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

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.

Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue

We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted

Arabidopsis RecQl4A suppresses homologous recombination and modulates DNA damage responses

The DNA damage response and DNA recombination are two interrelated mechanisms involved in maintaining the integrity of the genome, but in plants they are poorly understood. RecQ is a family of genes with conserved roles in the regulation of DNA recombination in eukaryotes; there are seven members in Arabidopsis. Here we report on the functional analysis of the Arabidopsis RecQl4A gene. Ectopic expression of Arabidopsis RecQl4A in yeast RecQ-deficient cells suppressed their hypersensitivity to the DNA-damaging drug methyl methanesulfonate (MMS) and enhanced their rate of homologous recombination (HR). Analysis of three recQl4A mutant alleles revealed no obvious developmental defects or telomere deregulation in plants grown under standard growth conditions. Compared with wild-type Arabidopsis, the recQl4A mutant seedlings were found to be hypersensitive to UV light and MMS, and more resistant to mitomycin C. The average frequency of intrachromosomal HR in recQl4A mutant plants was increased 7.5-fold over that observed in wild-type plants. The data reveal roles for Arabidopsis RecQl4A in maintenance of genome stability by modulation of the DNA damage response and suppression of HR.

Method for the identification of single mutations in large genomic regions using massive parallel sequencing

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

Positional information resolves structural variations and uncovers an evolutionarily divergent genetic locus in accessions of Arabidopsis thaliana.

Genome sequencing of closely related individuals has yielded valuable insights that link genome evolution to phenotypic variations. However, advancement in sequencing technology has also led to an escalation in the number of poor quality–drafted genomes assembled based on reference genomes that can have highly divergent or haplotypic regions. The self-fertilizing nature of Arabidopsis thaliana poses an advantage to sequencing projects because its genome is mostly homozygous. To determine the accuracy of an Arabidopsis drafted genome in less conserved regions, we performed a resequencing experiment on a 3 ~71-kb genomic interval in the Landsberg erecta (Ler-0) accession. We identified novel structural variations (SVs) between Ler-0 and the reference accession Col-0 using a long-range polymerase chain reaction approach to generate an Illumina data set that has positional information, that is, a data set with reads that map to a known location. Positional information is important for accurate genome assembly and the resolution of SVs particularly in highly duplicated or repetitive regions. Sixty-one regions with misassembly signatures were identified from the Ler-0 draft, suggesting the presence of novel SVs that are not represented in the draft sequence. Sixty of those were resolved by iterative mapping using our data set. Fifteen large indels (>100 bp) identified from this study were found to be located either within protein-coding regions or upstream regulatory regions, suggesting the formation of novel alleles or altered regulation of existing genes in Ler-0. We propose future genome-sequencing experiments to follow a clone-based approach that incorporates positional information to ultimately reveal haplotype-specific differences between accessions

CPR5: A Jack of all trades in plants

In our recent paper in Journal of Experimental Botany, we examined the effects of cpr5/old1 mutations and CPR5 overexpression on Arabidopsis growth and development.1 We found that CPR5 is important for early plant growth but promotes senescence at late development and hence proposed it as a senescence-regulatory gene as predicted by the Evolutionary Theory of Senescence derived from studies on animal ageing. One of the key unsolved issues is how CPR5 contributes to the early plant growth and development. Here we discuss the possible cellular functions of CPR5