Summary: An analysis of changes in global gene expression patterns during developmental leaf senescence in Arabidopsis has identified more than 800 genes that show a reproducible increase in transcript abundance. This extensive change illustrates the dramatic alterations in cell metabolism that underpin the developmental transition from a photosynthetically active leaf to a senescing organ which functions as a source of mobilizable nutrients. Comparison of changes in gene expression patterns during natural leaf senescence with those identified, when senescence is artificially induced in leaves induced to senesce by darkness or during sucrose starvation‐induced senescence in cell suspension cultures, has shown not only similarities but also considerable differences. The data suggest that alternative pathways for essential metabolic processes such as nitrogen mobilization are used in different senescent systems. Gene expression patterns in the senescent cell suspension cultures are more similar to those for dark‐induced senescence and this may be a consequence of sugar starvation in both tissues. Gene expression analysis in senescing leaves of plant lines defective in signalling pathways involving salicylic acid (SA), jasmonic acid (JA) and ethylene has shown that these three pathways are all required for expression of many genes during developmental senescence. The JA/ethylene pathways also appear to operate in regulating gene expression in dark‐induced and cell suspension senescence whereas the SA pathway is not involved. The importance of the SA pathway in the senescence process is illustrated by the discovery that developmental leaf senescence, but not dark‐induced senescence, is delayed in plants defective in the SA pathway.

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium -mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

Summary: Senescence is the final stage of leaf development. Although it means the loss of vitality of leaf tissue, leaf senescence is tightly controlled by the development to increase the fitness of the whole plant. The molecular mechanisms regulating the induction and progression of leaf senescence are complex. We used a cDNA microarray, containing 11 500 Arabidopsis DNA elements, and the whole‐genome Arabidopsis ATH1 Genome Array to examine global gene expression in dark‐induced leaf senescence. By monitoring the gene expression patterns at carefully chosen time points, with three biological replicates each time, we identified thousands of up‐ or down‐regulated genes involved in dark‐induced senescence. These genes were clustered and categorized according to their expression patterns and responsiveness to dark treatment. Genes with different expression kinetics were classified according to different biological processes. Genes showing significant alteration of expression patterns in all available biochemical pathways were plotted to envision the molecular events occurring in the processes examined. With the expression data, we postulated an innovative biochemical pathway involving pyruvate orthophosphate dikinase in generating asparagine for nitrogen remobilization in dark‐treated leaves. We also surveyed the alteration in expression of Arabidopsis transcription factor genes and established an apparent association of GRAS, bZIP, WRKY, NAC, and C2H2 transcription factor families with leaf senescence.

Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2 -derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a , - b , - c , TAS2 , and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock.