Receptor-like kinase-mediated cell signaling pathways play fundamental roles in many aspects of plant growth and development. A pair of Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinases (LRR-RLKs), HAESA (HAE) and HAESA-LIKE2 (HSL2), have been shown to activate the cell separation process that leads to organ abscission. Another pair of LRR-RLKs, EVERSHED (EVR) and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1, act as inhibitors of abscission, potentially by modulating HAE/HSL2 activity. Cycling of these RLKs to and from the cell surface may be regulated by NEVERSHED (NEV), a membrane trafficking regulator that is essential for organ abscission. We report here the characterization of CAST AWAY (CST), a receptor-like cytoplasmic kinase that acts as a spatial inhibitor of cell separation. Disruption of CST suppresses the abscission defects of nev mutant flowers and restores the discrete identity of the trans-Golgi network in nev abscission zones. After organ shedding, enlarged abscission zones with obscured boundaries are found in nev cst flowers. We show that CST is a dual-specificity kinase in vitro and that myristoylation at its amino terminus promotes association with the plasma membrane. Using the bimolecular fluorescence complementation assay, we have detected interactions of CST with HAE and EVR at the plasma membrane of Arabidopsis protoplasts and hypothesize that CST negatively regulates cell separation signaling directly and indirectly. A model integrating the potential roles of receptor-like kinase signaling and membrane trafficking during organ separation is presented.

SUMMARYThe shoot epidermis of land plants serves as a crucial interface between plants and the atmosphere: pavement cells protect plants from desiccation and other environmental stresses, while stomata facilitate gas exchange and transpiration. Advances have been made in our understanding of stomatal patterning and differentiation, and a set of 'master regulatory' transcription factors of stomatal development have been identified. However, they are limited to specifying stomatal differentiation within the epidermis. Here, we report the identification of an Arabidopsis homeodomain-leucine zipper IV (HD-ZIP IV) protein, HOMEODOMAIN GLABROUS2 (HDG2), as a key epidermal component promoting stomatal differentiation. HDG2 is highly enriched in meristemoids, which are transient-amplifying populations of stomatal-cell lineages. Ectopic expression of HDG2 confers differentiation of stomata in internal mesophyll tissues and occasional multiple epidermal layers. Conversely, a loss-of-function hdg2 mutation delays stomatal differentiation and, rarely but consistently, results in aberrant stomata. A closely related HD-ZIP IV gene, Arabidopsis thaliana MERISTEM LAYER1 (AtML1), shares overlapping function with HDG2: AtML1 overexpression also triggers ectopic stomatal differentiation in the mesophyll layer and atml1 mutation enhances the stomatal differentiation defects of hdg2. Consistently, HDG2 and AtML1 bind the same DNA elements, and activate transcription in yeast. Furthermore, HDG2 transactivates expression of genes that regulate stomatal development in planta. Our study highlights the similarities and uniqueness of these two HD-ZIP IV genes in the specification of protodermal identity and stomatal differentiation beyond predetermined tissue layers.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is a genome editing technology transforming the field of plant biology by virtue of the system's efficiency and specificity. The system has quickly evolved for many diverse applications including multiplex gene mutation, gene replacement and transcriptional control. As CRISPR/Cas9 is increasingly applied to plants, it is becoming clear that each component of the system can be modified to improve editing results. This review aims to highlight common considerations and options when conducting CRISPR/Cas9 experiments.

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. hk5 and hk6 single mutants affected root growth, leaf width, inflorescence architecture, and/or floral development. The double hk5 hk6 mutant had more substantial effects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering is delayed in the hk5 hk6 mutant and the panicle is 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.

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SUMMARY 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.

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Summary 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.