The H1/H5 domain contributes to OsTRBF2 phase separation and gene repression during rice development.

Transcription factors (TFs) tightly control plant development by regulating gene expression. The phase separation of TFs plays a vital role in gene regulation. Many plant TFs have the potential to form phase-separated protein condensates; however, little is known about which TFs are regulated by phase separation and how it affects their roles in plant development. Here, we report that the rice (Oryza sativa) single Myb TF TELOMERE REPEAT-BINDING FACTOR 2 (TRBF2) is highly expressed in fast-growing tissues at the seedling stage. TRBF2 is a transcriptional repressor that binds to the transcriptional start site of thousands of genes. Mutation of TRBF2 leads to pleiotropic developmental defects and misexpression of many genes. TRBF2 displays characteristics consistent with phase separation in vivo and forms phase-separated condensates in vitro. The H1/H5 domain of TRBF2 plays a crucial role in phase separation, chromatin targeting and gene repression. Replacing the H1/H5 domain by a phase-separated intrinsically disordered region from Arabidopsis (Arabidopsis thaliana) AtSERRATE partially recovers the function of TRBF2 in gene repression in vitro and in transgenic plants. We also found that TRBF2 is required for trimethylation of histone H3 Lys27 (H3K27me3) deposition at specific genes and genome-wide. Our findings reveal that phase separation of TRBF2 facilitates gene repression in rice development.

BIG coordinates auxin and SHORT ROOT to promote asymmetric stem cell divisions in Arabidopsis roots.

KEY MESSAGE: BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance in Arabidopsis roots. The formative divisions of cortex/endodermis initials (CEIs) and CEI daughter cells (CEIDs) in Arabidopsis roots are coordinately controlled by the longitudinal auxin gradient and the radial SHORT ROOT (SHR) abundance. However, the mechanism underlying this coordination remains poorly understood. In this study, we demonstrate that BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance. Mutations in BIG gene repressed cell cycle progression, delaying the formative divisions within the ground tissues and impairing the establishment of endodermal and cortical identities. In addition, we uncovered auxin's suppressive effect on BIG expression, triggering CYCLIND6;1 (CYCD6;1) activation in an SHR-dependent fashion. Moreover, the degradation of RETINOBLASTOMA-RELATED (RBR) is jointly regulated by BIG and CYCD6;1. The loss of BIG function led to RBR protein accumulation, detrimentally impacting the SHR/SCARECROW (SCR) protein complex and the CEI/CEID formative divisions. Collectively, these findings shed light on a fundamental mechanism wherein BIG intricately coordinates the interplay between SHR/SCR and auxin, steering ground tissue patterning within Arabidopsis root tissue.

WUSCHEL RELATED HOMEOBOX5 and 7 maintain callus development by promoting cell division in Arabidopsis.

In tissue culture, a high concentration of auxin in the callus induction medium (CIM) stimulates cell division and subsequent callus formation, which acquires root primordium-like characteristics necessary for cell pluripotency. In Arabidopsis, WUSCHEL-RELATED HOMEOBOX5 (WOX5) and its closest homolog WOX7, which are abundant in the middle cell layer of mature callus, play a crucial role in maintaining pluripotency by promoting auxin accumulation and enhancing cytokinin sensitivity. However, the mechanism by which WOX5/7 regulate callus formation remains unclear. In this study, we found that mutations in WOX5/7 resulted in a significant down-regulation of genes involved in the G2M and S phases during callus induction. Loss-of-function mutants of WOX5/7 exhibited reduced callus formation, which was correlated with decreased expression of CYCB1;1 compared to the wild-type. Furthermore, we provided evidence that WOX5 physically interacts with PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1), which spatio-temporally co-expresses with WOX5 in early-induced callus, and up-regulates a subset of cycle-regulating genes targeted by PAT1. Collectively, our findings suggest a critical role for the WOX5-PAT1 protein complex in regulating cell cycle progression, thereby promoting the continuous growth capacity of pluripotent callus.

BIG Modulates Stem Cell Niche and Meristem Development via SCR/SHR Pathway in Arabidopsis Roots.

BIG, a regulator of polar auxin transport, is necessary to regulate the growth and development of Arabidopsis. Although mutations in the BIG gene cause severe root developmental defects, the exact mechanism remains unclear. Here, we report that disruption of the BIG gene resulted in decreased quiescent center (QC) activity and columella cell numbers, which was accompanied by the downregulation of WUSCHEL-RELATED HOMEOBOX5 (WOX5) gene expression. BIG affected auxin distribution by regulating the expression of PIN-FORMED proteins (PINs), but the root morphological defects of big mutants could not be rescued solely by increasing auxin transport. Although the loss of BIG gene function resulted in decreased expression of the PLT1 and PLT2 genes, genetic interaction assays indicate that this is not the main reason for the root morphological defects of big mutants. Furthermore, genetic interaction assays suggest that BIG affects the stem cell niche (SCN) activity through the SCRSCARECROW (SCR)/SHORT ROOT (SHR) pathway and BIG disruption reduces the expression of SCR and SHR genes. In conclusion, our findings reveal that the BIG gene maintains root meristem activity and SCN integrity mainly through the SCR/SHR pathway.

Replication protein RPA2A regulates floral transition by cooperating with PRC2 in Arabidopsis.

RPA2A is a subunit of the conserved heterotrimeric replication protein A (RPA) in Arabidopsis, which is an essential replisome component that binds to single-stranded DNA during DNA replication. RPA2A controls a set of developmental processes, but the underlying mechanism is largely unknown. Here we show that RPA2A represses key flowering genes including FLOWERING LOCUS T (FT), AGAMOUS (AG) and AGAMOUS LIKE 71 (AGL71) to suppress floral transition by cooperating with the PRC2 complex. RPA2A is vigorously expressed in dividing cells and required for correct DNA replication. Mutation of RPA2A leads to early flowering, which is dependent on ectopic expression of key flowering genes including FT molecularly and genetically. RPA2A and PRC2 have common target genes including FT, AG and AGL71 supported using genetic analysis, transcriptome profiling and H3K27me3 ChIP-seq analysis. Furthermore, RPA2A physically interacts with PRC2 components CLF, EMF2 and MSI1, which recruits CLF to the chromatin loci of FT, AG and AGL71. Together, our results show that the replication protein RPA2A recruits PRC2 to key flowering genes through physical protein interaction, thereby repressing the expression of these genes to suppress floral transition in Arabidopsis.

Control of Root Stem Cell Differentiation and Lateral Root Emergence by CLE16/17 Peptides in Arabidopsis.

Secreted peptide-mediated cell-to-cell communication plays a crucial role in the development of multicellular organisms. A large number of secreted peptides have been predicated by bioinformatic approaches in plants. However, only a few of them have been functionally characterized. In this study, we show that two CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides CLE16/17 are required for both stem cell differentiation and lateral root (LR) emergence in Arabidopsis. We further demonstrate that the CLE16/17 peptides act through the CLAVATA1-ARABIDOPSIS CRINKLY4 (CLV1-ACR4) protein kinase complex in columella stem cell (CSC) differentiation, but not in LR emergence. Furthermore, we show that CLE16/17 promote LR emergence probably via activating the expression of HAESA/HAESA-LIKE2 (HAE/HSL2) required for cell wall remodeling. Collectively, our results reveal a CLV1-ACR4-dependent and -independent dual-function of the CLE16/17 peptides in root development.

Intersected functional zone of transcriptional regulators patterns stemness within stem cell niche of root apical meristem.

Root stem cell niche (SCN) consists of a quiescent center (QC) and surrounding stem cells. Disrupted symplastic communication leads to loss of stemness in the whole SCN. Several SCN regulators were reported to move between cells for SCN maintenance. However, single mutant of these regulators is insufficient to abolish QC stemness despite the high differentiation rate in surrounding stem cells. To dissect the mechanism behind such distinct stemness in SCN, we combined the mis-expression strategy with pWOX5:icals3m system in which QC is symplastically isolated. We found the starch accumulation in QC could be synergistically repressed by WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT). Like PLTs, other core regulators also exhibited dimorphic functions by inhibiting differentiation at a higher dose while promoting cell division at a low protein level. Being located in the center of the intersected expression zones, QC cells receive the highest level of core regulators, forming the most robust stemness within SCN. WUSCHEL-RELATED HOMEOBOX 5 was sufficient to activate PLT1/2 expression, contributing to the QC-enriched PLTs. Our results provide experimental evidence supporting the long-standing hypothesis that the combination of spatial expression, synergistic function and dosage effect of core regulators result in spatially distinct stemness in SCN.

Organizer-Derived WOX5 Signal Maintains Root Columella Stem Cells through Chromatin-Mediated Repression of CDF4 Expression.

Stem cells in plants and animals are maintained pluripotent by signals from adjacent niche cells. In plants, WUSCHEL HOMEOBOX (WOX) transcription factors are central regulators of stem cell maintenance in different meristem types, yet their molecular mode of action has remained elusive. Here we show that in the Arabidopsis root meristem, the WOX5 protein moves from the root niche organizer, the quiescent center, into the columella stem cells, where it directly represses the transcription factor gene CDF4. This creates a gradient of CDF4 transcription, which promotes differentiation opposite to the WOX5 gradient, allowing stem cell daughter cells to exit the stem cell state. We further show that WOX5 represses CDF4 transcription by recruiting TPL/TPR co-repressors and the histone deacetylase HDA19, which consequently induces histone deacetylation at the CDF4 regulatory region. Our results show that chromatin-mediated repression of differentiation programs is a common strategy in plant and animal stem cell niches.

ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture.

The inflorescence of flowering plants is a highly organized structure, not only contributing to plant reproductive processes, but also constituting an important part of the entire plant morphology. Previous studies have revealed that the class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP or KNAT1), KNAT2, and KNAT6 play essential roles in inflorescence architecture. Pedicel morphology is known to contribute greatly to inflorescence architecture, and BP negatively regulates KNAT2 and KNAT6 to ensure that pedicels have a normal upward-pointing orientation. These findings indicate that a genetic network exists in controlling pedicel orientation, but how this network functions in the developmental process remains elusive. Here it is reported that the ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) gene, which belongs to the BELL1-like homeodomain gene family, is a new member participating in regulating pedicel orientation in the class-I KNOX network. In a genetic screening for suppressors of isoginchaku-2D, a gain-of-function ASYMMETRIC LEAVES2 mutant that displays downward-pointing pedicels, a suppressor mutant was obtained. Characterization of this mutant revealed that the mutation corresponds to ATH1. Genetic analysis indicated that ATH1 acts mainly in the KNAT2 pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that ATH1 physically interacts with KNAT2. The data indicate that the ATH1-KNAT2 complex acts redundantly with KNAT6, both of which are negatively regulated by BP during pedicel development.

The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis.

The shoot apical meristem (SAM) of angiosperms comprises a group of undifferentiated cells which divide to maintain the meristem and also give rise to all the above-ground structures of the plant. Previous studies revealed that the Arabidopsis ARGONAUTE10 [AGO10, also called PINHEAD (PNH) or ZWILLE (ZLL)] gene is one of the critical SAM regulators, but the mechanism by which AGO10 modulates the SAM is unknown. In the present study we show that AGO10 genetically represses microRNA165/166 (miR165/166) for SAM maintenance as well as establishment of leaf adaxial-abaxial polarity. Levels of miR165/166 in leaves and embryonic SAMs of pnh/zll/ago10 mutants are abnormally elevated, leading to a reduction in the quantity of homeodomain-leucine zipper (HD-ZIP) III gene transcripts, the targets of miR165/166. This reduction is the primary cause of pnh/zll SAM and leaf defects, because the aberrant pnh/zll phenotypes were partially rescued by either increasing levels of HD-ZIP III transcripts or decreasing levels of miR165/166 in the SAM and leaf. Furthermore, plants with an abnormal apex were more frequent among pnh/zll rdr6 and pnh/zll ago7 double mutants and increased levels of miR165/166 were detected in rdr6 apices. These results indicate that AGO10 and RDR6/AGO7 may act in parallel in modulating accumulation of miR165/166 for normal plant development.

The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity.

Polarity formation is central to leaf morphogenesis, and several key genes that function in adaxial-abaxial polarity establishment have been identified and characterized extensively. We previously reported that Arabidopsis thaliana ASYMMERTIC LEAVES1 (AS1) and AS2 are important in promoting leaf adaxial fates. We obtained an as2 enhancer mutant, asymmetric leaves enhancer3 (ae3), which demonstrated pleiotropic plant phenotypes, including a defective adaxial identity in some leaves. The ae3 as2 double mutant displayed severely abaxialized leaves, which were accompanied by elevated levels of leaf abaxial promoting genes FILAMENTOUS FLOWER, YABBY3, KANADI1 (KAN1), and KAN2 and a reduced level of the adaxial promoting gene REVOLUTA. We identified AE3, which encodes a putative 26S proteasome subunit RPN8a. Furthermore, double mutant combinations of as2 with other 26S subunit mutations, including rpt2a, rpt4a, rpt5a, rpn1a, rpn9a, pad1, and pbe1, all displayed comparable phenotypes with those of ae3 as2, albeit with varying phenotypic severity. Since these mutated genes encode subunits that are located in different parts of the 26S proteasome, it is possible that the proteolytic function of the 26S holoenzyme is involved in leaf polarity formation. Together, our findings reveal that posttranslational regulation is essential in proper leaf patterning.

Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis.

In higher plants, class I KNOTTED1-like homeobox (KNOX) gene suppression and leaf polarity establishment are two processes crucial for leaf morphogenesis. The Arabidopsis genes, ASYMMETRIC LEAVES1 and 2 (AS1 and AS2), are required for repressing the class I KNOX genes and promoting leaf adaxial cell fates. In addition, the RNA-DEPENDENT RNA POLYMERASE6 (RDR6) gene acts synergistically with AS1 and AS2 to specify the adaxial polarity and repress the KNOX genes in leaves. It is known that RDR6 is one of the key components in plant post-transcriptional gene silencing (PTGS), and is likely to function with other silencing components in a genetic pathway in regulating leaf patterning. Here we report phenotypic analyses of double mutants combining as1 or as2 with other mutations relating to different RNA silencing pathways. We show that plants carrying rdr6, suppressor of gene silencing3 (sgs3) or zippy (zip, also called ago7) in combination with as1 or as2 demonstrate severe morphological defects, and the double mutant plants are generally similar to one another. Detailed phenotypic and molecular analyses reveal that leaves of rdr6 as2(1), sgs3 as2(1) and zip as2(1) all show an abnormal adaxial identity, and contain high levels of microRNA165/166 and FILAMENTOUS FLOWER (FIL) transcripts. These results suggest that RDR6, SGS3 and AGO7 act in the same pathway, which genetically interacts with the AS1-AS2 pathway for leaf development. The RDR6-SGS3-AGO7 pathway was previously identified as regulating the plant vegetative phase change. Our results reveal a new function of the pathway, which is also required for normal leaf morphogenesis.

Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity.

The shoot apical meristem (SAM) of seed plants is the site at which lateral organs are formed. Once organ primordia initiate from the SAM, they establish polarity along the adaxial-abaxial, proximodistal and mediolateral axes. Among these three axes, the adaxial-abaxial polarity is of primary importance in leaf patterning. In leaf development, once the adaxial-abaxial axis is established within leaf primordia, it provides cues for proper lamina growth and asymmetric development. It was reported previously that the Arabidopsis ASYMMETRIC LEAVES1 (AS1) and ASYMMETRIC LEAVES2 (AS2) genes are two key regulators of leaf polarity. In this work, we demonstrate a new function of the AS1 and AS2 genes in the establishment of adaxial-abaxial polarity by analyzing as1 and as2 alleles in the Landsberg erecta (Ler) genetic background. We provide genetic evidence that the Arabidopsis ERECTA (ER) gene is involved in the AS1-AS2 pathway to promote leaf adaxial fate. In addition, we show that AS1 and AS2 bind to each other, suggesting that AS1 and AS2 may form a complex that regulates the establishment of leaf polarity. We also report the effects on leaf polarity of overexpression of the AS1 or AS2 genes under the control of the cauliflower mosaic virus (CAMV) 35S promoter. Although plants with as1 and as2 mutations have very similar phenotypes, 35S::AS1/Ler and 35S::AS2/Ler transgenic plants showed dramatically different morphologies. A possible model of the AS1, AS2 and ER action in leaf polarity formation is discussed.