Summary: Cytokinins are hormones that regulate many developmental and physiological processes in plants. Recent work has revealed that the cytokinin signal is transduced by two‐component systems to the nucleus where target genes are activated. Most of the rapid transcriptional responses are unknown. We measured immediate‐early and delayed cytokinin responses through genome‐wide expression profiling with the Affymetrix ATH1 full genome array (Affymetrix Inc., Santa Clara, CA, USA). Fifteen minutes after cytokinin treatment of 5‐day‐old Arabidopsis seedlings, 71 genes were upregulated and 11 genes were downregulated. Immediate‐early cytokinin response genes include a high portion of transcriptional regulators, among them six transcription factors that had previously not been linked to cytokinin. Five plastid transcripts were rapidly regulated as well, indicating a rapid transfer of the signal to plastids or direct perception of the cytokinin signal by plastids. After 2 h of cytokinin treatment genes coding for transcriptional regulators, signaling proteins, developmental and hormonal regulators, primary and secondary metabolism, energy generation and stress reactions were over‐represented. A significant number of the responding genes are known to regulate light (PHYA, PSK1, CIP8, PAT1, APRR), auxin (Aux/IAA), ethylene (ETR2, EIN3, ERFs/EREBPs), gibberellin (GAI, RGA1, GA20 oxidase), nitrate (NTR2, NIA) and sugar (STP1, SUS1) dependent processes, indicating intense crosstalk with environmental cues, other hormones and metabolites. Analysis of cytokinin‐deficient 35S:AtCKX1 transgenic seedlings has revealed additional, long‐lasting cytokinin‐sensitive changes of transcript abundance. Comparative overlay‐analysis with the software tool mapman identified previously unknown cytokinin‐sensitive metabolic genes, for example in the metabolism of trehalose‐6‐phosphate. Taken together, we present a genome‐wide view of changes in cytokinin‐responsive transcript abundance of genes that might be functionally relevant for the many biological processes that are governed by cytokinins.

Background: The plant hormone cytokinin regulates growth and development of roots and shoots in opposite ways. In shoots it is a positive growth regulator whereas it inhibits growth in roots. It may be assumed that organ-specific regulation of gene expression is involved in these differential activities, but little is known about it. To get more insight into the transcriptional events triggered by cytokinin in roots and shoots, we studied genome-wide gene expression in cytokinin-treated and cytokinin-deficient roots and shoots.Results: It was found by principal component analysis of the transcriptomic data that the immediate-early response to a cytokinin stimulus differs from the later response, and that the transcriptome of cytokinin-deficient plants is different from both the early and the late cytokinin induction response. A higher cytokinin status in the roots activated the expression of numerous genes normally expressed predominantly in the shoot, while a lower cytokinin status in the shoot reduced the expression of genes normally more active in the shoot to a more root-like level. This shift predominantly affected nuclear genes encoding plastid proteins. An organ-specific regulation was assigned to a number of genes previously known to react to a cytokinin signal, including root-specificity for the cytokinin hydroxylase geneCYP735A2and shoot specificity for the cell cycle regulator geneCDKA;1. Numerous cytokinin-regulated genes were newly discovered or confirmed, including the meristem regulator genesSHEPHERDandCLAVATA1, auxin-related genes (IAA7,IAA13,AXR1, PIN2, PID), several genes involved in brassinosteroid (CYP710A1,CYP710A2,DIM/DWF) and flavonol (MYB12,CHS,FLS1) synthesis, various transporter genes (e.g.HKT1), numerous members of the AP2/ERF transcription factor gene family, genes involved in light signalling (PhyA,COP1,SPA1), and more than 80 ribosomal genes. However, contrasting with the fundamental difference of the growth response of roots and shoots to the hormone, the vast majority of the cytokinin-regulated transcriptome showed similar response patterns in roots and shoots.Conclusions: The shift of the root and shoot transcriptomes towards the respective other organ depending on the cytokinin status indicated that the hormone determines part of the organ-specific transcriptome pattern independent of morphological organ identity. Numerous novel cytokinin-regulated genes were discovered which had escaped earlier discovery, most probably due to unspecific sampling. These offer novel insights into the diverse activities of cytokinin, including crosstalk with other hormones and different environmental cues, identify the AP2/ERF class of transcriptions factors as particularly cytokinin sensitive, and also suggest translational control of cytokinin-induced changes.

The signal transduction of the phytohormone cytokinin is mediated by a multistep histidine-to-aspartate phosphorelay system. One component of this system are B-type response regulators, transcription factors mediating at least part of the response to cytokinin. In planta functional analysis of this family is hampered by the high level of functional redundancy of its 11 members. We generated a dominant repressor version of the Arabidopsis (Arabidopsis thaliana) response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology in order to study the extent of the contribution of B-type response regulators to cytokinin activities. In a protoplast test system, ARR1-SRDX suppressed ARR6:β-glucuronidase reporter gene activation by different B-type ARRs. 35S:ARR1-SRDX transgenic Arabidopsis plants showed phenotypic changes reminiscent of plants with a reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system, and larger seeds. Several bioassays showed that 35S:ARR1-SRDX plants have an increased resistance toward cytokinin. The rapid induction of a large part of the cytokinin response genes was dampened. The transcript levels of more than 500 genes were more than 2.5-fold reduced in 35S:ARR1-SRDX transgenic seedlings, suggesting a broad function of B-type ARRs. Collectively, the suppression of pleiotropic cytokinin activities by a dominant repressor version of a B-type ARR indicates that this protein family is involved in mediating most, if not all, of the cytokinin activities in Arabidopsis. In addition, a role for B-type ARRs in mediating cross talk with other pathways is supported by the resistance of 35S:ARR1-SRDX seeds to phytochrome B-mediated inhibition of germination by far-red light. This study demonstrates the usefulness of chimeric repressor silencing technology to overcome redundancy in transcription factor families for functional studies.

RNA editing occurs in the endosymbiont organelles of higher plants as C-to-U conversions of defined nucleotides. The availability of large quantities of RNA sequencing data makes it possible to identify RNA editing sites and to quantify their editing extent. We have investigated RNA editing in 34 protein-coding mitochondrial transcripts of four Populus species, a genus noteworthy for its remarkably small number of RNA editing sites compared to other angiosperms. 27 of these transcripts were subject to RNA editing in at least one species. In total, 355 RNA editing sites were identified with high confidence, their editing extents ranging from 10 to 100%. The most heavily edited transcripts were ccmC with the highest density of RNA editing sites (50.5 sites / kb) and ccmFn with the highest number of sites (39 sites). Most of the editing events are at position 1 or 2 of the codons, usually altering the encoded amino acid, and are highly conserved among the species, also with regard to their editing extent. However, one SNP was found in the newly sequenced and annotated mitochondrial genome of P. alba resulting in the loss of an RNA editing site compared to P. tremula and P. davidiana. This SNP causes a C-to-T transition and an amino acid exchange from Ser to Phe, highlighting the widely discussed role of RNA editing in compensating mutations.

The genome-wide transcriptional response of the model organism Arabidopsis thaliana to cytokinin has been investigated by different research groups as soon as large-scale transcriptomic techniques became affordable. Over the last ten years many transcriptomic datasets related to cytokinin have been generated using different technological platforms, some of which are published only in databases, culminating in an RNA sequencing experiment. Two approaches have been made to establish a core set of cytokinin-regulated transcripts by meta-analysis of these datasets using different preferences regarding their selection. Here we add another meta-analysis derived from an independent microarray platform (CATMA), combine all the meta-analyses available with RNAseq data in order to establish an advanced core set of cytokinin-regulated transcripts, and compare the results with the regulation of orthologous rice genes by cytokinin. We discuss the functions of some of the less known cytokinin-regulated genes indicating areas deserving further research to explore cytokinin function. Finally, we investigate the promoters of the core set of cytokinin-induced genes for the abundance and distribution of known cytokinin-responsive cis elements and identify a set of novel candidate motifs.

The phytohormone cytokinin plays a key role in regulating plant growth and development, and is involved in numerous physiological responses to environmental changes. The type-B response regulators, which regulate the transcription of cytokinin response genes, are a part of the cytokinin signaling system. Arabidopsis thaliana encodes 11 type-B response regulators (type-B ARRs), and some of them were shown to bind in vitro to the core cytokinin response motif (CRM) 5′-(A/G)GAT(T/C)-3′ or, in the case of ARR1, to an extended motif (ECRM), 5′-AAGAT(T/C)TT-3′. Here we obtained in planta proof for the functionality of the latter motif. Promoter deletion analysis of the primary cytokinin response gene ARR6 showed that a combination of two extended motifs within the promoter is required to mediate the full transcriptional activation by ARR1 and other type-B ARRs. CRMs were found to be over-represented in the vicinity of ECRMs in the promoters of cytokinin-regulated genes, suggesting their functional relevance. Moreover, an evolutionarily conserved 27 bp long T-rich region between −220 and −193 bp was identified and shown to be required for the full activation by type-B ARRs and the response to cytokinin. This novel enhancer is not bound by the DNA-binding domain of ARR1, indicating that additional proteins might be involved in mediating the transcriptional cytokinin response. Furthermore, genome-wide expression profiling identified genes, among them ARR16, whose induction by cytokinin depends on both ARR1 and other specific type-B ARRs. This together with the ECRM/CRM sequence clustering indicates cooperative action of different type-B ARRs for the activation of particular target genes.