Temperature-mediated flower size plasticity in Arabidopsis.

Organisms can rapidly mitigate the effects of environmental changes by changing their phenotypes, known as phenotypic plasticity. Yet, little is known about the temperature-mediated plasticity of traits that are directly linked to plant fitness such as flower size. We discovered substantial genetic variation in flower size plasticity to temperature both among selfing Arabidopsis thaliana and outcrossing A. arenosa individuals collected from a natural growth habitat. Genetic analysis using a panel of 290 A. thaliana accession and mutant lines revealed that MADS AFFECTING FLOWERING (MAF) 2-5 gene cluster, previously shown to regulate temperature-mediated flowering time, was associated to the flower size plasticity to temperature. Furthermore, our findings pointed that the control of plasticity differs from control of the trait itself. Altogether, our study advances the understanding of genetic and molecular factors underlying plasticity on fundamental fitness traits, such as flower size, in response to future climate scenarios.

A. thaliana Hybrids Develop Growth Abnormalities through Integration of Stress, Hormone and Growth Signaling.

Hybrids between Arabidopsis thaliana accessions are important in revealing the consequences of epistatic interactions in plants. F1 hybrids between the A. thaliana accessions displaying either defense or developmental phenotypes have been revealing the roles of the underlying epistatic genes. The interaction of two naturally occurring alleles of the OUTGROWTH-ASSOCIATED KINASE (OAK) gene in Sha and Lag2-2, previously shown to cause a similar phenotype in a different allelic combination in A. thaliana, was required for the hybrid phenotype. Outgrowth formation in the hybrids was associated with reduced levels of salicylic acid, jasmonic acid and abscisic acid in petioles and the application of these hormones mitigated the formation of the outgrowths. Moreover, different abiotic stresses were found to mitigate the outgrowth phenotype. The involvement of stress and hormone signaling in outgrowth formation was supported by a global transcriptome analysis, which additionally revealed that TCP1, a transcription factor known to regulate leaf growth and symmetry, was downregulated in the outgrowth tissue. These results demonstrate that a combination of natural alleles of OAK regulates growth and development through the integration of hormone and stress signals and highlight the importance of natural variation as a resource to discover the function of gene variants that are not present in the most studied accessions of A. thaliana.

Leaf chlorosis in Arabidopsis thaliana hybrids is associated with transgenerational decline and imbalanced ribosome number.

The interaction of two parental genomes can result in negative outcomes in offspring, also known as hybrid incompatibility. We have previously reported a case in which two recessively interacting alleles result in hybrid chlorosis in Arabidopsis thaliana. A DEAD-box RNA helicase 18 (AtRH18) was identified to be necessary for chlorosis. In this study, we use a sophisticated genetic approach to investigate genes underlying hybrid chlorosis. Sequence comparisons, DNA methylation inhibitor drug treatment and segregation analysis were used to investigate the epigenetic regulation of hybrid chlorosis. Relative rRNA numbers were quantified using real-time quantitative PCR. We confirmed the causality of AtRH18 and provided evidence for the involvement of the promoter region of AtRH18 in the hybrid chlorosis. Furthermore, AtMOM1 from the second parent was identified as the likely candidate gene on chromosome 1. Chlorotic hybrids displayed transgenerational decline in chlorosis, and DNA demethylation experiment restored chlorophyll levels in chlorotic hybrids. Quantification of rRNA indicated that hybrid chlorosis was associated with an imbalance in the ratio of cytosolic and plastid ribosomes. Our findings highlight that the epigenetic regulation of AtRH18 causes hybrid breakdown and provide novel information about the role of AtRH18 in plant development.

The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells.

Several metabolic processes tightly regulate growth and biomass accumulation. A highly conserved protein complex containing the target of rapamycin (TOR) kinase is known to integrate intra- and extracellular stimuli controlling nutrient allocation and hence cellular growth. Although several functions of TOR have been described in various heterotrophic eukaryotes, our understanding lags far behind in photosynthetic organisms. In the present investigation, we used the model alga Chlamydomonas reinhardtii to conduct a time-resolved analysis of molecular and physiological features throughout the diurnal cycle after TOR inhibition. Detailed examination of the cell cycle phases revealed that growth is not only repressed by 50%, but also that significant, non-linear delays in the progression can be observed. By using metabolomics analysis, we elucidated that the growth repression was mainly driven by differential carbon partitioning between anabolic and catabolic processes. Accordingly, the time-resolved analysis illustrated that metabolic processes including amino acid-, starch- and triacylglycerol synthesis, as well RNA degradation, were redirected within minutes of TOR inhibition. Here especially the high accumulation of nitrogen-containing compounds indicated that an active TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression.

Regulatory-associated protein of TOR (RAPTOR) alters the hormonal and metabolic composition of Arabidopsis seeds, controlling seed morphology, viability and germination potential.

Target of Rapamycin (TOR) is a positive regulator of growth and development in all eukaryotes, which positively regulates anabolic processes like protein synthesis, while repressing catabolic processes, including autophagy. To better understand TOR function we decided to analyze its role in seed development and germination. We therefore performed a detailed phenotypic analysis using mutants of the REGULATORY-ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), a conserved TOR interactor, acting as a scaffold protein, which recruits substrates for the TOR kinase. Our results show that raptor1b plants produced seeds that were delayed in germination and less resistant to stresses, leading to decreased viability. These physiological phenotypes were accompanied by morphological changes including decreased seed-coat pigmentation and reduced production of seed-coat mucilage. A detailed molecular analysis revealed that many of these morphological changes were associated with significant changes of the metabolic content of raptor1b seeds, including elevated levels of free amino acids, as well as reduced levels of protective secondary metabolites and storage proteins. Most of these observed changes were accompanied by significantly altered phytohormone levels in the raptor1b seeds, with increases in abscisic acid, auxin and jasmonic acid, which are known to inhibit germination. Delayed germination and seedling growth, observed in the raptor1b seeds, could be partially restored by the exogenous supply of gibberellic acid, indicating that TOR is at the center of a regulatory hub controlling seed metabolism, maturation and germination.

Knockout of the two evolutionarily conserved peroxisomal 3-ketoacyl-CoA thiolases in Arabidopsis recapitulates the abnormal inflorescence meristem 1 phenotype.

A specific function for peroxisomal ß-oxidation in inflorescence development in Arabidopsis thaliana is suggested by the mutation of the abnormal inflorescence meristem 1 gene, which encodes one of two peroxisomal multifunctional proteins. Therefore, it should be possible to identify other ß-oxidation mutants that recapitulate the aim1 phenotype. Three genes encode peroxisomal 3-ketoacyl-CoA thiolase (KAT) in Arabidopsis. KAT2 and KAT5 are present throughout angiosperms whereas KAT1 is a Brassicaceae-specific duplication of KAT2 expressed at low levels in Arabidopsis. KAT2 plays a dominant role in all known aspects of peroxisomal ß-oxidation, including that of fatty acids, pro-auxins, jasmonate precursor oxophytodienoic acid, and trans-cinnamic acid. The functions of KAT1 and KAT5 are unknown. Since KAT5 is conserved throughout vascular plants and expressed strongly in flowers, kat2 kat5 double mutants were generated. These were slow growing, had abnormally branched inflorescences, and ectopic organ growth. They made viable pollen, but produced no seed indicating that infertility was due to defective gynaecium function. These phenotypes are strikingly similar to those of aim1. KAT5 in the Brassicaceae encodes both cytosolic and peroxisomal proteins and kat2 kat5 defects could be complemented by the re-introduction of peroxisomal (but not cytosolic) KAT5. It is concluded that peroxisomal KAT2 and KAT5 have partially redundant functions and operate downstream of AIM1 to provide ß-oxidation functions essential for inflorescence development and fertility.

Peroxisomal ATP-binding cassette transporter COMATOSE and the multifunctional protein abnormal INFLORESCENCE MERISTEM are required for the production of benzoylated metabolites in Arabidopsis seeds.

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal ß-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of ß-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.

Conservation of two lineages of peroxisomal (Type I) 3-ketoacyl-CoA thiolases in land plants, specialization of the genes in Brassicaceae, and characterization of their expression in Arabidopsis thaliana.

Arabidopsis thaliana has three genes encoding type I 3-ketoacyl-CoA thiolases (KAT1, KAT2, and KAT5), one of which (KAT5) is alternatively transcribed to produce both peroxisomal and cytosolic proteins. To evaluate the potential importance of these four gene products, their evolutionary history in plants and their expression patterns in Arabidopsis were investigated. Land plants as a whole have gene lineages corresponding to KAT2 and KAT5, implying conservation of distinct functions for these two genes. By contrast, analysis of synteny shows that KAT1 arose by duplication of the KAT2 locus. KAT1 is found in the Brassicaceae family, including in the genera Arabidopsis, Capsella, Thellungiella (=Eutrema) and Brassica, but not in the more distantly related Caricaceae (order Brassicales), or other plants. Gene expression analysis using qRT-PCR and ß-glucuronidase reporter genes showed strong expression of KAT2 during germination and in many plant tissues throughout the life cycle, consistent with its observed dominant function in fatty acid ß-oxidation. KAT1 was expressed very weakly while KAT5 was most strongly expressed during flower development and in seedlings after germination. Isoform-specific qRT-PCR analysis and promoter ß-glucuronidase reporters revealed that the two splicing variants of KAT5 have similar expression profiles. Alternative splicing of KAT5 to produce cytosolic and peroxisomal proteins is specific to and ubiquitous in the Brassicaceae, and possibly had an earlier origin in the order Brassicales. This implies that an additional function for KAT5 arose between 43 and 115 mybp. We speculate that this KAT5 mutation was recruited for a cytosolic function in secondary metabolism.

Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins.

Indole-3-butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (2,4-DB) are metabolised by peroxisomal beta-oxidation to active auxins that inhibit root growth. We screened Arabidopsis mutants for resistance to IBA and 2,4-DB and identified two new 2,4-DB resistant mutants. The mutant genes encode a putative oxidoreductase (SDRa) and a putative acyl-activating enzyme (AAE18). Both proteins are localised to peroxisomes. SDRa is coexpressed with core beta-oxidation genes, but germination, seedling growth and the fatty acid profile of sdra seedlings are indistinguishable from wild type. The sdra mutant is also resistant to IBA, but aae18 is not. AAE18 is the first example of a gene required for response to 2,4-DB but not IBA. The closest relative of AAE18 is AAE17. AAE17 is predicted to be peroxisomal, but an aae17 aae18 double mutant responded similarly to aae18 for all assays. We propose that AAE18 is capable of activating 2,4-DB but IBA activating enzymes remain to be discovered. We present an updated model for peroxisomal pro-auxin metabolism in Arabidopsis that includes SDRa and AAE18.