Critical Reexamination of Palynological Characters Used to Delimit Asclepiadaceae in Comparison to the Molecular Phylogeny Obtained from PlastidmatK Sequences

The family Asclepiadaceae (Dicotyledones) was created by Brown in 1810 by splitting in two the family Apocynaceae of Jussieu established in 1789. The morphological characters used to make this distinction were mainly palynological, such as presence of tetrads or pollinia and number and orientation of pollinia. Those characters, still used in higher taxonomic delimitation (families, subfamilies, and tribes), are here critically reexamined and compared to a molecular phylogeny obtained with one of the more variable plastid genes (matK) of 46 species in the order Gentianales. In this molecular phylogeny, Asclepiadaceae form a monophyletic group derived from within Apocynaceae. Each of the subfamilies of Asclepiadaceae is monophyletic and based on reliable palynological characters, but palynological characters are not useful to delimit tribes of the subfamily Asclepiadoideae. Based on the molecular data, these tribes have undergone parallelisms in several reproductive traits.     

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.     

Population genetics of Cordia alliodora (Boraginaceae), a neotropical tree. 1. Genetic variation in natural populations

We provide an estimate of genetic differentiation within and among 11 populations of Cordia alliodora, an economically important timber tree Cordia alliodora is a widespread species that is distributed throughout Central and South America. Our survey of isozyme variation was conducted on material gathered for international provenance trials over approximately 1,000 km in Central America. Results from provenance trials indicate that there are significant differences between Atlantic and Pacific coast provenances for quantitative characters. Genetic data support some of these findings. Populations of C. alliodora show significant differences in allele frequency at various loci. Significant differences in multilocus allele frequencies occur at 13 of the 55 possible combinations. Eight of these 13 populations are situated on opposite coasts. This physical separation corresponds well with the results of provenance trials that indicate differentiation among the Atlantic and Pacific populations in quantitative morphological traits. We also found a significant negative correlation between levels of heterozygosity and the amount of rainfall, indicating that populations from the drier zone are genetically more heterogenous than populations from the wet zone. Our study indicates that in situ and ex situ conservation should accord high priority to the dry zone populations; furthermore, conservation of this widespread species would require preservation of multiple populations.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc–dependent mechanism

Recent work has shown that Staufen1 plays key roles in skeletal muscle, yet little is known about its pattern of expression during embryonic and postnatal development. Here we first show that Staufen1 levels are abundant in mouse embryonic muscles and that its expression decreases thereafter, reaching low levels in mature muscles. A similar pattern of expression is seen as cultured myoblasts differentiate into myotubes. Muscle degeneration/regeneration experiments revealed that Staufen1 increases after cardiotoxin injection before returning to the low levels seen in mature muscles. We next prevented the decrease in Staufen1 during differentiation by generating stable C2C12 muscle cell lines overexpressing Staufen1. Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay. However, levels of c-myc, a factor known to inhibit differentiation, were increased in C2C12 cells overexpressing Staufen1 through enhanced translation. By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts. Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.     

Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.     

A phylogenetic analysis of the Orchidaceae: evidence from rbcL nucleotide

Cladistic parsimony analyses of rbcL nucleotide sequence data from 171 taxa representing nearly all tribes and subtribes of Orchidaceae are presented here. These analyses divide the family into five primary monophyletic clades: apostasioid, cypripedioid, vanilloid, orchidoid, and epidendroid orchids, arranged in that order. These clades, with the exception of the vanilloids, essentially correspond to currently recognized subfamilies. A distinct subfamily, based upon tribe Vanilleae, is supported for Vanilla and its allies. The general tree topology is, for the most part, congruent with previously published hypotheses of intrafamilial relationships; however, there is no evidence supporting the previously recognized subfamilies Spiranthoideae, Neottioideae, or Vandoideae. Subfamily Spiranthoideae is embedded within a single clade containing members of Orchidoideae and sister to tribe Diurideae. Genera representing tribe Tropideae are placed within the epidendroid clade. Most traditional subtribal units are supported within each clade, but few tribes, as currently circumscribed, are monophyletic. Although powerful in assessing monophyly of clades within the family, in this case rbcL fails to provide strong support for the interrelationships of the subfamilies (i.e., along the spine of the tree). The cladograms presented here should serve as a standard to which future morphological and molecular studies can be compared.     

A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.