SummaryAn 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 biliprotein phytochrome regulates plant growth and developmental responses to the ambient light environment through an unknown mechanism. Biochemical analyses demonstrate that phytochrome is an ancient molecule that evolved from a more compact light sensor in cyanobacteria. The cyanobacterial phytochrome Cph1 is a light-regulated histidine kinase that mediates red, far-red reversible phosphorylation of a small response regulator, Rcp1 (response regulator for cyanobacterial phytochrome), encoded by the adjacent gene, thus implicating protein phosphorylation-dephosphorylation in the initial step of light signal transduction by phytochrome.

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 siRNAinitiating 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.gene silencing | microRNA | transacting siRNA S mall silencing RNAs (sRNAs) in plants and animals, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play important roles in the development and the response to pathogens and stresses. These RNAs are also valuable tools in functional genomics and biotechnology. The sRNAs associate with ARGONAUTE (AGO) and other proteins in silencing effector complexes, and they bind to a target nucleic acid via Watson-Crick base pairing. In most instances, the silencing is a direct consequence of this interaction, and the AGO effector mediates RNA-mediated DNA or histone methylation, endonucleolytic RNA cleavage, or translational inhibition. In a few instances, an sRNA interaction also triggers the production of secondary siRNAs. The targeted RNA is converted into double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASEs (RDRs), which is then cleaved into the secondary siRNAs by DICER-LIKE (DCL) nucleases (1). Several proteins are known to be required for this process, but until now, the reason why most sRNA interactions do not result in secondary siRNA production was unclear.The transacting siRNA (tasiRNA) pathway in plants involves secondary siRNA production (2). Noncoding transcripts encoded by TAS1-4 genes serve as the precursors of tasiRNAs (3-5). After miRNA-directed cleavage, part of the remaining transcript is converted into dsRNA by RDR6. DCL4 then cleaves the dsRNA and generates tasiRNAs in a 21-nt phase relative to positions 10 and 11 of the miRNA that defines the site of targeted cleavage. TAS1 and TAS2 are targets of miR173, and their tasiRNAs in turn can target mRNAs for pentatricopeptide repeat (PPR) proteins. In one instance, a small sRNA cascade is initiated by miR173 (6, 7), because a TAS2-derived tasiRNA can itself initiate secondary siRNA production on several PPR mRNAs. The initiator of TAS3 tasiRNA is miR390 (3,8), and the TAS3 targets are AUXIN RESPONSE FACTOR mRNAs that influence the change from juvenile phase to adult phase, leaf morphology,...

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SummarySenescence 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.

Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp. PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity. Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic. Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.

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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.massively parallel signature sequencing ͉ TAS S mall regulatory RNAs modulate transcriptional gene silencing (TGS), mRNA degradation (post-TGS; PTGS) and translational repression in a wide spectrum of organisms. These small regulatory RNAs include microRNAs (miRNAs), heterochromatic siRNAs (hc-siRNAs), repeat-associated siRNAs (ra-siRNAs), natural sense-antisense transcript siRNAs (1), trans-acting siRNAs (ta-siRNAs) (2, 3), and the recently identified Piwi-interacting RNAs (4).In Arabidopsis, miRNAs are processed from hairpin precursors to play important roles in development and stress responses by either targeted cleavage of mRNA or translational repression (for review see ref. 5). The biogenesis of miRNAs requires a specific RNase III enzyme, DICER-LIKE protein 1 (DCL1) (5). Arabidopsis hcsiRNAs or ra-siRNAs trigger DNA methylation and histone modification and are thus involved in the assembly of heterochromatin and the control of transposon movement. hc-siRNAs or ra-siRNAs are usually derived from genomic repeats or transposons, a process requiring DICER-LIKE 3 (DCL3) and a specific RNA-dependent RNA polymerase, RDR2 (for reviews, see refs. 6 and 7).The identification of ta-siRNAs in Arabidopsis bridged the miRNA and siRNA pathways previously considered independent (2, 3, 8-10). ta-siRNAs clustered in 21-nt increments in both sense and antisense strands of several noncoding TAS transcripts were first identified from the study of two genes involved in PTGS, RDR6 and suppressor of gene silencing 3 (SGS3) (3, 8). Interestingly, the production of phased ta-siRNA is initially triggered by the targeted cleavage of primary TAS transcripts by miRNAs (2). After cleavage, the 5Ј or 3Ј cleavage products are converted into dsRNA with the assistanc...