Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways.

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

Author Correction: A B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot development.

The original version of this Article contained an error in Fig. 3. Panel b was inadvertently duplicated and the correct panel c was originally omitted. This error has been corrected in both the PDF and HTML versions of the Article.

A B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot development.

Cytokinin fulfills its diverse roles in planta through a series of transcriptional responses. We identify the in vivo DNA binding site profiles for three genetically redundant type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs): ARR1, ARR10, and ARR12. The expression and genome-wide DNA binding locations of the three B-ARRs extensively overlap. Constructing a primary cytokinin response transcriptional network reveals a recurring theme of widespread cross-regulation between the components of the cytokinin pathway and other plant hormone pathways. The B-ARRs are found to have similar DNA binding motifs, though sequences flanking the core motif were degenerate. Cytokinin treatments amalgamate the three different B-ARRs motifs to identical DNA binding signatures (AGATHY, H(a/t/c), Y(t/c)) which suggests cytokinin may regulate binding activity of B-ARR family members. Furthermore, we find that WUSCHEL, a key gene required for apical meristem maintenance, is a cytokinin-dependent B-ARR target gene, demonstrating the importance of the cytokinin transcription factor network in shoot development.

Cytokinin stabilizes WUSCHEL by acting on the protein domains required for nuclear enrichment and transcription.

Concentration-dependent transcriptional regulation and the spatial regulation of transcription factor levels are poorly studied in plant development. WUSCHEL, a stem cell-promoting homeodomain transcription factor, accumulates at a higher level in the rib meristem than in the overlying central zone, which harbors stem cells in the shoot apical meristems of Arabidopsis thaliana. The differential accumulation of WUSCHEL in adjacent cells is critical for the spatial regulation and levels of CLAVATA3, a negative regulator of WUSCHEL transcription. Earlier studies have revealed that DNA-dependent dimerization, subcellular partitioning and protein destabilization control WUSCHEL protein levels and spatial accumulation. Moreover, the destabilization of WUSCHEL may also depend on the protein concentration. However, the roles of extrinsic spatial cues in maintaining differential accumulation of WUS are not understood. Through transient manipulation of hormone levels, hormone response patterns and analysis of the receptor mutants, we show that cytokinin signaling in the rib meristem acts through the transcriptional regulatory domains, the acidic domain and the WUSCHEL-box, to stabilize the WUS protein. Furthermore, we show that the same WUSCHEL-box functions as a degron sequence in cytokinin deficient regions in the central zone, leading to the destabilization of WUSCHEL. The coupled functions of the WUSCHEL-box in nuclear retention as described earlier, together with cytokinin sensing, reinforce higher nuclear accumulation of WUSCHEL in the rib meristem. In contrast a sub-threshold level may expose the WUSCHEL-box to destabilizing signals in the central zone. Thus, the cytokinin signaling acts as an asymmetric spatial cue in stabilizing the WUSCHEL protein to lead to its differential accumulation in neighboring cells, which is critical for concentration-dependent spatial regulation of CLAVATA3 transcription and meristem maintenance. Furthermore, our work shows that cytokinin response is regulated independently of the WUSCHEL function which may provide robustness to the regulation of WUSCHEL concentration.

A high-resolution gene expression map of the Arabidopsis shoot meristem stem cell niche.

The shoot apical meristem (SAM) acts as a reservoir for stem cells. The central zone (CZ) harbors stem cells. The stem cell progenitors differentiate in the adjacent peripheral zone and in the rib meristem located just beneath the CZ. The SAM is further divided into distinct clonal layers: the L1 epidermal, L2 sub-epidermal and L3 layers. Collectively, SAMs are complex structures that consist of cells of different clonal origins that are organized into functional domains. By employing fluorescence-activated cell sorting, we have generated gene expression profiles of ten cell populations that belong to different clonal layers as well as domains along the central and peripheral axis. Our work reveals that cells in distinct clonal layers exhibit greater diversity in gene expression and greater transcriptional complexity than clonally related cell types in the central and peripheral axis. Assessment of molecular functions and biological processes reveals that epidermal cells express genes involved in pathogen defense: the L2 layer cells express genes involved in DNA repair pathways and telomere maintenance, and the L3 layers express transcripts involved in ion balance and salt tolerance besides photosynthesis. Strikingly, the stem cell-enriched transcriptome comprises very few hormone-responsive transcripts. In addition to providing insights into the expression profiles of hundreds of transcripts, the data presented here will act as a resource for reverse genetic analysis and will be useful in deciphering molecular pathways involved in cell type specification and their functions.

Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis.

The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue. Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways. To identify additional components or mechanisms, we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern. Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines, the ahk3cre1 cytokinin receptor mutant, and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation, whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon. Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern. Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

Towards a functional understanding of cell growth dynamics in shoot meristem stem-cell niche.

Shoot apical meristems (SAMs) harbor a set of stem-cells which supply cells for the development of all above-ground structures. A precise spatio-temporal control of growth patterns in stem-cells and the differentiating progeny is critical to maintain a stable set of stem-cells. In recent years, an array of approaches including molecular genetics, transient perturbations, live-imaging, image processing and mathematical modeling have been employed to study the cellular dynamics. In this article, we highlight recent studies that link cell-cell communication mechanisms to cell mechanics and overall growth control that govern stem-cell homeostasis and morphogenesis in SAMs.

Protein-protein interactions of tandem affinity purified protein kinases from rice.

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

Gene expression map of the Arabidopsis shoot apical meristem stem cell niche.

Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a high-resolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell-cell communication networks and epigenetic modifications.

A rice kinase-protein interaction map.

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Structural and functional insights into the regulation of Arabidopsis AGC VIIIa kinases.

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta.

Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes.

Plant development is characterized by precise control of gene regulation, leading to the correct spatial and temporal tissue patterning. We have characterized the Arabidopsis jabba-1D (jba-1D) mutant, which displays multiple enlarged shoot meristems, radialized leaves, reduced gynoecia and vascular defects. The jba-1D meristem phenotypes require WUSCHEL (WUS) activity, and correlate with a dramatic increase in WUS expression levels. We demonstrate that the jba-1D phenotypes are caused by over-expression of miR166g, and require the activity of the RNase III helicase DCL1. miR166g over-expression in jba-1D plants affects the transcripts of several class III homeodomain-leucine zipper (AtHD-ZIP) family target genes. The expression of PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA) is significantly reduced in a jba-1D background, while REVOLUTA (REV) expression is elevated and ATHB8 is unchanged. In addition, we show that miR166 has a dynamic expression pattern in wild-type and jba-1D embryos. Our analysis demonstrates an indirect role for miRNAs in controlling meristem formation via regulation of WUS expression, and reveals complex regulation of the class III AtHD-ZIP gene family.

Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.

Both acidic and basic amino acids in an amphitropic enzyme, CTP:phosphocholine cytidylyltransferase, dictate its selectivity for anionic membranes.

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.