Characterization of the DHH1/DDX6-Like RNA Helicase Family in Arabidopsis thaliana

General cytoplasmic mRNA turnover in eukaryotes is governed by two major pathways: 5’-to-3’ decay initiated by the multi-subunit mRNA decapping complex and carried out by EXORIBONUCLEASE 1/4 and 3’-to-5’ decay executed by the RNA exosome or the 3’-to-5’ exoribonuclease SUPPRESSOR OF VARICOSE (SOV). In yeast and metazoa, DEAD-box RNA helicase DHH1/DDX6 proteins are associated with the decapping complex and involved in translational repression and/or mRNA degradation. In this dissertation, RNA HELICASE 6, 8 and 12 (RH6, RH8 and RH12) were identified as orthologs of the DHH1/DDX6 family in the plant Arabidopsis thaliana. Genetic analysis uncovered that the genes RH6, RH8 and RH12 are functionally redundant and indispensable for growth and development, as the triple homozygous rh6 rh8 rh12 mutant displays pleiotropic phenotypes including delayed germination, altered vein patterning, and severe maturation defects including infertility. By use of confocal microscopy, we found that all the three RHs are nucleocytoplasmic proteins, with the cytoplasmic portion forming stress-inducible cytoplasmic ribonucleoprotein complexes associated with both processing bodies (PBs) and stress granules (SGs). Evaluation of PB and SGs in the triple homozygous mutant rh6 rh8 rh12 indicated that the RHs are required for PB but not SG formation. Genome-wide analysis of RNA decay kinetics in the rh6 rh8 rh12 mutant by genome-scale RNA-sequencing revealed that the RHs mediate degradation of short-lived mRNAs with diverse biological functions, the majority of which are substrates of the 5’ to 3’ decapping-mediated decay pathway. Further genome-scale polyadenylated mRNA and polyadenylated ribosome-associated mRNA analysis revealed that co-disruption of these RHs perturbed transcriptome and translatome homeostasis, characterized by an increase in the steady-state mRNA abundance and translational status of stress- and defense-response transcripts and the simultaneous repression in accumulation of growth and development-associated transcripts. Genetic analysis determined that the growth defects of rh6 rh8 rh12 are not related to small interfering RNA production, but caused by a constitutive immune response linked with stabilization and enhanced ribosome occupancy of defense-related mRNAs. The phenotypes, constitutive elevation of stress/defense-response mRNAs and repression of growth-related mRNAs was alleviated by (over)expression of a functional SOV allele. This study determined the plant DDH1/DDX6 orthologs are a variable component of the 5’ to 3’ decay machinery that accelerates decay of growth limiting defense-associated transcripts under non-stress conditions

Characterization of the DHH1/DDX6-Like RNA Helicase Family in Arabidopsis thaliana

General cytoplasmic mRNA turnover in eukaryotes is governed by two major pathways: 5’-to-3’ decay initiated by the multi-subunit mRNA decapping complex and carried out by EXORIBONUCLEASE 1/4 and 3’-to-5’ decay executed by the RNA exosome or the 3’-to-5’ exoribonuclease SUPPRESSOR OF VARICOSE (SOV). In yeast and metazoa, DEAD-box RNA helicase DHH1/DDX6 proteins are associated with the decapping complex and involved in translational repression and/or mRNA degradation. In this dissertation, RNA HELICASE 6, 8 and 12 (RH6, RH8 and RH12) were identified as orthologs of the DHH1/DDX6 family in the plant Arabidopsis thaliana. Genetic analysis uncovered that the genes RH6, RH8 and RH12 are functionally redundant and indispensable for growth and development, as the triple homozygous rh6 rh8 rh12 mutant displays pleiotropic phenotypes including delayed germination, altered vein patterning, and severe maturation defects including infertility. By use of confocal microscopy, we found that all the three RHs are nucleocytoplasmic proteins, with the cytoplasmic portion forming stress-inducible cytoplasmic ribonucleoprotein complexes associated with both processing bodies (PBs) and stress granules (SGs). Evaluation of PB and SGs in the triple homozygous mutant rh6 rh8 rh12 indicated that the RHs are required for PB but not SG formation. Genome-wide analysis of RNA decay kinetics in the rh6 rh8 rh12 mutant by genome-scale RNA-sequencing revealed that the RHs mediate degradation of short-lived mRNAs with diverse biological functions, the majority of which are substrates of the 5’ to 3’ decapping-mediated decay pathway. Further genome-scale polyadenylated mRNA and polyadenylated ribosome-associated mRNA analysis revealed that co-disruption of these RHs perturbed transcriptome and translatome homeostasis, characterized by an increase in the steady-state mRNA abundance and translational status of stress- and defense-response transcripts and the simultaneous repression in accumulation of growth and development-associated transcripts. Genetic analysis determined that the growth defects of rh6 rh8 rh12 are not related to small interfering RNA production, but caused by a constitutive immune response linked with stabilization and enhanced ribosome occupancy of defense-related mRNAs. The phenotypes, constitutive elevation of stress/defense-response mRNAs and repression of growth-related mRNAs was alleviated by (over)expression of a functional SOV allele. This study determined the plant DDH1/DDX6 orthologs are a variable component of the 5’ to 3’ decay machinery that accelerates decay of growth limiting defense-associated transcripts under non-stress conditions

Commercial scale production of Yamabushitake mushroom (Hericium erinaceus (Bull.) Pers. 1797) using rubber and bamboo sawdust substrates in tropical regions

Abstract Yamabushitake (Hericium erinaceus) is one of the most sought out mushrooms that is widely used for both direct consumption and medicinal purposes. While its demand increases worldwide, cultivation of the mushroom is limited to temperate areas and its production in tropical regions has never been explored. The aim of this study was to test the utilization of rubber and bamboo sawdust, alone or as a substrate mixture, for industrial scale Yamabushitake mushroom production. Five substrate treatments with various ratios of the two sawdust were compared for their physicochemical properties in relation to mushroom productivity. The highest mushroom fresh and dry (113.22 and 23.25 g, respectively), biological efficiency (42.61%), and cap size (9.53 cm) were obtained from the substrates containing 100% rubber sawdust, with the mushroom yield decreasing proportional to the ratio of bamboo sawdust. The 100% rubber sawdust substrate provided a higher initial organic matter and carbon content together with C:N ratio at 63.2%, 36.7% and 65.48, respectively, whereas the 100% bamboo sawdust provided higher nitrogen content (1.03%), which was associated with lower mushroom yield but higher number of fruiting bodies. As in the 100% rubber sawdust substrate, a comparable mushroom yield and growth attributes were also obtained in the 3:1 rubber-bamboo sawdust mixture substrate. Principle component analysis of the measured variables indicated a strong influence of substrate C:N ratio before spawning and the change in substrate electrical conductivity and N content after cultivation to the variation in mushroom productivity among the treatments. The results demonstrate the applicability of rubber sawdust and its combination with up to 25% of bamboo sawdust for Yamabushitake mushroom cultivation and provide the basis for substrate optimization in the tropical Yamabushitake mushroom industry through a circular economy framework