SummaryThe onset of leaf senescence is controlled by leaf age and ethylene can promote leaf senescence within a specific age window. We exploited the interaction between leaf age and ethylene and isolated mutants with altered leaf senescence that are named as onset of leaf death (old) mutants. Early leaf senescence mutants representing three genetic loci were selected and their senescence syndromes were characterised using phenotypical, physiological and molecular markers. old1 is represented by three recessive alleles and displayed earlier senescence both in air and upon ethylene exposure. The etiolated old1 seedlings exhibited a hypersensitive triple response. old2 is a dominant trait and the mutant plants were indistinguishable from the wild-type when grown in air but showed an earlier senescence syndrome upon ethylene treatment. old3 is a semi-dominant trait and its earlier onset of senescence is independent of ethylene treatment. Analyses of the chlorophyll degradation, ion leakage and SAG expression showed that leaf senescence was advanced in ethylene-treated old2 plants and in both air-grown and ethylene-treated old1 and old3 plants. Epistatic analysis indicated that OLD1 might act downstream of OLD2 and upstream of OLD3 and mediate the interaction between leaf age and ethylene. A genetic model was proposed that links the three OLD genes and ethylene into a regulatory pathway controlling the onset of leaf senescence.

Early leaf senescence is associated with an altered cellular redox balance in Arabidopsis cpr5/old1 mutants Jing, H. -C.; Hebeler, R.; Oeljeklaus, S.; Sitek, B.; Stuehler, K.; Meyer, H. E.; Sturre, M. J. G.; Hille, Jacob; Warscheid, B.; Dijkwel, P. P. Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Jing, H. -C., Hebeler, R., Oeljeklaus, S., Sitek, B., Stuehler, K., Meyer, H. E., ... Stühler, K. (2008). Early leaf senescence is associated with an altered cellular redox balance in Arabidopsis cpr5/old1 mutants. Plant Biology, 10(1), 85-98. DOI: 10.111185-98. DOI: 10. /j.143885-98. DOI: 10. -8677.2008 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. INTRODUCTIONAerobic life depends on oxygen. However, oxygen atoms and oxygen-containing molecules can act as highly reactive molecules, commonly known as reactive oxygen species (ROS), when unpaired electrons are present. ROS include triplet and singlet oxygen, superoxide, nitric oxide and hydroxyl radicals, which can potentially damage and deteriorate various cellular components, such as proteins, DNA and lipids, by oxidation. Thus, oxygen is inherently detrimental to aerobic organisms. The dual effect of oxygen on shaping life-history traits and evolution of aerobic organisms is generally known as the 'Oxygen Paradox' (Davies 1995). ROS can be generated by internal organelles and other cellular components, including mitochondria, chloroplasts and microbodies, as well as by external stress conditions, such as ozone exposure and UV-B radiation. To cope with the danger imposed by ROS, cells have evolved sophisticated ROS scavenging systems. These include antioxidative enzymes such as catalase, superoxide dismutase and ascorbate peroxidase, as well as non-enzymatic antioxidants such as ascorbate, the tripeptide glutathione, tocopherol and carotenoids (Apel & Hirt 2004). In addition to being a 'wear-and-tear' force, ROS are important cellular signalling molecules in the cell cycle, hormonal signalling, programmed cell death, growth and development and responses to biotic and abiotc stresses (Finkel 2003;Foyer & Noctor 2005; Keywords Cell death; CPR5 ⁄ OLD1; leaf senescence; reactive oxygen species; redox balance. ABSTRACTReactive oxygen species (ROS) are the inevitable by-products of essential cellular metabolic and physiological activities. Plants have developed sophisticated gene networks of ROS generation and sc...

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before. Molecular & Cellular Proteomics 7:, 108 -120.A major focus of proteome research is the simultaneous identification and quantification of proteins in cells, tissues, or organisms in dependence on the developmental stage, different physiological conditions, environmental influences, or genotypes. This quantitative, mass spectrometry (MS)1 -based description of proteomes was facilitated by the development of various stable isotope labeling techniques that have since been applied to proteomics studies in a multitude of organisms (1, 2). In plant proteomics, however, the most frequently used method for comparative, quantitative studies so far has been two-dimensional PAGE (3). In traditional two-dimensional PAGE approaches, quantitative differences in protein abundance between biological samples are revealed by comparing spot patterns in individual gels based on densitometric analysis following silver or Coomassie Blue staining. Limitations of this method regarding reproducibility, sensitivity, and dynamic range of protein quantification were improved significantly by introducing the DIGE technology (4). The DIGE technique employs spectrally resolvable fluorescent cyanine dyes (CyDyes) to label proteins prior to separation by twodimensional PAGE (5). Using the minimal labeling approach, two distinct protein samples are separately labeled with the fluorescent dyes Cy3 and Cy5, respectively, while an internal standard consisting of equa...

Evolutionary theories of senescence predict that genes with pleiotropic functions are important for senescence regulation. In plants there is no direct molecular genetic test for the existence of such senescence-regulatory genes. Arabidopsis cpr5 mutants exhibit multiple phenotypes including hypersensitivity to various signalling molecules, constitutive expression of pathogen-related genes, abnormal trichome development, spontaneous lesion formation, and accelerated leaf senescence. These indicate that CPR5 is a beneficial gene which controls multiple facets of the Arabidopsis life cycle. Ectopic expression of CPR5 restored all the mutant phenotypes. However, in transgenic plants with increased CPR5 transcripts, accelerated leaf senescence was observed in detached leaves and at late development around 50 d after germination, as illustrated by the earlier onset of senescence-associated physiological and molecular markers. Thus, CPR5 has early-life beneficial effects by repressing cell death and insuring normal plant development, but late-life deleterious effects by promoting developmental senescence. As such, CPR5 appears to function as a typical senescence-regulatory gene as predicted by the evolutionary theories of senescence.

Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms.he circadian clock is a cell-based, regulatory network that controls processes from gene expression to behavior. These daily clocks, found in diverse organisms, share a set of signature properties (1). One of these is a free-running, circa 24-h (circadian) oscillation in constant conditions. The phenomenon of selfsustained rhythm, however, has never been the "aim" of evolution. It is per se not a prerequisite for the timing system but rather a consequence of how a daily timing system has developed in an environment that is utterly predictable in its alternation of light and darkness, warmer and colder temperatures, and numerous other qualities (2). Notably, many organisms do not show obvious freerunning rhythms. For instance, the ascomycete, Neurospora crassa, suppresses daily, rhythmic circadian spore formation when CO 2 accumulates (3). The accidental discovery of a mutant strain that makes "bands" of spores once every 22 h in constant darknesswithout exchanging the air to decrease CO 2 levels-permitted development of Neurospora as a clock model system (4). Even the banding strain of Neurospora appears arrhythmic in constant light, as do many animals. Yet, in the case of Neurospora, several transcript levels and the activity of the enzyme nitrate reductase are oscillating with a circa 24-h period despite no observable rhythms in spore formation (5, 6). When animals become arrhythmic in constant light, usually a decrease in irradiance will allow rhythmicity to emerge (7). These examples suggest that the expression of a free-running clock very much depends on conditions or that it is not a universal property of circadian clocks. The...

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2(2) resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2(2) resistance gene and the susceptible allele, tm-2. The Tm-2(20 gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2(2) gene survive. The Tm-2(2) locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2(2) sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2(2) allele. Between the tm-2 and the Tm-2(2) polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2(2) gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2(2) resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2(2)-encoded resistance is aimed at the Achilles' heel of the virus.

Map-based cloning of mutant genes is straightforward if the genome sequence and sufficient molecular markers are available. When a mutated gene in Arabidopsis causes a clear phenotype and is located in a genomic region where sufficient meiotic recombination takes place, the gene can be identified within 6-12 months. However, mutated genes that cause weak phenotypes are difficult to map to small genomic intervals due to faulty selection of F2 plants. Here, we describe a method that allows for rapid identification of roughly mapped genes by using a massive parallel sequencing strategy. A genomic region of 150 kb was PCR amplified in 7-17 kb pieces from an EMS Arabidopsis onset of leaf death (old) mutant and its wild-type accession Landsberg erecta (Ler-0). Massive parallel sequencing and subsequent de novo assembly of the short sequences reliably identified 253 polymorphisms in a 110-kb region between the reference Col-0 and Ler-0 sequence. The analysis further revealed potential mutations in the old mutant of which one was confirmed to be present in the mutant. Thus the described method can be used for accelerating the map-based cloning of genes that cause weak phenotypes. An accompanying advantage is that the amplified fragments can be cloned and used to complement the mutant.