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.

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