Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real‐time PCR

Populus euphratica is the only arboreal species that is established in the world's largest shifting‐sand desert in China and is well‐adapted to the extreme desert environment, so it is widely considered a model system for researching into abiotic stress resistance of woody plants. However, few P. euphratica reference genes (RGs) have been identified for quantitative real‐time polymerase chain reaction (qRT‐PCR) until now. Validation of suitable RGs is essential for gene expression normalization research. In this study, we screened 16 endogenous candidate RGs in P. euphratica leaves in six abiotic stress treatments, including abscisic acid (ABA), cold, dehydration, drought, short‐duration salt (SS) and long‐duration salt (LS) treatments, each with 6 treatment gradients. After calculation of PCR efficiencies, three different software tools, NormFinder, geNorm and BestKeeper, were employed to analyze the qRT‐PCR data systematically, and the outputs were merged by means of a non‐weighted unsupervised rank aggregation method. The genes selected as optimal for gene expression analysis of the six treatments were RPL17 (ribosomal protein L17) in ABA, EF1α (elongation factor‐1 alpha) in cold, HIS (histone superfamily protein H3) in dehydration, GIIα in drought and SS, and TUB (tubulin) in LS. The expression of 60S (the 60S ribosomal protein) varied the least during all treatments. To illustrate the suitability of these RGs, the relative quantifications of three stress‐inducible genes, PePYL1, PeSCOF‐1 and PeSCL7 were investigated with different RGs. The results, calculated using qBasePlus software, showed that compared with the least‐appropriate RGs, the expression profiles normalized by the recommended RGs were closer to expectations. Our study provided an important RG application guideline for P. euphratica gene expression characterization.     

Sequence and functional analyses of the aldehyde dehydrogenase 7B4 gene promoter in Arabidopsis thaliana and selected Brassicaceae: regulation patterns in response to wounding and osmotic stress

Key message

The core promoter of the antiquitin ALDH7B4 gene was compared between selected Brassicaceae. Conserved cis elements controlling osmotic stress and wound-induced expression were identified and analysed in Arabidopsis thaliana leaves and seeds.

Abstract

Aldehyde dehydrogenases metabolise a wide range of aliphatic and aromatic aldehydes, which become cytotoxic at high levels. Family 7 aldehyde dehydrogenase genes, often described as antiquitins or turgor-responsive genes in plants, are broadly conserved across all domains. Despite the high conservation of the plant ALDH7 proteins and their importance in stress responses, their regulation has not been investigated. Here, we compared ALDH7 genes of different Brassicaceae and found that, in contrast to the gene organisation and protein coding sequences, similarities in the promoter sequences were limited to the first few hundred nucleotides upstream of the translation start codon. The function of this region was studied by isolating the core promoter of the Arabidopsis thaliana ALDH7B4 gene, taken as model. The promoter was found to be responsive to wounding in addition to salt and dehydration stress. Cis-acting elements involved in stress responsiveness were analysed and two conserved ACGT-containing motifs proximal to the translation start codon were found to be essential for the responsiveness to osmotic stress in leaves and in seeds. The integrity of an upstream ACGT motif and a dehydration-responsive element/C-repeat—low temperature-responsive element was found to be necessary for ALDH7B4 expression in seeds and induction by salt, dehydration and ABA in leaves. The comparison of the gene expression in selected Arabidopsis mutants demonstrated that osmotic stress-induced ALDH7B4 expression in leaves and seeds involves both ABA- and lipid-signalling components.     

Analyse des artefacts lithiques du site de Longgupo

Excavation has enabled recovery of 854 artifacts within 30 archaeological levels in the south sector and 11 in the north (chapter 3). These levels are quite probably contemporaneous, or even the same. The quantitative disparity in the number of strata between the two sectors is simply due to the fact that only the lower half of the northern zone was completely investigated. Similarly, the number of artifacts recovered by level varies according to the surface area excavated, although is some cases the density of material is significant despite the small area excavated, for example stratum C IV 5 which contains 174 lithic artifacts in 2 m². Before undertaking the technological analysis of the artifacts, given the preceding polemics provoked by the great age of this site and its implications for the spread of the first populations out of Africa, it was deemed important to carry out a plurifactorial analysis combining all of the data related to: the stratigraphy, taphonomic processes (post-depositional disturbance), analysis of natural processes that may have produced eoliths, experimentation and techno-functional analysis of the material. The stratigraphy shows clear interstratifications of fine and coarse fluviatile levels with often very clear particle size sorting of the coarse fraction. The archaeological material is typically found at the interface of these strata, either at the base of a clayey matrix, overlying a preceding coarse level, or in the superficial part of a sandy-clay deposit underlying coarse deposits. Post-depositional disturbance revealed during the new excavations in 2003–2006 cannot alone be the cause of eoliths. Excavation of a 6 m² zone in the modern river bed, located below the site, has demonstrated that the technological traits of eoliths recovered cannot in any way be confused with the technological traits of the artifacts recovered at the site itself. Similarly, viewed quantitatively, the 6 m² zone excavated in the river bed yielded around 20 eoliths while the 30 m² zone excavated at the site yielded 854 artifacts, one stratum alone yielding 184 artifacts in a 3 m² zone. During the experimental phase, adopting the same conditions of procuring raw material, from the same river bed, we very quickly realized the rarity of types of adequate volume that had been generally used at the site and the need to use certain operational processes to create such a form. In addition, the hardness and presence of several natural fracture planes in the Triassic limestone explain the choice of different operational processes and the very high number of knapping accidents, including those occurring during bipolar percussion. Although 90% of the raw material used was cobbles or broken blocks of local Triassic limestone, 10% of the tools were made on exogenous raw materials – siliceous or gravelly limestone, quartzitic sandstone, chert, volcanic rock – that are absent from the immediate environment of the site. These raw materials were brought to the site in the form of tools: worked cobbles, large retouched flakes, backed double-truncated flakes, a plaquette with a lateral bifacial edge, etc. The 854 artifacts have been classified into six object classes: worked cobbles with transversal edge (39%), worked cobbles with lateral edge (2%), unipolar flakes (27%), bipolar objects (half-blocks, half-cobbles including some flat “split” cobbles, “orange slices”, flakes and diverse fragments) (17%) and fragments resulting from knapping of blocks or cobbles (13%), hammerstone (2%). When the frequencies of these classes are calculated for each of the sectors, percentages are similar, indicating a high degree of homogeneity in the archaeological assemblages at the site. The situation is somewhat different when assemblages are compared within a single sector. Slight differences appear in the percentages of bipolar pieces and unipolar flakes. These differences seem to be random, like the frequency rate of knapping accidents in bipolar reduction, or economic, such as the choice of operational schemes to create worked cobbles based on the availability of suitable raw materials. The technological affinity between each of the archaeological assemblages tends to demonstrate great stability in technological knowledge through time. The class of worked cobbles is by far the most important and, apart from a few flakes produced intentionally, it appears to group all of the tools. To avoid placing these tools in a restrictive, semantically meaningless, class, we prefer the concept of matrix to the term worked cobble. A matrix is a structured arrangement of a series of technological traits, in a form as close as possible to that of the future tool. The matrix phase leads to the tool production phase, which may be unnecessary if the matrix phase includes fictionalization. In other words, the concept of matrix enables separating the phase of preparing a predetermined volume, such as a blade, Levallois flake or bifacial piece, from the tool production phase, consisting in creating the type of transformative edge intended, if necessary. The tool is thus an artifact of a specific form with an integrated edge and an operational scheme both specific to the function attributed to it and means of use associated with the form. Observed variability relates to: the size of the volume ranging from to 20, morphology, the form of the line formed by the edge which can in frontal view be curved, linear, sinuous or denticulated, and in transversal view curved, linear, sinuous or saw-toothed, and the length of the edge ranging from 1 to 10. Matrices with a simple bevel are distinguished from double bevels. In the framework of the technological analysis of production schemes to produce matrices with a simple bevel, a broad range of variability in production schemes can be observed, divided arbitrarily into two stages. The first stage consists in creating as closely as possibly the technological traits of the future matrix due to five general schemes. The first scheme (A) consists in selecting a cobble or block naturally possessing at least some of the technological traits needed. The missing traits are added by various preparations, including bipolar percussion 3 times out of 5. The second scheme (B) consists in knapping a flake from the block with some of the technological traits required for the matrix present on one of its surfaces. The third scheme (C) consists in the choice of a plaquette from which a bipolar shock creates the main traits of the matrix. The fourth scheme (D) consists in choosing a volume very similar to the intended matrix. The fifth and final scheme (E) consists in knapping a flake with technological traits very different from those intended. Depending on the distance between intention and realization, a second stage may be necessary. In general, this second stage perfects or creates the intended active edge, which is rarely obtained in the first stage. To produce a matrix with a double bevel, it is sometimes necessary to add an intermediate stage in order to prepare the second bevel. The first stage remains the same, with the use of the five operational schemes. By contrast, a clear difference exists in the percentages for the use of these schemes. For a matrix with a simple bevel, scheme A is dominant, followed by scheme D, while the situation is reversed for a matrix with a double bevel, where scheme D is dominant. Unipolar flakes, representing 27% of the assemblage, are produced in three different ways. The most important is flakes resulting from matrix production. The two others are flakes produced during different knapping schemes, some flakes in relation to the few cores present, other flakes in exogenous raw materials produced elsewhere and generally much larger. The other classes are dominated by bipolar products resulting essentially from knapping accidents. To summarize, these assemblages are characterized by: the search for tools differentiated by form and active edges; more than 90% of the tools made on two kinds of supports: a matrix with a simple bevel or a double bevel; matrices obtained using different operational schemes successively associating if necessary a knapping stage and a shaping stage. While the Triassic limestone is hard and thus imposes a strong constraint on knapping, the range of operational schemes appears to have been a “cultural” response diversified to this constraint and the presence of tools on exogenous raw materials. At the scale of China, comparison of this industry is impossible since it is the only site of this age and to contain so much material. The site of Majuangou, the only site of similar caliber, is younger by several hundred thousand years and is located several thousand kilometers to the north, making comparisons meaningless. We note only that most of the tool supports at Majuangou are knapped flakes. On an inter-continental scale, the comparison of sites of equal age is more promising. But lithic analyses are based on different methods, preventing comparison of similar data. However, if we make a simple summary of the data available, we can first say that in Africa, during these periods, different development technological stages were present and stages that are considered more evolved are manifestly less common using our approach. While these stages are more or less contemporaneous, which counters the idea of uniqueness, they would more surely be evidence of populations that were not in direct contact and had separate lines of development. In Asia, the Longgupo industry evidences a different technological option than that of contemporaneous populations in Africa. By contrast, when we take into account its developmental stage, we realize that this is an “evolved” stage in which the form of the support of the future tool is predominant. If we compare Africa and Asia in terms of stages, we are a priori at the same stage with different options being selected.     

Engineering tocopherol biosynthetic pathway in lettuce

In order to increase tocopherol content, genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and γ-tocopherol methyltransferase (γ-TMT) were constitutively over-expressed in lettuce (Lactuca sativa L. var. logifolia), alone or in combination. Over-expression of hpt could increase total tocopherol content, while over-expression of γ-tmt could shift tocopherol composition in favor of α-tocopherol. Transgenic lettuce lines expressing both hpt and γ-tmt produced significantly higher amount of tocopherol and elevated α-/γ-tocopherol ratio compared with non-transgenic control and transgenic lines harboring a single gene (hpt or γ-tmt). The best line produced eight times more tocopherol than the non-transgenic control and more than twice than hpt single-gene transgenic line.     

High-resolution genetic mapping and candidate gene identification of the <Emphasis Type="Italic">SLP1</Emphasis> locus that controls glume development in rice

Stunted lemma palea 1 (slp1) is a rice mutant that displays dwarfism, shortened inflorescence lengths, severely degenerated lemmas/paleas, and sterility. The SLP1 locus was mapped between markers RM447 and D275 in the distal region of the long arm of chromosome 8, using the F2 progeny derived from the cross between the Slp1/slp1 mutant (Oryza sativa subsp. japonica) and the variety Taichung Native 1 (TN1, O. sativa subsp. indica). The SLP1 locus was further delimited to a 46.4-kb genomic region containing three putative genes: OsSPL16, OsMADS45, and OsMADS37. Comparisons of the sequence variations and expression levels of the three candidate genes between wild-type plants and homozygous slp1 mutants suggested that a missense mutation in the sixth amino acid of the OsSPL16 protein was likely responsible for the slp1 mutant phenotypes.     

Genomic data reject the hypothesis of a prosimian primate clade

The phylogenetic position of tarsiers within the primates has been a controversial subject for over a century. Despite numerous morphological and molecular studies, there has been weak support for grouping tarsiers with either strepsirrhine primates in a prosimian clade or with anthropoids in a haplorrhine clade. Here, we take advantage of the recently released whole genome assembly of the Philippine tarsier, Tarsius syrichta, in order to infer the phylogenetic relationship of Tarsius within the order Primates. We also present estimates of divergence times within the primates. Using a 1.26 million base pair multiple sequence alignment derived from 1078 orthologous genes, we provide overwhelming statistical support for the presence of a haplorrhine clade. We also present divergence date estimates using local relaxed molecular clock methods. The estimated time of the most recent common ancestor of extant Primates ranged from 64.9 Ma to 72.6 Ma, and haplorrhines were estimated to have a most recent common ancestor between 58.9 Ma and 68.6 Ma. Examination of rates of nucleotide substitution in the three major extant primate clades show that anthropoids have a slower substitution rate than either strepsirrhines or tarsiers. Our results provide the framework on which primate morphological, reproductive, and genomic features can be reconstructed in the broader context of mammalian phylogeny.     

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.     

The characteristics of allelic polymorphism in killer-immunoglobulin-like receptor framework genes in African Americans

The frequencies of alleles of killer cell immunoglobulin-like receptor genes, KIR3DL3 and KIR3DL2, and the carrier frequency of KIR2DL4 alleles have been determined from a population of African Americans (n = 100) by DNA sequencing of the coding regions. Fifty alleles of KIR3DL3 were observed with the most frequent, KIR3DL3*00901 (13%). KIR3DL2 was also diverse; 32 alleles with KIR3DL2*00103 the most frequent (17%). For KIR2DL4, of the 18 alleles observed, one allele, KIR2DL4*00103, was found in 64 of the 100 individuals. Thirty-six novel alleles encoding a total of 28 unique receptors are described. Pairwise comparisons among all of the alleles at each locus suggest a predominance of synonymous substitutions. The variation at all three framework loci fits a neutral model of evolution.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.