Arabidopsis transcription factor TCP4 controls the identity of the apical gynoecium.

The style and stigma at the apical gynoecium are crucial for flowering plant reproduction. However, the mechanisms underlying specification of the apical gynoecium remain unclear. Here, we demonstrate that Class II TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors are critical for apical gynoecium specification in Arabidopsis (Arabidopsis thaliana). The septuple tcp2 tcp3 tcp4 tcp5 tcp10 tcp13 tcp17 (tcpSEP) and duodecuple tcp2 tcp3 tcp4 tcp5 tcp10 tcp13 tcp17 tcp24 tcp1 tcp12 tcp18 tcp16 (tcpDUO) mutants produce narrower and longer styles, while disruption of TCPs and CRABS CLAW (CRC) or NGATHAs (NGAs) in tcpDUO crc or tcpDUO nga1 nga2 nga4 causes the apical gynoecium to be replaced by lamellar structures with indeterminate growth. TCPs are predominantly expressed in the apex of the gynoecium. TCP4 interacts with CRC to synergistically upregulate the expression level of NGAs, and NGAs further form high-order complexes to control the expression of auxin-related genes in the apical gynoecium by directly interacting with TCP4. Our findings demonstrate that TCP4 physically associates with CRC and NGAs to control auxin biosynthesis in forming fine structures of the apical gynoecium.

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Binding by the Polycomb complex component BMI1 and H2A monoubiquitination shape local and long-range interactions in the Arabidopsis genome.

Three-dimensional (3D) chromatin organization is highly dynamic during development and seems to play a crucial role in regulating gene expression. Self-interacting domains, commonly called topologically associating domains (TADs) or compartment domains (CDs), have been proposed as the basic structural units of chromatin organization. Surprisingly, although these units have been found in several plant species, they escaped detection in Arabidopsis (Arabidopsis thaliana). Here, we show that the Arabidopsis genome is partitioned into contiguous CDs with different epigenetic features, which are required to maintain appropriate intra-CD and long-range interactions. Consistent with this notion, the histone-modifying Polycomb group machinery is involved in 3D chromatin organization. Yet, while it is clear that Polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) helps establish local and long-range chromatin interactions in plants, the implications of PRC1-mediated histone H2A monoubiquitination on lysine 121 (H2AK121ub) are unclear. We found that PRC1, together with PRC2, maintains intra-CD interactions, but it also hinders the formation of H3K4me3-enriched local chromatin loops when acting independently of PRC2. Moreover, the loss of PRC1 or PRC2 activity differentially affects long-range chromatin interactions, and these 3D changes differentially affect gene expression. Our results suggest that H2AK121ub helps prevent the formation of transposable element/H3K27me1-rich long loops and serves as a docking point for H3K27me3 incorporation.

DNA-TCP complex structures reveal a unique recognition mechanism for TCP transcription factor families.

Plant-specific TCP transcription factors are key regulators of diverse plant functions. TCP transcription factors have long been annotated as basic helix-loop-helix (bHLH) transcription factors according to remote sequence homology without experimental validation, and their consensus DNA-binding sequences and protein-DNA recognition mechanisms have remained elusive. Here, we report the crystal structures of the class I TCP domain from AtTCP15 and the class II TCP domain from AtTCP10 in complex with different double-stranded DNA (dsDNA). The complex structures reveal that the TCP domain is a distinct DNA-binding motif and the homodimeric TCP domains adopt a unique three-site recognition mode, binding to dsDNA mainly through a central pair of ß-strands formed by the dimer interface and two basic flexible loops from each monomer. The consensus DNA-binding sequence for class I TCPs is a perfectly palindromic 11 bp (GTGGGNCCCAC), whereas that for class II TCPs is a near-palindromic 11 bp (GTGGTCCCCAC). The unique DNA binding mode allows the TCP domains to display broad specificity for a range of DNA sequences even shorter than 11 bp, adding further complexity to the regulatory network of plant TCP transcription factors.

MicroRNA775 regulates intrinsic leaf size and reduces cell wall pectin levels by targeting a galactosyltransferase gene in Arabidopsis.

Plants possess unique primary cell walls made of complex polysaccharides that play critical roles in determining intrinsic cell and organ size. How genes responsible for synthesizing and modifying the polysaccharides in the cell wall are regulated by microRNAs (miRNAs) to control plant size remains largely unexplored. Here we identified 23 putative cell wall-related miRNAs, termed as CW-miRNAs, in Arabidopsis thaliana and characterized miR775 as an example. We showed that miR775 post-transcriptionally silences GALT9, which encodes an endomembrane-located galactosyltransferase belonging to the glycosyltransferase 31 family. Over-expression of miR775 and deletion of GALT9 led to significantly enlarged leaf-related organs, primarily due to increased cell size. Monosaccharide quantification, confocal Raman imaging, and immunolabeling combined with atomic force microscopy revealed that the MIR775A-GALT9 circuit modulates pectin levels and the elastic modulus of the cell wall. We also showed that MIR775A is directly repressed by the transcription factor ELONGATED HYPOCOTYL5 (HY5). Genetic analysis confirmed that HY5 is a negative regulator of leaf size that acts through the HY5-MIR775A-GALT9 repression cascade to control pectin levels. These findings demonstrate that miR775-regulated cell wall remodeling is an integral determinant of intrinsic leaf size in A. thaliana. Studying other CW-miRNAs would provide more insights into cell wall biology.

TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

The Transcription Factors TCP4 and PIF3 Antagonistically Regulate Organ-Specific Light Induction of SAUR Genes to Modulate Cotyledon Opening during De-Etiolation in Arabidopsis.

Light elicits different growth responses in different organs of plants. These organ-specific responses are prominently displayed during de-etiolation. While major light-responsive components and early signaling pathways in this process have been identified, this information has yet to explain how organ-specific light responses are achieved. Here, we report that members of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factor family participate in photomorphogenesis and facilitate light-induced cotyledon opening in Arabidopsis (Arabidopsis thaliana). Chromatin immunoprecipitation sequencing and RNA sequencing analyses indicated that TCP4 targets a number of SMALL AUXIN UPREGULATED RNA (SAUR) genes that have previously been shown to exhibit organ-specific, light-responsive expression. We demonstrate that TCP4-like transcription factors, which are predominantly expressed in the cotyledons of both light- and dark-grown seedlings, activate SAUR16 and SAUR50 expression in response to light. Light regulates the binding of TCP4 to the promoters of SAUR14, SAUR16, and SAUR50 through PHYTOCHROME-INTERACTING FACTORs (PIFs). PIF3, which accumulates in etiolated seedlings and its levels rapidly decline upon light exposure, also binds to the SAUR16 and SAUR50 promoters, while suppressing the binding of TCP4 to these promoters in the dark. Our study reveals that the interplay between light-responsive factors PIFs and the developmental regulator TCP4 determines the cotyledon-specific light regulation of SAUR16 and SAUR50, which contributes to cotyledon closure and opening before and after de-etiolation.

TANDEM ZINC-FINGER/PLUS3 Is a Key Component of Phytochrome A Signaling.

Phytochrome A (phyA) is the primary plant photoreceptor responsible for perceiving and mediating various responses to far-red (FR) light and is essential for survival in canopy shade. In this study, we identified two Arabidopsis thaliana mutants that grew longer hypocotyls in FR light. Genetic analyses showed that they were allelic and their FR phenotypes were caused by mutations in the gene named TANDEM ZINC-FINGER/PLUS3 (TZP), previously shown to encode a nuclear protein involved in blue light signaling and phyB-dependent regulation of photoperiodic flowering. We show that the expression of TZP is dramatically induced by light and that TZP proteins are differentially modified in different light conditions. Furthermore, we show that TZP interacts with both phyA and FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and regulates the abundance of phyA, FHY1, and ELONGATED HYPOCOTYL5 proteins in FR light. Moreover, our data indicate that TZP is required for the formation of a phosphorylated form of phyA in the nucleus in FR light. Together, our results identify TZP as a positive regulator of phyA signaling required for phosphorylation of the phyA photoreceptor, thus suggesting an important role of phosphorylated phyA in inducing the FR light response.

The TIE1 transcriptional repressor controls shoot branching by directly repressing BRANCHED1 in Arabidopsis.

Shoot branching is a major determinant of plant architecture and is regulated by both endogenous and environmental factors. BRANCHED1 (BRC1) is a central local regulator that integrates signals controlling shoot branching. So far, the regulation of BRC1 activity at the protein level is still largely unknown. In this study, we demonstrated that TIE1 (TCP interactor containing EAR motif protein 1), a repressor previously identified as an important factor in the control of leaf development, also regulates shoot branching by repressing BRC1 activity. TIE1 is predominantly expressed in young axillary buds. The gain-of-function mutant tie1-D produced more branches and the overexpression of TIE1 recapitulated the increased branching of tie1-D, while disruption of TIE1 resulted in lower bud activity and fewer branches. We also demonstrated that the TIE1 protein interacts with BRC1 in vitro and in vivo. Expression of BRC1 fused with the C-terminus of the TIE1 protein in wild type caused excessive branching similar to that observed in tie1-D and brc1 loss-of-function mutants. Transcriptome analyses revealed that TIE1 regulated about 30% of the BRC1-dependent genes, including the BRC1 direct targets HB21, HB40 and HB53. These results indicate that TIE1 acts as a positive regulator of shoot branching by directly repressing BRC1 activity. Thus, our results reveal that TIE1 is an important shoot branching regulator, and provide new insights in the post-transcriptional regulation of the TCP transcription factor BRC1.

Correction: The TIE1 transcriptional repressor controls shoot branching by directly repressing BRANCHED1 in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1007296.].

The Arabidopsis RING-Type E3 Ligase TEAR1 Controls Leaf Development by Targeting the TIE1 Transcriptional Repressor for Degradation.

The developmental plasticity of leaf size and shape is important for leaf function and plant survival. However, the mechanisms by which plants form diverse leaves in response to environmental conditions are not well understood. Here, we identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) and found that it regulates leaf development by promoting the degradation of TCP INTERACTOR-CONTAINING EAR MOTIF PROTEIN1 (TIE1), an important repressor of CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are key for leaf development. TEAR1 contains a typical C3H2C3-type RING domain and has E3 ligase activity. We show that TEAR1 interacts with the TCP repressor TIE1, which is ubiquitinated in vivo and degraded by the 26S proteasome system. We demonstrate that TEAR1 is colocalized with TIE1 in nuclei and negatively regulates TIE1 protein levels. Overexpression of TEAR1 rescued leaf defects caused by TIE1 overexpression, whereas disruption of TEAR1 resulted in leaf phenotypes resembling those caused by TIE1 overexpression or TCP dysfunction. Deficiency in TEAR partially rescued the leaf defects of TCP4 overexpression line and enhanced the wavy leaf phenotypes of jaw-5D We propose that TEAR1 positively regulates CIN-like TCP activity to promote leaf development by mediating the degradation of the TCP repressor TIE1.

A Novel Imprinted Gene NUWA Controls Mitochondrial Function in Early Seed Development in Arabidopsis.

Imprinted genes display biased expression of paternal and maternal alleles and are only found in mammals and flowering plants. Compared to several hundred imprinted genes that are functionally characterized in mammals, very few imprinted genes were confirmed in plants and even fewer of them have been functionally investigated. Here, we report a new imprinted gene, NUWA, in plants. NUWA is an essential gene, because loss of its function resulted in reduced transmission through the female gametophyte and defective cell/nuclear proliferation in early Arabidopsis embryo and endosperm. NUWA is a maternally expressed imprinted gene, as only the maternal allele of NUWA is transcribed and translated from gametogenesis to the 16-cell globular embryo stage after fertilization, and the de novo transcription of the maternal allele of NUWA starts from the zygote stage. Different from other identified plant imprinted genes whose encoded proteins are mostly localized to the nucleus, the NUWA protein was localized to the mitochondria and was essential for mitochondria function. Our work uncovers a novel imprinted gene of a previously unidentified type, namely, a maternal-specific expressed nuclear gene with its encoded protein localizing to and controlling the function of the maternally inherited mitochondria. This reveals a unique mechanism of maternal control of the mitochondria and adds an extra layer of complexity to the regulation of nucleus-organelle coordination during early plant development.

The Regulation of CIN-like TCP Transcription Factors.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) family proteins are the plant-specific transcription factors extensively participating in diverse developmental processes by integrating external cues with internal signals. The roles of CINCINNATA (CIN)-like TCPs are conserved in control of the morphology and size of leaves, petal development, trichome formation and plant flowering. The tight regulation of CIN-like TCP activity at transcriptional and post-transcriptional levels are central for plant developmental plasticity in response to the ever-changing environmental conditions. In this review, we summarize recent progresses with regard to the function and regulation of CIN-like TCPs. CIN-like TCPs are regulated by abiotic and biotic cues including light, temperature and pathogens. They are also finely controlled by microRNA319 (miRNA319), chromatin remodeling complexes and auxin homeostasis. The protein degradation plays critical roles in tightly controlling the activity of CIN-like TCPs as well.

CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis.

The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1.

The Arabidopsis USL1 controls multiple aspects of development by affecting late endosome morphology.

The polar transport of auxin controls many aspects of plant development. However, the molecular mechanisms underlying auxin tranport regulation remain to be further elucidated. We identified a mutant named as usl1 (unflattened and small leaves) in a genetic screen in Arabidopsis thaliana. The usl1 displayed multiple aspects of developmental defects in leaves, embryogenesis, cotyledons, silique phyllotaxy and lateral roots in addition to abnormal leaves. USL1 encodes a protein orthologous to the yeast vacuolar protein sorting (Vps) 38p and human UV RADIATION RESISTANCE-ASSOCIATED GENE (UVRAG). Cell biology, Co-IP/MS and yeast two-hybrid were used to identify the function of USL1. USL1 colocalizes at the subcellular level with VPS29, a key factor of the retromer complex that controls auxin transport. The morphology of the VPS29-associated late endosomes (LE) is altered from small dots in the wild-type to aberrant enlarged circles in the usl1 mutants. The usl1 mutant synergistically interacts with vps29. We also found that USL1 forms a complex with AtVPS30 and AtVPS34. We propose that USL1 controls multiple aspects of plant development by affecting late endosome morphology and by regulating the PIN1 polarity. Our findings provide a new layer of the understanding on the mechanisms of plant development regulation.

The WRKY Transcription Factor WRKY71/EXB1 Controls Shoot Branching by Transcriptionally Regulating RAX Genes in Arabidopsis.

Plant shoot branching is pivotal for developmental plasticity and crop yield. The formation of branch meristems is regulated by several key transcription factors including REGULATOR OF AXILLARY MERISTEMS1 (RAX1), RAX2, and RAX3. However, the regulatory network of shoot branching is still largely unknown. Here, we report the identification of EXCESSIVE BRANCHES1 (EXB1), which affects axillary meristem (AM) initiation and bud activity. Overexpression of EXB1 in the gain-of-function mutant exb1-D leads to severe bushy and dwarf phenotypes, which result from excessive AM initiation and elevated bud activities. EXB1 encodes the WRKY transcription factor WRKY71, which has demonstrated transactivation activities. Disruption of WRKY71/EXB1 by chimeric repressor silencing technology leads to fewer branches, indicating that EXB1 plays important roles in the control of shoot branching. We demonstrate that EXB1 controls AM initiation by positively regulating the transcription of RAX1, RAX2, and RAX3. Disruption of the RAX genes partially rescues the branching phenotype caused by EXB1 overexpression. We further show that EXB1 also regulates auxin homeostasis in control of shoot branching. Our data demonstrate that EXB1 plays pivotal roles in shoot branching by regulating both transcription of RAX genes and auxin pathways.

ADP1 affects plant architecture by regulating local auxin biosynthesis.

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.

The TIE1 transcriptional repressor links TCP transcription factors with TOPLESS/TOPLESS-RELATED corepressors and modulates leaf development in Arabidopsis.

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development.

The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25.

Iron is an essential micronutrient for plants and animals, and plants are a major source of iron for humans. Therefore, understanding the regulation of iron homeostasis in plants is critical. We identified a T-DNA insertion mutant, yellow and sensitive to iron-deficiency 1 (yid1), that was hypersensitive to iron deficiency, containing a reduced amount of iron. YID1 encodes the Arabidopsis Mediator complex subunit MED16. We demonstrated that YID1/MED16 interacted with another subunit, MED25. MED25 played an important role in regulation of iron homeostasis by interacting with EIN3 and EIL1, two transcription factors in ethylene signaling associated with regulation of iron homeostasis. We found that the transcriptome in yid1 and med25 mutants was significantly affected by iron deficiency. In particular, the transcription levels of FIT, IRT1 and FRO2 were reduced in the yid1 and med25 mutants under iron-deficient conditions. The finding that YID1/MED16 and MED25 positively regulate iron homeostasis in Arabidopsis increases our understanding of the complex transcriptional regulation of iron homeostasis in plants.

A PP6-type phosphatase holoenzyme directly regulates PIN phosphorylation and auxin efflux in Arabidopsis.

The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation.