Induced Variations in Brassinosteroid Genes Define Barley Height and Sturdiness,and Expand the Green Revolution Genetic Toolkit

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.The introduction of dwarfing genes to increase culm sturdiness of cereal crops was crucial for the first Green Revolution (Hedden, 2003). The culms of tall cereal crops were not strong enough to support the heavy spikes of high-yielding cultivars, especially under high-nitrogen conditions. As a result, plants fell over, a process known as lodging. This caused losses in yield and grain-quality issues attributable to fungal infections, mycotoxin contamination, and preharvest germination (Rajkumara, 2008). Today, a second Green Revolution is on its way, to revolutionize the agricultural sector and to ensure food production for a growing world population. Concurrently, global climate change is expected to cause more frequent occurrences of extreme weather conditions, including thunderstorms with torrential rain and strong winds, thus promoting cereal culm breakage (Porter and Semenov, 2005; National Climate Assessment Development Advisory Committee, 2013). Accordingly, plant architectures that resist lodging remain a major crop-improvement goal and identification of genes that regulate culm length is required to enhance the genetic toolbox in order to facilitate efficient marker-assisted breeding. The mutations and the corresponding genes that enabled the Green Revolution in wheat (Triticum aestivum) and rice (Oryza sativa) have been identified (Hedden, 2003). They all relate to gibberellin metabolism and signal transduction. It is now known that other plant hormones such as brassinosteroids are also involved in the regulation of plant height. Knowledge of the molecular mechanisms underlying the effects of the two hormones on cell elongation and division has mainly come from studies in Arabidopsis (Arabidopsis thaliana; Bai et al., 2012). Mutant-based breeding strategies to fine-tune brassinosteroid metabolism and signaling pathways could improve lodging behavior in modern crops (Vriet et al., 2012) such as barley (Hordeum vulgare), which is the fourth most abundant cereal in both area and tonnage harvested (http://faostat.fao.org).A short-culm phenotype in crops is often accompanied by other phenotypic changes. Depending on the penetrance of such pleiotropic characters, but also the parental background and different scientific traditions and expertise, short-culmed barley mutants were historically divided into groups, such as brachytic (brh), breviaristatum (ari), dense spike (dsp), erectoides (ert), semibrachytic (uzu), semidwarf (sdw), or slender dwarf (sld; Franckowiak and Lundqvist, 2012). Subsequent mutant characterization was limited to intragroup screens and very few allelism tests between mutants from different groups have been reported (Franckowiak and Lundqvist, 2012). Although the total number of short-culm barley mutants exceeds 500 (Franckowiak and Lundqvist, 2012), very few have been characterized at the DNA level (Helliwell et al., 2001; Jia et al., 2009; Chandler and Harding, 2013; Houston et al., 2013). One of the first identified haplotypes was uzu barley (Chono et al., 2003). The Uzu1 gene encodes the brassinosteroid hormone receptor and is orthologous to the BRASSINOSTEROID-INSENSITIVE1 (BRI1) gene of Arabidopsis, a crucial promoter of plant growth (Li and Chory, 1997). The uzu1.a allele has been used in East Asia for over a century and is presently distributed in winter barley cultivars in Japan, the Korean peninsula, and China (Saisho et al., 2004). Its agronomic importance comes from the short and sturdy culm that provides lodging resistance, and an upright plant architecture that tolerates dense planting.Today, more than 50 different brassinosteroids have been identified in plants (Bajguz and Tretyn, 2003). Most are intermediates of the complex biosynthetic pathway (Shimada et al., 2001). Approximately nine genes code for the enzymes that participate in the biosynthetic pathway from episterol to brassinolide (Supplemental Fig. S1). Brassinosteroid deficiency is caused by down-regulation of these genes, but it can also be associated with brassinosteroid signaling. The first protein in the signaling network is the brassinosteroid receptor encoded by BRI1 (Li and Chory, 1997; Kim and Wang, 2010). In this work, we show how to visually identify brassinosteroid-mutant barley plants and we describe more than 20 relevant mutations in four genes of the brassinosteroid biosynthesis and signaling pathways that can be used in marker-assisted breeding strategies.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca²⁺ ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn²⁺ ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

Ordovician (Dobrotivá Formation) ostracodes and trilobites from Ejpovice (Bohemia) and their relations to faunas of northern and western Europe

For the first time a complete ostracode and trilobite fauna is described from the Dobrovitá Formation (Ordovician) of Perunica. In contrast to the trilobite fauna the ostracode fauna evidences close relations to both Armorica and Baltica. The trilobite fauna comprises 11 species out of 11 genera, the ostracode fauna comprises 15 (5 new) species out of 15 (1 new) genera.     

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.     

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Occurrence of 16s rRNA methylase ArmA producing Enterobacteriaceae in a general hospital in Warsaw, Poland

Resistance to gentamicin, amikacin and kanamycin was screened in 270 clinical isolates of Enterobacteriaceae originated from April 19 to May 19, 2010 in a regular hospital in Warsaw, Poland. Most of the isolated bacteria were considered pathogenic. Nineteen isolates (7%) were simultaneously resistant to two or three of the tested aminoglycosides. MICs of the three aminoglycosides ranged form 128 to 1024 mcg/ml for six isolates. These isolates were suspected to produce 16S rRNA methylase. Genes encoding for three methylases reported in Europe: ArmA, RmtB and RmtC were searched by PCR. The armA gene was detected in all of the six isolates. This group encompassed Enterobacter cloacae (n=4), Klebsiella pneumoniae (n=1) and Proteus mirabilis (n=1). Five isolates of this group carried the bla(CAX-M) gene for CTX-M type ESBL. The remaining isolate E. cloacae DM0340 was ESBL negative and lacked bla(CRX-M) that may suggest an altered genetic environment of the armA gene in this isolate. Our results showed that 2.2% of the tested isolates produced 16S rRNA methylase ArmA. This finding may argue for a high incidence of ArmA producing Enterobacteriaceae in Poland when compared to reports from other European countries.     

Alport syndrome: a genetic study of 31 families

Thirty one families with Alport syndrome including 3 families with associated syndromes were studied. The location of the COL4A5 gene, responsible for the Alport syndrome, was determined by linkage analysis with eight probes of the Xq arm and by a radiation hybrid panel. Concordant data indicated the localization of the Alport gene between DXS17 and DXS11. Four deletions and one single base mutation of the COL4A5 gene were detected. Homogeneity tests failed to show any evidence of genetic heterogeneity superimposed on clinical heterogeneity for ophthalmic signs and end-stage renal disease age.     

A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.