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

Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis

Although gibberellins (GAs) are well known for their growth control function, little is known about their effects on primary metabolism. Here the modulation of gene expression and metabolic adjustment in response to changes in plant (Arabidopsis thaliana) growth imposed on varying the gibberellin regime were evaluated. Polysomal mRNA populations were profiled following treatment of plants with paclobutrazol (PAC), an inhibitor of GA biosynthesis, and gibberellic acid (GA3) to monitor translational regulation of mRNAs globally. Gibberellin levels did not affect levels of carbohydrates in plants treated with PAC and/or GA3. However, the tricarboxylic acid cycle intermediates malate and fumarate, two alternative carbon storage molecules, accumulated upon PAC treatment. Moreover, an increase in nitrate and in the levels of the amino acids was observed in plants grown under a low GA regime. Only minor changes in amino acid levels were detected in plants treated with GA3 alone, or PAC plus GA3. Comparison of the molecular changes at the transcript and metabolite levels demonstrated that a low GA level mainly affects growth by uncoupling growth from carbon availability. These observations, together with the translatome changes, reveal an interaction between energy metabolism and GA-mediated control of growth to coordinate cell wall extension, secondary metabolism, and lipid metabolism

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

The Role and Regulation of Autophagy and the Proteasome During Aging and Senescence in Plants

Aging and senescence in plants has a major impact on agriculture, such as in crop yield, the value of ornamental crops, and the shelf life of vegetables and fruits. Senescence represents the final developmental phase of the leaf and inevitably results in the death of the organ. Still, the process is completely under the control of the plant. Plants use their protein degradation systems to maintain proteostasis and transport or salvage nutrients from senescing organs to develop reproductive parts. Herein, we present an overview of current knowledge about the main protein degradation pathways in plants during senescence: The proteasome and autophagy. Although both pathways degrade proteins, autophagy appears to prevent aging, while the proteasome functions as a positive regulator of senescence

ROS-mediated redox signaling during cell differentiation in plants

Schmidt R, Schippers JHM. ROS-mediated redox signaling during cell differentiation in plants. Biochimica et Biophysica Acta (BBA) - General Subjects. 2015;1850(8):1497-1508

Setting the PAS, the role of circadian PAS domain proteins during environmental adaptation in plants

The Per-Arnt-Sim (PAS) domain represents an ancient protein module that can be found across all kingdoms of life. The domain functions as a sensing unit for a diverse array of signals, including molecular oxygen, small metabolites, and light. In plants, several PAS domain-containing proteins form an integral part of the circadian clock and regulate responses to environmental change. Moreover, these proteins function in pathways that control development and plant stress adaptation responses. Here, we discuss the role of PAS domain-containing proteins in anticipation, and adaptation to environmental changes in plants

Role of Peroxiporins in Plant Stress Signaling and Development

Frohn S, Dreyer BH, Schmidt-Schippers R, Schippers JHM. Role of Peroxiporins in Plant Stress Signaling and Development. In: Peroxiporins. Redox Signal Mediators In and Between Cells. Boca Raton: CRC Press; 2023: 147-157

Redox and low-oxygen stress: signal integration and interplay

Sasidharan R, Schippers JHM, Schmidt R. Redox and low-oxygen stress: signal integration and interplay. Plant physiology. 2021: kiaa081.An analysis of the role of reactive oxygen species, reactive nitrogen species, and redox components in hypoxia signaling pathways and an outline of potential future research avenues. © The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please email: [email protected]