The structure of B-ARR reveals the molecular basis of transcriptional activation by cytokinin.

The phytohormone cytokinin has various roles in plant development, including meristem maintenance, vascular differentiation, leaf senescence, and regeneration. Prior investigations have revealed that cytokinin acts via a phosphorelay similar to the two-component system by which bacteria sense and respond to external stimuli. The eventual targets of this phosphorelay are type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs), containing the conserved N-terminal receiver domain (RD), middle DNA binding domain (DBD), and C-terminal transactivation domain. While it has been established for two decades that the phosphoryl transfer from a specific histidyl residue in ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEINS (AHPs) to an aspartyl residue in the RD of B-ARRs results in a rapid transcriptional response to cytokinin, the underlying molecular basis remains unclear. In this work, we determine the crystal structures of the RD-DBD of ARR1 (ARR1RD-DBD) as well as the ARR1DBD-DNA complex from Arabidopsis. Analyses of the ARR1DBD-DNA complex have revealed the structural basis for sequence-specific recognition of the GAT trinucleotide by ARR1. In particular, comparing the ARR1RD-DBD and ARR1DBD-DNA structures reveals that unphosphorylated ARR1RD-DBD exists in a closed conformation with extensive contacts between the RD and DBD. In vitro and vivo functional assays have further suggested that phosphorylation of the RD weakens its interaction with DBD, subsequently permits the DNA binding capacity of DBD, and promotes the transcriptional activity of ARR1. Our findings thus provide mechanistic insights into phosphorelay activation of gene transcription in response to cytokinin.

Redundant and specific roles of individual MIR172 genes in plant development.

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

A spatiotemporally regulated transcriptional complex underlies heteroblastic development of leaf hairs in Arabidopsis thaliana.

Heteroblasty refers to a phenomenon that a plant produces morphologically or functionally different lateral organs in an age-dependent manner. In the model plant Arabidopsis thaliana, the production of trichomes (epidermal leaf hairs) on the abaxial (lower) side of leaves is a heteroblastic mark for the juvenile-to-adult transition. Here, we show that the heteroblastic development of abaxial trichomes is regulated by a spatiotemporally regulated complex comprising the leaf abaxial fate determinant (KAN1) and the developmental timer (miR172-targeted AP2-like proteins). We provide evidence that a short-distance chromatin loop brings the downstream enhancer element into close association with the promoter elements of GL1, which encodes a MYB transcription factor essential for trichome initiation. During juvenile phase, the KAN1-AP2 repressive complex binds to the downstream sequence of GL1 and represses its expression through chromatin looping. As plants age, the gradual reduction in AP2-like protein levels leads to decreased amount of the KAN1-AP2 complex, thereby licensing GL1 expression and the abaxial trichome initiation. Our results thus reveal a novel molecular mechanism by which a heteroblastic trait is governed by integrating age and leaf polarity cue in plants.

AP2/ERF Transcription Factors Integrate Age and Wound Signals for Root Regeneration.

Age and wounding are two major determinants for regeneration. In plants, the root regeneration is triggered by wound-induced auxin biosynthesis. As plants age, the root regenerative capacity gradually decreases. How wounding leads to the auxin burst and how age and wound signals collaboratively regulate root regenerative capacity are poorly understood. Here, we show that the increased levels of three closely-related miR156-targeted Arabidopsis (Arabidopsis thaliana) SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, SPL2, SPL10, and SPL11, suppress root regeneration with age by inhibiting wound-induced auxin biosynthesis. Mechanistically, we find that a subset of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors including ABSCISIC ACID REPRESSOR1 and ERF109 is rapidly induced by wounding and serves as a proxy for wound signal to induce auxin biosynthesis. In older plants, SPL2/10/11 directly bind to the promoters of AP2/ERFs and attenuates their induction, thereby dampening auxin accumulation at the wound. Our results thus identify AP2/ERFs as a hub for integration of age and wound signal for root regeneration.

A Two-Step Model for de Novo Activation of WUSCHEL during Plant Shoot Regeneration.

Plant cells are totipotent and competent to regenerate from differentiated organs. It has been known for six decades that cytokinin-rich medium induces shoot regeneration from callus cells. However, the underlying molecular mechanism remains elusive. The homeodomain transcription factor WUSCHEL (WUS) is essential for de novo establishment of the shoot stem cell niche in Arabidopsis thaliana We found that WUS-positive (WUS+) cells mark the shoot progenitor region during regeneration. A cytokinin-rich environment initially promotes the removal of the repressive histone mark H3K27me3 at the WUS locus in a cell cycle-dependent manner. Subsequently, the B-type ARABIDOPSIS RESPONSE REGULATORs (ARRs) ARR1, ARR2, ARR10, and ARR12, which function as transcriptional activators in the cytokinin signaling pathway, spatially activate WUS expression through binding with microRNA165/6-targeted HD-ZIP III transcription factors. Thus, our results provide important insights into the molecular framework for cytokinin-directed shoot regeneration and reveal a two-step mechanism for de novo activation of WUS.

An intrinsic microRNA timer regulates progressive decline in shoot regenerative capacity in plants.

Plant cells are totipotent and competent to regenerate from differentiated organs. It has been shown that two phytohormones, auxin and cytokinin, play critical roles within this process. As in animals, the regenerative capacity declines with age in plants, but the molecular basis for this phenomenon remains elusive. Here, we demonstrate that an age-regulated microRNA, miR156, regulates shoot regenerative capacity. As a plant ages, the gradual increase in miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors leads to the progressive decline in shoot regenerative capacity. In old plants, SPL reduces shoot regenerative capacity by attenuating the cytokinin response through binding with the B-type ARABIDOPSIS RESPONSE REGULATORs, which encode the transcriptional activators in the cytokinin signaling pathway. Consistently, the increased amount of exogenous cytokinin complements the reduced shoot regenerative capacity in old plants. Therefore, the recruitment of age cues in response to cytokinin contributes to shoot regenerative competence.

Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA.

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.

Red-light-dependent interaction of phyB with SPA1 promotes COP1-SPA1 dissociation and photomorphogenic development in Arabidopsis.

Arabidopsis phytochromes (phyA-phyE) are photoreceptors dedicated to sensing red/far-red light. Phytochromes promote photomorphogenic developments upon light irradiation via a signaling pathway that involves rapid degradation of PIFs (PHYTOCHROME INTERACTING FACTORS) and suppression of COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) nuclear accumulation, through physical interactions with PIFs and COP1, respectively. Both phyA and phyB, the two best characterized phytochromes, regulate plant photomorphogenesis predominantly under far-red light and red light, respectively. It has been demonstrated that SPA1 (SUPPRESSOR OF PHYTOCHROME A 1) associates with COP1 to promote COP1 activity and suppress photomorphogenesis. Here, we report that the mechanism underlying phyB-promoted photomorphogenesis in red light involves direct physical and functional interactions between red-light-activated phyB and SPA1. We found that SPA1 acts genetically downstream of PHYB to repress photomorphogenesis in red light. Protein interaction studies in both yeast and Arabidopsis demonstrated that the photoactivated phyB represses the association of SPA1 with COP1, which is mediated, at least in part, through red-light-dependent interaction of phyB with SPA1. Moreover, we show that phyA physically interacts with SPA1 in a Pfr-form-dependent manner, and that SPA1 acts downstream of PHYA to regulate photomorphogenesis in far-red light. This study provides a genetic and biochemical model of how photoactivated phyB represses the activity of COP1-SPA1 complex through direct interaction with SPA1 to promote photomorphogenesis in red light.

Temporal control of leaf complexity by miRNA-regulated licensing of protein complexes.

The tremendous diversity of leaf shapes has caught the attention of naturalists for centuries. In addition to interspecific and intraspecific differences, leaf morphologies may differ in single plants according to age, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the progression from the juvenile to the adult phase is characterized by increased leaf serration. A similar trend is seen in species with more complex leaves, such as the A. thaliana relative Cardamine hirsuta, in which the number of leaflets per leaf increases with age. Although the genetic changes that led to the overall simpler leaf architecture in A. thaliana are increasingly well understood, less is known about the events underlying age-dependent changes within single plants, in either A. thaliana or C. hirsuta. Here, we describe a conserved miRNA transcription factor regulon responsible for an age-dependent increase in leaf complexity. In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana and of leaflets in C. hirsuta. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs. These findings point to posttranslational interaction between unrelated miRNA-targeted transcription factors as a core feature of these regulatory circuits.

Sugar is an endogenous cue for juvenile-to-adult phase transition in plants.

The transition from the juvenile to adult phase in plants is controlled by diverse exogenous and endogenous cues such as age, day length, light, nutrients, and temperature. Previous studies have shown that the gradual decline in microRNA156 (miR156) with age promotes the expression of adult traits. However, how age temporally regulates the abundance of miR156 is poorly understood. We show here that the expression of miR156 responds to sugar. Sugar represses miR156 expression at both the transcriptional level and post-transcriptional level through the degradation of miR156 primary transcripts. Defoliation and photosynthetic mutant assays further demonstrate that sugar from the pre-existing leaves acts as a mobile signal to repress miR156, and subsequently triggers the juvenile-to-adult phase transition in young leaf primordia. We propose that the gradual increase in sugar after seed germination serves as an endogenous cue for developmental timing in plants. DOI:http://dx.doi.org/10.7554/eLife.00269.001.

Molecular basis of age-dependent vernalization in Cardamine flexuosa.

Plants flower in response to many varied cues, such as temperature, photoperiod, and age. The floral transition of Cardamine flexuosa, a herbaceous biennial-to-perennial plant, requires exposure to cold temperature, a treatment known as vernalization. C. flexuosa younger than 5 weeks old are not fully responsive to cold treatment. We demonstrate that the levels of two age-regulated microRNAs, miR156 and miR172, regulate the timing of sensitivity in response to vernalization. Age and vernalization pathways coordinately regulate flowering through modulating the expression of CfSOC1, a flower-promoting MADS-box gene. The related annual Arabidopsis thaliana, which has both vernalization and age pathways, does not possess an age-dependent vernalization response. Thus, the recruitment of age cue in response to environmental signals contributes to the evolution of life cycle in plants.