Emergence timing and fitness consequences of variation in seed oil composition in Arabidopsis thaliana

Early seedling emergence can increase plant fitness under competition. Seed oil composition (the types and relative amounts of fatty acids in the oils) may play an important role in determining emergence timing and early growth rate in oilseeds. Saturated fatty acids provide more energy per carbon atom than unsaturated fatty acids but have substantially higher melting points (when chain length is held constant). This characteristic forms the basis of an adaptive hypothesis that lower melting point seeds (lower proportion of saturated fatty acids) should be favored under colder germination temperatures due to earlier germination and faster growth before photosynthesis, while at warmer germination temperatures, seeds with a higher amount of energy (higher proportion of saturated fatty acids) should be favored. To assess the effects of seed oil melting point on timing of seedling emergence and fitness, high‐ and low‐melting point lines from a recombinant inbred cross of Arabidopsis thaliana were competed in a fully factorial experiment at warm and cold temperatures with two different density treatments. Emergence timing between these lines was not significantly different at either temperature, which aligned with warm temperature predictions, but not cold temperature predictions. Under all conditions, plants competing against high‐melting point lines had lower fitness relative to those against low‐melting point lines, which matched expectations for undifferentiated emergence times.     

The nuclear protein Poly(ADP‐ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP‐ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear‐localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3‐1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col‐0 wild‐type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability.     

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

The PP2A‐interactor TIP41 modulates ABA responses in Arabidopsis thaliana

Modulation of growth in response to environmental cues is a fundamental aspect of plant adaptation to abiotic stresses. TIP41 (TAP42 INTERACTING PROTEIN OF 41 kDa) is the Arabidopsis thaliana orthologue of proteins isolated in mammals and yeast that participate in the Target‐of‐Rapamycin (TOR) pathway, which modifies cell growth in response to nutrient status and environmental conditions. Here, we characterized the function of TIP41 in Arabidopsis. Expression analyses showed that TIP41 is constitutively expressed in vascular tissues, and is induced following long‐term exposure to NaCl, polyethylene glycol and abscisic acid (ABA), suggesting a role of TIP41 in adaptation to abiotic stress. Visualization of a fusion protein with yellow fluorescent protein indicated that TIP41 is localized in the cytoplasm and the nucleus. Abolished expression of TIP41 results in smaller plants with a lower number of rosette leaves and lateral roots, and an increased sensitivity to treatments with chemical TOR inhibitors, indicating that TOR signalling is affected in these mutants. In addition, tip41 mutants are hypersensitive to ABA at germination and seedling stage, whereas over‐expressing plants show higher tolerance. Several TOR‐ and ABA‐responsive genes are differentially expressed in tip41, including iron homeostasis, senescence and ethylene‐associated genes. In yeast and mammals, TIP41 provides a link between the TOR pathway and the protein phosphatase 2A (PP2A), which in plants participates in several ABA‐mediated mechanisms. Here, we showed an interaction of TIP41 with the catalytic subunit of PP2A. Taken together, these results offer important insights into the function of Arabidopsis TIP41 in the modulation of plant growth and ABA responses.     

Autophagy mediates caloric restriction‐induced lifespan extension in Arabidopsis

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy‐related genes results in the short lifespan and suppression of the lifespan‐extending effect of the CR. Our data demonstrate that the autophagy‐dependent mechanism of CR‐induced lifespan extension is conserved between autotrophs and heterotrophs.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Salt hypersensitivity is associated with excessive xylem development in a thermospermine‐deficient mutant of Arabidopsis thaliana

In Arabidopsis, spermine is produced in most tissues and has been implicated in stress response, while its structural isomer thermospermine is only in xylem precursor cells. Studies on acaulis5 (acl5), a mutant defective in the biosynthesis of thermospermine, have revealed that thermospermine plays a repressive role in xylem development through enhancement of mRNA translation of the SAC51 family. In contrast, the pao5 mutant defective in the degradation of thermospermine has high levels of thermospermine and shows increased salt tolerance, suggesting a role of thermospermine in salt stress response. Here we compared acl5 with a mutant of spermine synthase, spms, in terms of abiotic stress tolerance and found that acl5 was much more sensitive to sodium than the wild‐type and spms. A double‐mutant of acl5 and sac51‐d, which suppresses the excessive xylem phenotype of acl5, recovered normal sensitivity, while a quadruple T‐DNA insertion mutant of the SAC51 family, which has an increased thermospermine level but shows excessive xylem development, showed increased salt sensitivity, unlike pao5. Together with the result that the salt tolerance of both wild‐type and acl5 seedlings was improved by long‐term treatment with thermospermine, we suggest a correlation of the salt tolerance with reduced xylem development rather than with the thermospermine level. We further found that the mutants containing high thermospermine levels showed increased tolerance to drought and heat stress, suggesting another role of thermospermine that may be common with that of spermine and secondary to that in restricting excess xylem development associated with salt hypersensitivity.     

Structural,mutagenic and in silico studies of xyloglucan fucosylation in Arabidopsis thaliana suggest a water‐mediated mechanism

The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2‐fucosyl residues to xyloglucan side chains – a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X‐ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X‐ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water‐mediated fucosylation mechanism facilitated by an H‐bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

The plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry‐based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over‐producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.     

The outer membrane Omp85‐like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana

• The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C‐terminal β‐barrel‐shaped membrane domain and one or more N‐terminal polypeptide transport‐associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80.
• We analysed expression of the gene and characterised two T‐DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome.
• We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants.
• We conclude that the function of the plastid‐localised and vein‐specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full‐grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.