to the drb2/mARF10 plant line (Figure 4D). Compared to drb2 plants, miR160 abundance was significantly elevated in drb2/mARF10 roots which readily accounted for the significantly reduced level of ARF16 (Figure 4I) and ARF17 expression (Figure S4G) in the root system of this transformant line. Considering the known role for ARF16 and ARF17 [61,63] in negatively regulating the activity of ARF6 and/or ARF8, it is unsurprising that an increase in adventitious root formation was observed in drb2/mARF10 plants.
4.3. The Interaction of miR160 with Its eTM Regulatory Transcript, eTM160-1, Appears to Be via Distinct Mechanisms in the drb1 and drb2 Mutant Backgrounds
The first functional eTM identified in plants was INDUCED BY PHOSPHATE STARVATION1 (IPS1), a non-cleavable ncRNA which was shown to add to the regulatory complexity of the miR399/PHOSPHATE2 expression module [64]. Since this initial discovery, numerous other eTMs have been identified in Arabidopsis [56], and in other plants species such as rice and upland cotton [56,65,66]. The Arabidopsis miR160 is one such miRNA for which an eTM has been identified and shown to add regulatory complexity to the miR160–ARF target gene interaction [56]. More specifically, the Arabidopsis miR160 expression module is under additional regulatory scrutiny by eTM160-1 and eTM160-2, which sequester miR160 activity in a spatiotemporal manner [56]. The RT-qPCR analyses presented here show that in unmodified drb1 roots, reduced miR160 abundance was associated with decreased expression of both miR160-specific eTMs (Figure 1H–J). Similarly, in drb2 roots, an elevated level of miR160 was associated with increased abundance of the eTM160-1 transcript (Figure 1H,I). However, eTM160-2 expression was reduced in response to elevated miR160 abundance in drb2 roots (Figure 1J). This initial profiling exercise of eTM expression in unmodified drb1 and drb2 plants, indicated that in the root system of 21-day-old Arabidopsis plants, the eTM160-1 ncRNA occupied the role of the primary non-cleavable target decoy of miR160-directed ARF target gene expression regulation via scaling in its abundance in accordance with its regulated miRNA (Figure 1H,I).
In response to elevated miR160 abundance in Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 roots (Figure 2F), the level of the eTM160-1 transcript was again observed to scale closely with that of its regulated miRNA (Figure 2G). This shared expression trend again suggested that a feedback mechanism was operating in the roots of Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 plants with elevated miR160 abundance promoting the transcriptional activity of the locus from which the eTM160-1 is transcribed. In the roots of drb1/mARF10 and drb1/mARF16 transformants, eTM160-1 levels were again observed to scale in accordance with miR160 abundance. The abundance of the eTM160-1 ncRNA was also elevated in the roots of the drb1/MIR160B transformant line even though miR160 abundance was determined to remain unchanged from its levels in unmodified drb1 plants (Figure 3F,G). However, in this transformant line, the abundance of the PRE-MIR160B precursor transcript was dramatically increased by 79-fold (Figure 3E) to potentially suggest that eTM160-1 expression may be responsive to both a change in mature miR160 abundance, and the transcriptional activity of the MIR genes from which miR160 precursor transcripts are transcribed. Such multitiered responsive would ensure that the tight regulatory control over the miR160 expression module is maintained in the Arabidopsis root system. Interestingly, in contrast to unmodified Col-0, drb1 and drb2 roots, and the roots of the MIR160B, mARF10 and mARF16 transformant lines in the Col-0 and drb1 genetic backgrounds, an opposing abundance trend for the miR160 and eTM160-1 transcripts was documented for the three transformant lines generated in the drb2 mutant background (Figure 4F,G). This finding indicates that the molecular alterations introduced into the miR160 expression module had a more pronounced negative impact on this regulatory transcript than did the identical molecular alterations following their introduction into either Col-0 plants or the drb1 mutant. More specifically, the introduction of the MIR160B, mARF10 and mARF16 transgenes into the drb2 mutant background and the in planta expression of these transgenes in a plant line where DRB2 function is absent, somehow rendered the regulatory capacity of the eTM160-1 transcript to the miR160 sRNA defective. This finding indicates that a higher level of complexity may be required to appropriately control the involvement of DRB2 in the miR160 expression module in Arabidopsis roots.
5. Conclusions
In this study, we used a molecular modification approach to determine the degree of involvement of DRB2 in regulating the miR160 expression module with our analyses revealing that DRB2 adds an additional layer of regulatory complexity to miR160 production from its precursor transcripts. Namely, DRB2 acts synergistically to the DRB1/DCL1 partnership for miR160 processing from PRE-MIR160A and PRE-MIR160C, and is antagonistic to DRB1/DCL1 function for miR160 production from PRE-MIR160B. The molecular alterations introduced into the Col-0, drb1 and drb2 plant lines, altered the architecture of the Arabidopsis root system, with the in planta expression of the MIR160B, mARF10 and mARF16 transgenes promoting the elongation of the primary root and enhancing lateral and adventitious root formation. Molecular assessment of the individual components of the miR160 expression module in transformants Col-0/MIR160B, Col-0/mARF10, Col-0/mARF16, drb1/MIR160B, drb1/mARF10, drb1/mARF16, drb2/MIR160B, drb2/mARF10 and drb2/mARF16, and comparison of the obtained molecular profiles to those constructed for the root systems of unmodified Col-0, drb1 and drb2 plants, indicated that promotion of primary root elongation was the result of enhanced miR160-directed expression repression of ARF17 expression. Molecular profiling of the miR160 expression module in this transformant series also showed that promotion of lateral root development was likely the result of altered miR160-directed expression regulation of ARF17, and to a lesser degree, ARF10 and ARF16. Similarly, enhanced miR160-directed expression regulation of ARF10, ARF16 and/or ARF17, was the likely initiator of promoting adventitious root development in individual transformant lines expressing either the MIR160B, mARF10 or mARF17 transgene. Finally, we also show that the ability of the eTM, eTM160-1, to sequester the activity of miR160 in Arabidopsis roots is impaired when further molecular modifications to the miR160 expression module are introduced into the drb2 mutant background: a finding that readily demonstrates the high degree of regulatory complexity involved in controlling the miR160 expression module in Arabidopsis roots.
A.L.E. proposed the research. K.Z., J.L.P. and J.M.J.O. performed the experiments. K.Z. analyzed the data. D.A.C., M.-B.W., C.P.L.G. and A.L.E. supervised the project. K.Z. and A.L.E. drafted the manuscript. J.L.P., J.M.J.O., D.A.C., M.-B.W., C.P.L.G. and A.L.E. revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
All data reported here are available from the authors upon request.
The authors would like to thank the fellow members of the Centre for Plant Science at the University of Newcastle for their guidance and advice on the growth and care of Arabidopsis thaliana plants.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Phenotypic analysis of root system architecture of 3-week-old Col-0, drb1 and drb2 plants and molecular assessment of the miR160/ARF10/ARF16/ARF17 expression module. (A) Root system development of 3-week-old Col-0, drb1 and drb2 plants. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C) and adventitious root number (D) in 3-week-old Col-0, drb1 and drb2 plants. (E–M) RT-qPCR quantification of the level of expression of PRE-MIR160A (E), PRE-MIR160B (F), PRE-MIR160C (G), miR160 (H), eTM160-1 (I), eTM160-2 (J), ARF10 (K), ARF16 (L) and ARF17 (M) in the roots of 3-week-old Col-0, drb1 and drb2 plants. (E–M) Fold changes were determined by the 2−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for Col-0 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Enlarge this image.Figure 2. Phenotypic and molecular assessment of the miR160 expression module in Col-0 plants and the Col-0/MIR160B, Col-0/mARF10, and Col-0/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old Col-0 plants and the Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old Col-0, Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 plants. (E–J) RT-qPCR quantification of the level of expression of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old Col-0, Col-0/MIR160B, Col-0/mARF10 and Col-0/mARF16 plants. (E–J) Fold changes were determined by the 2−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for Col-0 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Enlarge this image.Figure 3. Phenotypic and molecular assessment of the miR160 expression module in the drb1 single mutant and the drb1/MIR160B, drb1/mARF10, and drb1/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old drb1 plants and the drb1/MIR160B, drb1/mARF10 and drb1/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old drb1, drb1/MIR160B, drb1/mARF10 and drb1/mARF16 plants. RT-qPCR quantification of the level of expression of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old drb1, drb1/MIR160B, drb1/mARF10 and drb1/mARF16 plants. (E–J) Fold changes were determined by the 2−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for drb1 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Enlarge this image.Figure 4. Phenotypic and molecular assessment of the miR160 expression module in the drb2 single mutant and the drb2/MIR160B, drb2/mARF10, and drb2/mARF16 transformant lines. (A) Typical root system architecture displayed by 3-week-old drb2 plants and the drb2/MIR160B, drb2/mARF10 and drb2/mARF16 transformant lines. Scale bar = 1.0 cm. Quantification of primary root length (B), lateral root number (C), and adventitious root number (D) in 3-week-old drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 plants. (E–J) RT-qPCR quantification of transcript abundance of PRE-MIR160B (E), miR160 (F), eTM160-1 (G), eTM160-2 (H), ARF10 (I), and ARF16 (J) in the roots of 3-week-old drb2, drb2/MIR160B, drb2/mARF10 and drb2/mARF16 plants. (E–J) Fold changes were determined by the 2−∆∆CT method with the use of three biological replicates which contained six pooled plants per replicate. Averages of expression are represented as a fold change for each assessed transcript and were compared to the values obtained for drb2 plants by a standard two-tailed t-test. Error bars represent the standard error of the mean (SEM) and asterisks show * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Supplementary Materials
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References
1. Jones-Rhoades, M.W.; Bartel, D.P. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell; 2004; 14, pp. 787-799. [DOI: https://dx.doi.org/10.1016/j.molcel.2004.05.027] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15200956]
2. Allen, E.; Xie, Z.; Gustafson, A.M.; Carrington, J.C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell; 2005; 121, pp. 207-221. [DOI: https://dx.doi.org/10.1016/j.cell.2005.04.004] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15851028]
3. Borsani, O.; Zhu, J.; Verslues, P.E.; Sunkar, R.; Zhu, J.K. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell; 2005; 123, pp. 1279-1291. [DOI: https://dx.doi.org/10.1016/j.cell.2005.11.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16377568]
4. Vaucheret, H. Post-transcriptional small RNA pathways in plants: Mechanisms and regulations. Genes. Dev.; 2006; 20, pp. 759-771. [DOI: https://dx.doi.org/10.1101/gad.1410506] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16600909]
5. Axtell, M.J. Classification and comparison of small RNAs from plants. Ann. Rev. Plant Biol.; 2013; 64, pp. 137-159. [DOI: https://dx.doi.org/10.1146/annurev-arplant-050312-120043] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23330790]
6. Vazquez, F.; Gasciolli, V.; Crété, P.; Vaucheret, H. The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol.; 2004; 14, pp. 346-351. [DOI: https://dx.doi.org/10.1016/j.cub.2004.01.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14972688]
7. Adenot, X.; Elmayan, T.; Lauressergues, D.; Boutet, S.; Bouche, N.; Gasciolli, V.; Vaucheret, H. DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr. Biol.; 2006; 16, pp. 927-932. [DOI: https://dx.doi.org/10.1016/j.cub.2006.03.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16682354]
8. Kurihara, Y.; Takashi, Y.; Watanabe, Y. The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis. RNA; 2006; 12, pp. 206-212. [DOI: https://dx.doi.org/10.1261/rna.2146906]
9. Hiraguri, A.; Itoh, R.; Kondo, N.; Nomura, Y.; Aizawa, D.; Murai, Y.; Koiwa, H.; Seki, M.; Shinozaki, K.; Fukuhara, T. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol. Biol.; 2005; 57, pp. 173-188. [DOI: https://dx.doi.org/10.1007/s11103-004-6853-5]
10. Fagard, M.; Boutet, S.; Morel, J.B.; Bellini, C.; Vaucheret, H. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc. Natl. Acad. Sci. USA; 2000; 97, pp. 11650-11654. [DOI: https://dx.doi.org/10.1073/pnas.200217597]
11. Morel, J.B.; Godon, C.; Mourrain, P.; Béclin, C.; Boutet, S.; Feuerbach, F.; Proux, F.; Vaucheret, H. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell; 2002; 14, pp. 629-639. [DOI: https://dx.doi.org/10.1105/tpc.010358] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11910010]
12. Vaucheret, H.; Vazquez, F.; Crété, P.; Bartel, D.P. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes. Dev.; 2004; 18, pp. 1187-1197. [DOI: https://dx.doi.org/10.1101/gad.1201404] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15131082]
13. Baumberger, N.; Baulcombe, D.C. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA; 2005; 102, pp. 11928-11933. [DOI: https://dx.doi.org/10.1073/pnas.0505461102] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16081530]
14. Guddeti, S.; Zhang, D.C.; Li, A.L.; Leseberg, C.H.; Kang, H.; Li, X.G.; Zhai, W.X.; Johns, M.A.; Mao, L. Molecular evolution of the rice miR395 gene family. Cell Res.; 2005; 15, pp. 631-638. [DOI: https://dx.doi.org/10.1038/sj.cr.7290333] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16117853]
15. Warthmann, N.; Das, S.; Lanz, C.; Weigel, D. Comparative analysis of the MIR319a microRNA locus in Arabidopsis and related Brassicaceae. Mol. Biol. Evol.; 2008; 25, pp. 892-902. [DOI: https://dx.doi.org/10.1093/molbev/msn029] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18296705]
16. Jiang, D.; Yin, C.; Yu, A.; Zhou, X.; Liang, W.; Yuan, Z.; Xu, Y.; Yu, Q.; Wen, T.; Zhang, D. Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice. Cell Res.; 2006; 16, pp. 507-518. [DOI: https://dx.doi.org/10.1038/sj.cr.7310062] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16699546]
17. Meyers, B.C.; Green, P.J.; Lu, C. miRNAs in the plant genome: All things great and small. Genome Dyn.; 2008; 4, pp. 108-118. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18756081]
18. Xie, Z.; Khanna, K.; Ruan, S. Expression of microRNAs and its regulation in plants. Semin. Cell Dev. Biol.; 2010; 21, pp. 790-797. [DOI: https://dx.doi.org/10.1016/j.semcdb.2010.03.012]
19. Yang, L.; Liu, Z.; Lu, F.; Dong, A.; Huang, H. SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. Plant J.; 2006; 47, pp. 841-850. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2006.02835.x]
20. Han, M.H.; Goud, S.; Song, L.; Fedoroff, N. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl. Acad. Sci. USA; 2004; 101, pp. 1093-1098. [DOI: https://dx.doi.org/10.1073/pnas.0307969100]
21. Fang, Y.; Spector, D.L. Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr. Biol.; 2007; 17, pp. 818-823. [DOI: https://dx.doi.org/10.1016/j.cub.2007.04.005] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17442570]
22. Fujioka, Y.; Utsumi, M.; Ohba, Y.; Watanabe, Y. Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in Arabidopsis. Plant Cell Physiol.; 2007; 48, pp. 1243-1453. [DOI: https://dx.doi.org/10.1093/pcp/pcm099]
23. Dong, Z.; Han, M.H.; Fedoroff, N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl. Acad. Sci. USA; 2008; 105, pp. 9970-9975. [DOI: https://dx.doi.org/10.1073/pnas.0803356105]
24. Chen, X.; Liu, J.; Cheng, Y.; Jia, D. HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development; 2002; 129, pp. 1085-1094. [DOI: https://dx.doi.org/10.1242/dev.129.5.1085]
25. Li, J.; Yang, Z.; Yu, B.; Liu, J.; Chen, X. Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr. Biol.; 2005; 15, pp. 1501-1507. [DOI: https://dx.doi.org/10.1016/j.cub.2005.07.029] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16111943]
26. Eamens, A.L.; Smith, N.A.; Curtin, S.J.; Wang, M.B.; Waterhouse, P.M. The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA; 2009; 15, pp. 2219-2235. [DOI: https://dx.doi.org/10.1261/rna.1646909]
27. Lanet, E.; Delannoy, E.; Sormani, R.; Floris, M.; Brodersen, P.; Crété, P.; Voinnet, O.; Robaglia, C. Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell; 2009; 21, pp. 1762-1768. [DOI: https://dx.doi.org/10.1105/tpc.108.063412]
28. Curtin, S.J.; Watson, J.M.; Smith, N.A.; Eamens, A.L.; Blanchard, C.L.; Waterhouse, P.M. The roles of plant dsRNA-binding proteins in RNAi-like pathways. FEBS Lett.; 2008; 582, pp. 2753-2760. [DOI: https://dx.doi.org/10.1016/j.febslet.2008.07.004]
29. Pélissier, T.; Clavel, M.; Chaparro, C.; Pouch-Pélissier, M.N.; Vaucheret, H.; Deragon, J.M. Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis. RNA; 2011; 17, pp. 1502-1510. [DOI: https://dx.doi.org/10.1261/rna.2680711] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21700726]
30. Eamens, A.L.; Kim, K.W.; Curtin, S.J.; Waterhouse, P.M. DRB2 is required for microRNA biogenesis in Arabidopsis thaliana. PLoS ONE; 2012; 7, e35933. [DOI: https://dx.doi.org/10.1371/journal.pone.0035933]
31. Eamens, A.L.; Kim, K.W.; Waterhouse, P.M. DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana. Plant Signal Behav.; 2012; 7, pp. 1224-1229. [DOI: https://dx.doi.org/10.4161/psb.21518] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22902697]
32. Rajagopalan, R.; Vaucheret, H.; Trejo, J.; Bartel, D.P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes. Dev.; 2006; 20, pp. 3407-3425. [DOI: https://dx.doi.org/10.1101/gad.1476406] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17182867]
33. Reis, R.S.; Hart-Smith, G.; Eamens, A.L.; Wilkins, M.R.; Waterhouse, P.M. Gene regulation by translational inhibition is determined by Dicer partnering proteins. Nat. Plants.; 2015; 1, 14027. [DOI: https://dx.doi.org/10.1038/nplants.2014.27] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27246880]
34. Reis, R.S.; Hart-Smith, G.; Eamens, A.L.; Wilkins, M.R.; Waterhouse, P.M. MicroRNA Regulatory mechanisms play different roles in Arabidopsis. J. Proteome Res.; 2015; 14, pp. 4743-4751. [DOI: https://dx.doi.org/10.1021/acs.jproteome.5b00616] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26387911]
35. Jones, B.; Gunneras, S.A.; Petersson, S.V.; Tarkowski, P.; Graham, N.; May, S.; Dolezal, K.; Sandberg, G.; Ljung, K. Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction. Plant Cell; 2010; 22, pp. 2956-2969. [DOI: https://dx.doi.org/10.1105/tpc.110.074856] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20823193]
36. Tao, Y.; Ferrer, J.L.; Ljung, K.; Pojer, F.; Hong, F.; Long, J.A.; Li, L.; Moreno, J.E.; Bowman, M.E.; Ivans, L.J. et al. Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell; 2008; 133, pp. 164-176. [DOI: https://dx.doi.org/10.1016/j.cell.2008.01.049] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18394996]
37. Zhao, Y. Auxin biosynthesis and its role in plant development. Ann. Rev. Plant Biol.; 2010; 61, pp. 49-64. [DOI: https://dx.doi.org/10.1146/annurev-arplant-042809-112308]
38. Rashotte, A.M.; Brady, S.R.; Reed, R.C.; Ante, S.J.; Muday, G.K. Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol.; 2000; 122, pp. 481-490. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10677441]
39. Stowe-Evans, E.L.; Luesse, D.R.; Liscum, E. The enhancement of phototropin-induced phototropic curvature in Arabidopsis occurs via a photoreversible phytochrome Adependent modulation of auxin responsiveness. Plant Physiol.; 2001; 126, pp. 826-834.
40. Bainbridge, K.; Guyomarc’h, S.; Bayer, E.; Swarup, R.; Bennett, M.; Mandel, T.; Kuhlemeier, C. Auxin influx carriers stabilize phyllotactic patterning. Genes. Dev.; 2008; 22, pp. 810-823. [DOI: https://dx.doi.org/10.1101/gad.462608]
41. Krizek, B.A. Auxin regulation of Arabidopsis flower development involves members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) family. J. Exp. Bot.; 2011; 62, pp. 3311-3319. [DOI: https://dx.doi.org/10.1093/jxb/err127] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21511900]
42. Liu, X.; Zhang, H.; Zhao, Y.; Feng, Z.; Li, Q.; Yang, H.Q.; Luan, S.; Li, J.; He, Z.H. Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis. Proc. Natl. Acad. Sci. USA; 2013; 110, pp. 15485-15490. [DOI: https://dx.doi.org/10.1073/pnas.1304651110] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23986496]
43. Park, J.; Kim, Y.S.; Kim, S.G.; Jung, J.H.; Woo, J.C.; Park, C.M. Integration of auxin and salt signals by the NAC transcription factor NTM2 during seed germination in Arabidopsis. Plant Physiol.; 2001; 156, pp. 537-549. [DOI: https://dx.doi.org/10.1104/pp.111.177071]
44. Laskowski, M.J.; Williams, M.E.; Nusbaum, C.; Sussex, I.M. Formation of lateral root meristems is a two-stage process. Development; 1995; 121, pp. 3303-3310. [DOI: https://dx.doi.org/10.1242/dev.121.10.3303]
45. Benjamins, R.; Quint, A.; Weijers, D.; Hooykaas, P.; Offringa, R. The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development; 2001; 128, pp. 4057-4067. [DOI: https://dx.doi.org/10.1242/dev.128.20.4057]
46. Sabatini, S.; Beis, D.; Wolkenfelt, H.; Murfett, J.; Guilfoyle, T.J.; Malamy, J.; Benfey, P.; Leyser, O.; Bechtold, N.; Weisbeek, P. et al. An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell; 1999; 99, pp. 463-472. [DOI: https://dx.doi.org/10.1016/S0092-8674(00)81535-4]
47. Casimiro, I.; Marchant, A.; Bhalerao, R.P.; Beeckman, T.; Dhooge, S.; Swarup, R.; Graham, N.; Inze, D.; Sandberg, G.; Casero, P.J. et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell; 2001; 13, pp. 843-852. [DOI: https://dx.doi.org/10.1105/tpc.13.4.843]
48. Chapman, E.J.; Estelle, M. Mechanism of auxin-regulated gene expression in plants. Annu. Rev. Genet.; 2009; 43, pp. 265-285. [DOI: https://dx.doi.org/10.1146/annurev-genet-102108-134148] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19686081]
49. Wang, J.W.; Wang, L.J.; Mao, Y.B.; Cai, W.J.; Xue, H.W.; Chen, X.Y. Control of root cap formation by microRNA-targeted auxin response factors in Arabidopsis. Plant Cell; 2005; 17, pp. 2204-2216. [DOI: https://dx.doi.org/10.1105/tpc.105.033076]
50. Piya, S.; Shrestha, S.K.; Binder, B.; Stewart, C.N.; Hewezi, T., Jr. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis. Front. Plant Sci.; 2014; 5, 744. [DOI: https://dx.doi.org/10.3389/fpls.2014.00744]
51. Coutu, C.; Brandle, J.; Brown, D.; Brown, K.; Miki, B.; Simmonds, J.; Hegedus, D.D. pORE: A modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res.; 2007; 16, pp. 771-778. [DOI: https://dx.doi.org/10.1007/s11248-007-9066-2]
52. Gleave, A.P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol.; 1992; 20, pp. 1203-1207. [DOI: https://dx.doi.org/10.1007/BF00028910]
53. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.; 1998; 16, pp. 735-743. [DOI: https://dx.doi.org/10.1046/j.1365-313x.1998.00343.x]
54. Pegler, J.L.; Oultram, J.M.J.; Nguyen, D.Q.; Grof, C.P.L.; Eamens, A.L. MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana. Plants; 2021; 10, 130. [DOI: https://dx.doi.org/10.3390/plants10010130]
55. Marin, E.; Jouannet, V.; Herz, A.; Lokerse, A.S.; Weijers, D.; Vaucheret, H.; Nussaume, L.; Crespi, M.D.; Maizel, A. miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. Plant Cell.; 2010; 22, pp. 1104-1117. [DOI: https://dx.doi.org/10.1105/tpc.109.072553]
56. Wu, H.J.; Wang, Z.M.; Wang, M.; Wang, X.J. Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants. Plant Physiol.; 2013; 161, pp. 1875-1884. [DOI: https://dx.doi.org/10.1104/pp.113.215962]
57. Wang, J.W.; Czech, B.; Weigel, D. miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell; 2009; 138, pp. 738-749. [DOI: https://dx.doi.org/10.1016/j.cell.2009.06.014]
58. Wu, G.; Park, M.Y.; Conway, S.R.; Wang, J.W.; Weigel, D.; Poethig, R.S. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell; 2009; 138, pp. 750-759. [DOI: https://dx.doi.org/10.1016/j.cell.2009.06.031]
59. Zhao, L.; Kim, Y.; Dinh, T.T.; Chen, X. miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems. Plant J.; 2007; 51, pp. 840-849. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2007.03181.x]
60. Mallory, A.C.; Bartel, D.P.; Bartel, B. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell; 2005; 17, pp. 1360-1375. [DOI: https://dx.doi.org/10.1105/tpc.105.031716]
61. Couzigou, J.M.; Combier, J.P. Plant microRNAs: Key regulators of root architecture and biotic interactions. New Phytol.; 2016; 212, pp. 22-35. [DOI: https://dx.doi.org/10.1111/nph.14058]
62. Roychoudhry, S.; Del Bianco, M.; Kieffer, M.; Kepinski, S. Auxin controls gravitropic setpoint angle in higher plant lateral branches. Curr. Biol.; 2013; 23, pp. 1497-1504. [DOI: https://dx.doi.org/10.1016/j.cub.2013.06.034] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23891109]
63. Gutierrez, L.; Bussell, J.D.; Pacurar, D.I.; Schwambach, J.; Pacurar, M.; Bellini, C. Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell; 2009; 21, pp. 3119-3132. [DOI: https://dx.doi.org/10.1105/tpc.108.064758] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19820192]
64. Franco-Zorrilla, J.M.; Valli, A.; Todesco, M.; Mateos, I.; Puga, M.I.; Rubio-Somoza, I.; Leyva, A.; Weigel, D.; García, J.A.; Paz-Ares, J. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet.; 2007; 39, pp. 1033-1037. [DOI: https://dx.doi.org/10.1038/ng2079] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17643101]
65. Wang, M.; Wu, H.J.; Fang, J.; Chu, C.; Wang, X.J. A long noncoding RNA involved in rice reproductive development by negatively regulating Osa-miR160. Sci. Bull.; 2017; 62, pp. 470-475. [DOI: https://dx.doi.org/10.1016/j.scib.2017.03.013]
66. Zhang, X.; Shen, J.; Xu, Q.; Dong, J.; Song, L.; Wang, W.; Shen, F. Long noncoding RNA lncRNA354 functions as a competing endogenous RNA of miR160b to regulate ARF genes in response to salt stress in upland cotton. Plant Cell Environ.; 2021; 44, pp. 3302-3321. [DOI: https://dx.doi.org/10.1111/pce.14133]
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; Oultram, Jackson M J 1
; Collings, David A 2
; Ming-Bo, Wang 3 ; Grof, Christopher P L 4 ; Eamens, Andrew L 5
1 Centre for Plant Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment University of Newcastle, Callaghan, NSW 2308, Australia;
2 Centre for Plant Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment University of Newcastle, Callaghan, NSW 2308, Australia;
3 CSIRO Agriculture and Food, Canberra, ACT 2601, Australia;
4 Centre for Plant Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment University of Newcastle, Callaghan, NSW 2308, Australia;
5 Seaweed Research Group, School of Health, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia