Rye B chromosomes differently influence the expression of A chromosome-encoded genes depending on the host species.

The B chromosome (B) is a dispensable component of the genome in many species. To evaluate the impact of Bs on the transcriptome of the standard A chromosomes (A), comparative RNA-seq analyses of rye and wheat anthers with and without additional rye Bs were conducted. In both species, 5-6% of the A-derived transcripts across the entire genomes were differentially expressed in the presence of 2Bs. The GO term enrichment analysis revealed that Bs influence A chromosome encoded processes like "gene silencing"; "DNA methylation or demethylation"; "chromatin silencing"; "negative regulation of gene expression, epigenetic"; "post-embryonic development"; and "chromosome organization." 244 B chromosome responsive A-located genes in + 2B rye and + B wheat shared the same biological function. Positively correlated with the number of Bs, 939 and 1391 B-specific transcripts were identified in + 2B and + 4B wheat samples, respectively. 85% of B-transcripts in + 2B were also found in + 4B transcriptomes. 297 B-specific transcripts were identified in + 2B rye, and 27% were common to the B-derived transcripts identified in + B wheat. Bs encode mobile elements and housekeeping genes, but most B-transcripts were without detectable similarity to known genes. Some of these genes are involved in cell division-related functions like Nuf2 and might indicate their importance in maintaining Bs. The transcriptome analysis provides new insights into the complex interrelationship between standard A chromosomes and supernumerary B chromosomes.

Tissue-Specific Transcriptome Analysis Reveals Candidate Transcripts Associated with the Process of Programmed B Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

Supernumerary B chromosomes of Aegilops speltoides undergo precise elimination in roots early in embryo development.

Not necessarily all cells of an organism contain the same genome. Some eukaryotes exhibit dramatic differences between cells of different organs, resulting from programmed elimination of chromosomes or their fragments. Here, we present a detailed analysis of programmed B chromosome elimination in plants. Using goatgrass Aegilops speltoides as a model, we demonstrate that the elimination of B chromosomes is a strictly controlled and highly efficient root-specific process. At the onset of embryo differentiation B chromosomes undergo elimination in proto-root cells. Independent of centromere activity, B chromosomes demonstrate nondisjunction of chromatids and lagging in anaphase, leading to micronucleation. Chromatin structure and DNA replication differ between micronuclei and primary nuclei and degradation of micronucleated DNA is the final step of B chromosome elimination. This process might allow root tissues to survive the detrimental expression, or overexpression of B chromosome-located root-specific genes with paralogs located on standard chromosomes.

Depletion of KNL2 Results in Altered Expression of Genes Involved in Regulation of the Cell Cycle, Transcription, and Development in Arabidopsis.

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

Extracting genotype information of Arabidopsis thaliana recombinant inbred lines from transcript profiles established with high-density oligonucleotide arrays.

KEY MESSAGE: Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A. thaliana accessions. Individual probes of the GeneChip® that show sequence polymorphisms between different A. thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A. thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A. thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Tailoring high-density oligonucleotide arrays for transcript profiling of different Arabidopsis thaliana accessions using a sequence-based approach.

KEY MESSAGE: Excluding polymorphic probes from GeneChip ® transcript profiling experiments via a sequence-based approach results in improved detection of differentially expressed genes in developing seeds of Arabidopsis thaliana accessions Col-0 and C24. GeneChip® arrays represent a powerful tool for transcript profiling experiments. The ATH1 GeneChip® has been designed based on the sequence of the Arabidopsis thaliana reference genome Col-0, hence the features on the array exactly match the sequences of Col-0 transcripts. In contrast, transcripts of other A. thaliana accessions or related species may show nucleotide differences and/or insertions/deletions when compared to the corresponding Col-0 transcripts, therefore, comparisons of transcript abundance involving different A. thaliana accessions or related species may be compromised for a certain number of transcripts. To tackle this limitation, a sequence-based strategy was developed. Only features on the array that were identical in sequence for the specimen to be compared were considered for transcript profiling. The impact of the proposed strategy was evaluated for transcript profiles that were established for developing seeds of A. thaliana accessions Col-0 and C24.

Genetic dissection of metabolite variation in Arabidopsis seeds: evidence for mQTL hotspots and a master regulatory locus of seed metabolism.

To gain insight into genetic factors controlling seed metabolic composition and its relationship to major seed properties, an Arabidopsis recombinant inbred line (RIL) population, derived from accessions Col-0 and C24, was studied using an MS-based metabolic profiling approach. Relative intensities of 311 polar primary metabolites were used to identify associated genomic loci and to elucidate their interactions by quantitative trait locus (QTL) mapping. A total of 786 metabolic QTLs (mQTLs) were unequally distributed across the genome, forming several hotspots. For the branched-chain amino acid leucine, mQTLs and candidate genes were elucidated in detail. Correlation studies displayed links between metabolite levels, seed protein content, and seed weight. Principal component analysis revealed a clustering of samples, with PC1 mapping to a region on the short arm of chromosome IV. The overlap of this region with mQTL hotspots indicates the presence of a potential master regulatory locus of seed metabolism. As a result of database queries, a series of candidate regulatory genes, including bZIP10, were identified within this region. Depending on the search conditions, metabolic pathway-derived candidate genes for 40-61% of tested mQTLs could be determined, providing an extensive basis for further identification and characterization of hitherto unknown genes causal for natural variation of Arabidopsis seed metabolism.

Transcriptome analysis of the aquaporin AtPIP1;2 deficient line in Arabidopsis thaliana.

Atmospheric CO2 impacts all aspects of plant development. It has changed in the past and is predicted to change further on. Studies on the response of crop plants to low and elevated CO2 concerning growth, productivity and physiological processes are intense. In contrast, the molecular mechanisms of cellular CO2 exchange are still under discussion. At the same time it becomes more and more accepted that carbon dioxide is transported across cellular biomembranes by CO2 conducting aquaporins. Our recent study (Boudichevskaia et al., 2015) demonstrates that the lack of a single gene product - aquaporin AtPIP1;2 - resulted in massive transcriptional reprogramming in Arabidopsis as a consequence of reduced tissue CO2 diffusion rates. Therefore, the transcriptome data of the aquaporin AtPIP1;2 deficient line can be used in the comparative expression analyses for better understanding the role of aquaporins with regard to CO2 and water transport in plants. Here we describe a gene expression dataset generated for three biological replicates per genotype on Affymetrix platform. We provide detailed methods and analysis on microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE62167. Additionally, we provide the R code for data preprocessing and quality control.

T-DNA insertion in aquaporin gene AtPIP1;2 generates transcription profiles reminiscent of a low CO2 response.

Results from CO2 diffusion studies and characterization of Arabidopsis thaliana aquaporin AtPIP1;2 T-DNA insertion lines support the idea that specific aquaporins facilitate the diffusion of CO2 through biological membranes. However, their function as CO2 diffusion facilitators in plant physiology is still a matter of debate. Assuming that a lack of AtPIP1;2 causes a characteristic transcriptional response, we compared data from a AtPIP1;2 T-DNA insertion line obtained by Illumina sequencing, Affymetrix chip analysis and quantitative RT-PCR to the transcriptome of plants grown under drought stress or under low CO2 conditions. The plant reaction to the deficit of AtPIP1;2 was unlike drought stress responses but comparable with that of low CO2 conditions. In addition, we observed a phenotype characteristic to plants grown under low CO2 . The findings support the hypothesis that the AtPIP1;2 function in plant physiology is not to facilitate water but CO2 diffusion.

Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids.

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids.