Cytokinin: From autoclaved DNA to two-component signaling.

Since its first identification in the 1950s as a regulator of cell division, cytokinin has been linked to many physiological processes in plants, spanning growth and development and various responses to the environment. Studies from the last two and one-half decades have revealed the pathways underlying the biosynthesis and metabolism of cytokinin and have elucidated the mechanisms of its perception and signaling, which reflects an ancient signaling system evolved from two-component elements in bacteria. Mutants in the genes encoding elements involved in these processes have helped refine our understanding of cytokinin functions in plants. Further, recent advances have provided insight into the mechanisms of intracellular and long-distance cytokinin transport and the identification of several proteins that operate downstream of cytokinin signaling. Here, we review these processes through a historical lens, providing an overview of cytokinin metabolism, transport, signaling, and functions in higher plants.

Ethylene-triggered subcellular trafficking of CTR1 enhances the response to ethylene gas.

The phytohormone ethylene controls plant growth and stress responses. Ethylene-exposed dark-grown Arabidopsis seedlings exhibit dramatic growth reduction, yet the seedlings rapidly return to the basal growth rate when ethylene gas is removed. However, the underlying mechanism governing this acclimation of dark-grown seedlings to ethylene remains enigmatic. Here, we report that ethylene triggers the translocation of the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling, from the endoplasmic reticulum to the nucleus. Nuclear-localized CTR1 stabilizes the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor by interacting with and inhibiting EIN3-BINDING F-box (EBF) proteins, thus enhancing the ethylene response and delaying growth recovery. Furthermore, Arabidopsis plants with enhanced nuclear-localized CTR1 exhibited improved tolerance to drought and salinity stress. These findings uncover a mechanism of the ethylene signaling pathway that links the spatiotemporal dynamics of cellular signaling components to physiological responses.

Integrated omics reveal novel functions and underlying mechanisms of the receptor kinase FERONIA in Arabidopsis thaliana.

The receptor kinase FERONIA (FER) is a versatile regulator of plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new FER functions, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of Arabidopsis (Arabidopsis thaliana) wild-type and fer-4 loss-of-function mutant plants. Gene ontology terms for phytohormone signaling, abiotic stress, and biotic stress were significantly enriched among differentially expressed transcripts, differentially abundant proteins, and/or misphosphorylated proteins, in agreement with the known roles for FER in these processes. Analysis of multiomics data and subsequent experimental evidence revealed previously unknown functions for FER in endoplasmic reticulum (ER) body formation and glucosinolate biosynthesis. FER functions through the transcription factor NAI1 to mediate ER body formation. FER also negatively regulates indole glucosinolate biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors is hypophosphorylated in the fer-4 mutant and demonstrated that FER acts through the transcription factor ABA INSENSITIVE5 (ABI5) to negatively regulate the ABA response during cotyledon greening. Our integrated omics study, therefore, reveals novel functions for FER and provides new insights into the underlying mechanisms of FER function.

Meta-analysis of transcriptomic studies of cytokinin-treated rice roots defines a core set of cytokinin response genes.

Cytokinins regulate diverse aspects of plant growth and development, primarily through modulation of gene expression. The cytokinin-responsive transcriptome has been thoroughly described in dicots, especially Arabidopsis, but much less so in monocots. Here, we present a meta-analysis of five different transcriptomic analyses of rice (Oryza sativa) roots treated with cytokinin, including three previously unpublished experiments. We developed a treatment method in which hormone is added to the media of rice seedlings grown in sterile hydroponic culture under a continuous airflow, which resulted in minimal perturbation of the seedlings, thus greatly reducing changes in gene expression in the absence of exogenous hormone. We defined a core set of 205 upregulated and 86 downregulated genes that were differentially expressed in at least three of the transcriptomic datasets. This core set includes genes encoding the type-A response regulators (RRs) and cytokinin oxidases/dehydrogenases, which have been shown to be primary cytokinin response genes. GO analysis revealed that the upregulated genes were enriched for terms related to cytokinin/hormone signaling and metabolism, while the downregulated genes were significantly enriched for genes encoding transporters. Variations of type-B RR binding motifs were significantly enriched in the promoters of the upregulated genes, as were binding sites for other potential partner transcription factors. The promoters of the downregulated genes were generally enriched for distinct cis-acting motifs and did not include the type-B RR binding motif. This analysis provides insight into the molecular mechanisms underlying cytokinin action in a monocot and provides a useful foundation for future studies of this hormone in rice and other cereals.

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis.

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.

Functional Analysis of the Rice Type-B Response Regulator RR22.

The phytohormone cytokinin plays a critical role in regulating growth and development throughout the life cycle of the plant. The primary transcriptional response to cytokinin is mediated by the action of the type-B response regulators (RRs), with much of our understanding for their functional roles being derived from studies in the dicot Arabidopsis. To examine the roles played by type-B RRs in a monocot, we employed gain-of-function and loss-of-function mutations to characterize RR22 function in rice. Ectopic overexpression of RR22 in rice results in an enhanced cytokinin response based on molecular and physiological assays. Phenotypes associated with enhanced activity of RR22 include effects on leaf and root growth, inflorescence architecture, and trichome formation. Analysis of four Tos17 insertion alleles of RR22 revealed effects on inflorescence architecture, trichomes, and development of the stigma brush involved in pollen capture. Both loss- and gain-of-function RR22 alleles affected the number of leaf silica-cell files, which provide mechanical stability and improve resistance to pathogens. Taken together, these results indicate that a delicate balance of cytokinin transcriptional activity is necessary for optimal growth and development in rice.

EXO70D isoforms mediate selective autophagic degradation of type-A ARR proteins to regulate cytokinin sensitivity.

The phytohormone cytokinin influences many aspects of plant growth and development, several of which also involve the cellular process of autophagy, including leaf senescence, nutrient remobilization, and developmental transitions. The Arabidopsis type-A response regulators (type-A ARR) are negative regulators of cytokinin signaling that are transcriptionally induced in response to cytokinin. Here, we describe a mechanistic link between cytokinin signaling and autophagy, demonstrating that plants modulate cytokinin sensitivity through autophagic regulation of type-A ARR proteins. Type-A ARR proteins were degraded by autophagy in an AUTOPHAGY-RELATED (ATG)5-dependent manner, and this degradation is promoted by phosphorylation on a conserved aspartate in the receiver domain of the type-A ARRs. EXO70D family members interacted with type-A ARR proteins, likely in a phosphorylation-dependent manner, and recruited them to autophagosomes via interaction of the EXO70D AIM with the core autophagy protein, ATG8. Consistently, loss-of-function exo70D1,2,3 mutants exhibited compromised targeting of type-A ARRs to autophagic vesicles, have elevated levels of type-A ARR proteins, and are hyposensitive to cytokinin. Disruption of both type-A ARRs and EXO70D1,2,3 compromised survival in carbon-deficient conditions, suggesting interaction between autophagy and cytokinin responsiveness in response to stress. These results indicate that the EXO70D proteins act as selective autophagy receptors to target type-A ARR cargos for autophagic degradation, demonstrating modulation of cytokinin signaling by selective autophagy.

The HK5 and HK6 cytokinin receptors mediate diverse developmental pathways in rice.

The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice (Oryza sativa) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.

Mutagenomics: A Rapid, High-Throughput Method to Identify Causative Mutations from a Genetic Screen.

Genetic screens are powerful tools to dissect complex biological processes, but a rate-limiting step is often the cloning of targeted genes. Here, we present a strategy, "mutagenomics," to identify causal mutations from a screen in a high throughput fashion in the absence of backcrossing. Mutagenomics is initiated by sequencing the genomes of the mutants identified, which are then subjected to a three-stage pipeline. The first stage identifies sequence changes in genes previously linked to the targeted pathway. The second stage uses heuristics derived from a simulation strategy to identify genes that are represented by multiple independent alleles more often than expected by chance. The third stage identifies candidate genes for the remaining lines by sequencing multiple lines of common descent. Our simulations indicate that sequencing as few as three to four sibling lines generally results in fewer than five candidate genes. We applied mutagenomics to a screen for Arabidopsis (Arabidopsis thaliana) mutants involved in the response to the phytohormone cytokinin. Mutagenomics identified likely causative genes for many of the mutant lines analyzed from this screen, including 13 alleles of the gene encoding the ARABIDOPSIS HIS KINASE4 cytokinin receptor. The screen also identified 1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE7, an ACC synthase homolog involved in ethylene biosynthesis, and ELONGATED HYPOCOTYL5 (HY5), a master transcriptional regulator of photomorphogenesis. HY5 was found to mediate a subset of the transcriptional response to cytokinin. Mutagenomics has the potential to accelerate the pace and utility of genetic screens in Arabidopsis.

Dynamic Construction, Perception, and Remodeling of Plant Cell Walls.

Plant cell walls are dynamic structures that are synthesized by plants to provide durable coverings for the delicate cells they encase. They are made of polysaccharides, proteins, and other biomolecules and have evolved to withstand large amounts of physical force and to resist external attack by herbivores and pathogens but can in many cases expand, contract, and undergo controlled degradation and reconstruction to facilitate developmental transitions and regulate plant physiology and reproduction. Recent advances in genetics, microscopy, biochemistry, structural biology, and physical characterization methods have revealed a diverse set of mechanisms by which plant cells dynamically monitor and regulate the composition and architecture of their cell walls, but much remains to be discovered about how the nanoscale assembly of these remarkable structures underpins the majestic forms and vital ecological functions achieved by plants.

Type-B response regulators of rice play key roles in growth, development and cytokinin signaling.

Cytokinins are plant hormones with crucial roles in growth and development. Although cytokinin signaling is well characterized in the model dicot Arabidopsis, we are only beginning to understand its role in monocots, such as rice (Oryza sativa) and other cereals of agronomic importance. Here, we used primarily a CRISPR/Cas9 gene-editing approach to characterize the roles of a key family of transcription factors, the type-B response regulators (RRs), in cytokinin signaling in rice. Results from the analysis of single rr mutants as well as higher-order rr21/22/23 mutant lines revealed functional overlap as well as subfunctionalization within members of the gene family. Mutant phenotypes associated with decreased activity of rice type-B RRs included effects on leaf and root growth, inflorescence architecture, flower development and fertilization, trichome formation and cytokinin sensitivity. Development of the stigma brush involved in pollen capture was compromised in the rr21/22/23 mutant, whereas anther development was compromised in the rr24 mutant. Novel as well as conserved roles for type-B RRs in the growth and development of a monocot compared with dicots were identified.

The Regulation of Cellulose Biosynthesis in Plants.

Cell walls define the shape of plant cells, controlling the extent and orientation of cell elongation, and hence organ growth. The main load-bearing component of plant cell walls is cellulose, and how plants regulate its biosynthesis during development and in response to various environmental perturbations is a central question in plant biology. Cellulose is synthesized by cellulose synthase (CESA) complexes (CSCs) that are assembled in the Golgi apparatus and then delivered to the plasma membrane (PM), where they actively synthesize cellulose. CSCs travel along cortical microtubule paths that define the orientation of synthesis of the cellulose microfibrils. CSCs recycle between the PM and various intracellular compartments, and this trafficking plays an important role in determining the level of cellulose synthesized. In this review, we summarize recent findings in CESA complex organization, CESA posttranslational modifications and trafficking, and other components that interact with CESAs. We also discuss cell wall integrity maintenance, with a focus on how this impacts cellulose biosynthesis.

Using indCAPS to Detect CRISPR/Cas9 Induced Mutations.

Cleaved amplified polymorphic sequences (CAPS) assays are useful tools for detecting small mutations such as single nucleotide polymorphisms (SNPs) or insertion/deletions (indels) present in an amplified DNA fragment. A mutation that disrupts or creates a restriction site will prevent cleavage by a restriction enzyme, allowing discrimination of wild-type and mutant alleles. In cases where no convenient restriction site is present, a derived Cleaved Amplified Polymorphic Sequence (dCAPS) assay can be used, where mismatches in the primer are used to create a diagnostic restriction site. No special design constraints are present for a CAPS assay, but cases where CAPS assays can be used are infrequent. A dCAPS assay can be burdensome to design by hand, but it is more broadly applicable. This protocol will describe the use of the indCAPS tool for the design of CAPS and dCAPS primers. The indCAPS tool was designed to be compatible with indel alleles, which prior tools struggled with but have increased importance since the rise of CRISPR/Cas9 mutagenesis methods.

1-Aminocyclopropane 1-Carboxylic Acid and Its Emerging Role as an Ethylene-Independent Growth Regulator.

1-Aminocyclopropane 1-carboxylic acid (ACC) is the direct precursor of the plant hormone ethylene. ACC is synthesized from S-adenosyl-L-methionine (SAM) by ACC synthases (ACSs) and subsequently oxidized to ethylene by ACC oxidases (ACOs). Exogenous ACC application has been used as a proxy for ethylene in numerous studies as it is readily converted by nearly all plant tissues to ethylene. However, in recent years, a growing body of evidence suggests that ACC plays a signaling role independent of the biosynthesis. In this review, we briefly summarize our current knowledge of ACC as an ethylene precursor, and present new findings with regards to the post-translational modifications of ACS proteins and to ACC transport. We also summarize the role of ACC in regulating plant development, and its involvement in cell wall signaling, guard mother cell division, and pathogen virulence.

Cytokinin modulates context-dependent chromatin accessibility through the type-B response regulators.

The phytohormone cytokinin regulates diverse aspects of plant growth and development, probably through context-dependent transcriptional regulation that relies on a dynamic interplay between regulatory proteins and chromatin. We employed the assay for transposase accessible chromatin with sequencing to profile changes in the chromatin landscape of Arabidopsis roots and shoots in response to cytokinin. Our results reveal differentially accessible chromatin regions indicative of dynamic regulation in response to cytokinin. These changes in chromatin occur preferentially upstream of cytokinin-regulated genes. The changes also largely overlap with binding sites for the type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs), transcription factors that mediate the primary response to cytokinin. Furthermore, the type-B ARRs were found to be necessary for the changes in chromatin state in response to cytokinin. Last, we identified context-dependent responses by comparing root and shoot profiles. This study provides new insight into the dynamics between cytokinin and chromatin with regard to directing transcriptional programmes and how cytokinin mediates its pleiotropic effects.

SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane.

Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.

Coordination of Chloroplast Development through the Action of the GNC and GLK Transcription Factor Families.

Fundamental questions regarding how chloroplasts develop from proplastids remain poorly understood despite their central importance to plant life. Two families of nuclear transcription factors, the GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and GOLDEN TWO-LIKE (GLK) families, have been implicated in directly and positively regulating chloroplast development. Here, we determined the degree of functional overlap between the two transcription factor families in Arabidopsis (Arabidopsis thaliana), characterizing their ability to regulate chloroplast biogenesis both alone and in concert. We determined the DNA-binding motifs for GNC and GLK2 using protein-binding microarrays; the enrichment of these motifs in transcriptome datasets indicates that GNC and GLK2 are repressors and activators of gene expression, respectively. ChIP-seq analysis of GNC identified PHYTOCHROME INTERACTING FACTOR and brassinosteroid activity genes as targets whose repression by GNC facilitates chloroplast biogenesis. In addition, GNC targets and represses genes involved in ERECTA signaling and thereby facilitates stomatal development. Our results define key regulatory features of the GNC and GLK transcription factor families that contribute to the control of chloroplast biogenesis and photosynthetic activity, including areas of independence and cross talk.

Role of BASIC PENTACYSTEINE transcription factors in a subset of cytokinin signaling responses.

Cytokinin plays diverse roles in plant growth and development, generally acting by modulating gene transcription in target tissues. The type-B Arabidopsis response regulators (ARR) transcription factors have emerged as primary targets of cytokinin signaling and are required for essentially all cytokinin-mediated changes in gene expression. The diversity of cytokinin function is likely imparted by the activity of various transcription factors working with the type-B ARRs to alter specific sets of target genes. One potential set of co-regulators modulating the cytokinin response are the BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family of plant-specific transcription factors. Here, we show that disruption of multiple BPCs results in reduced sensitivity to cytokinin. Further, the BPCs are necessary for the induction of a subset of genes in response to cytokinin. We identified direct in vivo targets of BPC6 using ChIP-Seq and found an enrichment of promoters of genes differentially expressed in response to cytokinin. Further, a significant number of BPC6 regulated genes are also direct targets of the type-B ARRs. Potential cis-binding elements for a number of other transcription factors linked to cytokinin action are enriched in the BPC binding fragments, including those for the cytokinin response factors (CRFs). In addition, several BPCs interact with a subset of type-A ARRs. Consistent with these results, a significant number of genes whose expression is altered in bpc mutant roots are also mis-expressed in crf1,3,5,6 and type-A arr3,4,5,6,7,8,9,15 mutant roots. These results suggest that the BPCs are part of a complex network of transcription factors that are involved in the response to cytokinin.