CaMV-35S promoter sequence-specific DNA methylation in lettuce.

KEY MESSAGE: We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

Museum-archived and recent acquisition nitrates from the Atacama Desert, Chile, South America: refinement of the dual isotopic compositions (δ15N vs. δ18O).

Sodium nitrate ores from the Atacama Desert in South America were economically important as they represented huge natural resources for the fertilizer and explosives industries during the early nineteenth to early twentieth centuries. Nitrogen and oxygen isotope ratios (δ15N and δ18O) of these desert nitrates generally show unique compositions (from close to 0 and up to ca. +50 ‰, respectively). The nitrates indicate the provenance as atmospheric in origin due to the mass-independent photochemical reaction of nitric oxide (NO) with ozone (O3) in the atmosphere to produce nitrate (NO3-). This paper examines the previously existing isotope data for specimens acquired from the Atacama Desert. It then reports new data from dual isotope analysis of historic nitrate specimens archived in museums in the UK. In the stable isotope signatures for nitrates from two areas of the Atacama Desert, Tarapacá in the north and Antofagasta in the south, were examined, and this analysis enabled a more detailed definition of their isotopic compositional ranges. This improved database is useful for tracing the provenance of the historic nitrates used in gunpowder and saltpetre, and also the cause of nitrate pollution in natural environments for which routine chemistry alone cannot provide the definite evidence for the origin.

Dual isotopic (δ15N-δ18O) characterization of saltpetre currently prevailing in Lao PDR and its global compilation: new insight into isotope fractionation during production processes.

Saltpetre (KNO3; potassium nitrate) is one of the major ingredients of gunpowder. Simplex saltpetre (total 126 samples) together with gunpowder (total 93 samples) commodities which are currently prevailing in local markets as used for wildlife hunting and rocket festivals (local name in Lao: Boun Ban Fai) were collected from throughout Lao PDR. Dual isotopic composition of nitrates (δ15N, δ18O) was analyzed by the microbial denitrification method. Binary plotting of the data was conducted to characterize the mode of formation. In Lao PDR, almost all the commodities were imported from neighbouring Thailand, and to a lesser extent from Israel. Binary plotting of δ15N vs. δ18O of nitrates shows their intrinsic origin manufactured by Haber-Bosch and subsequently Ostwald processes. We observed an inverse correlation (δ15Nnitrate: up to + 12 ‰; δ18Onitrate: down to + 15 ‰), deviating from the reservoir values (free air) of δ15N (dinitrogen; up to 0 ‰) and δ18O (oxygen; +23.5 ‰), interpreted this as an indication of isotope fractionation during manufacture. The most plausible interpretation for this is the fractionation associated with formation of NO following NO2 and finally NO3 products. A nearly comparable inverse relationship is also observed for nitrates in pyrotechnics manufactured in China.

Dual (oxygen and nitrogen) isotopic characterization of the museum archived nitrates from the United States of America, South Africa and Australia.

Dual (oxygen and nitrogen) isotopic composition of the museum archived nitrates from the United States of America, South Africa and Australia was studied. The analyzed specimens were collected in middle 19th to early 20th centuries, and represent world-wide acquisition of the Smithsonian Institution Natural Museum of Natural History (Washington, D. C., USA) and the Natural History Museum (London, UK). The samples consist of transparent to semi-transparent aggregates of minute nitrate, euhedral crystallites which imply precipitation from percolating fluids under ample space and dry regimes. The major nitrate chemistry is saltpetre (KNO3) with minor nitratine (NaNO3). A binary plot of δ15N vs. δ18O of almost all nitrates indicates a trend, reflecting microbial origin through nitrification of ammonium. The diagram excludes the contribution of meteoric origin formed by mass-independent, photochemical reaction of NO with ozone in stratosphere. Calculated paleo-ambient fluid compositions responsible for microbial nitrification imply extreme evaporative concentration of relevant fluids under dry climatic regimes in the Northern Cape Province (South Africa) and in the Northern Territory (central Australia), and even throughout the United States of America. The dual isotopic characterization provides direct evidence to the origin of the museum archived nitrates.

A 64-bp sequence containing the GAAGA motif is essential for CaMV-35S promoter methylation in gentian.

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (-90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (-298 to -241 and -148 to -85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two "GAAGA" motifs (-268 to -264 and -135 to -131) were replaced by "GTTCA" in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (-148 to -85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.