Summary: N6‐adenosine methylation (m⁶A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m⁶A writer proteins in Arabidopsis thaliana. The components required for m⁶A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m⁶A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m⁶A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m⁶A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

The apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

Dirigent (DIR) proteins were found to mediate regio- and stereoselectivity of bimolecular phenoxy radical coupling during lignan biosynthesis. Here we summarize the current knowledge of the importance of DIR proteins in lignan and lignin biosynthesis and highlight their possible importance in plant development. We focus on the still rather enigmatic Arabidopsis DIR gene family, discussing the few members with known functional importance. We comment on recent discoveries describing the detailed structure of two DIR proteins with implications in the mechanism of DIR-mediated catalysis. Further, we summarize the ample evidence for stress-induced dirigent gene expression, suggesting the role of DIRs in adaptive responses. In the second part of our work, we present a preliminary bioinformatics-based characterization of the AtDIR family. The phylogenetic analysis of AtDIRs complemented by comparison with DIR proteins of mostly known function from other species allowed us to suggest possible roles for several members of this family and identify interesting AtDIR targets for further study. Finally, based on the available metadata and our in silico analysis of AtDIR promoters, we hypothesize about the existence of specific transcriptional controls for individual AtDIR genes and implicate them in various stress responses, hormonal regulations, and developmental processes.

Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta . Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1^H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.