Project description:Leaf senescence is an essential developmental process that involves altered regulation of thousands of genes and changes in many metabolic and signaling pathways resulting in massive physiological and structural changes in the leaf. The regulation of senescence is complex and although several senescence regulatory genes have been identified and characterized there is little information on how these individual regulators function globally in the control of the process. In this paper we use microarray analysis to obtain a high-resolution time course profile of gene expression during development of a single leaf over a three week period from just before full expansion to senescence. The multiple time points enable the use of highly informative clustering tools to reveal distinct time points at which signaling and metabolic pathways change during senescence. Analysis of motif enrichment in co-regulated gene clusters identifies clear groups of transcription factors active at different stages of leaf development and senescence. A novel experimental design strategy (A Mead et al, in preparation), based on the principle of the “loop design”, was developed to enable efficient extraction of information about key sample comparisons using a two-colour hybridisation experimental system. With 88 distinct samples (four biological replicates at each of 22 time points) to be compared, the experimental design included 176 two-colour microarray slides, allowing four technical replicates of each sample to be observed. Half of the slides were devoted to assessment of changes in gene expression between time points, using a simple loop design to link 11 samples from either the 7h time points or the 14h time points across the 11 sampling days, directly comparing samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, 27 DAS with 29 DAS, etc.), and directly comparing the samples collected at 39 DAS with those collected at 19 DAS. Four separate loops were constructed for the 7h time points and for the 14h time points, using the arbitrary biological replicate labelling to identify the samples to be included in each loop. The remaining slides provided assessment of differences between the 7h and 14h samples and between the arbitrarily labelled biological replicates, with some further assessment of changes between sampling days. All direct comparisons (pairs of samples hybridised together on a slide) were between 7h and 14h samples collected on adjacent sampling days (i.e. 19 DAS with 21 DAS, etc.), including comparisons between samples collected at 39 DAS and at 19 DAS, and different arbitrarily labelled biological replicates. These 88 comparisons formed a single loop connecting all 88 treatments, therefore ensuring that the design was fully connected (allowing each sample to be compared with every other sample).

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