Histone Acetyltransferase GCN5 Affects Auxin Transport during Root Growth by Modulating Histone Acetylation and Gene Expression of PINs

General Control Non-Derepressible 5 (GCN5) is a histone acetyltransferase that targets multiple genes and is essential for the acetylation of Lysine residues in the N-terminal tail of histone H3 in Arabidopsis. GCN5 interacts with the transcriptional coactivator Alteration/Deficiency in Activation 2b (ADA2b), which enhances its activity functioning in multiprotein complexes, such as the Spt-Ada-Gcn5-Acetyltransferase complex (SAGA). Mutations in GCN5 and ADA2b result in pleiotropic phenotypes, including alterations in the growth of roots. Auxin is known to regulate root development by modulating gene expression patterns. Auxin moves polarly during plant growth via the Pin-formed (PIN) auxin efflux transport proteins. The effect of GCN5 and ADA2b on auxin distribution at different stages of early root growth (4 to 7 days post-germination) was studied using the reporter lines DR5rev::GFP and PIN1::PIN1-GFP. In wild-type plants, auxin efflux transporter PIN1 expression increases from the fourth to the seventh day of root growth. The PIN1 expression was reduced in the roots of gcn5-1 and ada2b-1 compared to the wild type. The expression of PIN1 in ada2b-1 mutants is confined only to the meristematic zone, specifically in the stele cells, whereas it is almost abolished in the elongation zone. Gene expression analysis showed that genes associated with auxin transport, PIN1, PIN3 and PIN4, are downregulated in gcn5-1 and ada2b-1 mutants relative to the wild type. As a result, auxin accumulation was also reduced in gcn5-1 and ada2b-1 compared to wild-type roots. Furthermore, acetylation of Lysine 14 of histone H3 (H3K14) was also affected in the promoter and coding region of PIN1, PIN3 and PIN4 genes during root growth of Arabidopsis in gcn5 mutants. In conclusion, GCN5 acts as a positive regulator of auxin distribution in early root growth by modulating histone H3 acetylation and the expression of auxin efflux transport genes.


Introduction
The plant hormone auxin affects multiple developmental processes, such as embryogenesis, organogenesis, cell elongation, division, and differentiation and responses to environmental stimuli [1,2]. Auxin is unique among plant hormones because it has a directional, cell-to-cell transport implemented by membrane-bound protein efflux transporters [2,3]. This directional auxin transport is regulated by the asymmetric localization of the auxin efflux transporter of the PIN family [4][5][6]. In Arabidopsis thaliana, the PIN gene family has eight members [7], divided into two subfamilies. The first includes PIN1-4, 6,

The Histone Acetyltransferase GCN5 Affects Auxin Transport during Early Root Growth
In previous work we have shown that histone acetyltransferase GCN5 and the associated transcriptional adaptor ADA2b, are necessary for auxin-induced cytokinin signaling during early root growth [23]. Since auxin is directionally transported to the sites needed by the action of the PIN family of auxin efflux carriers [1,4] and GCN5 has been shown to promote the expression of PIN1 auxin transporter during gynoecium development [31], we hypothesized that GCN5 could also affect PIN1 expression during early root growth. To test this hypothesis, we crossed the gcn5-1 mutant with the translational fusion reporter line PIN1:PIN1-GFP [31,32] and monitored PIN1-GFP expression from the fourth to the seventh day after germination. The PIN1 pattern of expression has been previously de-scribed [33][34][35]. PIN1 is basally localized in the stele cells in the root meristematic zone ( Figure 1A,D). PIN1 is detected at the pericycle and the endodermis, as well as in the quiescent center (QC) and the four cortex and epidermis initial cells ( Figure 1A, D). In wild-type plants, the GFP signal, indicating the amount of PIN1 protein, increases from the fourth to the seventh day ( Figure 1A). On the seventh day, the signal was detected from the QC towards the central cylinder of the root ( Figure 1A). The signal had a stable intensity along the meristematic zone up to the elongation zone ( Figure 1A). In the gcn5-1 mutants, the GFP signal is significantly reduced ( Figure 1B,D). Interestingly, the GFP signal was not increased in the gcn5-1 mutant from the fourth day to the seventh day of root growth ( Figure 1B) but in fact it was dwindling with time ( Figure 1C). A quantification analysis of the fluorescence signal confirms that the GFP was reduced significantly in gcn5-1 roots ( Figure 1C,D). These results suggest that GCN5 is a positive regulator of auxin transport during root growth, having a synergistic positive effect on the expression of PIN1 transporter during root growth.
needed by the action of the PIN family of auxin efflux carriers [1,4] and GCN5 has been shown to promote the expression of PIN1 auxin transporter during gynoecium development [31], we hypothesized that GCN5 could also affect PIN1 expression during early root growth. To test this hypothesis, we crossed the gcn5-1 mutant with the translational fusion reporter line PIN1:PIN1-GFP [31,32] and monitored PIN1-GFP expression from the fourth to the seventh day after germination. The PIN1 pattern of expression has been previously described [33][34][35]. PIN1 is basally localized in the stele cells in the root meristematic zone ( Figure 1A,D). PIN1 is detected at the pericycle and the endodermis, as well as in the quiescent center (QC) and the four cortex and epidermis initial cells ( Figure 1A, D). In wild-type plants, the GFP signal, indicating the amount of PIN1 protein, increases from the fourth to the seventh day ( Figure 1A). On the seventh day, the signal was detected from the QC towards the central cylinder of the root ( Figure 1A). The signal had a stable intensity along the meristematic zone up to the elongation zone ( Figure 1A). In the gcn5-1 mutants, the GFP signal is significantly reduced ( Figure 1B,D). Interestingly, the GFP signal was not increased in the gcn5-1 mutant from the fourth day to the seventh day of root growth ( Figure 1B) but in fact it was dwindling with time ( Figure 1C). A quantification analysis of the fluorescence signal confirms that the GFP was reduced significantly in gcn5-1 roots ( Figure 1C,D). These results suggest that GCN5 is a positive regulator of auxin transport during root growth, having a synergistic positive effect on the expression of PIN1 transporter during root growth. (C) Graphical representation of fluorescence density between WT and gcn5-1. The bars represent the range of the two quadrants. The horizontal line in the bar is the median, while the terminals are the minimum and maximum values of the data. Asterisks above bars of gcn5-1 indicate statistical significance compared to the same d.a.g. of WT. Asterisks between brackets show the difference between fourth and the seventh d.a.g., using an independent samples t-test: * p < 0.05, ** p < 0.01, and *** p < 0.001. (D) Confocal microscopy for PIN1::PIN1-GFP expression in 7-day-old roots of WT and gcn5-1 after PI staining. Scale bars are 20 µm.

ADA2b Affects Auxin Transport during Early Root Growth
To test if the GCN5-associated protein ADA2b also affects PIN1 expression, we crossed ada2b-1 mutants with the PIN1:PIN1-GFP line. Similarly, to GCN5, ADA2b also promotes PIN1 expression during root growth. The GFP signal is significantly reduced in ada2b-1 mutants compared to the wild type on all days examined (Figure 2A,B,D). In ada2b-1 mutants, not only the levels of expression of PIN1 are reduced but also the expression pattern is limited ( Figure 2D). The GFP signal is detected only in the central stele cells and is absent from the pericycle and endodermis cells ( Figure 2D). The expression of PIN1 in ada2b-1 mutants is also confined only to the meristematic zone and is almost abolished in the elongation zone (Figure 2A,B,D). Quantifying the signal showed that it was significantly reduced in the ada2b-1 compared to the wild type in all four days. Contrary to gcn5-1 mutants, PIN1 expression in ada2b-1 mutants was increased from the fourth to seventh days of root growth ( Figure 2C). These results show that the transcriptional adaptor ADA2b also functions as a positive regulator of auxin flow by enhancing the expression of PIN1 during root growth.
significance compared to the same d.a.g. of WT. Asterisks between brackets show the difference between fourth and the seventh d.a.g., using an independent samples t-test: * p < 0.05, ** p < 0.01, and *** p < 0.001. (D) Confocal microscopy for PIN1::PIN1-GFP expression in 7-day-old roots of WT and gcn5-1 after PI staining. Scale bars are 20 μm.

ADA2b Affects Auxin Transport during Early Root Growth
To test if the GCN5-associated protein ADA2b also affects PIN1 expression, we crossed ada2b-1 mutants with the PIN1:PIN1-GFP line. Similarly, to GCN5, ADA2b also promotes PIN1 expression during root growth. The GFP signal is significantly reduced in ada2b-1 mutants compared to the wild type on all days examined (Figure 2A,B,D). In ada2b-1 mutants, not only the levels of expression of PIN1 are reduced but also the expression pattern is limited ( Figure 2D). The GFP signal is detected only in the central stele cells and is absent from the pericycle and endodermis cells ( Figure 2D). The expression of PIN1 in ada2b-1 mutants is also confined only to the meristematic zone and is almost abolished in the elongation zone (Figure 2A,B,D). Quantifying the signal showed that it was significantly reduced in the ada2b-1 compared to the wild type in all four days. Contrary to gcn5-1 mutants, PIN1 expression in ada2b-1 mutants was increased from the fourth to seventh days of root growth ( Figure 2C). These results show that the transcriptional adaptor ADA2b also functions as a positive regulator of auxin flow by enhancing the expression of PIN1 during root growth.

The Histone Acetyltransferase GCN5 and the Transcriptional Adaptor ADA2b Promote the Expression of PINs in the Root of Arabidopsis
The role of GCN5 and ADA2b on PIN family gene expression was monitored in the roots of seven-day-old plants. PIN1, PIN3 and PIN4 gene expression are downregulated by almost 50% in the roots of both gcn5-1 and ada2b-1 mutants compared to wild-type plants ( Figure 3A,C,D). In contrast, PIN2 and PIN7 expression showed no significant change in their levels ( Figure 3B,E), suggesting that GCN5/ADA2b's effect on the expression of PINs is gene-specific.
represent the range of the two quadrants. The horizontal line in the bar is the median, while the terminals are the minimum and maximum values of the data. Asterisks above bars of ada2b-1 indicate statistical significance compared to the same d.a.g. of WT. Asterisks between brackets show the difference between the fourth and seventh d.a.g., using an independent samples t-test: ** p < 0.01, and *** p < 0.001. (D) Confocal microscopy for PIN1::PIN1-GFP expression in 7-day-old roots of WT and ada2b-1 after PI staining. Scale bars are 20 μm.

The Histone Acetyltransferase GCN5 and the Transcriptional Adaptor ADA2b Promote the Expression of PINs in the Root of Arabidopsis
The role of GCN5 and ADA2b on PIN family gene expression was monitored in the roots of seven-day-old plants. PIN1, PIN3 and PIN4 gene expression are downregulated by almost 50% in the roots of both gcn5-1 and ada2b-1 mutants compared to wild-type plants ( Figure 3A,C,D). In contrast, PIN2 and PIN7 expression showed no significant change in their levels ( Figure 3B,E), suggesting that GCN5/ADA2b's effect on the expression of PINs is gene-specific.

GCN5 and ADA2b Affect Auxin Distribution at the Root Tip during Early Root Growth
To test if the changes in the expression of PINs observed in gcn5-1 and ada2b-1 mutants result in altered auxin distribution in the root tip, we monitored the expression of the auxin-responsive DR5rev::GFP reporter line in the gcn5 and ada2b background from the fourth to the seventh day after germination. On the fourth and fifth days after germination, the DRrev::GFP signal in gcn5-1 and ada2b-1 was mostly similar to the wild-type plants ( Figure 4A-C). On the sixth day after germination, gcn5 and ada2b display altered distribution of the GFP signal; the signal becomes more intense in the QC and less intense

GCN5 and ADA2b Affect Auxin Distribution at the Root Tip during Early Root Growth
To test if the changes in the expression of PINs observed in gcn5-1 and ada2b-1 mutants result in altered auxin distribution in the root tip, we monitored the expression of the auxin-responsive DR5rev::GFP reporter line in the gcn5 and ada2b background from the fourth to the seventh day after germination. On the fourth and fifth days after germination, the DRrev::GFP signal in gcn5-1 and ada2b-1 was mostly similar to the wild-type plants ( Figure 4A-C). On the sixth day after germination, gcn5 and ada2b display altered distribution of the GFP signal; the signal becomes more intense in the QC and less intense in the columella cells ( Figure 4A,B). On the seventh day, the changes in the DR5 signal become even more apparent, with a significant enhancement of the signal in the QC and a great reduction in the columella cells ( Figure 4A-C). The differences in DR5 expression are more pronounced in the ada2b-1 mutant than in the gcn5-1 ( Figure 4A-C). In conclusion, these results suggest that changes in the expression of auxin transport genes observed in gcn5 and ada2b mutants lead to changes in the distribution of auxin in the root tip.
in the columella cells ( Figure 4A，B). On the seventh day, the changes in the DR5 signal become even more apparent, with a significant enhancement of the signal in the QC and a great reduction in the columella cells ( Figure 4A-C). The differences in DR5 expression are more pronounced in the ada2b-1 mutant than in the gcn5-1 ( Figure 4A-C). In conclusion, these results suggest that changes in the expression of auxin transport genes observed in gcn5 and ada2b mutants lead to changes in the distribution of auxin in the root tip.  Scale bars are 20 µm. (C) Fluorescence density between wild-type and gcn5-1 (upper graph) and wildtype and ada2b-1 (lower graph) during root growth. The bars represent the range of the two quadrants. The horizontal line in the bar is the median, while the terminals are the minimum and maximum values of the data. Asterisks above bars of ada2b-1 indicate statistical significance compared to the same d.a.g. of wild-type. Asterisks between brackets show the difference between 4th and seventh d.a.g., using an independent samples t-test: ** p < 0.01, and *** p < 0.001.

GCN5 and ADA2b Affect the Expression of Genes Involved in Auxin Biosynthesis and Signaling
The reduced levels of auxin distribution in the root of gcn5-1 and ada2b-1 mutants suggest that the transcriptional adaptors GCN5 and ADA2b may also affect, besides auxin transport, auxin biosynthesis and signaling. To further characterize the molecular role of GCN5 and ADA2b in the auxin pathway, the expression levels of auxin-related genes in roots of seven-day-old wild-type, gcn5-1 and ada2b-1 plants were analyzed.
Specifically, YUCCA 5, 8 and 9 were studied since their expression was reported in the root cap cells [35]. The expression of YUCCA 5 was only increased in ada2b-1, in comparison to gcn5-1 and wild-type plants. YUCCA 8 was increased, while YUCCA 9 was decreased in both gcn5-1 and ada2b-1 relative to the wild type. (Figure 5A-C). The expression of TAA1, the first enzyme of the auxin biosynthesis pathway [36], was reduced in ada2b-1 but not in gcn5-1 mutants, as the reduction was statistically significant only for ada2b-1 ( Figure 5D). Therefore, we conclude that GCN5 and ADA2b regulate the auxin biosynthesis pathway in the root. The GH3.3 gene is critical for auxin homeostasis [37]; in both gcn5-1 and ada2b-1 GH3.3 was overexpressed relative to the wild-type ( Figure 5F), indicating root auxin availability in gcn5-1 and ada2b-1 may be reduced. In addition, the IAA3 gene, which represses auxin signaling [38], was downregulated in the gcn5-1 and ada2b-1 mutants ( Figure 5E), indicating that auxin responses were also reduced in both gcn5-1 and ada2b-1 mutants. and wild-type and ada2b-1 (lower graph) during root growth. The bars represent the range of the two quadrants. The horizontal line in the bar is the median, while the terminals are the minimum and maximum values of the data. Asterisks above bars of ada2b-1 indicate statistical significance compared to the same d.a.g. of wild-type. Asterisks between brackets show the difference between 4th and seventh d.a.g., using an independent samples t-test: ** p < 0.01, and *** p < 0.001.

GCN5 and ADA2b Affect the Expression of Genes Involved in Auxin Biosynthesis and Signaling
The reduced levels of auxin distribution in the root of gcn5-1 and ada2b-1 mutants suggest that the transcriptional adaptors GCN5 and ADA2b may also affect, besides auxin transport, auxin biosynthesis and signaling. To further characterize the molecular role of GCN5 and ADA2b in the auxin pathway, the expression levels of auxin-related genes in roots of seven-day-old wild-type, gcn5-1 and ada2b-1 plants were analyzed.
Specifically, YUCCA 5, 8 and 9 were studied since their expression was reported in the root cap cells [35]. The expression of YUCCA 5 was only increased in ada2b-1, in comparison to gcn5-1 and wild-type plants. YUCCA 8 was increased, while YUCCA 9 was decreased in both gcn5-1 and ada2b-1 relative to the wild type. (Figure 5A-C). The expression of TAA1, the first enzyme of the auxin biosynthesis pathway [36], was reduced in ada2b-1 but not in gcn5-1 mutants, as the reduction was statistically significant only for ada2b-1 ( Figure 5D). Therefore, we conclude that GCN5 and ADA2b regulate the auxin biosynthesis pathway in the root. The GH3.3 gene is critical for auxin homeostasis [37]; in both gcn5-1 and ada2b-1 GH3.3 was overexpressed relative to the wild-type ( Figure 5F), indicating root auxin availability in gcn5-1 and ada2b-1 may be reduced. In addition, the IAA3 gene, which represses auxin signaling [38], was downregulated in the gcn5-1 and ada2b-1 mutants ( Figure 5E), indicating that auxin responses were also reduced in both gcn5-1 and ada2b-1 mutants.

GCN5 and ADA2b Affect Histone H3K14 Acetylation on PIN Genomic Loci
To examine whether the observed altered expression of PIN genes in the gcn5-1 and ada2b-1 single mutants could result from altered histone acetylation of their loci, we performed ChIP analysis in the roots of seven-day-old seedlings using antibodies against histone H3 acetylated lysine 14 (H3K14Ac) and non-acetylated histone H3. H3K14 is the GCN5 target for acetylation [39,40]. For each genomic locus, two regions were examined, the promoter region, spanning from the transcription start site (+1) roughly up to about 200 bases upstream (−200), and an additional region inside the second exon. At the promoter region of the PIN1 locus, H3K14 acetylation does not change in the gcn5-1 roots, whereas it is increased in ada2b-1 roots. In the coding region of PIN1, the gcn5-1 mutants display significantly reduced H3K14 acetylation compared to the wild type and ada2b mutants ( Figure 6A). A similar pattern is exhibited in the PIN3 locus; gcn5-1 mutants have reduced H3K14 acetylation in the coding region but not in the promoter. Ada2b-1 shows increased acetylation in both regions compared to wild-type roots ( Figure 6B). In the PIN4 locus, gcn5-1 has reduced H3K14 acetylation in both the promoter and coding region. In contrast, ada2b-1 displays increased H3K14 acetylation in both regions ( Figure 6C). These results suggest that GCN5 is a positive modulator of histone H3K14 acetylation on the genomic loci of PIN genes. Furthermore, ADA2b is a positive regulator of PIN gene expression by a different mechanism than GCN5.

Discussion
Herein, we explored the role of histone acetyltransferase GCN5 and the transcriptional adaptor ADA2b in auxin flow during early root growth. We found that GCN5 and ADA2b regulate early root growth in Arabidopsis by modulating the expression and histone acetylation of members of the PIN family of auxin efflux transporters.
Auxins are involved in various developmental responses in plants, including root apical meristem formation and maintenance [41]. The auxin gradient is established and maintained by auxin transport proteins that are essential for root organogenesis [32,33]. Auxin concentration along the roots arises from collective activities and topology of the PIN proteins, the AUX1/LAX family proteins [10,42,43], and the multidrug-resistant/Pglycoprotein family proteins [44].
The roots of gcn5-1 and ada2b-1 mutants at the beginning of growth show the same levels of auxin signaling as the wild-type plants, implying that auxin is present during the embryogenesis of these plants. However, during root growth, auxin levels, as monitored by the DR5 reporter, decrease and accumulate in the QC [45]. The auxin accumulation in the QC observed in gcn5, and ada2b mutants could arise for various reasons.
First, fewer auxin molecules could be biosynthesized in the cells produced during root growth. This scenario is supported by the reduced expression of TAA1 and YUCCA9 in the gcn5-1 and ada2b-1 mutants. Therefore, GCN5 and ADA2b are important for proper auxin biosynthesis during root growth.
Second, as root growth proceeds, the initial amount of auxin produced during embryogenesis in gcn5-1 and ada2b-1 is reduced relative to the wild-type. This could be an effect of increased auxin degradation/inactivation in the mutants. Indeed, the GH3.3 auxin conjugation gene is overexpressed in gcn5-1 and ada2b-1 [this study , 22], indicating increased auxin inactivation. Thus, in the absence of both GCN5 and ADA2b, auxin degradation is promoted. However, beyond GCN5 and ADA2b there are other component(s) implicated in auxin biosynthesis and regulation that await to be unveiled.
Third, our data indicate that GCN5 and ADA2b affect auxin transport. The root cap cells affect the availability and distribution of auxin in the root meristem by affecting auxin transport through PIN proteins and, ultimately, the location of the auxin minimum and transition zone [46]. Indeed, the auxin efflux transporter PIN1 is expressed in the meristematic zone at a lower intensity in gcn5-1 and ada2b-1 mutants than in the wild type. In ada2b-1, PIN1 expression disappears from the pericycle and endodermis cell layers. Therefore, our data indicate that GCN5 and ADA2b are positive regulators of PIN1 expression in the early days of root growth. Furthermore, assuming that the initial available amount of auxin is catabolized in the cap cells, then because of reduced auxin transport through PIN1, it is impossible to re-infuse the cells with auxin, resulting in reduced auxin signaling. Gene expression experiments also support the latter. The expression of PIN1, PIN3 and PIN4 is reduced in gcn5-1 and ada2b-1 compared to the wild type.
In contrast, PIN2 expression is not affected; thus, auxin is typically transported from the root tip to the elongation zone in gcn5-1 and ada2b-1 mutants. These results suggest that GCN5 and ADA2b contribute to the proper transport of auxin through the central cylinder to the root tip and the QC, resulting in a sufficient amount of auxin being channeled for root growth. Therefore, the absence of GCN5 and ADA2b leads to disruption of auxin transport, which ultimately accounts for the reduced root size observed in the gcn5-1 and ada2b-1 mutants. A similar mechanism has been found to occur in Arabidopsis gynoecium. Specifically, GCN5 and ADA2b contribute to proper female development by positively regulating auxin transport through PIN1 [31]. However, locally, auxin biosynthesis in the QC is necessary and sufficient for the maintenance of the root meristem when the auxin polar transport system is affected [47].
Auxin regulates QC's maintenance and the root meristem's activity through PLETHORA transcription factors (PLTs) [14]. GCN5 and ADA2b regulate stem cell maintenance by regulating PLT expression, while PLT2 overexpression reverts the defective stem cell phenotype in gcn5-1 and ada2b-1 [22]. Expression of PLTs is induced by PIN-driven auxin gradient. Conversely, the transcription of PINs that stabilizes the position of the stem cell niche is maintained by PLTs [10,42,48]. Moreover, on the fifth day of root growth, maximal meristem size and stabilization of the transition zone are established by the auxin signaling via PLT action/cytokinin signaling network [49].
Both GCN5 and ADA2b promote the expression of PIN1, PIN3 and PIN4 to higher levels necessary for the physiological needs of the growing root. ADA2b has been shown to interact with transcription factors involved in auxin responses, recruiting GCN5 to the genomic regions needed [25,50]. In addition, ADA2b is required for histone acetylation in response to auxin [50,51]. The expression of PIN4 was upregulated after treatment with trichostatin A (TSA), a potent histone deacetylase inhibitor, suggesting that this gene could be regulated by histone acetylation [50]. These data suggest that PIN4 induction requires GCN5-specific histone acetylation. In our results, PIN4 is one of the genes affected by GCN5 and ADA2b since its expression is reduced in mutants.
Interestingly, PIN4 shows reduced histone H3K14 acetylation in both the promoter and the coding region, compared to PIN1 and PIN3, which only show a reduction in the coding region in gcn5 mutants. Therefore, PIN4 needs histone acetylation in its genomic locus for proper induction, and this acetylation could be accomplished by GCN5. In our results, GCN5 but not ADA2b affected the H3K14 acetylation at PIN1, 3 and 4 loci, although all three genes are downregulated in gcn5 and ada2b mutants. Therefore, GCN5-dependent histone acetylation is required to adequately express genes involved in auxin transport in the early days of root growth.
Maximal meristem size and stabilization of the transition zone are established by the auxin/PLT/ARR-B network five days after germination [49]. GCN5 and ADA2b also affect cytokinin levels by regulating cytokinin biosynthesis and catabolism [23]. Furthermore, auxin-induced cytokinin signaling depends on GCN5-ADA2b action [23]. Cytokinins were shown to control auxin transport, unitizing transcriptional and post-transcriptional regulation of PIN efflux carriers, thus controlling intercellular auxin distribution in the root tips [52][53][54]. Our results suggest that the synergistic network of auxin and cytokinin signaling requires a GCN5 and ADA2b complex that modulates histone acetylation of auxin efflux transporters during the early days of root growth. Moreover, it indicates an essential role of GCN5-dependent histone acetylation in the interaction between auxin transport, auxin signaling, cytokinin signaling and regulatory circuits during root growth. Root meristem size is in a perpetual change in response to external and internal conditions, signaled by the changing levels of histone acetylation and other chromatin modifications to modulate hormone responses. Integrating of all this information to alter root growth dynamically and the coordination required across the whole root system is still an open question for further studies.

Conclusions
In this manuscript we describe a biological role for GCN5 histone acetyltransferase and ADA2b transcriptional adaptor, during early root growth. Both proteins promote the expression of PIN1, PIN3 and PIN4 auxin transporters of the PIN family and help maintain a proper auxin distribution for normal root growth. These results place GCN5 and ADA2b as important players in the regulatory module that maintains an active root, affecting both auxin and cytokinin responses as well as their intertwined network of interactions. As root meristems integrate internal and external cues to guide root growth in the most effective way for the plant, factors that affect their function are of the utmost importance. Since the study of root growth and development is challenging in economically important crops and even more so in tree species, the study of model organisms such as Arabidopsis thaliana provides valuable insights on the hormonal and molecular mechanisms that could find their way into horticultural practice.

Plant Materials and Growth
The Arabidopsis thaliana (L) Heynh. gcn5-1 and ada2b-1 mutants in Wassilewskija-2 (Ws-2) background were acquired from the University of Wisconsin Arabidopsis Knockout Facility as previously described in [20], and were available to the lab. The PIN1:PIN1-GFP reporter line, was acquired by NASC (N23889). The PIN1:PIN1-GFP gcn5-1/+ line was created by crossing the PIN1:PIN1-GFP line with the gcn5-1 mutant as described by [31], and was available to the lab. Commercially available bleach was used for the sterilization of the seed surface. Seeds were stratified at 4 • C for 3-4 days in the dark. For plating, the Gamborg B5 (GB5) medium, including vitamins (Duchefa Biochemie, Amsterdam, Netherlands) supplemented with 1% sucrose (Duchefa) and 0.8% phytoagar (Duchefa) and adjusted at pH 5.7, was used. Plants were cultivated at an average of 22 • C with 70-80 mol m −2 s −1 cool-white, fluorescent lamps in long days (16 h light/8 h dark). Commercially available soil, Terrahum ® (Deutsche Kompost Handelsgesellschaft), was used for cultivation.

Genetic Analysis and Genotyping
The PIN1:PIN1-GFP ada2b-1/+ line was created using PIN1:PIN1-GFP pollen to fertilize ada2b-1/+ gynoecia. The F1 was left to self-fertilize, and the various genotypes were identified in the F2 using a combination of phenotypic and PCR-based methods (Supplemental Table S1 for primers). The gcn5-1 and ada2b-1 mutant alleles were tracked using PCR with specific primers.

Gene Expression Analysis
Ws-2, gcn5-1 and ada2b-1 plants were grown vertically in GB5 containing-medium plates for seven days for gene expression assays. Whole roots were collected and flashfrozen in liquid nitrogen, and RNA was extracted using the Nucleospin ® RNA Plant kit (Macherey-Nagel, Duren, Germany). In at least three independent biological repeats, reverse transcription was performed using 0.5 µg of total RNA with the PrimeScript TM first strand cDNA Synthesis kit (TaKaRa, Shiga, Japan). Quantitative reverse-transcription polymerase chain reactions (RT-qPCRs) were prepared with the AMPLIFYME SG Universal Mix (AM02) (BLIRT SA, Gdansk, Poland) at the ABI StepOne™ system (Applied Biosystems, Foster City, CA, USA). Three technical repeats were run for each sample. The At4g26410 gene was used as an endogenous control (Supplemental Table S1). Data were analyzed with the ∆∆Ct method using StepOne Software 2.1. Statistical significance was calculated using a one-way ANOVA, with Fisher's Least Significant Difference (LSD) and post-hoc test with a 95% confidence interval using IBM SPSS Statistics software 23.0 (Statistical Product and Service Solutions, USA).

Microscopy
A Zeiss AxioImager.Z2 (Carl Zeiss AG, Munich, Germany) equipped with a digital AxioCam MRc 5 camera for green fluorescent protein (GFP) detection was used for most observations. At a minimum, 30 seedlings were observed on 4, 5 and 6 days, and 80 were observed on the seventh day. The integrated intensity of GFP was measured by ImageJ software, and values were normalized as previously described [55]. Statistical significance was calculated using an independent samples t-test, with a 95% confidence interval, using IBM SPSS Statistics software 23.0 (Statistical Product and Service Solutions, USA).
For confocal microscopy, seven-day-old seedlings were stained with 10 µg/mL propidium iodide (PI) (Sigma-Aldrich, St Louis, MO, USA) and mounted directly in the PI solution. Root caps were visualized under a Zeiss Observer.Z1 microscope (Carl Zeiss AG, Munich, Germany), equipped with an LSM780 confocal laser scanning module to detect GFP. Imaging was achieved with ZEN2011 software according to the manufacturer's instructions.

Chromatin Immunoprecipitation (ChIP)
As previously described, a ChIP assay was performed with minor modifications [23,56]. Seven-day-old seedlings were harvested after selection, and a total of approximately 250, 650, and 900 roots for wild-type Ws-2, gcn5-1, and ada2b-1, respectively, were collected. The ada2b-1 roots were selected from a segregating population of more than 4000 seedlings. The total amount of tissue used for this experiment was 25 mg per sample. The exact amount of tissue was used for all three genotypes. Antibodies against acetylated histone H3K14 (Anti-Histone H3 (Lys14), EMD Millipore #07-353, Burlington, MA, USA) and non-acetylated H3 (ChIPAb + Histone H3 C-term, EMD Millipore #17-10046) were used. Immunoprecipitated DNA was diluted and analyzed by RT-qPCR (Supplemental Table S1 for primers used). RT-qPCRs were performed using the AMPLIFYME SG Universal Mix (AM02) (BLIRT SA, Gdánsk, Poland) at the ABI StepOne™system (Applied Biosystems, Foster City, CA, USA). Samples were analyzed and calibrated as previously described [20,23]. Statistical significance was calculated using a one-way ANOVA, with Fisher's Least Significant Difference (LSD) and post-hoc test with a 95% confidence interval using IBM SPSS Statistics software (Statistical Product and Service Solutions, USA).