Integration of Experiments across Diverse Environments Identifies the Genetic Determinants of Variation in Sorghum bicolor Seed Element Composition

Seedling establishment and seed nutritional quality require the sequestration of sufficient element nutrients. The identification of genes and alleles that modify element content in the grains of cereals, including sorghum (Sorghum bicolor), is fundamental to developing breeding and selection methods aimed at increasing bioavailable element content and improving crop growth. We have developed a high-throughput work flow for the simultaneous measurement of multiple elements in sorghum seeds. We measured seed element levels in the genotyped Sorghum Association Panel, representing all major cultivated sorghum races from diverse geographic and climatic regions, and mapped alleles contributing to seed element variation across three environments by genome-wide association. We observed significant phenotypic and genetic correlation between several elements across multiple years and diverse environments. The power of combining high-precision measurements with genome-wide association was demonstrated by implementing rank transformation and a multilocus mixed model to map alleles controlling 20 element traits, identifying 255 loci affecting the sorghum seed ionome. Sequence similarity to genes characterized in previous studies identified likely causative genes for the accumulation of zinc, manganese, nickel, calcium, and cadmium in sorghum seeds. In addition to strong candidates for these five elements, we provide a list of candidate loci for several other elements. Our approach enabled the identification of single-nucleotide polymorphisms in strong linkage disequilibrium with causative polymorphisms that can be evaluated in targeted selection strategies for plant breeding and improvement.Sorghum (Sorghum bicolor) is a globally cultivated source of food, feed, and fiber. Contrasting needs for elemental nutrient accumulation limit crop yield and quality for sorghum marketed to different sectors. The seed-bearing reproductive organs, or panicles, in sorghum represent up to 30% of the total dry matter yield (Amaducci et al., 2004). Plant-based diets, in which grains compose the major food source, require the accumulation of bioavailable essential elements in the plant seeds. Currently, iron (Fe) and zinc (Zn) deficiencies negatively affect the health of over two billion people worldwide (World Health Organization, 2002). Increased bioavailable elemental nutrient content in the edible portions of sorghum for human and animal nutrition could ameliorate this nutritional crisis (Graham et al., 1999; World Health Organization, 2002). Additional global health benefits could be achieved by increasing magnesium (Mg), selenium (Se), calcium (Ca), and copper (Cu; White and Broadley, 2005) while reducing the concentration of toxic elements, including arsenic (As) and cadmium (Cd; Ma et al., 2008).Seed element accumulation results from interconnected biological processes, including element uptake by the roots, translocation and remobilization within the plant, and ultimately import, deposition, and assimilation/storage in the seeds. Element availability is further affected by the accumulation of metabolites in seeds (Vreugdenhil et al., 2004). High-throughput ionomic analysis, or concurrent measurement of multiple elements, allows for the quantitative and simultaneous measurement of an organism’s elemental composition, providing a snapshot of the functional state of an organism under different experimental conditions (Salt et al., 2008). Most studies of the plant ionome utilize inductively coupled plasma mass spectroscopy (ICP-MS). Briefly, inductively coupled plasma (ICP) functions to ionize the analyte into atoms, which are then detected by mass spectroscopy. Reference standards are used to identify and quantitate each element of interest in the sample. ICP-MS analysis can be accomplished in as little as 1 min per sample, which allows for high-throughput processing of thousands of samples (Salt et al., 2008). Previous studies have demonstrated that several elements, including Fe, manganese (Mn), Zn, cobalt (Co), and Cd, share mechanisms of accumulation (Yi and Guerinot, 1996; Vert et al., 2002; Connolly et al., 2003). Ionomic signatures derived from multiple elements also have been shown to better predict plant physiological status for some elements than the measure of the element’s concentration, including essential nutrients like Fe (Baxter et al., 2008). Holistically examining the ionome provides significant insights into the networks underlying ion homeostasis beyond single-element studies (Baxter and Dilkes, 2012).There are over 45,000 catalogued lines of sorghum at the U.S. Department of Agriculture Germplasm Resource Information Network. This diverse collection of sorghum germplasm contains genetic variation with undiscovered impact on seed element composition (Das et al., 1997). Mapping quantitative trait loci for seed element concentration has been successful in a number of species, including Arabidopsis (Arabidopsis thaliana; Vreugdenhil et al., 2004; Waters and Grusak, 2008; Buescher et al., 2010), rice (Oryza sativa; Norton et al., 2010; Zhang et al., 2014), wheat (Triticum aestivum; Shi et al., 2008; Peleg et al., 2009), and maize (Zea mays; Simić et al., 2012; Baxter et al., 2013, 2014). Genome-wide association (GWA) mapping is well suited for uncovering the genetic basis for complex traits, including seed element accumulation. One of the key strengths of association mapping is that a priori knowledge is not necessary to identify new loci associated with the trait of interest. Furthermore, a GWA mapping population is composed of lines that have undergone numerous recombination events, allowing for a narrower mapping interval. Previous GWA studies in maize (Tian et al., 2011), rice (Huang et al., 2010), and sorghum (Morris et al., 2013) have been successful in identifying the genetic basis for various agronomic traits. Here, we analyzed the seed ionome from a community-generated association panel to identify potential loci underlying seed element accumulation in sorghum.     

High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer

Background

The characterization of copy number alteration patterns in breast cancer requires high-resolution genome-wide profiling of a large panel of tumor specimens. To date, most genome-wide array comparative genomic hybridization studies have used tumor panels of relatively large tumor size and high Nottingham Prognostic Index (NPI) that are not as representative of breast cancer demographics.

Results

We performed an oligo-array-based high-resolution analysis of copy number alterations in 171 primary breast tumors of relatively small size and low NPI, which was therefore more representative of breast cancer demographics. Hierarchical clustering over the common regions of alteration identified a novel subtype of high-grade estrogen receptor (ER)-negative breast cancer, characterized by a low genomic instability index. We were able to validate the existence of this genomic subtype in one external breast cancer cohort. Using matched array expression data we also identified the genomic regions showing the strongest coordinate expression changes ('hotspots'). We show that several of these hotspots are located in the phosphatome, kinome and chromatinome, and harbor members of the 122-breast cancer CAN-list. Furthermore, we identify frequently amplified hotspots on 8q22.3 (EDD1, WDSOF1), 8q24.11-13 (THRAP6, DCC1, SQLE, SPG8) and 11q14.1 (NDUFC2, ALG8, USP35) associated with significantly worse prognosis. Amplification of any of these regions identified 37 samples with significantly worse overall survival (hazard ratio (HR) = 2.3 (1.3-1.4) p = 0.003) and time to distant metastasis (HR = 2.6 (1.4-5.1) p = 0.004) independently of NPI.

Conclusion

We present strong evidence for the existence of a novel subtype of high-grade ER-negative tumors that is characterized by a low genomic instability index. We also provide a genome-wide list of common copy number alteration regions in breast cancer that show strong coordinate aberrant expression, and further identify novel frequently amplified regions that correlate with poor prognosis. Many of the genes associated with these regions represent likely novel oncogenes or tumor suppressors.     

The adaptive value of functional and life-history traits across fertility treatments in an annual plant

Background and Aims

Plant functional traits are assumed to be adaptive. As selection acts on individuals and not on traits, interpreting the adaptive value of a trait not may be straightforward. For example, productive leaves are associated with fertile environments. However, it is not clear if productive leaves confer an advantage in these habitats, or if they are an advantage as part of a suite of coordinated traits.

Methods

Genotypes of Arabidopsis thaliana were grown in high and low nutrient treatments and low, neutral and high pH treatments. Nutrient availability is reduced in acidic or basic soils relative to neutral pH soils. pH treatments were used to alter the availability of resources rather than the amount of resources.

Key Results

Leaf function (specific leaf area, SLA) and life history (size at reproduction, age at reproduction) were variable across genotypes and were plastic. High nutrient availability induced higher SLA and larger size at reproduction. Genotypes that reproduced at large size in high nutrient conditions at neutral pH had the greatest fruit production. SLA was only indirectly related to fruit production through a causal relationship with rosette size; in high nutrient conditions, plants with high SLA were large at reproduction and had higher fruit production. In high nutrient and high pH treatments, plants were large at reproduction, but large size at reproduction was associated with low fecundity. This suggests that large size is adaptive under high nutrient availability.

Conclusions

Interpreting the adaptive value of functional traits will sometimes only be possible when these traits are considered as a suite of correlated and coordinated traits. Leaf functional traits may be important in defining adaptive strategies in A. thaliana but only through how they affect plant life history. Finally, manipulating soil pH can be a valuable tool in assessing adaptive plasticity on nutrient gradients.     

Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.     

TRAIL signaling promotes entosis in colorectal cancer

Entosis is a form of nonphagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumors show evidence of entosis; however, factors controlling entosis remain to be elucidated. Here, we find that besides inducing apoptosis, TRAIL signaling is a potent activator of entosis in colon cancer cells. Initiation of both apoptosis and entosis requires TRAIL receptors DR4 and DR5; however, induction of apoptosis and entosis diverges at caspase-8 as its structural presence is sufficient for induction of entosis but not apoptosis. Although apoptosis and entosis are morphologically and biochemically distinct, knockout of Bax and Bak, or inhibition of caspases, also inhibits entotic cell death and promotes survival and release of inner cells. Analysis of colorectal cancer tumors reveals a significant association between TRAIL signaling and CIC structures. Finally, the presence of CIC structures in the invasive front regions of colorectal tumors shows a strong correlation with adverse patient prognosis.     

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.     

A computational and experimental approach toward a priori identification of alternatively spliced exons

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data.