SeedTransNet: a directional translational network revealing regulatory patterns during seed maturation and germination.

Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

Novel pipeline identifies new upstream ORFs and non-AUG initiating main ORFs with conserved amino acid sequences in the 5' leader of mRNAs in Arabidopsis thaliana.

Eukaryotic mRNAs contain a 5' leader sequence preceding the main open reading frame (mORF) and, depending on the species, 20%-50% of eukaryotic mRNAs harbor an upstream ORF (uORF) in the 5' leader. An unknown fraction of these uORFs encode sequence conserved peptides (conserved peptide uORFs, CPuORFs). Experimentally validated CPuORFs demonstrated to regulate the translation of downstream mORFs often do so in a metabolite concentration-dependent manner. Previous research has shown that most CPuORFs possess a start codon context suboptimal for translation initiation, which turns out to be favorable for translational regulation. The suboptimal initiation context may even include non-AUG start codons, which makes CPuORFs hard to predict. For this reason, we developed a novel pipeline to identify CPuORFs unbiased of start codon using well-annotated sequence data from 31 eudicot plant species and rice. Our new pipeline was able to identify 29 novel Arabidopsis thaliana (Arabidopsis) CPuORFs, conserved across a wide variety of eudicot species of which 15 do not initiate with an AUG start codon. In addition to CPuORFs, the pipeline was able to find 14 conserved coding regions directly upstream and in frame with the mORF, which likely initiate translation on a non-AUG start codon. Altogether, our pipeline identified highly conserved coding regions in the 5' leaders of Arabidopsis transcripts, including in genes with proven functional importance such as LHY, a key regulator of the circadian clock, and the RAPTOR1 subunit of the target of rapamycin (TOR) kinase.

Seed-Stored mRNAs that Are Specifically Associated to Monosomes Are Translationally Regulated during Germination.

The life cycle of many organisms includes a quiescent stage, such as bacterial or fungal spores, insect larvae, or plant seeds. Common to these stages is their low water content and high survivability during harsh conditions. Upon rehydration, organisms need to reactivate metabolism and protein synthesis. Plant seeds contain many mRNAs that are transcribed during seed development. Translation of these mRNAs occurs during early seed germination, even before the requirement of transcription. Therefore, stored mRNAs are postulated to be important for germination. How these mRNAs are stored and protected during long-term storage is unknown. The aim of this study was to investigate how mRNAs are stored in dry seeds and whether they are indeed translated during seed germination. We investigated seed polysome profiles and the mRNAs and protein complexes that are associated with these ribosomes in seeds of the model organism Arabidopsis (Arabidopsis thaliana). We showed that most stored mRNAs are associated with monosomes in dry seeds; therefore, we focus on monosomes in this study. Seed ribosome complexes are associated with mRNA-binding proteins, stress granule, and P-body proteins, which suggests regulated packing of seed mRNAs. Interestingly, ∼17% of the mRNAs that are specifically associated with monosomes are translationally up-regulated during seed germination. These mRNAs are transcribed during seed maturation, suggesting a role for this developmental stage in determining the translational fate of mRNAs during early germination.

Extensive translational regulation during seed germination revealed by polysomal profiling.

This work investigates the extent of translational regulation during seed germination. The polysome occupancy of each gene is determined by genome-wide profiling of total mRNA and polysome-associated mRNA. This reveals extensive translational regulation during Arabidopsis thaliana seed germination. The polysome occupancy of thousands of individual mRNAs changes to a large extent during the germination process. Intriguingly, these changes are restricted to two temporal phases (shifts) during germination, seed hydration and germination. Sequence features, such as upstream open reading frame number, transcript length, mRNA stability, secondary structures, and the presence and location of specific motifs correlated with this translational regulation. These features differed significantly between the two shifts, indicating that independent mechanisms regulate translation during seed germination. This study reveals substantial translational dynamics during seed germination and identifies development-dependent sequence features and cis elements that correlate with the translation control, uncovering a novel and important layer of gene regulation during seed germination.

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

BACKGROUND: Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. RESULTS: Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. CONCLUSIONS: This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.