Global effects of the small RNA biogenesis machinery on the Arabidopsis thaliana transcriptome.

In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation.

Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.

In Arabidopsis, the network responsible for the maintenance of the shoot apical meristem (SAM) is built on a negative feedback loop involving the peptide ligand CLAVATA3 (CLV3) and the homeodomain transcription factor WUSCHEL (WUS). The local WUS/CLV3 regulatory module is linked to the organism-wide cytokinin signalling system by direct transcriptional control of A-type ARABIDOPSIS RESPONSE REGULATOR genes (ARRs) by WUS. Here we investigate two A-type ARR genes, ARR7 and ARR15, which are negative regulators of cytokinin signalling. We show that the expression of ARR7, WUS and CLV3 is dependent on cytokinin signalling. While ARR7 expression strongly responds to variations in cytokinin activity, WUS and CLV3 appeared to be much more buffered against this type of variation. As earlier studies had shown that pertubation of A-type ARR activity only causes mild effects on SAM function, we employed tissue regeneration assays as a sensitised background. Root explants pre-treated on auxin and cytokinin-rich callus-inducing medium showed severely suppressed shoot regeneration when ARR7 and ARR15 were overexpressed, whereas loss of function of these genes had a strongly promoting effect. This phenotype was even aggravated in the arr3,4,5,6,7,8,9 septuple mutant. Futhermore, loss-of A-type ARR function in arr7 and arr3,4,5,6,7,8,9 mutants strongly stimulated callus development, indicating that cell proliferation is repressed by A-type ARRs. To elucidate the mechanisms underlying the enhanced capacity of the arr3,4,5,6,7,8,9 septuple mutant to develop shoot tissue in culture, we used whole-genome expression profiling. Among the transcripts with increased abundance in arr3,4,5,6,7,8,9 inflorescence apices a strong enrichment for functions in pollen development was apparent, while the reduced transcripts showed a more heterogeneous distribution of functional categories, ranging from development to pathogen defence.

Requirement of B2-type cyclin-dependent kinases for meristem integrity in Arabidopsis thaliana.

To maintain proper meristem function, cell division and differentiation must be coordinately regulated in distinct subdomains of the meristem. Although a number of regulators necessary for the correct organization of the shoot apical meristem (SAM) have been identified, it is still largely unknown how their function is integrated with the cell cycle machinery to translate domain identity into correct cellular behavior. We show here that the cyclin-dependent kinases CDKB2;1 and CDKB2;2 are required both for normal cell cycle progression and for meristem organization. Consistently, the CDKB2 genes are highly expressed in the SAM in a cell cycle-dependent fashion, and disruption of CDKB2 function leads to severe meristematic defects. In addition, strong alterations in hormone signaling both at the level of active hormones and with respect to transcriptional and physiological outputs were observed in plants with disturbed CDKB2 activity.