Stable Carbon Isotope Discrimination Is under Genetic Control in the C4 Species Maize with Several Genomic Regions Influencing Trait Expression

In plants with C₄ photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ13C) are largely unknown, and genetic components influencing Δ13C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ13C is under genetic control in this C₄ species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ13C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ13C was detected under field and greenhouse conditions. For several introgression library lines, Δ13C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ13C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ13C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ13C. Plant development stage had no effect on Δ13C expression, as phenotypic as well as genotypic correlations between Δ13C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ13C to be under polygenic control in the C₄ species maize.During photosynthesis, plants use light energy to convert atmospheric CO₂ and water into carbohydrates. For the element carbon, there are two stable isotopes, ¹²C and ¹³C. Due to the physical and chemical properties of photosynthetic CO₂ fixation, plants are depleted in ¹³C compared with atmospheric CO₂. In C₃ plants, this discrimination of stable carbon isotopes (Δ13C) has long been used to detect genetic differences of water use efficiency and has been shown to be an accurate predictor for yield under drought (Rebetzke et al., 2002). As Δ13C is linearly related to the ratio of intercellular to atmospheric CO₂ partial pressure (Farquhar et al., 1982), stomatal closure under drought stress is associated with reduced Δ13C. For C₄ plants, our knowledge about the mechanisms underlying Δ13C and about its association with water use efficiency is much more limited. Differences in Δ13C between genotypes of C₄ species have been reported, among others, for sorghum (Sorghum bicolor; Hubick et al., 1990) and maize (Zea mays; Monneveux et al., 2007). However, comprehensive studies analyzing the inheritance of Δ13C have not been performed to date.In C₃ plants, the important steps of CO₂ uptake include the diffusion of atmospheric CO₂ through the boundary layer and the stomata. Subsequently, CO₂ is taken up by the cell and enters the chloroplast, where carboxylation by Rubisco takes place. During photosynthetic carbon fixation, the strongest fractionation of carbon isotopes occurs during the carboxylation reaction of Rubisco (Roeske and O’Leary, 1984). A theoretical model of Δ13C in C₃ photosynthesis has been described by Farquhar et al. (1982), in which Δ13C depends linearly on the ratio of intercellular to ambient partial pressure of CO₂ (p_i p_a−1), and thus provides an indication of stomatal conductance and photosynthetic capacity. Additionally, the model includes the dependency of Δ13C on the fractionation of carbon isotopes during CO₂ diffusion in the air and on the enzymatic properties of the Rubisco enzyme.For rice (Oryza sativa), tomato (Solanum lycopersicum), and wheat (Triticum aestivum), it has been shown that genetic variation for Δ13C is quantitative, genotype-by-environment interaction is small, and the trait heritability is high (Condon and Richards, 1992; Rebetzke et al., 2002; Comstock et al., 2005; Impa et al., 2005). Quantitative trait loci (QTL) for Δ13C have been mapped (Handley et al., 1994; Price et al., 2002; Rebetzke et al., 2008), and in the model plant Arabidopsis (Arabidopsis thaliana), four genes have been identified that are associated with Δ13C. Two are involved in stomatal patterning and thus influence stomatal conductance (Masle et al., 2005; Nilson and Assmann, 2010), and one of them influences photosynthetic capacity as well (Masle et al., 2005). One gene plays a role in cuticular wax composition and is also associated with stomatal conductance (Lü et al., 2012), whereas the fourth gene encodes a cellulose synthase subunit, and mutations in this gene lead to decreased Δ13C. Presumably, this is the result of a decreased cell turgor due to a decreased water transport capacity of the xylem (Liang et al., 2010).For C₄ plants, our knowledge about the genetic mechanisms and physiological processes underlying Δ13C is much more limited, due to the more complex mechanism of CO₂ fixation. The first carboxylation step in C₄ plants takes place in mesophyll cells, in which CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC). During this reaction, combined with the fractionation of carbon isotopes during HCO₃⁻ formation, carbon is actually enriched in ¹³C (Farquhar, 1983). The C₄ organic acid formed by PEPC is transported to the bundle sheath cells, where CO₂ is released to be fixed by Rubisco in the second step. However, a fraction of CO₂ released in the bundle sheath can diffuse back to the mesophyll cells. The proportion of carbon fixed by PEPC that subsequently leaks out of the bundle sheath cells is termed leakiness (ϕ) and reduces the opportunity of Rubisco to discriminate against ¹³C in C₄ plants. According to the theoretical model by Farquhar (1983), Δ13C and p_i p_a−1 are also linearly related in C₄ plants, but the regression slope is determined by ϕ. Consequently, there can be a positive or a negative correlation of Δ13C and p_i p_a−1 depending on ϕ (Hubick et al., 1990). Regarding the entire fixation process, discrimination against ¹³C in C₄ plants is not as strong as in C₃ plants, and so far there have been few studies reporting a genetic variation of Δ13C in C₄ plants. In sorghum, small but significant differences in Δ13C have been observed among 12 cultivars (Hubick et al., 1990), and similar to C₃ plants, Δ13C has been shown to be correlated with transpiration efficiency (Henderson et al., 1998). Additionally, it has been shown for maize and sugarcane (Saccharum officinarum) that stress conditions lead to an increase in Δ13C (Bowman et al., 1989; Meinzer et al., 1994; Ranjith et al., 1995; Buchmann et al., 1996). Experiments under drought and under well-watered conditions showed higher values for Δ13C in drought-tolerant maize hybrids than in susceptible checks (Monneveux et al., 2007).The use of Δ13C as an indirect trait in breeding for drought tolerance in C₄ species would be highly beneficial, given a stable trait expression and high heritability similar to that in C₃ plants. To assess whether Δ13C can also be used in C₄ plants as an indirect selection trait for drought-tolerant lines, it needs to be shown that Δ13C is under genetic control, although the physiology and molecular mechanisms of Δ13C are not yet fully understood. In this study, we used an introgression library (IL; Eshed and Zamir, 1994) derived from two elite parents to analyze the genetic variation in Δ13C under well-watered conditions. ILs have been successfully used in genetics to identify QTL for various qualitatively and quantitatively inherited traits. An IL is a defined set of nearly isogenic inbred lines derived from repeated backcrosses with one of the parents (recurrent parent [RP]) and marker-assisted selection for single fragments (Supplemental Fig. S1). Ideally, each IL line carries a single chromosome fragment of a donor parent (DP) in the genetic background of an RP. Taken together, the different segments cover the whole donor genome, allowing estimation of the effects of single donor fragments in an otherwise identical genetic background (Eshed and Zamir, 1994). The RP of the IL under investigation originates from southeastern Europe and is an elite inbred line of the maize dent pool. As DP, we chose an unrelated maize line representative of the European flint pool. The IL (IL_01–IL_89) was genotyped using the Illumina MaizeSNP50 Bead Chip (Ganal et al., 2011) carrying 56,110 single-nucleotide polymorphism (SNP) markers.Kernel Δ13C of 77 IL lines was measured in the field and in the greenhouse (Δ13C is genetically controlled in the C₄ species maize. Our specific goals were (1) to characterize the genetic architecture of Δ13C (i.e. to determine the number of genomic regions associated with Δ13C), (2) to localize genomic regions influencing Δ13C, and (3) to assess the extent to which genotypic variation in Δ13C might be the result of differences in plant development.

Table I.

Overview of the experiments and experimental designs

Eosinophils and Megakaryocytes Support the Early Growth of Murine MOPC315 Myeloma Cells in Their Bone Marrow Niches

Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.     

Tracking Functional Brain Changes in Patients with Depression under Psychodynamic Psychotherapy Using Individualized Stimuli

Objective

Neurobiological models of depression posit limbic hyperactivity that should normalize after successful treatment. For psychotherapy, though, brain changes in patients with depression show substantial variability. Two critical issues in relevant studies concern the use of unspecific stimulation experiments and relatively short treatment protocols. Therefore changes in brain reactions to individualized stimuli were studied in patients with depression after eight months of psychodynamic psychotherapy.

Methods

18 unmedicated patients with recurrent major depressive disorder were confronted with individualized and clinically derived content in a functional MRI experiment before (T1) and after eight months (T2) of psychodynamic therapy. A control group of 17 healthy subjects was also tested twice without intervention. The experimental stimuli were sentences describing each participant''s dysfunctional interpersonal relationship patterns derived from clinical interviews based on Operationalized Psychodynamic Diagnostics (OPD).

Results

At T1 patients showed enhanced activation compared to controls in several limbic and subcortical regions, including amygdala and basal ganglia, when confronted with OPD sentences. At T2 the differences in brain activity between patients and controls were no longer apparent. Concurrently, patients had improved significantly in depression scores.

Conclusions

Using ecologically valid stimuli, this study supports the model of limbic hyperactivity in depression that normalizes after treatment. Without a control group of untreated patients measured twice, though, changes in patients'' brain activity could also be attributed to other factors than psychodynamic therapy.     

Soil microarthropods respond differently to simulated drought in organic and conventional farming systems

In Central Europe, summer droughts are increasing in frequency which threatens production and biodiversity in agroecosystems. The potential of different farming systems to mitigate detrimental drought effects on soil animals is largely unknown. We investigated the effects of simulated drought on the abundance and community composition of soil microarthropods (Collembola and Oribatida and Meso‐, Pro‐, and Astigmata) in winter wheat fields under long‐term conventional and organic farming in the DOK trial, Switzerland. We simulated drought by excluding 65% of the ambient precipitation during the wheat‐growing season from March to June 2017. The abundance of Collembola and Oribatida declined more consistently in conventionally managed fields compared to organically managed fields under simulated drought. The abundance of Collembola as well as Meso‐, Pro‐ and Astigmata, but not the abundance of Oribatida, increased in deeper soil layers due to simulated drought, suggesting vertical migration as a drought avoidance strategy. The species composition of Oribatida communities, but not of Collembola communities, differed significantly between drought treatments and between farming systems. Soil carbon content was a major factor structuring Oribatida communities. Our results suggest that organic farming buffers negative effects of drought on soil microarthropods, presumably due to higher soil carbon content and associated higher soil moisture and improved soil structure. This potential of organic farming systems to mitigate consequences of future droughts on soil biodiversity is promising and needs further exploration across larger climatic and spatial scales and should be extended to other groups of soil biota.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine

The biosynthesis of non-ribosomal peptides, many of which have pharmaceutical activities, is an evolutionary privilege of microorganisms. Capitalizing on the universal set of the Streptomyces lavendulae non-ribosomal peptide synthase BpsA and the Streptomyces verticillus 4'-phosphopantetheinyl transferase Svp, we have engineered Escherichia coli as well as mammalian cells, including human stem cells, to produce the blue 3,3'-bipyridyl pigment keto-indigoidine and the reduced colorless but fluorescent leuco-isoform. Detailed characterization of a tailored substrate-free chromogenic assay and FACS analysis showed that indigoidine's blue color and fluorescence could be reliably profiled in bacteria and mammalian cells using standard multiwell-compatible detection equipment. Besides serving as an inexpensive, reliable, versatile and easy-to-assay cross-kingdom reporter system, the potential of having mammalian cells produce non-ribosomal peptides, preferably ones with biopharmaceutical activities, may provide novel treatment opportunities in future gene- and cell-based therapies.     

Basics in paleodemography: a comparison of age indicators applied to the early medieval skeletal sample of Lauchheim

Recent advances in the methods of skeletal age estimation have rekindled interest in their applicability to paleodemography. The current study contributes to the discussion by applying several long established as well as recently developed or refined aging methods to a subsample of 121 adult skeletons from the early medieval cemetery of Lauchheim. The skeletal remains were analyzed by 13 independent observers using a variety of aging techniques (complex method and other multimethod approaches, Transition Analysis, cranial suture closure, auricular surface method, osteon density method, tooth root translucency measurement, and tooth cementum annulation counting). The age ranges and mean age estimations were compared and results indicate that all methods showed smaller age ranges for the younger individuals, but broader age ranges for the older age groups.