Genetic dissection of early-season cold tolerance in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench)

Soil temperatures at 15°C or below limit germination and seedling establishment for warm season cereal crops such as sorghum (Sorghum bicolor (L.) Moench) during early-season planting. To better understand the genetics of early-season cold tolerance in sorghum, mapping of quantitative trait loci (QTL) associated with germination, emergence and vigor using a recombinant inbred mapping population was carried out. A mapping population consisting of 171 F₇–F₈ recombinant inbred lines (RILs) derived from the cross between RTx430 (cold-sensitive) and PI610727 (cold-tolerant) was developed and a genetic map was constructed using 141 microsatellites or simple sequence repeat (SSR) markers. The RILs were evaluated for cold and optimal temperature germinability in the laboratory, field emergence, and seedling vigor in two locations during early-season planting. Two or more QTL were detected for all traits, except for seedling vigor, with only one QTL was detected in the population. A QTL for cold germinability (Germ 12-2.1) showed the highest LOD value and was also associated with optimal germinability. One of the QTL for field emergence, Fearlygerm-9.3, a contribution from PI610727, was found significant in both locations used for the study. This study showed alignment of QTL in SBi1 (Fearlygerm-1.2 and FGerm30-1.2) with previously reported QTL associated with late field emergence identified from a different mapping population. This indicates that PI617027 shares some common loci with other known early-season cold-tolerant sorghum germplasm but also harbors novel QTL that could be useful in introgression of enhanced laboratory germination and early-season field emergence.     

Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species

Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver) lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells) was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS). We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.     

TRAIL/MEKK4/p38/HSP27/Akt survival network is biphasically modulated by the Src/CIN85/c-Cbl complex

Previously, we showed that mitogen-activated protein kinase/extracellular signal-related kinase 4 (MEKK4) is responsible for p38 activation and that its activation during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment also increases the catalytic activity of Akt. Here, we further investigated how the TRAIL-induced MEKK4/p38/heat shock protein (HSP27)/Akt survival network is modulated by the Src/c-Cbl interacting protein of 85 kDa (CIN85)/c-Cbl complex. TRAIL-induced activation of Akt catalytic activity and phosphorylation were highly correlated with p38/HSP27 phosphorylation, whereas the phosphorylation of p38/HSP27 increased further during incubation with curcumin and TRAIL, which caused significant apoptotic cell death. CIN85, a c-Cbl-binding protein, plays an essential role in connecting cell survival to cell death. The interaction of CIN85 with MEKK4 was increased during the late phase of TRAIL incubation, suggesting that sustained p38 and HSP27 phosphorylation protects cells by preventing further cell death. However, further increases in p38/HSP27 phosphorylation induced by cotreatment with curcumin and TRAIL converted cell fate to death. Taken together, these data demonstrate that phosphorylated p38/HSP27 as biphasic modulators act in conjunction with CIN85 to determine whether cells survive or die in response to apoptotic stress.     

Complete genome sequence of the metabolically versatile plant growth-promoting endophyte Variovorax paradoxus S110

Variovorax paradoxus is a microorganism of special interest due to its diverse metabolic capabilities, including the biodegradation of both biogenic compounds and anthropogenic contaminants. V. paradoxus also engages in mutually beneficial interactions with both bacteria and plants. The complete genome sequence of V. paradoxus S110 is composed of 6,754,997 bp with 6,279 predicted protein-coding sequences within two circular chromosomes. Genomic analysis has revealed multiple metabolic features for autotrophic and heterotrophic lifestyles. These metabolic diversities enable independent survival, as well as a symbiotic lifestyle. Consequently, S110 appears to have evolved into a superbly adaptable microorganism that is able to survive in ever-changing environmental conditions. Based on our findings, we suggest V. paradoxus S110 as a potential candidate for agrobiotechnological applications, such as biofertilizer and biopesticide. Because it has many associations with other biota, it is also suited to serve as an additional model system for studies of microbe-plant and microbe-microbe interactions.     

Sirtuin 1 (SIRT1) protein degradation in response to persistent c-Jun N-terminal kinase 1 (JNK1) activation contributes to hepatic steatosis in obesity

SIRT1 is involved in the pathogenesis of obesity, diabetes, and aging. However, it is not clear how SIRT1 activity is regulated by intracellular kinases in cells. In this study, we investigated SIRT1 phosphorylation and protein degradation in response to JNK1 activation in obese mice. Mouse SIRT1 is phosphorylated by JNK1 at Ser-46 (Ser-47 in human SIRT1), which is one of the four potential residues targeted by JNK1. The phosphorylation induces a brief activation of SIRT1 function and degradation of SIRT1 thereafter by the proteasome. Ubiquitination occurs in SIRT1 protein after the phosphorylation. Mutation of Ser-46 to alanine prevents the phosphorylation, ubiquitination, and degradation. In vivo, SIRT1 undergoes an extensive degradation in hepatocytes in obesity as a consequence of persistent activation of JNK1. The degradation leads to inhibition of SIRT1 function, which contributes to development of hepatic steatosis. The degradation disappears in obesity when JNK1 is inactivated in mice. JNK2 exhibits an opposite activity in the regulation of SIRT1 degradation. The JNK1-SIRT1 pathway provides a new molecular mechanism for the pathogenesis of hepatic steatosis in obesity.     

Regulated AtHKT1 gene expression by a distal enhancer element and DNA methylation in the promoter plays an important role in salt tolerance

Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.     

Label-free measurement of cell viability via counting cells attached on affinity substrates

The commonly used trypan blue dye exclusion method and other modified cell viability methods, such as fluorescein dye and tetrazolium dye exclusion, artificially introduce toxic chemicals to cells and, thus, alter cellular organelles when measuring cell viability. Therefore, cell viability could be affected by the processes currently used to observe viability. In this study, the cell viability of Chinese hamster ovary (CHO) cells was measured by simply counting attached cells after the cultured CHO cells were attached on a Concanavalin A (Con A) substrate. The efficiency of cell attachment to Con A surfaces was different for live and dead cells allowing the cell viability of CHO cells to be measured without any chemical modifications to the cells.