Ageing in plants:Conserved strategies and novel pathways

Ageing increases chaos and entropy and ultimately leads to the death of living organisms. Nevertheless, single gene mutations substantially alter lifespan, revealing that ageing is subject to genetic control. In higher plants, ageing is most obviously manifested by the senescence of leaves, and recent molecular genetic studies, in particular the isolation of Arabidopsis mutants with altered leaf senescence, have greatly advanced our understanding of ageing regulation in plants. This paper provides an overview of the identified genes and their respective molecular pathways. Hormones, metabolic flux, reactive oxygen species and protein degradation are prominent strategies employed by plants to control leaf senescence. Plants predominantly use similar ageing-regulating strategies as yeast and animals but have evolved different molecular pathways. The senescence window concept is proposed to describe the age-dependent actions of the regulatory genes. It is concluded that the similarities and differences in ageing between plants and other organisms are deeply rooted in the evolution of ageing and we hope to stimulate discussion and research in the fascinating field of leaf senescence

ABA signalling manipulation suppresses senescence of a leafy vegetable stored at room temperature

Postharvest senescence and associated stresses limit the shelf life and nutritional value of vegetables. Improved understanding of these processes creates options for better management. After harvest, controlled exposure to abiotic stresses and/or exogenous phytohormones can enhance nutraceutical, organoleptic and commercial longevity traits. With leaf senescence, abscisic acid (ABA) contents progressively rise, but the actual biological functions of this hormone through senescence still need to be clarified. Postharvest senescence of detached green cabbage leaves (Brassica oleracea var. capitata) was characterized under cold (4 °C) and room temperature (25 °C) storage conditions. Hormonal profiling of regions of the leaf blade (apical, medial, basal) revealed a decrease in cytokinins contents during the first days under both conditions, while ABA only increased at 25 °C. Treatments with ABA and a partial agonist of ABA (pyrabactin) for 8 days did not lead to significant effects on water and pigment contents, but increased cell integrity and altered 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and cytokinins contents. Transcriptome analysis showed transcriptional regulation of ABA, cytokinin and ethylene metabolism and signalling; proteasome components; senescence regulation; protection of chloroplast functionality and cell homeostasis; and suppression of defence responses (including glucosinolates and phenylpropanoids metabolism). It is concluded that increasing the concentration of ABA (or its partial agonist pyrabactin) from the start of postharvest suppresses senescence of stored leaves, changes the transcriptional regulation of glucosinolates metabolism and down‐regulates biotic stress defence mechanisms. These results suggest a potential for manipulating ABA signalling for improving postharvest quality of leafy vegetables stored at ambient temperature

ABA signalling manipulation suppresses senescence of a leafy vegetable stored at room temperature

Postharvest senescence and associated stresses limit the shelf life and nutritional value of vegetables. Improved understanding of these processes creates options for better management. After harvest, controlled exposure to abiotic stresses and/or exogenous phytohormones can enhance nutraceutical, organoleptic and commercial longevity traits. With leaf senescence, abscisic acid (ABA) contents progressively rise, but the actual biological functions of this hormone through senescence still need to be clarified. Postharvest senescence of detached green cabbage leaves (Brassica oleracea var. capitata) was characterized under cold (4 degrees C) and room temperature (25 degrees C) storage conditions. Hormonal profiling of regions of the leaf blade (apical, medial, basal) revealed a decrease in cytokinins contents during the first days under both conditions, while ABA only increased at 25 degrees C. Treatments with ABA and a partial agonist of ABA (pyrabactin) for 8 days did not lead to significant effects on water and pigment contents, but increased cell integrity and altered 1-aminocyclopropane-1-carboxylic acid (ACC) and cytokinins contents. Transcriptome analysis showed transcriptional regulation of ABA, cytokinin and ethylene metabolism and signalling; proteasome components; senescence regulation; protection of chloroplast functionality and cell homeostasis; and suppression of defence responses (including glucosinolates and phenylpropanoids metabolism). It is concluded that increasing the concentration of ABA (or its partial agonist pyrabactin) from the start of postharvest suppresses senescence of stored leaves, changes the transcriptional regulation of glucosinolates metabolism and down-regulates biotic stress defence mechanisms. These results suggest a potential for manipulating ABA signalling for improving postharvest quality of leafy vegetables stored at ambient temperature

Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro

Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order

Trapping DNA Replication Origins from the Human Genome

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks

A mutation in Arabidopsis SAL1 alters its in vitro activity against IP3 and delays developmental leaf senescence in association with lower ROS levels

Key message: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP 3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP 3. Abstract: Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H 2O 2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3′ (2′), 5′-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP 3), where it removes the 1-phosphate group from the IP 3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP 3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP 3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence

A mutation in the cytosolic O-acetylserine (thiol) lyase induces a genome-dependent early leaf death phenotype in Arabidopsis

Background: Cysteine is a component in organic compounds including glutathione that have been implicated in the adaptation of plants to stresses. O-acetylserine (thiol) lyase (OAS-TL) catalyses the final step of cysteine biosynthesis. OAS-TL enzyme isoforms are localised in the cytoplasm, the plastids and mitochondria but the contribution of individual OAS-TL isoforms to plant sulphur metabolism has not yet been fully clarified. Results: The seedling lethal phenotype of the Arabidopsis onset of leaf death3-1 (old3-1) mutant is due to a point mutation in the OAS-A1 gene, encoding the cytosolic OAS-TL. The mutation causes a single amino acid substitution from Gly162 to Glu162, abolishing old3-1 OAS-TL activity in vitro. The old3-1 mutation segregates as a monogenic semi-dominant trait when backcrossed to its wild type accession Landsberg erecta (Ler-0) and the Di-2 accession. Consistent with its semi-dominant behaviour, wild type Ler-0 plants transformed with the mutated old3-1 gene, displayed the early leaf death phenotype. However, the old3-1 mutation segregates in an 11:4:1 (wild type: semi-dominant: mutant) ratio when backcrossed to the Colombia-0 and Wassilewskija accessions. Thus, the early leaf death phenotype depends on two semi-dominant loci. The second locus that determines the old3-1 early leaf death phenotype is referred to as odd-ler (for old3 determinant in the Ler accession) and is located on chromosome 3. The early leaf death phenotype is temperature dependent and is associated with increased expression of defence-response and oxidative-stress marker genes. Independent of the presence of the odd-ler gene, OAS-A1 is involved in maintaining sulphur and thiol levels and is required for resistance against cadmium stress. Conclusions: The cytosolic OAS-TL is involved in maintaining organic sulphur levels. The old3-1 mutation causes genome-dependent and independent phenotypes and uncovers a novel function for the mutated OAS-TL in cell death regulation.

Comparative analysis of DNA loop length in nontransformed and transformed hamster cells

The size of the looped, matrix-attached DNA domains was estimated in both nontransformed and transformed hamster cells. In BHK cells it was observed that in interphase as well as during mitosis, transformed cells, on the average, have shorter loops than nontransformed cells. In CHO cells, however, no alteration of the length of the looped DNA domains was found to accompany transformation. The implications of these observations are discussed in relation to the process of transformation

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3–related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome