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. 2018 Jan 18;30(2):347–359. doi: 10.1105/tpc.17.00856

GIF Transcriptional Coregulators Control Root Meristem Homeostasis

María Florencia Ercoli a, Antonella Ferela a, Juan Manuel Debernardi a, Ana Paula Perrone a, Ramiro E Rodriguez a,b, Javier F Palatnik a,b,1
PMCID: PMC5868699  PMID: 29352064

GRF-INTERACTING FACTORs, including ANGUSTIFOLIA3, GIF2, and GIF3, control root meristem and quiescent center organization through interactions with GRF transcription factors and BRAHMA complexes.

Abstract

In the root meristem, the quiescent center (QC) is surrounded by stem cells, which in turn generate the different cell types of the root. QC cells rarely divide under normal conditions but can replenish damaged stem cells. In the proximal meristem, the daughters of stem cells, which are referred to as transit-amplifying cells, undergo additional rounds of cell division prior to differentiation. Here, we describe the functions of GRF-INTERACTING FACTORs (GIFs), including ANGUSTIFOLIA3 (AN3), in Arabidopsis thaliana roots. GIFs have been shown to interact with GRF transcription factors and SWI/SNF chromatin remodeling complexes. We found that combinations of GIF mutants cause the loss of QC identity. However, despite their QC impairment, GIF mutants have a significantly enlarged root meristem with additional lateral root cap layers. We show that the increased expression of PLETHORA1 (PLT1) is at least partially responsible for the large root meristems of an3 mutants. Furthermore, we found that GIFs are necessary for maintaining the precise expression patterns of key developmental regulators and that AN3 complexes bind directly to the promoter regions of PLT1 as well as SCARECROW. We propose that AN3/GIFs participate in different pathways that control QC organization and the size of the meristem.

INTRODUCTION

The indeterminate growth of the Arabidopsis thaliana root is supported by the meristem at the tip of the organ. This root meristem is organized into defined domains. The quiescent center (QC), characterized by the expression of the homeodomain transcription factor gene WOX5 (Petricka et al., 2012a), is surrounded by stem cells, which in turn are responsible for the different cell types that comprise the stereotypical Arabidopsis root. QC cells barely divide under normal conditions, although their proliferation can be activated after the stem cells are damaged (Cruz-Ramírez et al., 2013; Heyman et al., 2013; Vilarrasa-Blasi et al., 2014). The QC and adjacent stem cells are specified by two parallel routes directed by the transcription factors PLETHORA (PLT) and SHORTROOT (SHR)/SCARECROW (SCR) (Petricka et al., 2012a; Heyman et al., 2014). Shootward from the QC, in the proximal meristem, the stem cell progeny undergoes rapid, transient-amplifying cell divisions that provide the necessary number of cells for organ growth (Scheres, 2007; Heidstra and Sabatini, 2014).

Transcription factors of the GROWTH-REGULATING FACTOR (GRF) class are defined by the presence of a WRC and QLQ domain, which are necessary for DNA binding and protein-protein interactions, respectively. In Arabidopsis roots, the GRFs are specifically expressed in transit-amplifying cells located in the proximal meristem, where they promote rapid cell divisions while repressing genes that are active in stem cells (Rodriguez et al., 2015). This precise localization of the GRFs in transit-amplifying cells is accomplished through posttranscriptional repression by the microRNA miR396, which excludes the GRFs from the stem cell region (Rodriguez et al., 2015).

The GRFs interact with small cofactors called GRF-INTERACTING FACTORs (GIFs). In Arabidopsis, the GIF family is composed of three members: ANGUSTIFOLIA3 (AN3; also known as GIF1), GIF2, and GIF3, which promote the growth of leaves and aerial organs (Kim and Kende, 2004; Horiguchi et al., 2005). GIFs cannot bind to DNA per se, but they can function as coactivators of the GRFs (Kim and Kende, 2004; Horiguchi et al., 2005; Debernardi et al., 2014; Nelissen et al., 2015; Rodriguez et al., 2016). In addition, AN3 can interact with BRAHMA, a central component of the SWI/SNF chromatin remodeling complexes (Debernardi et al., 2014; Vercruyssen et al., 2014; Nelissen et al., 2015). GIFs are defined by an N-terminal domain that is homologous to the SNH domain present in human SYNOVIAL TRANSLOCATION (SYT) (Kim and Kende, 2004; Horiguchi et al., 2005), which mediates the interaction with human BRAHMA and BRAHMA RELATED GENE1 (Nagai et al., 2001; Perani et al., 2003). Therefore, while the interactions between GIFs/SYT and BRAHMA complexes are conserved between animals and plants, the partnership between GIFs and GRFs has been acquired more recently in evolution, as the GRFs are plant-specific transcription factors.

In the aerial part of the plant, the downregulation of GRF activity through miR396 overexpression or loss-of-function mutants in AN3 and other GIFs have similar effects, including a reduction in meristem and leaf size (Kim and Kende, 2004; Horiguchi et al., 2005; Rodriguez et al., 2010; Lee et al., 2014). Here, we describe the functions of the GIF coregulators in root development and show that they play a broad role in maintaining homeostasis of the root meristem. Most conspicuously, GIFs are necessary to maintain the QC, a function that is independent of GRF transcription factors. Surprisingly, gif mutants that have a distorted QC also have significantly enlarged root meristems. We propose that GIFs act through different partners, including the GRFs and BRAHMA complexes, to maintain the homeostasis of the root meristem. Furthermore, we show that AN3 complexes directly regulate PLT1 and SCR, highlighting the importance of this system in fine-tuning cellular programs during root development.

RESULTS

Control of the QC by GIF Coregulators

The three members in the Arabidopsis GIF family of transcriptional coregulators (Figure 1A) encode polypeptides harboring a SNH and QG domain (Kim and Kende, 2004). We characterized the root tip architecture in individual, double, and triple mutants (Figures 1B and 1C). Single mutants, as well as an3 gif3 and gif2 gif3 double mutants, presented a normal cellular organization in the QC (Figures 1B and 1C; Supplemental Figure 1). However, an3 gif2 and an3 gif2 gif3 displayed a strong disorganization of the QC (Figures 1C and 1D; Supplemental Figure 1). Unlike the two to three cells that are well defined in size and shape in a longitudinal section of the wild-type QC, we found no regular organization in the presumptive QC region of the an3 gif2 or an3 gif2 gif3 mutants (Figures 1C and 1D; Supplemental Figure 1). All an3 gif2 gif3 triple mutants analyzed and ∼70% of an3 gif2 double mutants displayed a strong distortion of the QC area (Figures 1C and 1D; Supplemental Figure 1). We analyzed the expression of QC marker ProWOX5:GFP (Sarkar et al., 2007) by crossing the reporter line to the different mutants and found it to be severely affected in an3 gif2, which is in good agreement with the disorganization of the QC (Figures 1E and 1F).

Figure 1.

Figure 1.

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GIFs Are Necessary to Maintain the QC.

(A) Schematic comparison of the gene structures of different members of the Arabidopsis GIF family. The light-gray boxes represent the untranslated region, while the dark boxes are the exon sequences.

(B) QC organization in roots of the wild type and single mutants an3, gif2, and gif3 at 7 d after sowing. The white arrowheads mark the position of the QC. Bars = 50 µm.

(C) QC organization in roots of multiple GIF mutants gif2 gif3, an3 gif3, an3 gif2, and an3 gif2 gif3 7 d after sowing. The white arrowheads mark the position of the QC. Bars = 50 µm.

(D) Percentage of roots with strong QC distortion in different mutant backgrounds. Wild-type and an3 gif2 QC organization was assayed in more than 140 plants. In the other cases, at least 50 roots were evaluated for each background.

(E) Expression of ProWOX5:GFP in the wild type, an3, gif2, AN3/an3 gif2/gif2, and an3 gif2. The white arrowheads mark the position of the QC. Bars = 50 µm.

(F) Combined box/violin plot diagram of GFP fluorescence intensity from the ProWOX5:GFP reporter in the wild-type, an3, gif2, AN3/an3 gif2/gif2, and an3 gif2 background. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). The data show the GFP fluorescent intensity scored in 25 different plants of each genotype. The combined box/violin plot was produced in R using the ggplot2 library with default parameters. Outliers are plotted as individual dots.

(G) QC organization in roots of the wild type transformed with a dominant repressor version of AN3 (ProAN3:AN3-SRDX) or an empty vector (wt) at 7d after sowing. Below, the expression of ProWOX5:GFP in the wild type and ProAN3:AN3-SRDX. The white arrowheads mark the position of the QC. A schematic representation of ProAN3:AN3-SRDX is shown (light-gray boxes represent untranslated regions, dark boxes represent exons, and the pink box indicates the SRDX sequence). Bars = 50 µm.

To validate our observation that the distortion of the QC in an3 gif2 and an3 gif2 gif3 was caused by reduced GIF activity, we generated a dominant repressor version of AN3 by fusing the EAR-repression motif (SRDX) (Hiratsu et al., 2003) to the C terminus of the protein. Plants harboring ProAN3:AN3-SRDX, in which AN3-SRDX is expressed under the control of AN3 regulatory regions, displayed a strong distortion of the QC and a strong reduction in ProWOX5:GFP expression in 26 out of 44 transgenic plants analyzed (Figure 1G). Taken together, these results indicate that GIF coregulators are necessary to maintain the QC in Arabidopsis roots.

Control of Root Meristem Size by GIF Coregulators

Analysis of primary root growth in gif2, gif3, and gif2 gif3 mutants showed no obvious differences compared with wild-type plants (Figures 2A and 2B; Supplemental Figures 2A and 2B). In contrast, an3 single mutants had longer roots than the wild type, a phenotype that was confirmed in mutants of two different accessions (Figures 2A to 2C; Supplemental Figure 3A). Interestingly, an3 gif2 double mutants had shorter roots than the wild type (Figures 2A and 2B), and an3 gif2 gif3 triple mutants were even more affected (Figures 2A to 2C). Next, we determined the root meristem size in the different GIF mutants. We found that an3, an3 gif3, an3 gif2, and an3 gif2 gif3 mutants displayed significantly larger meristems than wild-type plants (Figures 2D and 2F; Supplemental Figures 2 and 3). In all cases, the larger meristem was a consequence of a higher number of meristematic cells (Figure 2G; Supplemental Figures 2D and 3C). Overall, an3 single mutants had an enlarged root meristem and longer roots than wild-type plants, while an3 gif2 or an3 gif2 gif3 mutants, which had larger meristems together with a strong disorganization of the QC, had shorter roots. We estimated the cell cycle duration in the different mutants and found it to be delayed in an3 gif2 gif3 mutants compared with wild-type roots (Supplemental Figure 4). Next, we overexpressed the three Arabidopsis GIFs (i.e., AN3, GIF2, and GIF3) under the control of the 35S promoter. In all cases, we found that high GIF levels caused a reduction in meristem length (Figures 2E and 2H) due to a decrease in cell number (Figure 2I), a phenotype opposite to that observed in gif mutants. Altogether, these results indicate that GIFs quantitatively control root meristem size, while a strong downregulation of GIF activity (e.g., combination of mutants or AN3-SRDX) impairs the QC.

Figure 2.

Figure 2.

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GIFs Regulate Root Meristem Size and Growth.

(A) Root growth phenotype 6 d after sowing in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. Bar = 1 cm.

(B) Root elongation rate (mm/h) in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. The data shown are mean ± se of the root elongation rate measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(C) Root elongation (mm) in wild type, an3 and an3 gif2 gif3. DAS (days after sowing). The data shown are mean ± se of the root elongation over time measured in 25 different roots of each genotype

(D) Root tip architecture 7 d after sowing in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(E) Root tip architecture 7 d after sowing in the wild type, Pro35S:AN3, Pro35S:GIF2, and Pro35S:GIF3. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(F) Meristematic zone length (μm) in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. The data shown are mean ± se of the meristematic zone length measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(G) Number of cortex cells in the root meristem (Nm) in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. The data shown are mean ± se of the number of meristematic cortex cells scored from 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(H) Meristematic zone length (μm) in the wild type, Pro35S:AN3, Pro35S:GIF2, and Pro35S:GIF3. The data shown are mean ± se of the meristematic zone length measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(I) Number of cortex cells in the root meristem (Nm) in the wild type, Pro35S:AN3, Pro35S:GIF2, and Pro35S:GIF3. The data shown are mean ± se of the number of meristematic cortex cells scored from 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

To investigate whether the effects of the an3 gif2 mutants in the root meristem were reversible, we generated a ProAN3:AN3-GR-GFP (AN3-GR) fusion, in which AN3 activation is triggered by dexamethasone (DEX), and introduced it into an3 gif2 mutants. After 30 h of DEX treatment, the size of the root meristem of the an3 gif2 mutants harboring AN3-GR began to recover and was fully complemented 40 h after DEX treatment (Figures 3A and 3B). Interestingly, 40 h after DEX treatment, the QC organization was recovered in an3 gif2 mutants harboring AN3-GR in most cases (only 15% of an3 gif2 double mutants showed a strong QC distortion; Supplemental Table 1; Figure 3C). Altogether, these results confirm that the defects observed in the AN3/GIF mutants were caused by low levels of these transcriptional coregulators and were at least partially reversible upon activation of AN3.

Figure 3.

Figure 3.

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Inducible AN3-GR Rescues the Root Meristem Defects in the an3 gif2 Mutants.

(A) and (B) Root meristem size changes in an3 gif2 mutants harboring AN3-GR after DEX treatment.

(A) Root tip architecture at 6 d after sowing in the wild type and an3 gif2 AN3-GR. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(B) Meristematic zone length (μm) in the wild type and an3 gif2 AN3-GR. The data shown are mean ± se of the meristematic zone length measured in at least 30 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(C) QC phenotypes in roots of an3 gif2 plants harboring AN3-GR treated with DEX for 20, 30, and 40 h. See Supplemental Table 1 for a quantification of roots with QC defects upon treatment. Bar = 50 μm.

GIF Coregulators and Their Partners Are Expressed in Different Root Domains

Prompted by the observation that the overexpression of AN3, GIF2, and GIF3 caused similar phenotypes, but only an3 single mutants were affected in the root meristem, we decided to analyze their expression patterns in Arabidopsis roots. We generated reporters for the three GIFs by fusing GFP to the C-terminal part of each gene. We found that ProAN3:AN3-GFP (AN3:GFP; Figure 4D) was strongly expressed in the root meristem and QC region, while ProGIF2:GIF2-GFP (GIF2:GFP; Figure 4E) was more broadly expressed, including the elongation zone and columella cells. ProGIF3:GIF3-GFP (GIF3:GFP) expression was restricted to the vascular region (Figure 4F), indicating that the three genes have overlapping but not identical expression patterns. Of the 40 independent reporter lines analyzed for each gene, these patterns of expression were observed in more than 90% of the lines for AN3:GFP (Figure 4D) and GIF2:GFP (Figure 4E), and in more than 60% for GIF3:GFP, which was detected at lower levels (Figure 4F). In addition, only AN3 showed a strong peak of expression in the QC and stem cell region (Figure 4D, inset), which is in agreement with previous reports that identify AN3 as a QC-enriched gene (Nawy et al., 2005), and with the defects in the QC of an3 gif2 and an3 gif2 gif3 mutants, but not of gif2 gif3 (Figures 1C and 1D). Finally, we measured AN3 and GIF2 transcript levels in an3, gif2, and an3 gif2 mutants (Figures 4B and 4C) and found that AN3 transcript levels increased in gif2 mutants (Figure 4B), suggesting the existence of a compensatory mechanism to maintain GIF activity.

Figure 4.

Figure 4.

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Expression Patterns of GIFs, BRM, and GRFs in the Root Meristem.

(A) Schematic comparison of the domain structures of AN3, a GRF, and BRM.

(B) Relative transcript levels of AN3 in wild-type, an3, gif2, and an3 gif2 roots, as determined by RT-qPCR. The data shown are means ± se of three biological replicates. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(C) Transcript level of GIF2 in wild-type, an3, gif2, and an3 gif2 roots, as determined by RT-qPCR. The data shown are means ± se of three biological replicates. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(D) to (I) Expression of GFP reporters in AN3 (D), GIF2 (E), GIF3 (F), BRM (G), GRF2 (H), and GRF3 (I). These reporter lines harbor C-terminal translational fusions of GFP to the complete gene, including introns. These constructs are expressed under the control of their own promoter sequences. The figures on the right side of each panel show GFP intensity profile analysis of the regions highlighted with rectangles. Pink shapes indicate the QC and the stem cell region. White and cyan bars = 50 µm.

As GIFs are known to interact with GRF transcription factors and BRAHMA complexes (Kim and Kende, 2004; Horiguchi et al., 2005; Debernardi et al., 2014; Vercruyssen et al., 2014; Nelissen et al., 2015) (Figure 4A), we compared the expression patterns of GRF2, GRF3, and BRAHMA, which have been described before (Smaczniak et al., 2012), to those of AN3, GIF2, and GIF3. Both GRF2 and GRF3 were specifically expressed in transit-amplifying cells and outside of the stem cell region (Figures 4H and 4I), corresponding to previous results (Rodriguez et al., 2015). In contrast, BRAHMA was expressed throughout the meristem, with a peak in the QC-stem cell area, overlapping with the peak of expression of AN3 (Figure 4G).

The AN3 GRF Pathway Controls Root Meristem Size

We investigated the genetic interactions between AN3 and its partners. Since the GRFs function redundantly (Rodriguez et al., 2015), we analyzed Pro35S:miR396 plants, which have an overall reduction of GRF activity (Rodriguez et al., 2010, 2015; Ercoli et al., 2016). Previous studies have shown that Pro35S:miR396 roots have a large meristem that can be rescued via the expression of a miR396-resistant version of GRF3 (rGRF3) that is insensitive to the repression by the microRNA (Rodriguez et al., 2015). We examined 50 independent lines expressing Pro35S:miR396. Thirty-six of these lines had a meristem larger than wild-type plants, and 10 of them had a meristem similar to or even larger than an3 gif2 mutants (Supplemental Figure 5A). That the overexpression of miR396 had a larger effect on root meristem size than an3 gif2 was not unexpected, as a decrease in the activity of the GRF transcription factors might have a stronger effect than the loss of their coactivators. In agreement with this possibility, it has been shown that rGRF3 can partially rescue the leaf size defects of an3 (Debernardi et al., 2014). Despite these large effects on meristem size, Pro35S:miR396 plants did not show alterations in the QC region (Supplemental Figures 5A and 5B). We then analyzed the effects of overexpression of miR396 in an3 in 50 independent primary transgenic plants. As previously observed (Rodriguez et al., 2010; Mecchia et al., 2013), we found many plants with defects in the shoot apical meristem, leaf polarity, and size (Figures 5A to 5D). We analyzed the roots of 20 Pro35S:miR396 an3 plants with strong leaf defects (Figure 5D) and found that the QC was unaffected (Figure 5D). To confirm that AN3 controls root meristem size through the GRFs, we analyzed the effects of Pro35S:AN3 in plants overexpressing miR396. While Pro35S:AN3 roots showed a reduction in root meristem size, Pro35S:miR396 caused the opposite effect (Figures 5E and 5F). Plants harboring both Pro35S:AN3 and Pro35S:miR396 obtained by crossing the parental lines had a large root meristem similar to that of Pro35S:miR396 plants (Figures 5E and 5F), confirming that AN3 requires the GRFs to modify root meristem size. These results indicate that AN3 together with the GRFs control root meristem length, but they do not seem to act together to maintain the QC.

Figure 5.

Figure 5.

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AN3 Participates in Different Genetic Pathways to Control Specific Zones of the Root Meristem.

(A) to (D) Shoot apices (upper figure) and root QC (lower figure) upon overexpression of miR936 in wild-type (S96 accession) and an3 (an3-1) plants. The yellow arrowheads indicate severely affected leaves, while the white arrowheads mark the position of the QC. Gray bar = 1 mm and white bar = 50 µm.

(E) Root meristem size after overexpression of AN3 driven by the 35S promoter (Pro35S:AN3) in wild-type and miR396-overexpressing plants. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(F) Meristematic zone length (μm) in wild-type, Pro35S:miR396, Pro35S:AN3 (−), and Pro35S:AN3 Pro35S:miR396 plants. The data shown are mean ± se of the meristematic zone length measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

Partial Rescue of brahma Defects by an3

Unlike Pro35S:miR396 plants, brahma mutants have a strong distortion of the QC and a rather short root meristem (Figures 6A, 6B, and 6E) (Yang et al., 2015). We analyzed the genetic interaction between an3 and brahma and found that an3 restored the root meristem size of brahma mutants to a wild-type size (Figures 6A and 6B). As expected, analysis of an3 brahma crosses revealed that they also exhibited strong disorganization of the QC area (Figures 6A and 6F). Furthermore, we found that an3/an3 BRAHMA/brahma roots had a distorted QC in 77% of the roots analyzed (Figures 6A and 6G; Supplemental Table 2), while an3 or BRAHMA/brahma did not show QC disorganization per se (Figures 6C and 6D; Supplemental Table 2), indicating that both genes cooperate in maintaining QC homeostasis. Therefore, while an3 enhanced the QC defects of brahma, it partially restored the meristem size. These results are in agreement with the role of AN3 together with the GRFs in the control of the root meristem size and AN3 together with BRAHMA in the maintenance of the QC.

Figure 6.

Figure 6.

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Control of the Root Meristem and QC by AN3 and BRAHMA.

(A) Root tip architecture at 7 d after sowing in the wild type, an3, brm (brm-1), an3 brm, and an3/an3 BRM/brm. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(B) Meristematic zone length (μm) in the wild type, an3, brm, and an3 brm. The data shown are mean ± se of the meristematic zone length measured in 25 different roots of each genotype. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(C) to (G) QC organization of roots 7 d after sowing in an3 (C), BRM/brm (D), brm (E), an3 brm (F), and an3/ an3 BRM/brm (G). The white arrowheads mark the position of the QC. See Supplemental Table 2 for a detailed analysis of the frequency of roots with QC defects for these genotypes. Bar = 50 μm.

GIFs Fine-Tune Gene Expression in Different Root Domains

To characterize the alterations in the QC and the meristem observed in an3/gif mutants, we analyzed the expression of cellular markers. We used an AGL42 reporter (ProAGL42:GFP) that is expressed in the QC and the surrounding stele and ground tissue stem cells (Nawy et al., 2005). In an3, the activity of the AGL42 reporter expanded into the transit-amplifying cells (Figures 7A and 7B). A similar effect was previously described in miR396 overexpressors (Figures 7K and 7L). However, in AN3/an3 gif2/gif2 and an3 gif2 double mutants, AGL42 expression was specifically downregulated in the QC area, while the ectopic expression of this gene in transit-amplifying cells remained active (Figures 7C and 7D; Supplemental Table 3). That only gif mutants but not miR396 overexpressors affected AGL42 expression in the QC is consistent with the notion that AN3/GIFs functions in QC maintenance independently of the GRFs.

Figure 7.

Figure 7.

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Definition of the Root Developmental Domains by GIFs.

(A) to (H) Expression of the reporters ProAGL242:GFP ([A] to [D]) and ProPLT1:CFP ([E] to [H]) in the wild-type ([A] and [E]), an3 ([B] and [F]), AN3/an3 gif2/gif2 ([C] and [G]), and an3 gif2 background ([D] and [H]). The white arrowheads mark the position of the QC. See Supplemental Table 3 for a detailed analysis of the expression of the reporter ProAGL42:GFP in these mutants. Bars = 50 µm.

(I) and (J) Profile of fluorescence intensity in root tips of the ProPLT1:CFP reporter in the wild type, an3 gif2 (I), and Pro35S:miR396 (J) roots. The value of CFP intensity was normalized to the maximum value measured in the wild type. The intensity profile corresponds to the average of 10 plants per genotype.

(K) to (N) Expression of marker lines ProAGL242:GFP ([K] and [L]) and ProPLT1:CFP ([M] and [N]) in the wild type ([K] and [M]) and Pro35S:miR396 background ([L] and [N]).

We then analyzed the expression of the transcriptional reporters PLT1 (ProPLT1:CFP) and PLT2 (ProPLT2:CFP), which are highly active in the stem cell area and less active in proliferating cells (Galinha et al., 2007; Mähönen et al., 2014). Both the PLT1 and PLT2 expression patterns were expanded in an3 root meristems, although the changes in PLT1 expression were more obvious (Figure 7F; Supplemental Figure 6). In the an3 gif2 double mutants, PLT1 expression decreased in the QC but expanded shootward to a broader area (Figures 7F to 7H), which is consistent with the observed distortion of the QC region and the enlarged proximal meristem. As a consequence, the well-defined gradient of ProPLT1:CFP expression along the longitudinal axis was blurred to a larger domain, with rather homogenous expression levels (Figure 7I). Interestingly, an expansion of the expression pattern of ProPLT1:CFP in transit-amplifying cells was also observed in Pro35S:miR396 plants. However, in that case, PLT1 expression was unaffected in the QC (Figures 7J, 7M, and 7N). These results support the notion that GRFs and AN3/GIFs play a role in the regulation of meristem size and that AN3/GIFs have additional functions in the maintenance of the QC. Altogether, our findings show that AN3/GIFs help generate the specific gene expression patterns in the different root domains.

Mechanistic Links between AN3 and the Major Root Regulators PLT and SCR

Obtaining a readout of the PLT gradient is essential for determining the root domains, as the peak of PLT expression determines the stem cell niche, while lower levels are necessary to promote cell proliferation in the root meristem (Galinha et al., 2007; Mähönen et al., 2014). We then performed a more detailed analysis of the genetic interaction between AN3 and PLT1, whose expression is significantly affected in an3/gif mutants (Figures 7F and 7I). In contrast to an3, which has a larger meristem, the loss of PLT1 led to a reduction in root meristem size (Figures 8A and 8C). Analysis of an3 plt1 revealed a meristem size identical to that of plt1, suggesting that the larger meristem size observed in the an3 mutants depends on the misregulation of PLT1 expression (Figures 8A and 8C). As the ectopic expression of PLT is associated with an increase in the number of lateral root cap (LRC) layers (Galinha et al., 2007), we analyzed an3 and an3 gif2 mutants and found that their roots showed this phenotype (Figures 8B and 8D), which was confirmed by examining the expression of a SOMBRERO reporter (ProSMB:GFP) (Fendrych et al., 2014). Interestingly, the number of LRC layers in the an3 plt1 double mutants was similar to that in wild-type roots (Figure 8E), further indicating the importance of PLT1 misregulation in the developmental defects in an3 roots.

Figure 8.

Figure 8.

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Direct Regulation of PLT1 and SCR by AN3 Complexes.

(A) Root tip architecture 7 d after sowing in the wild type, an3, plt1, and an3 plt1. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. Bar = 50 μm.

(B) Expression of an LRC-specific marker (ProSMB:GFP) in wild-type, an3, and an3 gif2 roots. The white arrowheads mark the position of the QC, and the yellow arrowheads mark the end of the meristem where cells start to elongate. The inset magnifies the same position in each root showing the increase in the number of LRC layers. The white stars mark the LRC layers. Bar = 50 μm.

(C) Meristematic zone length (μm) in the wild type, an3, plt1, and an3 plt1. The data shown are mean ± se of the meristematic zone length measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(D) Number of LRC layers measured 35 μm above the QC in the wild type, an3, gif2, gif3, an3 gif2, and an3 gif2 gif3. The data shown are means ± se of the number of lateral root cap measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(E) Number of LRC layers measured 35 μm above the QC in the wild type, an3, plt1, and an3 plt1. The data shown are means ± se of the number of lateral root cap measured in 25 different roots of each genotype. Different letters indicate significant differences, as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

(F) Expression of AN3:GFP in wild-type (Wassilewskija accession) and plt1 plt2 mutants. The white arrowheads mark the position of the QC. Bars = 50 μm.

(G) Relative transcript level of PLT1 in wild-type and an3 roots, as determined by RT-qPCR. The data shown are means ± se of three biological replicates. Asterisks indicate significant differences from the wild type, as determined by Student’s t test (P < 0.05).

(H) Relative transcript level of PLT1 in an3 AN3-GR roots after 2 h of DEX induction (+DEX; 25 μM), as determined by RT-qPCR. The data shown are means ± se of three biological replicates. Asterisks indicate significant differences from mock-treated plants (− DEX), as determined by Student’s t test (P < 0.05).

(I) ChIP-qPCR (left) for three specific regions in the PLT1 gene named A, B, and C and one specific region in the SCR gene named D. The data shown are from two biological replicates. ChIP assays were performed using anti-GFP antibodies on 6-d-old an3 roots expressing GFP-tagged AN3-GR vector treated with DEX or mock. Right, scheme of AN3 target genes, PLT1 and SCR. A GRF binding site in the PLT1 promoter and an AN3-complex binding site in the SCR promoter are indicated with red rectangles. The green arrowheads show the position of the primers used in the ChIP-qPCR experiment.

(J) Relative transcript level of SCR in an3 x AN3-GR roots after 2 h of DEX induction (+ DEX; 25 μM), as determined by RT-qPCR. The data shown are mean ± se of three biological replicates. Asterisks indicate significant differences from mock-treated plants, as determined by Student’s t test (P < 0.05).

(K) Expression of ProSCR:SCR-GFP in wild type (accession Wassilewskija) and plants transformed with a dominant repressor version of AN3 (ProAN3:AN3-SRDX) 7 d after sowing. The white arrowheads mark the position of the QC. Bars = 50 µm.

As PLT1 was misexpressed in the an3/gif mutants (Figures 7F to 7I), we hypothesized that AN3 might be a regulator of PLT1. To test this hypothesis, we measured PLT1 transcript levels in an3 and found that this gene was upregulated in the root meristem (Figure 8G). We then turned to the DEX-inducible an3 AN3-GR system and studied the response of PLT1 2 h after AN3 induction with DEX. We found that PLT1 transcript levels were significantly repressed upon AN3 induction (Figure 8H). To confirm that PLT1 is directly regulated by AN3 complexes, we performed chromatin immunoprecipitation (ChIP) assays using an3 AN3-GR-expressing lines. Analysis of the PLT1 promoter region identified a potential GRF binding site, TGTCAGA (O’Malley et al., 2016), ∼4 kb upstream of the coding sequence. We designed primers matching flanking regions of this site, as well as a control region in the PLT1 coding sequence (Figure 8I). ChIP experiments performed 30 min after DEX treatment of an3 AN3-GR with primers designed against PLT1 promoter regions revealed a strong enrichment, in contrast to the control region (Figure 8I), demonstrating that AN3 complexes can directly bind to the PLT1 promoter regions and regulate the expression of this gene. In turn, previous studies have shown that AN3 is among the genes directly regulated by PLT2 (Santuari et al., 2016). We therefore crossed an AN3:GFP reporter line to the plt1 plt2 mutant and confirmed that AN3 was significantly downregulated in the progeny (Figure 8F). These results indicate that AN3 and PLT mutually regulate each other.

To explore the additional functions of AN3 in the QC region, we analyzed the results of a previously described AN3 ChIP-seq experiment performed in Arabidopsis suspension cell cultures (Vercruyssen et al., 2014). Among the 2836 peaks identified in the Arabidopsis genome, we found a potential binding site for an AN3 complex in the SCR promoter. We designed primers flanking this putative binding site and confirmed the binding of the AN3 complex to this region by ChIP-PCR using an3 AN3-GR plants treated with or without DEX (Figure 8I). Next, we determined the transcript levels of SCR after treating an3 AN3-GR lines with DEX (Figure 8J) and found an increase in SCR levels, further confirming the regulation of SCR by AN3. Finally, we expressed the repressor version of AN3, ProAN3:AN3-SRDX, in ProSCR:SCR-GFP reporter lines. Of the 40 independent transgenic lines analyzed, 22 had a decrease in SCR-GFP signal in the QC area (Figure 8K). Altogether, these results indicate that there is a link between AN3 and SCR.

DISCUSSION

The cellular organization of the root meristem is essential for establishing the radial patterning of the root and promoting root growth. Two main pathways, which are directed by SHR/SCR and PLT transcription factors, have been shown to play essential roles in root meristem organization. Mutations in these transcription factors affect the QC and the surrounding stem cells, reduce meristem size, and impair root growth (reviewed in Petricka et al., 2012b; Heyman et al., 2014). Here, we describe the functions of GIF coregulators in root meristem homeostasis. In contrast to other regulators of the root meristem that affect the QC and reduce meristem size (Sabatini et al., 2003; Aida et al., 2004; Ortega-Martínez et al., 2007; Vanstraelen et al., 2009; Chen et al., 2011; González-García et al., 2011; Sang et al., 2012; Moubayidin et al., 2013), the loss of GIF genes affects the QC area but increases the size of the meristem.

Expression of GIF Coregulators in Arabidopsis Roots

AN3, GIF2, and GIF3 have overlapping expression patterns, which likely explains their redundant functions. While GIF2 is expressed rather homogenously throughout the root meristem, GIF3 expression is enriched in the vascular region. AN3 has a peak of expression in the stem cell region, with a gradient extending toward the transit amplifying cells. That AN3 is expressed at higher levels in the stem cell area explains why its mutation affects WOX5 expression and QC maintenance, especially in combination with other GIFs. Interestingly, the pattern of AN3 expression is reminiscent of the gradient of PLT transcription factors (Aida et al., 2004; Galinha et al., 2007; Mähönen et al., 2014). The gene network controlled by PLTs has recently been studied in detail, and AN3 was consistently detected as a PLT target gene (Santuari et al., 2016). Furthermore, our results showing that overall AN3 expression was significantly reduced in plt1 plt2 confirm the biological relevance of this regulation (Figure 8F).

Mechanistic Links between AN3 and Major Root Regulators

In turn, we found that PLT1 and PLT2 expression was misregulated in an3/gif mutants. The precise expression pattern of PLT1, with a peak in the stem cell area that extended toward the proximal meristem, became blurred in the an3 gif2 double mutant. While the peak of expression of PLT1 in the stem cell area was reduced in this mutant, its domain of expression extended shootward. The large meristem and ectopic lateral root layers observed in an3 mutant are consistent with phenotypes caused by the ectopic expression of PLT genes (Galinha et al., 2007). The finding that an3 plt1 crosses suppressed the an3 phenotypes corroborates the notion that ectopic PLT1 expression was responsible for these defects. Actually, our AN3-GR ChIP PCR analysis showed that AN3 complexes bind directly to the PLT1 promoter, providing a mechanistic link between AN3 and PLT genes. In this context, AN3/GIFs coregulators and PLTs might be regulating each other’s expression to fine-tune their expression patterns. Mutations in an3/gif compromise the expression of other genes in the QC such as WOX5 and AGL42. At least AGL42 expression was upregulated in the meristem, suggesting that AN3/GIF coregulators are necessary for the homeostasis of the root meristem. In addition, we found that AN3 complexes can bind to the SCR promoter to regulate its expression. Therefore, AN3 can fine-tune the expression of two genes encoding major regulators of root development, the PLT and SCR transcription factors. AN3 is then required for the expression of SCR in the QC area, while it represses PLT1 in proliferating cells.

Functions of AN3 and GIF2/3 in Leaves and Roots

Loss of function of AN3 reduces leaf size (Kim and Kende, 2004; Horiguchi et al., 2005), while additional mutations in GIF2 and GIF3 significantly impair leaf growth and the shoot apical meristem (Kim and Kende, 2004; Horiguchi et al., 2005; Lee et al., 2014). Furthermore, simultaneous mutations of AN3 and HANABA TANARU lead to ectopic root formation in cotyledons (Kanei et al., 2012), highlighting the role of the GIF coregulators in the control of shoot development. However, their functions in the above- or belowground parts of the plant appear to differ. In the aerial parts, miR396 overexpressors and grf mutants have similar phenotypes to an3/gif mutants. Furthermore, an3 Pro35S:miR396 plants have an affected shoot apical meristem and develop leaves with polarity defects (Rodriguez et al., 2010; Mecchia et al., 2013). In contrast, Pro35S:miR396 only affects the size of the root meristem without affecting the QC, and an3 Pro35S:miR396 plants, which have severely affected shoot development, have roots with large meristems (our data). These results suggest that AN3/GIFs have additional functions in roots, especially in QC maintenance, that go beyond the activities of the GRFs.

Control of the QC Region by AN3 and GIF2/3

Detailed studies have shown that AN3 can interact with GRFs (Kim and Kende, 2004; Horiguchi et al., 2005) and BRAHMA chromatin remodeling complexes (Kim and Kende, 2004; Debernardi et al., 2014; Vercruyssen et al., 2014; Nelissen et al., 2015). Furthermore, AN3 is thought to represent a link between the GRFs (or other transcription factors) and chromatin remodeling complexes during leaf development (Vercruyssen et al., 2014; Nelissen et al., 2015). BRAHMA complexes, in turn, are important for the organization of the QC and for modifying the expression of members of the PIN auxin efflux protein family (Yang et al., 2015).

Previous studies have shown that miR396 overexpression does not affect the organization of the QC or SCR expression levels (Rodriguez et al., 2015). Here, we showed that AN3 complexes regulate SCR, which is required to maintain the QC and stem cells. It is likely that AN3 interacts with BRAHMA to maintain the QC region, but it is plausible that a BRAHMA-AN3 complex also interacts with other transcription factors. It has been proposed that the composition of AN3 complexes dynamically changes along a developing maize (Zea mays) leaf (Nelissen et al., 2015), and a similar process might occur in roots as well. Recent studies have described in detail the regulation of cell-specific functions through different protein complexes involving SHORTROOT, SCR, and JACKDAW (Long et al., 2017). We speculate that different AN3 complexes could also be involved in fine-tuning cellular programs in different domains of the root meristem.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana accession Col-0 was used throughout this study unless otherwise indicated. See Supplemental Table 4 for a list and description of the mutants and reporter lines used in this study. Plants were grown in long photoperiods (16 h light/8 h dark) using fluorescent bulbs (triphosphor code #840, 100 µmol quanta m−2 s−1) at 21°C. For root analysis, surface-sterilized seeds were sown on solid medium containing 1× Murashige and Skoog salt mixture, 1% sucrose, and 2.3 mM MES (pH 5.8) in 1% agar. After stratifying the seeds in the dark (4°C) for 2 to 3 d, the Petri dishes were placed in a vertical orientation inside growing chambers. For chemical treatments, DEX (D4902-1G; Sigma-Aldrich) was stored as 25 mM stocks in ethanol and used at 25 μM for the indicated periods (2 h and from 20 to 40 h).

Cloning and Generation of Transgenic Lines

See Supplemental Table 5 for a detailed description of the constructs used in this study. For the analysis of expression pattern of AN3, GIF2, and GIF3, the reporter lines were generated by fusing GFP to the C terminus of each protein. A DNA fragment consisting of the promoter region and the genomic sequences (including introns) was used in each case. The ProAN3:AN3-SRDX and ProAN3:AN3-GFP-GR constructs were obtained in a similar way by fusing the SRDX repression domain or the glucocorticoid receptor coding sequence, respectively, to the C terminus. Arabidopsis plants were transformed using the floral dip method (Clough and Bent, 1998).

Confocal Microscopy

Confocal laser scanning microscopy was performed throughout the study using a Plan Apochromat 20×, 0.8-NA lens on a Zeiss LSM880 microscope. Roots were stained with 10 μg/mL propidium iodide for 2 min, rinsed, mounted in water, and visualized after excitation by an argon 488-nm laser line. The fluorescence emission was collected from 590 to 700nm (propidium iodide), 496 to 542nm (GFP), and 465 to 570nm (CFP). Cellular parameters and fluorescence signal intensity were analyzed with Fiji (Schindelin et al., 2012).

Analysis of Root Growth

For root elongation measurements, seedlings were grown vertically for 10 days. Starting from day 6 after germination until the end of the experiment, a dot was drawn at the position of the root tip. Finally, plates were photographed, and the root length was measured over time with Fiji (Schindelin et al., 2012). Root growth rate, expressed in mm/h, was estimated from the slope of the graph plots of root length (mm) versus plant age (expressed as days after sowing).

Meristematic zone length and meristematic cell number were determined according to the file of cortex cells from the confocal microscopy images. The meristematic zone was defined as the region of isodiametric cells from the QC up to the cell that was twice the length of the immediately preceding cell (Dello Ioio et al., 2008; Long et al., 2017). The average duration of the cell cycle (T) for meristematic cortex cells was calculated as described previously (Rodriguez et al., 2015). For each individual root, the following equation was utilized: Inline graphic, where Nm is the number of meristematic cells in one file of the cortex, Le is the average length of fully elongated cortex cells expressed in nm, and V is the root growth rate calculated as described previously in mm/h. ANOVA results are provided in Supplemental Table 6.

Gene Expression Analysis via RT-qPCR

Total RNA was isolated from root tissue using Tripure isolation reagent (Roche). Approximately 100 root tips (∼2 mm) were collected from each sample at 6 DAG. The numbers of biological replicates, together with the treatment applied, are stated in the corresponding figure legends. Total RNA (0.5 µg) was treated with RQ1 RNase-free DNase (Promega). First-strand cDNA synthesis was performed using SuperScript III Reverse Transcriptase (Invitrogen). PCR was performed in a Mastercycler ep realplex thermal cycler (Eppendorf) using SYBR Green I (Roche) to monitor double-stranded DNA synthesis. The relative transcript level was determined for each sample and normalized using the PROTEIN PHOSPHATASE2A cDNA level (Czechowski et al., 2005). The normalization was performed as described previously (Livak and Schmittgen, 2001). Melting curve analyses at the end of the process and “no template controls” were performed to ensure product-specific amplification without primer-dimer artifacts. Primer sequences are given in Supplemental Table 7.

ChIP

ChIP experiments were performed as described by Kaufmann et al. (2010) with some modifications. The an3 AN3-GR-GFP assay was performed using root tips of 6-d-old seedlings using anti-GFP antibody (ChIP Grade; Abcam ab290, lot GR76757-1). Briefly, the seedlings were transferred to plates containing mock medium (DEX −) or medium supplemented with DEX (DEX +) (D4902-1G; Sigma-Aldrich) by lifting the nylon mesh holding the roots with forceps. After 30 min of treatment, the roots were collected (∼1500 root tips per sample). Following fixation in 1% (v/v) formaldehyde, the tissues were homogenized and the nuclei isolated and lysed. Cross-linked chromatin was sonicated using a Bioruptor UCD-200 (Diagenode) water bath (15 s on/45 s off pulses; three times). The complexes were immunoprecipitated with 1 μL antibody for 1 h at 4°C with gentle shaking, followed by incubation for 50 min at 4°C with 50 mL of Protein A agarose beads (Invitrogen). Immunoprecipitated DNA was then recovered and analyzed by qPCR. An aliquot of untreated sonicated chromatin was processed in parallel as the total input DNA control. Fold enrichment was calculated with or without the addition of DEX for the indicated regions. The primers used in the ChIP experiments are listed in Supplemental Table 7.

Accession Numbers

Accession numbers (Arabidopsis Genome Initiative locus identifiers) for the genes described in this article are provided in Supplemental Table 7.

Supplemental Data

Acknowledgments

We thank Ben Scheres for the ProPLT1:CFP and ProPLT2CFP reporters; Jen Hoe Kim for the an3/gif1, gif2, and gif3 mutants; Cezary Smaczniak and Kerstin Kauffman for the BRAHMA reporter; Yuhai Cui for the brm-1 mutant; Miguel Moreno-Risueño, Julia Baulies, and Carla Schommer for comments on the manuscript; and Rodrigo Vena, Camila Goldy, and Enrique Morales for help with microscope imaging. M.F.E., J.M.D., A.F., and A.P.P. were supported by fellowships from CONICET. R.E.R. and J.P. are members of CONICET. R.E.R. was supported by ANPCyT (PICT 2012/1686). This work was also supported by grants to J.F.P. (ANPCyT PICT 2015-3557 and 2016-0761).

AUTHOR CONTRIBUTIONS

M.F.E., R.E.R., and J.P. conceived and designed the research. M.F.E., J.M.D., R.E.R., A.F., and A.P. performed the experiments. M.F.E., R.E.R., and J.F.P. analyzed data. M.F.E. and J.P. wrote the manuscript.

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