Identification of KIN10-target genes in Arabidopsis mesophyll cells

The goal of this experiment was to explore the extent of KIN10 (At3g01090) transcriptional regulation and identify its early target genes in Arabidopsis mesophyll protoplasts. Results suggest that KIN10 targets a remarkably broad array of genes that orchestrate transcription networks, promote catabolism and autophagy, and suppress anabolism and ribosome biogenesis. The transient expression condition ruled out secondary or long-term effects of metabolism and growth, and circumvented experimental limitations caused by redundancy and embryonic lethality observed in mammals and plants. Keywords: KIN10, KIN11, darkness, hypoxia, starvation, stress, sugar signalling, Arabidopsis, SnRK1 Analysis of the transcript changes induced by transient KIN10 (At3g01090) expression (6h) in Arabidopsis mesophyll protoplasts using protoplasts similarly transfected with control plasmid DNA for comparison. To maximize the significance of the duplicated experimental data for defining differentially expressed genes, the biologically independent experiments were carefully performed by two individuals using different soil, plants, growth chambers, DNAs and microarray facilities for hybridization and scanning to cover potential technical and biological variations. For each pair (control & KIN10) of large-scale protoplast transfection experiments, about 3 million protoplasts were isolated from the fifth and sixth leaves of 36 randomly picked plants and pooled. RNA samples isolated from control and KIN10-transfected protoplasts were subjected to quality controls, including RNA quality and quantity inspection by gel electrophoresis and staining as well as consistent gene expression changes by duplicated real-time quantitative RT-PCR analyses of selected marker genes.