Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis.

Cytokinins are N(6)-substituted adenine derivatives that play diverse roles in plant growth and development. We sought to define a robust set of genes regulated by cytokinin as well as to query the response of genes not represented on microarrays. To this end, we performed a meta-analysis of microarray data from a variety of cytokinin-treated samples and used RNA-seq to examine cytokinin-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Microarray meta-analysis using 13 microarray experiments combined with empirically defined filtering criteria identified a set of 226 genes differentially regulated by cytokinin, a subset of which has previously been validated by other methods. RNA-seq validated about 73% of the up-regulated genes identified by this meta-analysis. In silico promoter analysis indicated an overrepresentation of type-B Arabidopsis response regulator binding elements, consistent with the role of type-B Arabidopsis response regulators as primary mediators of cytokinin-responsive gene expression. RNA-seq analysis identified 73 cytokinin-regulated genes that were not represented on the ATH1 microarray. Representative genes were verified using quantitative reverse transcription-polymerase chain reaction and NanoString analysis. Analysis of the genes identified reveals a substantial effect of cytokinin on genes encoding proteins involved in secondary metabolism, particularly those acting in flavonoid and phenylpropanoid biosynthesis, as well as in the regulation of redox state of the cell, particularly a set of glutaredoxin genes. Novel splicing events were found in members of some gene families that are known to play a role in cytokinin signaling or metabolism. The genes identified in this analysis represent a robust set of cytokinin-responsive genes that are useful in the analysis of cytokinin function in plants.

De-etiolated 1 and damaged DNA binding protein 1 interact to regulate Arabidopsis photomorphogenesis.

BACKGROUND: Plant development is exquisitely sensitive to light. Seedlings grown in the dark have a developmentally arrested etiolated phenotype, whereas in the light they develop leaves and complete their life cycle. Arabidopsis de-etiolated 1 (det1) mutants develop like light-grown seedlings even when grown in the dark. DET1 encodes a nuclear protein that appears to act downstream from multiple photoreceptors to regulate morphogenesis and gene expression in response to light. However, its function has remained unknown. RESULTS: We used microarrays to examine defects in transcription in dark-grown det1 seedlings. We found extensive changes in gene expression, including many of the transcriptional responses observed in light-treated wild-type seedlings. We used an epitope-tagging approach to determine the basis of DET1 function. GFP-DET1 rescues the det1 phenotype, is localized to the nucleus, and forms an approximately 350 kDa complex, which is required for full DET1 activity. We affinity-purified the DET1 complex and identified an approximately 120 kDa copurifying protein that is the plant homolog of UV-Damaged DNA Binding Protein 1 (DDB1), a protein implicated in the human disease xeroderma pigmentosa. A null mutation in Arabidopsis DDB1A results in no obvious phenotype on its own, yet it enhances the phenotype of a weak det1 allele. CONCLUSIONS: DET1 and DDB1 interact both biochemically and genetically. In animal cells, DDB1 interacts with histone acetyltransferase complexes. The DET1/DDB1 complex may regulate gene expression in response to light via recruitment of HAT activity. Thus, DET1, whose sequence is conserved in both animals and plants, may play a direct role in the regulation of many genes.

Cytokinin regulates type-A Arabidopsis Response Regulator activity and protein stability via two-component phosphorelay.

The plant hormone cytokinin regulates many aspects of growth and development. Cytokinin signaling involves His kinase receptors that perceive cytokinin and transmit the signal via a multistep phosphorelay similar to bacterial two-component signaling systems. The final targets of this phosphorelay are a set of Arabidopsis thaliana Response Regulator (ARR) proteins containing a receiver domain with a conserved Asp phosphorylation site. One class of these, the type-A ARRs, are negative regulators of cytokinin signaling that are rapidly transcriptionally upregulated in response to cytokinin. In this study, we tested the role of phosphorylation in type-A ARR function. Our results indicate that phosphorylation of the receiver domain is required for type-A ARR function and suggest that negative regulation of cytokinin signaling by the type-A ARRs most likely involves phosphorylation-dependent interactions. Furthermore, we show that a subset of the type-A ARR proteins are stabilized in response to cytokinin in part via phosphorylation. These studies shed light on the mechanism by which type-A ARRs act to negatively regulate cytokinin signaling and reveal a novel mechanism by which cytokinin controls type-A ARR function.

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling.

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.

Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis.

Type-B Arabidopsis thaliana response regulators (ARRs) are transcription factors that function in the final step of two-component signaling systems. To characterize their role in plant growth and development, we isolated T-DNA insertions within six of the genes (ARR1, ARR2, ARR10, ARR11, ARR12, and ARR18) from the largest subfamily of type-B ARRs and also constructed various double and triple combinations of these mutations. Higher order mutants revealed progressively decreased sensitivity to cytokinin, including effects on root elongation, lateral root formation, callus induction and greening, and induction of cytokinin primary response genes. The triple mutant arr1,10,12 showed almost complete insensitivity to cytokinin under many of the assay conditions used. By contrast, no significant change in the sensitivity to ethylene was found among the mutants examined. These results indicate that there is functional overlap among the type-B ARRs and that they act as positive regulators of cytokinin signal transduction.

HY5, Circadian Clock-Associated 1, and a cis-element, DET1 dark response element, mediate DET1 regulation of chlorophyll a/b-binding protein 2 expression.

DET1 is a pleiotropic regulator of Arabidopsis development and controls the expression of many light-regulated genes. To gain a better understanding of the mechanism by which DET1 controls transcription from light-regulated promoters, we identified elements in the chlorophyll a/b-binding protein 2 (CAB2) promoter that are required for DET1-mediated expression. Using a series of reporter constructs in which the luciferase gene is controlled by CAB2 promoter fragments, we defined two DET1-responsive elements in the CAB2 promoter that are essential for proper CAB2 transcription. A 40-bp DET1 dark-response element (DtRE) is required for both dark and root-specific repression of CAB2, whereas the known CAB upstream factor-1 element is required for DET1 activation-associated effects in the light and repression in the roots. HY5, a factor that binds CAB upstream factor-1, is also required for DET1 effects in the light. DtRE binds two distinct activities in Arabidopsis seedling extracts: a novel activity with binding site CAAAACGC that we have named CAB2 DET1-associated factor 1 plus an activity that is likely to be the myb transcription factor Circadian Clock-Associated 1. Both activities are altered in dark-grown det1 extracts as compared with wild type, correlating a change in extractable DNA binding activity with a major change in CAB2 expression. We conclude that DET1 represses the CAB2 promoter in the dark by regulating the binding of two factors, CAB2 DET1-associated factor 1 and Circadian Clock-Associated 1, to the DtRE.