A SNP associated with alternative splicing of RPT5b causes unequal redundancy between RPT5a and RPT5b among Arabidopsis thaliana natural variation

<p>Abstract</p> <p>Background</p> <p>The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in <it>Arabidopsis thaliana</it>; <it>RPT5a </it>and <it>RPT5b</it>. We showed previously that <it>RPT5a </it>and <it>RPT5b </it>are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), <it>RPT5b </it>does not complement the effect of a strong <it>rpt5a </it>mutation in the male gametophyte, and only partially complements <it>rpt5a </it>mutation in the sporophyte. <it>RPT5b^Col-0</it>and <it>RPT5b^Ws-4</it>differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.</p> <p>Results</p> <p>By exploiting natural variation at <it>RPT5b </it>we determined that the SNP located in <it>RPT5b </it>intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the <it>RPT5b </it>pre-mRNA mis-splicing and the lack of ability to complement an <it>rpt5a </it>mutant phenotype.</p> <p>Conclusion</p> <p>The accession-dependent unequal redundancy between <it>RPT5a </it>and <it>RPT5b </it>genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.</p

Arabidopsis seed content QTL mapping using high-throughput phenotyping: the assets of Near Infrared Spectroscopy

Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of hundreds of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analysed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 x Col-0 recombinant inbred line population. Some QTL co-localised with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and Genome Wide Association Studies

Improving seed oil and protein content in Brassicaceae: some new genetic insights from Arabidopsis thaliana

Western Europe oleoproteaginous species like rapeseed mainly accumulate oil and protein in their seeds. To become competitive with soybean, seed protein quantity and quality should be improved in rapeseed. The negative correlation existing between seed protein and oil content apparently prevents the possibility to increase protein content without affecting oil content. Exploration of natural and induced genetic variability in the model plant Arabidopsis thaliana allows the identification of several genotypes impaired in this negative correlation. Different genetic approaches have been undertaken in order to isolate genetic factors responsible for the tight control of seed oil and protein homeostasis and this negative correlation. Once isolated in this model plant, such genetic determinants will be identified in important crops such as rapeseed or other oilseed crops in order to manipulate both components independently and thus produce on purposed seeds. In the long term, this research will help breed new varieties that could contribute to reduce Europe’s dependence on US soybean import

Improving seed oil and protein content in

Western Europe oleoproteaginous species like rapeseed mainly accumulate oil and protein in their seeds. To become competitive with soybean, seed protein quantity and quality should be improved in rapeseed. The negative correlation existing between seed protein and oil content apparently prevents the possibility to increase protein content without affecting oil content. Exploration of natural and induced genetic variability in the model plant Arabidopsis thaliana allows the identification of several genotypes impaired in this negative correlation. Different genetic approaches have been undertaken in order to isolate genetic factors responsible for the tight control of seed oil and protein homeostasis and this negative correlation. Once isolated in this model plant, such genetic determinants will be identified in important crops such as rapeseed or other oilseed crops in order to manipulate both components independently and thus produce on purposed seeds. In the long term, this research will help breed new varieties that could contribute to reduce Europe’s dependence on US soybean import

QTL meta-analysis in Arabidopsis reveals an interaction between leaf senescence and resource allocation to seeds

Abstract Sequential and monocarpic senescence are observed at vegetative and reproductive stages, respectively. Both facilitate nitrogen (N) remobilization and control the duration of carbon (C) fixation. Genetic and environmental factors control N and C resource allocation to seeds. Studies of natural variation in Arabidopsis thaliana revealed differences between accessions for leaf senescence phenotypes, seed N and C contents, and N remobilization efficiency-related traits. Here, a quantitative genetics approach was used to gain a better understanding of seed filling regulation in relation to leaf senescence and resource allocation. For that purpose, three Arabidopsis recombinant inbred line populations (Ct-1×Col-0, Cvi-0×Col-0, Bur-0×Col-0) were used to map QTL (quantitative trait loci) for ten traits related to senescence, resource allocation, and seed filling. The use of common markers across the three different maps allowed direct comparisons of the positions of the detected QTL in a single consensus map. QTL meta-analysis was then used to identify interesting regions (metaQTL) where QTL for several traits co-localized. MetaQTL were compared with positions of candidate genes known to be involved in senescence processes and flowering time. Finally, investigation of the correlation between yield and seed N concentration in the three populations suggests that leaf senescence disrupts the negative correlation generally observed between these two traits

The Arabidopsis Proteasome RPT5 Subunits Are Essential for Gametophyte Development and Show Accession-Dependent Redundancy[W]

We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAA-ATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression

Bay and Sha alleles at <i>CLR.2</i> diversely complement <i>kcs18-1</i> mutant.

<p>CLR was determined for each of the four allelic combinations obtained in F1 seeds, generated by crossing arHIF196_[Bay] or arHIF196_[Sha] with a <i>kcs18-1</i> mutant or its wild type background (Col-0). For each combination, 10 independent crosses have been done and CLR was determined from 100 seeds of each cross. Each data point represents the mean +/− SE (n = 10). CLR values for Col-0 and <i>kcs18-1</i> are indicated by an arrow along the y axis. The QTL genotype x gene allele interaction term is very highly significant (p<0.1%).</p

One SNP in <i>KCS18</i> coding sequence is sufficient to modulate chain length ratio.

<p>A, Promoters and coding sequences of the <i>KCS17</i> and <i>KCS18</i> genes. SNPs identified between Bay-0 and Shahdara are indicated with letters and their respective positions are indicated by arrows. B, Regions covering SNPs shown in A have been sequenced in the indicated accessions. The nucleotide for each SNP is shown for all accessions. CLR values are indicated at the right in addition to <i>CLR.2</i> segregation in crosses with Col-0 or Cvi-0.</p

Confirmation of the <i>CLR.2</i> QTL with heterogeneous inbred families (HIFs).

<p>A, F6 recombinant inbred lines (RILs) from the Bay-0 x Shahdara cross showing residual heterozygosity in the region of the <i>CLR.2</i> QTL. Numbers on the top designate the RIL, with the hatched black and grey bar beneath indicating the region still segregating. Recombination breakpoints delimiting heterozygous regions are arbitrarily depicted in the middle of the marker interval. The vertical black line represents chromosome 4 with markers indicated on the left. Marker positions and identity can be found at <a href="http://www.inra.fr/vast/in" target="_blank">www.inra.fr/vast/in</a> the MSAT database. B, Comparison of CLR for HIFs fixed for the Bay (black) or the Sha (grey) allele at the segregating region. CLR was determined from 20 seeds per plants. Bars represent SE values (n = 8, 4 repetitions were done on 2 plants). Significance in t-test, ***p<10⁻⁸.</p