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. 2021 Jan 25;185(4):1798–1812. doi: 10.1093/plphys/kiab014

ARF2 represses expression of plant GRF transcription factors in a complementary mechanism to microRNA miR396

Matías Beltramino 1, Juan Manuel Debernardi 1, Antonella Ferela 1, Javier F Palatnik 1,2,✉,2
PMCID: PMC8133599  PMID: 33580700

A switch in the repression mechanisms controlling expression of transcription factors generates a link between two growth regulating pathways.

Abstract

Members of the GROWTH REGULATING FACTOR (GRF) family of transcription factors play key roles in the promotion of plant growth and development. Many GRFs are post-transcriptionally repressed by microRNA (miRNA) miR396, an evolutionarily conserved small RNA, which restricts their expression to proliferative tissue. We performed a comprehensive analysis of the GRF family in eudicot plants and found that in many species all the GRFs have a miR396-binding site. Yet, we also identified GRFs with mutations in the sequence recognized by miR396, suggesting a partial or complete release of their post-transcriptional repression. Interestingly, Brassicaceae species share a group of GRFs that lack miR396 regulation, including Arabidopsis GRF5 and GRF6. We show that instead of miR396-mediated post-transcriptional regulation, the spatiotemporal control of GRF5 is achieved through evolutionarily conserved promoter sequences, and that AUXIN RESPONSE FACTOR 2 (ARF2) binds to such conserved sequences to repress GRF5 expression. Furthermore, we demonstrate that the unchecked expression of GRF5 in arf2 mutants is responsible for the increased cell number of arf2 leaves. The results describe a switch in the repression mechanisms that control the expression of GRFs and mechanistically link the control of leaf growth by miR396, GRFs, and ARF2 transcription factors.

Introduction

Leaves account for most of the photosynthetic carbon fixation in terrestrial ecosystems and exhibit a huge morphological diversity [recently reviewed in Chitwood and Sinha, 2016; Du et al., 2018; Maugarny-Cales and Laufs, 2018; Nikolov et al., 2019]. Leaves first emerge at the flanks of the shoot apical meristem (SAM), which harbors a group of pluripotent cells that are responsible for the generation of the above-ground organs of the plant. In the model plant Arabidopsis, cell proliferation occurs first throughout the leaf primordium and subsequently becomes restricted to the base of the growing organ, whereas cell expansion and differentiation occur after cells leave the proliferative zone and locate toward the distal part of the organ (reviewed in Gonzalez and Inze, 2015; Du et al., 2018). At a certain stage, cell proliferation stops, and leaf growth becomes the result of cell expansion.

Organ growth and shape depend on the precise expression of key regulatory genes, and work in Arabidopsis has identified many transcription factors that control leaf growth and development. Some of them repress cell proliferation such as PEAPOD (White, 2006), CIN-TCPs (Nath et al., 2003; Palatnik et al., 2003; Efroni et al., 2008; Schommer et al., 2014), and AUXIN RESPONSE FACTOR 2 (ARF2; Ellis et al., 2005; Okushima et al., 2005; Schruff et al., 2006). Others like AINTEGUMENTA (ANT; Elliott et al., 1996; Mizukami and Fischer, 2000), JAGGED (Dinneny et al., 2004; Ohno et al., 2004), and members of the GROWTH-REGULATING FACTOR (GRF) family (Kim et al., 2003; Horiguchi et al., 2005; Lee et al., 2009; Rodriguez et al., 2010; Debernardi et al., 2014) promote leaf growth.

The GRFs are plant-specific transcriptions factors defined by the presence of a WRC domain involved in DNA-binding and a QLQ domain involved in protein–protein interactions (reviewed in Kim, 2019; Liebsch and Palatnik, 2020). In Arabidopsis, seven out of the nine GRFs (GRF1–4 and GRF7–9) are post-transcriptionally repressed by microRNA (miRNA) miR396 (Liu et al., 2009; Rodriguez et al., 2010; Wang et al., 2011a). MiR396 becomes highly expressed in expanding and differentiating cells, which results in the degradation of GRF transcripts and their localized expression in proliferative tissue (Rodriguez et al., 2010; Debernardi et al., 2012; Schommer et al., 2014), a situation that is conserved in many species with different patterns of leaf growth (Das Gupta and Nath, 2015). The introduction of silent mutations in the miR396-binding site of GRF2 (Rodriguez et al., 2010) or GRF3 (Debernardi et al., 2014; Beltramino et al., 2018) leads to the ectopic expression of GRFs and larger leaves due to an increase in cell proliferation, similar to the phenotypes caused by inactivation of Arabidopsis miR396 through target mimicry (Beltramino et al., 2018) or targeted mutations in MIR396 loci (Hou et al., 2019). Interestingly, overexpression of Arabidopsis GRF5, which is not regulated by miR396, also increases leaf size (Horiguchi et al., 2005; Vercruyssen et al., 2014, 2015). Conversely, loss-of-function mutations in GRF5 (Horiguchi et al., 2005) mimic the reduced leaf size observed in miR396-regulated GRF1–4 loss-of-function mutants (Kim et al., 2003; Lee et al., 2009; Debernardi et al., 2014) or mild miR396 overexpressors (Liu et al., 2009; Rodriguez et al., 2010; Wang et al., 2011a).

Here, we show that in many eudicots all genes encoding GRFs have a predicted miR396-binding site, yet Brassicaceae species share a group of GRFs with homology to Arabidopsis GRF5 and GRF6 that lack miR396 regulation. We show that the precise pattern of expression of GRF5 requires evolutionarily conserved promoter elements that control GRF5 transcription. We demonstrate that ARF2 binds to a conserved region in the GRF5 promoter and represses its expression. Furthermore, we show that the increased cell number of arf2 leaves is caused by the ectopic expression of GRF5. Our results reveal a switch in the repression mechanisms ensuring GRF expression in plants and provide a mechanistic link for two known pathways that control leaf development.

Results

Conservation and divergence of the miR396–GRF network

We analyzed publicly available data of 36 eudicot species belonging to 18 families for the presence of genes encoding GRF transcription factors (Phytozome v12, PlantTFDB; Figure 1, A; Supplemental Table S1 and Supplemental Data set S1). These species harbor 7–22 GRFs (Figure 1, A and Supplemental Table S1). We searched for a miR396-binding site in these sequences and found that 365 out of the 413 GRFs shared an exact 20-nucleotide sequence motif that is complementary to miR396 (Figure 1, A and B and Supplemental Table S1). The interaction between this motif and miR396 results in nearly perfect interaction with a bulged nucleotide between positions 7 and 8 of the miRNA, whose identity is variable (Figure 1, B). In 14 out of the 36 species analyzed, all GRFs have this evolutionarily conserved 20-nucleotide motif perfectly conserved (Figure 1, A and B). We also detected GRFs with modifications in this sequence and, with respect to the conserved miR396-binding motif, classified them as having three changes or less (Figure 1, A, light blue boxes) or more than three changes (Figure 1, A, brown boxes). Based on the current knowledge of miRNA-target recognition in plants (Allen et al., 2005; Schwab et al., 2005; Chorostecki et al., 2012; Liu et al., 2014), and that the miR396–GRF interaction already has a bulged nucleotide, we reasoned that more than three additional mismatches will likely result in a non-functional miR396-binding site.

Figure 1.

Figure 1

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Analysis of the miR396 target site in GRF transcription factors. A, Tree representation of 36 species with publicly available genome sequences (Adapted from Phytozome v12.1); each box denotes a gene encoding a GRF transcription factor. Blue boxes represent GRFs harboring the consensus site for miR396, as indicated in (B); light blue boxes indicate a GRF harboring a miR396-binding site with three changes or less with respect to the consensus sequence, whereas brown boxes show GRFs with more than three changes. B, Scheme representing a typical GRF gene. The consensus target site for miR396 (including the amino acids encoded) and the interaction with Arabidopsis miR396a is shown below. Mfe ratio: mfe ratio between each site and a perfectly complementary site. C, Interaction of miR396 with GRFs that have deviations from the miR396-consensus site (three changes or less). The amino acids encoded by the miR396-binding sites are indicated on the left. Genes with the same changes in the miR396-binding site are presented together. Gorai004G204600 (G. raimondii); Manes05G043700, Manes12G117600 (M. esculenta); Solyc08g79800, Solyc09g009200 (S. lycopersicum); DCAR_011951, DCAR_008567, DCAR_013369 (D. carota); SapurV1A.0045s0430 (S. purpurea); Potri.003G118100, Potri.019G042300 (P. trichocarpa), Cucsa.141640 (C. sativus); MDP0000842815 (M. domestica); Kaladp0094s0052 (K. laxiflora); XP_09133081(Bra) (B. rapa); and evm.model.supercontig_25.118 (C. papaya).

In our analysis, we found 17 GRFs belonging to 11 species that had three mismatches or less with respect to the conserved miR396-binding site (Figure 1, A and C). We inspected the sequences of the mature miR396 of these species (miRBase release 22.1) and did not find any compensatory change in the miRNA (Supplemental Figure S1), indicating that these changes decrease interaction with the endogenous miR396. The position and identity of the changes varied among most of these GRFs (Figure 1, C), suggesting that these are independently acquired mutations. We calculated the minimum free energy (mfe) for each interaction, as well as the ratio between the mfe of each site and the mfe of a perfectly complementary site (Figure 1, C). Many of the sites have a mfe ratio above 0.75 (Figure 1, C), as has been shown to occur in functionally active miRNA-target interactions (Liu et al., 2014). Conversely, all GRFs with more than three mismatches with respect to the consensus binding site have mfe ratios of approximately 0.5 or less (Figure 2, A and Supplementary Figure S2, A) confirming that they are not candidates to be regulated by miR396.

Figure 2.

Figure 2

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Conservation and function of GRF5. A, Nucleotide and amino acid sequences of the remnants of a miR396-binding site in GRF5- and GRF6-like sequences in Brassicaceae. Red letters in the nucleotide sequences indicate bases that change in all the Brassicaceae GRFs lacking a miR396-binding sequence. Violet and green letters indicate positions with changes in the GRF5 and GRF6 clade, respectively. See Supplemental Table S1 for details about the species analyzed. Mfe: mfe of the interaction with Arabidopsis miR396a. Mfe ratio: mfe ratio between each site and a perfectly complementary site. B–E, Rosettes of wild-type (B), grf3 (C), grf5 (D), and grf3 grf5 (E) plants. Bar = 1 cm. F, Area and pictures of fully expanded first leaf of wild-type, grf3, grf5, and grf3 grf5 plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05; n ≥ 12). Leaves were digitally extracted for comparison. Bar = 1 cm. G–L, Effects of miR396 over-expression on wild-type (G, J), grf5 (H, K), and grf3 grf5 (I, L) in 14-d-old seedlings. Bar = 0.5 cm.

Overall, the results indicate that post-transcriptional control of the GRFs through an evolutionarily conserved miR396-binding site is a highly common scenario in eudicots; however, some GRFs display changes in the miR396-binding site that might modulate the miRNA-dependent control or lack this regulation entirely.

Characterization of Brassicaceae GRFs lacking miR396 regulation

Whereas GRFs with three or more mismatches with respect to the consensus miR396-target site were found in several species belonging to different families (Figure 1, A and Supplemental Figure S2, A), all the Brassicaceae species analyzed have at least two GRFs lacking a miR396-binding site (Figure 1, A). Phylogenetic analysis revealed that these genes can be grouped into two clades related to Arabidopsis GRF5 and GRF6, respectively (Figure 2, A). A closer inspection of these GRFs allowed the identification of a sequence resembling the consensus miR396-binding site. Changes at positions 15, 17, and 20 with respect to the consensus miR396-binding site were common to all Brassicaceae GRF5- and GRF6-like sequences (Figure 2, A, red letters), whereas additional modifications were found to be specific to GRF5 or GRF6 clades (Figure 2, A, violet and green letters, respectively).

Interestingly, the changes in the miR396-binding site of GRF6 introduced important modifications in the protein sequence, including the insertion of two amino acids. This region, located at the end of the miR396-binding site is conserved in GRFs from species that lack miR396, such as Physcomitrella patens (Supplemental Figure S2, B), suggesting that these changes might alter GRF6 activity.

Previous work has shown that GRF5 and miR396-regulated GRF1–4 control leaf growth (Kim et al., 2003; Horiguchi et al., 2005; Rodriguez et al., 2010; Lee et al., 2013; Vercruyssen et al., 2015). In good agreement, GRF5 has a similar expression profile to GRF1–4 in Arabidopsis tissues, in contrast to GRF6 that has low expression levels (Supplemental Figure S3). Therefore, we thought to explore in more detail the regulation and function of GRF5.

We crossed loss-of-function mutants in GRF5 and GRF3, two GRFs that efficiently promote organ growth after overexpression in Arabidopsis (Kim et al., 2003; Horiguchi et al., 2005; Debernardi et al., 2014; Vercruyssen et al., 2015; Beltramino et al., 2018). We found that grf3 grf5 mutants have additive effects in the reduction of leaf growth (Figure 2, B–F). However, overexpression of miR396 in grf5 or grf3 grf5 mutants caused a severe reduction of leaf growth and developmental defects (Figure 2, G–L), including frequent pin-like structures (Figure 2, L), suggesting that there are additive or synergistic effects on plant growth depending on the total level of GRF5 and miR396-regulated GRFs.

Evolutionary conservation of GRF5 upstream regulatory sequences

Whereas miR396 controls the spatiotemporal expression of certain GRFs (Rodriguez et al., 2010, 2015), the mechanisms controlling the spatiotemporal expression of GRF5, and therefore GRF5-controlled cell division, are currently unknown. To obtain insights into the regulation of GRF5, we prepared reporters to compare the promoter activity of GRF5 (pGRF5:GUS) and GRF3 (pGRF3:GUS) by fusing approximately 2-kb upstream regulatory sequences to GUS (Figure 3, A and B). GRF5 upstream promoter sequences were sufficient to drive the expression of the reporter in young developing tissues, including leaves and flowers, as well as in carpels (18 out of 20 primary transgenic plants, Figure 3, A), in good agreement with previous results (Horiguchi et al., 2005).

Figure 3.

Figure 3

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Transcriptional regulation of GRF5. A and B, GUS staining showing promoter activity of GRF5 (pGRF5:GUS) (A) and GRF3 (pGRF3:GUS) (B) in 14-d-old seedlings, inflorescences, and flowers (from left). Bar = 1 mm. C and D, VISTA plot of pair-wise comparisons of Arabidopsis GRF5 (C) and GRF3 (D) with orthologs from Brassicaceae species. The analyzed regions correspond to 2-kb sequences upstream of the ATG. See Supplemental Table S1 for details about the species analyzed. E, Alignment of a conserved promoter region present in nine Brassicaceae species. ARF2-binding sites predicted by a FIMO analysis (P < 0.01) with the ARF2-binding motifs highlighted in blue.

On the other hand, upstream regulatory sequences of GRF3 were broadly active in plant tissues, including leaves and flowers (20 out of 20 primary transgenic plants, Figure 3, B). These results are in line with previous data showing that a reporter for GRF2 containing a mutated miR396-binding site is expressed in a broader domain than a GRF2 reporter regulated by miR396 (Supplemental Figure S4, A and B; Rodriguez et al., 2010).

The specific transcriptional pattern of the GRF5 promoter prompted us to study it in more detail. We compared the promoter sequences of GRF5 orthologs from crucifers using VISTA plots and found an extensive conservation among the different species (Figure 3, C). By contrast, the promoter of GRF3 (Figure 3, D) or other GRFs regulated by miR396 (Supplemental Figure S4, C–E) showed limited conservation restricted to a region near the start codon across the same species. We reasoned that the differences in the promoter conservation pattern between GRF5 and miR396-regulated GRF1–4 might reflect differences in the mechanisms that control their expression: whereas GRF1–4 rely mostly on the post-transcriptional repression by miR396, GRF5 is controlled at the transcriptional level.

In order to identify putative transcription factors that interact with GRF5 and GRF6 promoters, we analyzed publicly available data of the Arabidopsis cistrome (O’Malley et al., 2016). We found a total of 70 binding peaks corresponding to 51 transcription factors on the GRF5 promoter (Supplemental Figure S5, A and Supplemental Table S2) and 33 binding peaks corresponding to 14 transcription factors on the GRF6 promoter (Supplemental Figure S5, B and Supplemental Table S3). To select the best candidates, we sorted these potential regulators according to their binding strength and focused on the transcription factors that bind to evolutionarily conserved regions (Supplemental Figure S5). Among the top potential candidates found in GRF5 promoter, we identified ARF2 (Supplemental Figure S5, A). Several putative ARF2-binding sites were located in a conserved region 700 bp upstream of the ATG (Figure 3, E), which prompted us to study this connection in more detail. In contrast to GRF5, the GRF6 promoter has low level of conservation across Brassicaceae species and did not have ARF2-binding sites (Supplemental Figures S4, F, S5, B).

ARF2 directly represses GRF5 transcription

arf2 loss-of-function mutants have large dark-green leaves (Figure 4, A and C; Ellis et al., 2005; Okushima et al., 2005; Schruff et al., 2006; Lim et al., 2010), similar to GRF5 overexpressors (Figure 4, B; Horiguchi et al., 2005; Vercruyssen et al., 2015). To test whether ARF2 binds to the GRF5 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays. We used plants expressing ARF2-GFP from its own endogenous regulatory sequences (Rademacher et al., 2011) and GFP as a tag for immunoprecipitation of the chromatin complexes. We found a four-fold enrichment in the ARF2-GFP ChIP samples for the evolutionarily conserved region of the GRF5 promoter containing the putative ARF2-binding sites (Figure 4, D), supporting a direct binding of ARF2 to this region in vivo.

Figure 4.

Figure 4

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ARF2 regulates GRF5 expression. A–C, Rosettes of wild-type (A), p35S:GRF5 (B), and arf2 (C) 25-d-old plants. Rosettes were digitally extracted for comparison. Bar = 1 cm. D, Binding of ARF2 at the GRF5 promoter. Data are means ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05). At the right, GRF5 gene schematic representation. Pink box indicates localization of putative ARF2-binding sites in the GRF5 promoter, as detailed in Figure 3, E. The black arrowheads show the position of the primers used in the ChIP-qPCR experiments, with A and B indicating amplicons as shown to the left. E and F, GUS staining of typical 14-d-old transgenic plants harboring reporters for wild-type GRF5 promoter (E) and a mutant version with a deletion in the putative ARF2-binding sites (F). Figures of the seedlings in their entirely shows composite images assembled from multiple photos. Bar = 1 mm. G, Relative expression levels of GRF5 in arf2 and wild-type third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05). H, Relative expression levels of ARF2 in p35S:GRF5 and wild-type third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05).

Next, we generated a mutant version of the GRF5 promoter (pGRF5mut) by deleting the conserved region with the ARF2-binding sites (Figures 3, E, 4, F). The GUS expression driven by pGRF5mut, lacking the ARF2-binding motif, expanded throughout the leaf and roots in 20 out of 25 independent transgenic plants (Figure 4, F), in contrast to the localized expression of the GRF5 wild-type promoter (Figure 4, E).

We analyzed the transcript levels of GRF5 in the arf2 mutant and found a more than three-fold increase with respect to wild-type plants (Figure 4, G). Conversely, the transcript levels of ARF2 increased only marginally in p35S:GRF5 plants (Figure 4, H). Overall, these results demonstrate that ARF2 binds directly to the GRF5 promoter to repress its expression. Most interestingly, the mutation of this evolutionarily conserved region in the GRF5 promoter mimics mutations in miR396-binding site of the miRNA-regulated GRFs, as they both cause the ectopic expression of the transcription factors in a broad range of tissues and organs (Figures 3, A and B, 4, E and F and Supplemental Figure S4, A and B; Rodriguez et al., 2010, 2015).

A promoter from a miR396-targeted GRF5-like gene from cacao drives expression in proliferative tissue

In many species, such as Theobroma cacao, all GRFs have a miRNA-binding site (Figure 1, A). Thus, we thought it would be interesting to test whether a GRF5-like transcription factor that harbors a miR396-binding site is under similar transcriptional regulation as Arabidopsis GRF5. As a proof of principle, we chose Thecc1EG029130, which harbors a perfectly conserved miR396-binding site and clustered together with Arabidopsis GRF5 in our phylogenetic analysis (Figure 5, A and B). We prepared a reporter by fusing the Thecc1EG029130 promoter to GUS and transformed Arabidopsis plants. Interestingly, the activity of pThecc1EG029130:GUS was detected in young developing leaves with a proximo-distal expression gradient, as well as in carpels (18 out of 20 plants, Figure 5, D). Overall, the expression pattern of pThecc1EG029130:GUS was remarkably similar to pGRF5:GUS with the exception of leaf trichomes, where we only detected activity of pThecc1EG029130:GUS (Figure 5, C and D). We searched for motives in the promoter sequences of Brassicaceae GRF5 and Thecc1EG029130 using Multiple Em for Motif Elicitation (MEME; Bailey et al., 2015). The top motif was the ARF2-binding site identified in Figure 3, E, which was also present in Thecc1EG029130 (Supplemental Figure S6).

Figure 5.

Figure 5

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Transcriptional regulation of Thecc1EG029130, a GRF5-like transcription factor that harbors a miR396-binding site, in Arabidopsis plants. A, Phylogenetic tree of the 9 Arabidopsis GRFs and the 10 GRFs from T. cacao. Arabidopsis GRF5 and GRF6 (highlighted in violet and green, respectively) are the only GRFs lacking the consensus miR396-binding site. B, Scheme of the GRF5-like gene Thecc1EG029130 (highlighted in gray in the phylogenetic tree (A)) with detailed localization and sequence of miR396 target site. C and D, GUS staining showing promoter activity of Arabidopsis GRF5 (C) and Thecc1EG029130 (D) in typical 14-d-old seedlings and inflorescences. Bar = 1 mm.

ARF2 regulates cell number in leaves through the control of GRF5

To test the biological significance of the regulation of GRF5 by ARF2, we evaluated their genetic interaction. arf2 mutants have larger leaves (Figure 6, A–E) due to a combination of an increased cell number (Figure 6, G) and cell size (Figure 6, F; Ellis et al., 2005; Okushima et al., 2005; Schruff et al., 2006). In turn, grf5 mutants have lanceolate leaves (Figure 6, B and E) with a reduction in cell number (Figure 6, G; Horiguchi et al., 2005). The arf2 grf5 double mutants showed lanceolate leaves resembling grf5 (Figure 6, D and E). Cell number was also similar in arf2 grf5 and grf5, albeit the double mutant having slightly more cells (Figure 6, G). On the other hand, the average cell size of arf2 grf5 leaves was larger than wild-type and similar to arf2 (Figure 6, F). These results indicate that increased cell number in arf2, but not cell size, is caused by increased levels of GRF5.

Figure 6.

Figure 6

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Genetic interaction between arf2 and grf5. A–D, Rosettes of 25-d-old wild-type (A), grf5 (B), arf2 (C), and arf2 grf5 (D) plants. Rosettes were digitally extracted for comparison. Bar = 1 cm. E, Area and pictures of fully expanded first leaf of wild-type, arf2, grf5, and arf2 grf5 plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05; n = 15). Leaves were digitally extracted for comparison. Bar = 1 cm. F and G, Cell size (F) and estimation of palisade cell number per leaf (G) in wild-type, arf2, grf5, and arf2 grf5 plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05).

Regulatory feedback loops fine-tune gene expression levels in an ARF2–miR396–GRF network

As GRF5 acts in concert with other GRFs regulated by miR396, we thought to study in more detail the interplay between ARF2 and the miR396/GRF regulatory network. To do this, we first determined the effect of arf2 on miR396 and miR396-regulated GRFs. We analyzed a pMIR396b:GUS transcriptional reporter (Figure 7, A and B) and found that it had decreased expression during leaf development of arf2 (Figure 7, B). We measured miR396 levels by qPCR in arf2 and confirmed that miR396 levels were reduced in this mutant (Figure 7, C). Then, we determined the transcript levels of GRF1–4 and GRF6 in arf2 and found that all of them showed a mild increase except for GRF6 (Figure 7, D). On the other hand, we found a small decrease in GRF3 and GRF4 transcript levels in p35S:GRF5 plants (Figure 7, N).

Figure 7.

Figure 7

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Feedback loops in the ARF2–miR396–GRF regulatory network. A and B, Typical GUS staining of third leaves in 14-d-old transgenic plants harboring reporters for pMIR396b:GUS in wild-type (A) and arf2 mutant backgrounds (B). Bar = 0.5 mm. C, Relative expression levels of miR396 in arf2 third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05). D, Relative expression levels of GRF1, 2, 3, 4, and 6 in arf2 third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05). E–H, Rosette of 25-d-old wild-type (E), arf2 (F), rGRF3 (G), and arf2 rGRF3 (H) plants. Rosettes were digitally extracted for comparison. Bar = 1 cm. I, Area and pictures of fully expanded first leaf of wild-type, arf2, rGRF3, and arf2 rGRF3 plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05; n ≥ 15). Leaves were digitally extracted for comparison. Bar = 1 cm. J–K, Cell size (J) and estimation of palisade cell number per leaf (K) in the first leaves of wild-type, arf2, rGRF3, and arf2 rGRF3 plants. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). L, Relative expression levels of GRF5 in wild-type, rGRF3, arf2, and arf2 rGRF3 plants. The data shown are mean ± SEM of three biological replicates. Different letters indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05). M, Relative expression levels of miR396 in rGRF3 third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05). N, Relative expression levels of GRF1-6 in p35S:GRF5 third leaves of 12-d-old plants. The data shown are mean ± SEM of three biological replicates. Asterisks indicate significant differences from the wild type as determined by Student’s t test (P < 0.05).

A GRF3 gene decoupled from the post-transcriptional repression of miR396 through mutations in the miRNA-binding site (rGRF3) has high GRF activity and promotes cell proliferation and leaf size (Figure 7, G, I, J, K, and L; Debernardi et al., 2014; Beltramino et al., 2018). We crossed rGRF3 expressing plants and arf2 mutants, and found that arf2 rGRF3 plants had an additional increase of leaf size compared with the parental lines (Figure 7, E–I), an effect that was caused by an increase in cell number (Figure 7, J and K). The additive effects of rGRF3 and arf2 on leaf size indicate that the decrease of miR396 in arf2 single mutants was not sufficient to fully release the expression of miRNA-regulated GRFs. In addition, we found that GRF5 transcript levels were increased in rGRF3 plants, and that rGRF3 and arf2 had additive effect on GRF5 expression (Figure 7, L). We also measured miR396 levels in rGRF3 plants and found that they were decreased by approximately 40% (Figure 7, M), which is in agreement with previous results showing that miR396 levels decrease in p35S:GRF3 or p35S:GRF1 roots (Hewezi and Baum, 2012). Overall, these results show that ARF2, GRF5, and miR396/GRFs are interconnected through regulatory loops, and that additive effects on the promotion of leaf growth are obtained by combining arf2 and rGRF3.

The GRFs work together with GRF-INTERACTING FACTORs (GIFs; Kim and Kende, 2004; Horiguchi et al., 2005). GIF1, also known as ANGUSTIFOLIA3 (AN3), is a GRF co-regulator that has high affinity for GRF5 and GRF3 (Debernardi et al., 2014; Vercruyssen et al., 2014). Mutation of GIF1/AN3 causes a reduction in leaf size as a result of decreased cell proliferation (Kim and Kende, 2004; Horiguchi et al., 2005). To evaluate a general down-regulation of GRF activity in arf2 mutants, we generated arf2 gif1 double mutants. The arf2 gif1 mutants displayed small leaves (Supplemental Figure S7) due to a strong reduction in cell number (Supplemental Figure S7), indicating that the increase in leaf size observed in arf2 requires functional GRF–GIF complexes.

Discussion

Transcription factors are key components of gene regulatory networks that govern the patterns of cell proliferation and differentiation, which are ultimately responsible for the elaboration of the complex body of multicellular organisms. Re-wiring gene networks or their co-option into novel contexts can be associated with changes in growth and shape observed during evolution. Recent work has highlighted the importance of the GRF transcription factors in the control of plant growth in different species, including crops (reviewed in Kim, 2019; Liebsch and Palatnik, 2020). Here, we describe the diversification of repression mechanisms that control GRF expression. Whereas the spatiotemporal expression of some GRFs is post-transcriptionally regulated by miR396, we show that, in the case of GRF5, this spatiotemporal control relies on transcriptional regulation, at least partially, through repression by ARF2. GRF5 and certain miR396-regulated GRFs, such as GRF1–4, have similar functions in the control of leaf development, so that the simultaneous modification of miR396 and ARF2 repression pathways resulted in an enhanced promotion of organ growth (Figure 8).

Figure 8.

Figure 8

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A proposed model for the regulation of Arabidopsis growth by the ARF2–miR396–GRF regulatory network. GRFs are repressed by diverse mechanisms: while GRF1–4 are post-transcriptionally repressed by miR396, GRF5 is repressed transcriptionally by ARF2. These transcription factors interact with the co-activator GIF1 and promote cell proliferation in leaves. In addition, ARF2 levels have a mild effect on miR396 and GRF1–4.

Dodging the post-transcriptional repression by miR396

The spatiotemporal control of GRFs by miR396 observed in Arabidopsis (Rodriguez et al., 2010; Debernardi et al., 2012) is conserved between species with divergent patterns of growth (Das Gupta and Nath, 2015), with miR396 accumulating in expanding and differentiating cells. The analysis of the miR396–GRF network revealed that the miR396-binding site is highly conserved and that many eudicotyledonous plants likely rely on a general regulation of the GRF family by miR396. At least in Arabidopsis, miR396 is directly activated by TCP4 (Schommer et al., 2014), a transcription factor known to repress proliferation and induce differentiation (Palatnik et al., 2003; Efroni et al., 2008; Sarvepalli and Nath, 2011).

Apart from this extensive post-transcriptional regulation of the GRFs, we found that some of these transcription factors lack an obvious miR396-binding site. These GRFs are scattered among species belonging to different families, except for the analyzed crucifers, which all share at least two GRFs lacking a miR396-recognition site. These genes can be grouped into two clades corresponding to GRF5 and GRF6 of Arabidopsis thaliana. Studies in Arabidopsis have shown that mutations in the miR396-binding site of GRF2 and GRF3 cause the ectopic expression of the transcription factors in a broader domain (Rodriguez et al., 2010, 2015; Debernardi et al., 2012), similar to the effect caused by the mutation of the ARF2-binding site in the GRF5 promoter. Therefore, these two motives fulfill similar functions, albeit at different levels, restricting the expression of the GRFs to proliferative tissue. It will be interesting to study the phenotypic effects caused by GRF5 uncoupled from ARF2 regulation.

The occurrence and artificial selection of natural alleles of GRFs with changes in the miR396-binding site have been reported in domesticated plant varieties. A rare naturally occurring allele of OsGRF4 from rice (Oryza sativa ssp.) harbors two mutations in the miR396-binding site, resulting in an increased level of OsGRF4, larger grain size, and enhanced grain yield, similar to the effects caused by a decrease in miR396 activity (Che et al., 2015; Duan et al., 2015; Gao et al., 2015; Hu et al., 2015; Li et al., 2016, 2018). In addition, loose cluster bunches of Vitis vinifera “Pinot noir” clones are associated with point mutations in the miR396-binding site of VvGRF4, which increases GRF activity and thus promotes pedicel elongation (Rossmann et al., 2020). Therefore, it seems that a moderate release of the repression by miR396 can be associated with the promotion of growth of certain organs or tissues in different plant species. We found several GRFs from different species that have acquired only one or two mutations in the miR396-binding site. Thus, it might be plausible to consider that these changes could also be linked to the promotion of certain organs in these species.

Interestingly, monocots have an additional miR396 variant that has an insertion of one base in the small RNA sequence, so that there is no bulge in the interaction between the miRNA and the GRFs (Debernardi et al., 2012). This monocot-specific miR396 is more efficient in its inactivation of GRFs (Debernardi et al., 2012), as expected from the better interaction. Therefore, albeit targeting of GRFs by miR396 seems to be broadly distributed among angiosperms, the strength of the regulation appears also to be, at least to certain extent, dynamic.

The ARF2–miR396–GRF regulatory network

The results presented here provide a mechanistic link between two well-known growth-regulating genes. ARF2 has pleiotropic roles in plant development and is one of the few genes known that regulate organ growth by controlling both cell number and cell size (this work; Ellis et al., 2005; Okushima et al., 2005; Schruff et al., 2006). ARF2 has been shown to act through HB33 and ANT in the response to abscisic acid (Wang et al., 2011b; Meng et al., 2015). Here, we demonstrate that ARF2 is a direct transcriptional repressor of GRF5, and that ARF2 controls cell number through GRF5.

At the protein level, the GRFs can interact with DELLA proteins, especially during cold stress (Li et al., 2018; Lantzouni et al., 2020). They form specific complexes with small transcriptional co-activators of the GIF family (Kim and Kende, 2004; Horiguchi et al., 2005), which in turn interact with SWI2/SNF2 chromatin remodeling complexes (Debernardi et al., 2014; Vercruyssen et al., 2014). We found that GIF1/AN3 is required for the large leaves observed in arf2, indicating that functional GRF–GIF complexes participate in the process. Interestingly, it has been demonstrated that the induction of GIF1/AN3 activates the expression of GRF3 and GRF5 (Vercruyssen et al., 2014) and we have shown that the induction of GRF3 activates at least GRF5, suggesting a feedforward regulation of the GRF–GIF module during leaf development. These regulatory loops also include miR396, as the miRNA is reduced in plants with high GRF levels (this work; Hewezi and Baum, 2012) or in arf2 mutants. A ChIP analysis of Arabidopsis GRF-overexpressing lines revealed their binding to the ARF2 promoter (Piya et al., 2020); however, here we only observed a minor effect of the GRFs on ARF2 transcript levels. Nevertheless, the extensive evolutionary conservation of the GRF5 promoter suggests that other transcriptional regulators might participate in this network. One such regulator might be the PLATZ transcription factor ORESARA15, whose ectopic expression activates GRF transcription (Kim et al., 2018). The spatiotemporal control of the promoter of a GRF5-like gene from T. cacao, which harbors a miR396-binding site, suggests that members of the GRF family might be under tight transcriptional and post-transcriptional control in certain species. The miR396–GRF/GIF network has been shown to participate in the control of plant growth and yield in crucifers as well as in monocots, such as rice (reviewed in Liebsch and Palatnik, 2020). Therefore, deciphering the regulation of the miR396–GRF network might have potential technological applications. The simultaneous targeted modification of miR396 and ARF2 shown here results in additive effects in the promotion of plant growth in Arabidopsis and, therefore, might be worth testing in crops.

Materials and methods

Plant material, growth conditions, and leaf analysis

Arabidopsis ecotype Col-0 was used for all the experiments. See Supplemental Table S5 for a description of the plasmids used to generate the transgenic lines characterized in this study. Plants were grown either on soil or 1× Murashige and Skoog (MS) medium and 0.8% agar, in long photoperiods (16-h light/8-h dark) at 23°C. Leaf areas were measured using FIJI (Schindelin et al., 2012) after dissection of the first leaf of 25-d-old plants. The mutant alleles arf2-8 (Salk_108995; Okushima et al., 2005), grf5-1 (Salk_086597; Horiguchi et al., 2005), grf3-1 (Salk_026786), and gif1-1 (Salk_150407; Kim and Kende, 2004) were obtained from the Arabidopsis stock center. pARF2:ARF2-GFP plants were kindly provided by Prof. Dr. Dolf Weijers (Rademacher et al., 2011).

Microscopy

To obtain paradermal views of palisade cells, leaves were fixed with 70% v/v ethanol and cleared with lactic acid. Palisade cells were observed using differential interference contrast microscopy in an Olympus BH2 microscope. Cell areas were measured using FIJI. The density of palisade cells per unit area was determined, and the area of the leaf blade was divided by this value to calculate the total number of palisade cells in the sub-epidermal layer. To determine the cell area, 20 palisade cells were measured in the center of each leaf. Measurements were carried out in 5–10 leaves.

Expression analysis

Total RNA was isolated using Tripure isolation reagent (Roche) following the manufacturer’s protocol. Samples were obtained for three biological replicates. Then, 0.5–1.0 μg of total RNA was treated with RQ1 RNase-free DNase (Promega) to remove genomic DNA. First-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase (Promega) with the appropriate primers. PCR was performed in a Mastercycler ep realplex thermal cycler (Eppendorf) or in an AriaMx real-time PCR (qPCR) System (Agilent Technologies) using SYBR Green I (Roche) to monitor double-stranded DNA synthesis. The relative transcript level was determined for each sample and normalized to PROTEIN PHOSPHATASE2A (PP2A). Mature miR396 levels were determined by stem loop RT-qPCR as described previously (Debernardi et al., 2012). Primer sequences are given in Supplemental Table S4.

To visualize GUS reporter activity, the plant tissue of transgenic plants was fixed in 90% v/v acetone on ice for 20 min. Then, samples were transferred to the 5-bromo-4-chloro-3-indolyl-b-d-glucuronide (X-gluc) buffer solution (750 mg/ml X-Gluc, 100 mM NaPO4 [pH 7], 3 mM K3Fe3(CN)6, 10 mM EDTA, 0.1% v/v Triton) and infiltrated under vacuum (500 mm-Hg) on ice for 20 min. Finally, it was incubated at 37°C. The stained tissue was cleared and stored in 70% v/v ethanol (Donnelly et al., 1999).

ChIP-qPCR

A quantity of 0.5 g of 12-d-old apex tissue of ARF2-GFP and wild-type were crosslinked and chromatin was extracted, as described before (Kaufmann et al., 2010). Immunoprecipitation was done using anti-GFP antibody (Chip Grade, Abcam Ab290). Antibody–protein/DNA complexes were isolated using protein-A agarose beads (Invitrogene). Then, the DNA was isolated by reverse crosslinking with proteinase K (Invitrogene) and purified. An aliquot of sonicated chromatin was processed in parallel as the total input DNA control sample. Fold enrichment was calculated by dividing the qPCR signal derived from ChIP samples against the one of each input DNA samples. Relative fold enrichment was calculated by normalizing the individual fold enrichment of amplicons to that of wild-type sample within each trial. Primers used are listed in Supplemental Table S4.

Sequence analysis

The complete CDS and protein sequences of GRFs were obtained from the PlantTFDB (http://planttfdb.cbi.pku.edu.cn; Jin et al., 2017) and one isoform was selected for each gene (Supplemental Data set S1). miRNA-binding site identification was carried out with RNAhybrid (Rehmsmeier et al., 2004; Supplemental Data set S1). The promoter sequences (2 kb upstream from ATG) were obtained from Phytozome web resource (https://phytozome.jgi.doe.gov; Goodstein et al., 2012). VISTA alignments of the isolated promoter sequences were done using the online available VISTA tools (http://genome.lbl.gov/vista; Frazer et al., 2004). The GRFs analyzed in the VISTA alignments were selected by using reciprocal BLAST with the corresponding AtGRF. Multiple sequence alignments were performed using T-Coffee (http://tcoffee.crg.cat; Notredame et al., 2000). Evolutionary analysis was conducted in MEGA5X (Kumar et al., 2018). The phylogenetic trees were constructed by the maximum-likelihood method with full length amino-acid sequences. Bootstrap values were estimated with 1,000 replicates, those greater than 50 were indicated on nodes. To find the binding motives of ARF2 transcription factor, we used ARF2 logo (O’Malley et al., 2016) and the software FIMO (Grant et al., 2011; P-value < 0.01).

In the analysis of Arabidopsis Cistrome (O’Malley et al., 2016), we selected all the transcription factors with a significant peak in GRF5 promoter (2 kb upstream of ATG). We calculated the mean value of significant peaks in all the genome for each transcription factor. Next, we obtained a relative signal value for each peak identified, as the ratio between the signal value of the peak and the mean value for the corresponding transcription factor. Motif discovery in Brassicaceae and cacao GRFs was performed using MEME (http://meme-suite.org/; Bailey et al., 2015).

Accession numbers

Sequence data from this article can be found in arabidopsis.org under accession numbers indicated in Supplemental Table S4 and in Supplemental Data set S1.

Supplemental data

Supplemental Figure S1. Conservation and divergence of miR396.

Supplemental Figure S2. GRFs with more than three mismatches in the miR396-binding site outside Brassicaceae and WRC domain conservation.

Supplemental Figure S3. GRF expression profiles in Arabidopsis.

Supplemental Figure S4. Expression of GRF2 and conservation of upstream promoter region in several GRFs.

Supplemental Figure S5. Analysis of putative transcription factors binding to the GRF5 and GRF6 promoters.

Supplemental Figure S6. Identification of a conserved motif in Thecc1EG029130 and Brassicaceae GRF5 promoters.

Supplemental Figure S7. Genetic interaction between ARF2 and GIF1.

Supplemental Table S1. Summary of the genes analyzed

Supplemental Table S2. Transcription factors binding to the promoter of GRF5

Supplemental Table S3. Transcription factors binding to the promoter of GRF6

Supplemental Table S4. Relevant gene identifiers and oligonucleotide primers used in qPCR

Supplemental Table S5. Binary plasmids used in this study

Supplemental Data set S1. Sequences of GRF transcription factors.

Supplementary Material

kiab014_Supplementary_Data
Click here for additional data file. (2.5MB, docx)

Acknowledgments

The authors would like to thank Dolf Weijers for sharing pARF2:ARF2-GFP seeds. They also thank Ramiro Rodriguez and Daniela Liebsch for comments on the manuscript, and Diego Aguirre for invaluable technical assistance.

Funding

M.B., A.F., and J.M.D. were supported with fellowships from CONICET and ANPCyT. J.F.P. is a member of the same institution. These studies were supported by grants to J.F.P. from ANPCyT PICT-2016-0761 and ICGEB CRP/ARG17-01.

Conflict of interest statement. None declared.

 

M.B., J.F.P., and J.M.D. planed and designed the research. M.B., A.F., and J.M.D. performed the experiments. M.B. and J.F.P performed the data analysis and interpretation, and wrote the manuscript. All authors reviewed the final version of the manuscript.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Javier F. Palatnik (palatnik@ibr-conicet.gov.ar).

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