Differential coupling of gibberellin responses by Rht-B1c suppressor alleles and Rht-B1b in wheat highlights a unique role for the DELLA N-terminus in dormancy

During the Green Revolution, substantial increases in wheat (Triticum aestivum) yields were realized, at least in part, through the introduction of the Reduced height (Rht)-B1b and Rht-D1b semi-dwarfing alleles. In contrast to Rht-B1b and Rht-D1b, the Rht-B1c allele is characterized by extreme dwarfism and exceptionally strong dormancy. Recently, 35 intragenic Rht-B1c suppressor alleles were created in the spring wheat cultivar Maringa, and termed overgrowth (ovg) alleles. Here, 14 ovg alleles with agronomically relevant plant heights were reproducibly classified into nine tall and five semi-dwarf alleles. These alleles differentially affected grain dormancy, internode elongation rate, and coleoptile and leaf lengths. The stability of these ovg effects was demonstrated for three ovg alleles in different genetic backgrounds and environments. Importantly, two semi-dwarf ovg alleles increased dormancy, which correlated with improved pre-harvest sprouting (PHS) resistance. Since no negative effects on grain yield or quality were observed, these semi-dwarf ovg alleles are valuable for breeding to achieve adequate height reduction and protection of grain quality in regions prone to PHS. Furthermore, this research highlights a unique role for the first 70 amino acids of the DELLA protein, encoded by the Rht-1 genes, in grain dormancy

A molecular timetable for apical bud formation and dormancy induction in poplar

The growth of perennial plants in the temperate zone alternates with periods of dormancy that are typically initiated during bud development in autumn. In a systems biology approach to unravel the underlying molecular program of apical bud development in poplar (Populus tremula 3 Populus alba), combined transcript and metabolite profiling were applied to a high-resolution time course from short-day induction to complete dormancy. Metabolite and gene expression dynamics were used to reconstruct the temporal sequence of events during bud development. Importantly, bud development could be dissected into bud formation, acclimation to dehydration and cold, and dormancy. To each of these processes, specific sets of regulatory and marker genes and metabolites are associated and provide a reference frame for future functional studies. Light, ethylene, and abscisic acid signal transduction pathways consecutively control bud development by setting, modifying, or terminating these processes. Ethylene signal transduction is positioned temporally between light and abscisic acid signals and is putatively activated by transiently low hexose pools. The timing and place of cell proliferation arrest (related to dormancy) and of the accumulation of storage compounds (related to acclimation processes) were established within the bud by electron microscopy. Finally, the identification of a large set of genes commonly expressed during the growth-to-dormancy transitions in poplar apical buds, cambium, or Arabidopsis thaliana seeds suggests parallels in the underlying molecular mechanisms in different plant organs

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

<p>Abstract</p> <p>Background</p> <p>Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.</p> <p>Results</p> <p>Here different dyes for viability estimation considering membrane potential (DiOC₂(3), DiBAC₄(3), DiOC₆(3)) and cell integrity (DiBAC₄(3)/PI, Syto9/PI) were successfully evaluated for <it>Bacillus megaterium </it>cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by <it>B. megaterium </it>was studied.</p> <p>Conclusions</p> <p>It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.</p

N-terminal truncated RHT-1 proteins generated by translational reinitiation cause semi-dwarfing of wheat Green Revolution alleles

The unprecedented wheat yield increases during the Green Revolution were achieved through the introduction of the Reduced height (Rht)-B1b and Rht-D1b semi-dwarfing alleles. These Rht-1 alleles encode growth-repressing DELLA genes containing a stop codon within their open reading frame that confers gibberellin (GA)-insensitive semi-dwarfism. In this study, we successfully took the hurdle of detecting wild-type RHT-1 proteins in different wheat organs and confirmed their degradation in response to GAs. We further demonstrated that Rht-B1b and Rht-D1b produce N-terminal truncated proteins through translational reinitiation. Expression of these N-terminal truncated proteins in transgenic lines and in Rht-D1c, an allele containing multiple Rht-D1b copies, demonstrated their ability to cause strong dwarfism, resulting from their insensitivity to GA-mediated degradation. N-terminal truncated proteins were detected in spikes and nodes, but not in the aleurone layers. Since Rht-B1b and Rht-D1b alleles cause dwarfism but have wild-type dormancy, this finding suggests that tissue-specific differences in translational reinitiation may explain why the Rht-1 alleles reduce plant height without affecting dormancy. Taken together, our findings not only reveal the molecular mechanism underlying the Green Revolution but also demonstrate that translational reinitiation in the main open reading frame occurs in plants

Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pall and pal2 single mutants and with limited phenotypic alterations in the pall pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids

Comparing PTSD symptom networks in type I vs. type II trauma survivors

Background: Network analysis has gained increasing attention as a new framework to study complex associations between symptoms of post-traumatic stress disorder (PTSD). A number of studies have been published to investigate symptom networks on different sets of symptoms in different populations, and the findings have been inconsistent. Objective: We aimed to extend previous research by testing whether differences in PTSD symptom networks can be found in survivors of type I (single event; sudden and unexpected, high levels of acute threat) vs. type II (repeated and/or protracted; anticipated) trauma (with regard to their index trauma). Method: Participants were trauma-exposed individuals with elevated levels of PTSD symptomatology, most of whom (94%) were undergoing assessment in preparation for PTSD treatment in several treatment centres in Germany and Switzerland (n = 286 with type I and n = 187 with type II trauma). We estimated Bayesian Gaussian graphical models for each trauma group and explored group differences in the symptom network. Results: First, for both trauma types, our analyses identified the edges that were repeatedly reported in previous network studies. Second, there was decisive evidence that the two networks were generated from different multivariate normal distributions, i.e. the networks differed on a global level. Third, explorative edge-wise comparisons showed moderate or strong evidence for specific 12 edges. Edges which emerged as especially important in distinguishing the networks were between intrusions and flashbacks, highlighting the stronger positive association in the group of type II trauma survivors compared to type I survivors. Flashbacks showed a similar pattern of results in the associations with detachment and sleep problems (type II > type I). Conclusion: Our findings suggest that trauma type contributes to the heterogeneity in the symptom network. Future research on PTSD symptom networks should include this variable in the analyses to reduce heterogeneity

Comparing PTSD symptom networks in type I vs. type II trauma survivors

Background: Network analysis has gained increasing attention as a new framework to study complex associations between symptoms of post-traumatic stress disorder (PTSD). A number of studies have been published to investigate symptom networks on different sets of symptoms in different populations, and the findings have been inconsistent. Objective: We aimed to extend previous research by testing whether differences in PTSD symptom networks can be found in survivors of type I (single event; sudden and unexpected, high levels of acute threat) vs. type II (repeated and/or protracted; anticipated) trauma (with regard to their index trauma). Method: Participants were trauma-exposed individuals with elevated levels of PTSD symptomatology, most of whom (94%) were undergoing assessment in preparation for PTSD treatment in several treatment centres in Germany and Switzerland (n = 286 with type I and n = 187 with type II trauma). We estimated Bayesian Gaussian graphical models for each trauma group and explored group differences in the symptom network. Results: First, for both trauma types, our analyses identified the edges that were repeatedly reported in previous network studies. Second, there was decisive evidence that the two networks were generated from different multivariate normal distributions, i.e. the networks differed on a global level. Third, explorative edge-wise comparisons showed moderate or strong evidence for specific 12 edges. Edges which emerged as especially important in distinguishing the networks were between intrusions and flashbacks, highlighting the stronger positive association in the group of type II trauma survivors compared to type I survivors. Flashbacks showed a similar pattern of results in the associations with detachment and sleep problems (type II > type I). Conclusion: Our findings suggest that trauma type contributes to the heterogeneity in the symptom network. Future research on PTSD symptom networks should include this variable in the analyses to reduce heterogeneity

Overcoming challenges in variant calling : exploring sequence diversity in candidate genes for plant development in perennial ryegrass (Lolium perenne)

Revealing DNA sequence variation within the Lolium perenne genepool is important for genetic analysis and development of breeding applications. We reviewed current literature on plant development to select candidate genes in pathways that control agronomic traits, and identified 503 orthologues in L. perenne. Using targeted resequencing, we constructed a comprehensive catalogue of genomic variation for a L. perenne germplasm collection of 736 genotypes derived from current cultivars, breeding material and wild accessions. To overcome challenges of variant calling in heterogeneous outbreeding species, we used two complementary strategies to explore sequence diversity. First, four variant calling pipelines were integrated with the VariantMetaCaller to reach maximal sensitivity. Additional multiplex amplicon sequencing was used to empirically estimate an appropriate precision threshold. Second, a de novo assembly strategy was used to reconstruct divergent alleles for each gene. The advantage of this approach was illustrated by discovery of 28 novel alleles of LpSDUF247, a polymorphic gene co-segregating with the S-locus of the grass self-incompatibility system. Our approach is applicable to other genetically diverse outbreeding species. The resulting collection of functionally annotated variants can be mined for variants causing phenotypic variation, either through genetic association studies, or by selecting carriers of rare defective alleles for physiological analyses