Genome size and sensitivity to DNA damage by X-rays-plant comets tell the story.

Among several factors affecting radiation sensitivity, genome size has received limited attention during the last 50 years since research at Brookhaven National Laboratory (USA) and other locations demonstrated substantial differences in radiation sensitivities, e.g. between tree species with large (e.g. conifers such as pines) versus small (e.g. dicots such as oaks) genome sizes. Taking advantage of the wide range of genome sizes among species, we investigated radiation sensitivity which we define in this study as DNA damage (break frequency) measured with the alkaline comet assay in isolated nuclei exposed to X-rays. As a starting point, we considered two possible explanations for the high radiation sensitivity of plants with large genome sizes: (i) inherently higher sensitivity of larger genomes and/or (ii) impaired DNA repair. In terms of genome size effects, experiments exposing isolated nuclei from six different plant species to X-rays, varying in genome sizes from 2.6 to 19.2 Gbp, showed that larger genomes are more sensitive to DNA damage by a relationship approximating the cube-root of the nuclear volume; e.g. a 10-fold increase in genome size increases sensitivity by about 2-fold. With regard to DNA repair, two conifer species, Sawara cypress (Chamaecyparis pisifera, 8.9 Gbp genome size) and Scots pine (Pinus sylvestris, 20 Gbp genome size), both effectively repaired DNA damage within 50 and 70 min, respectively, after acute X-ray exposures. Both species also showed delayed repair of double-strand DNA breaks, as we previously showed with Arabidopsis thaliana and Lolium multiflorum.

DNA repair after X-irradiation: lessons from plants.

The effects of low-dose radiation causing DNA damage continue to be subjects of interest. Problems with existing approaches to low-dose DNA damage are that single-strand breaks (the predominant radiation-induced lesion) are very rapidly repaired and that results using current methods for measuring DNA damage can be difficult to interpret. As a novel approach, we conducted studies using plants (rye grass and the model plant Arabidopsis) exposed to X-rays and used the alkaline comet assay to measure DNA damage and repair after exposures. Consistent with previous studies, we detected so-called 'rapid' and 'slow' phases of DNA repair after acute exposures of 5 and 15 Gy. After exposures corresponding to 2 Gy and lower, 'rapid' repair was so fast that it was difficult to detect. We also found that the so-called 'slow' phase in both plants actually consisted of two components; an initial period of negligible repair lasting 80-120 min followed by a period of rapid repair lasting <30 min. Using Arabidopsis mutants homozygous for both ATM and BRCA1, we found that both of these genes are required for DNA repair during the 3-h period of our experiments, indicating that the 'slow' phase involves a homologous repair (HR) of double-strand breaks and clustered single-strand breaks. The lag of repair in the 'slow' phase presumably involves induction of expression of genes involved in HR repair such as BRCA1 and RAD51.

Trace element mobility and transfer to vegetation within the Ethiopian Rift Valley lake areas.

To evaluate critical trace element loads in native vegetation and calculate soil-to-plant transfer factors (TFs), 11 trace elements (Cr, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Pb and Mn) have been determined in leaves of 9 taxonomically verified naturally growing terrestrial plant species as well as in soil samples collected around 3 Ethiopian Rift Valley lakes (Koka, Ziway and Awassa). The Cr concentration in leaves of all the plant species was higher than the "normal" range, with the highest level (8.4 mg per kg dw) being observed in Acacia tortilis from the Lake Koka area. Caper species (Capparis fascicularis) and Ethiopian dogstooth grass (Cynodon aethiopicus) from Koka also contained exceptionally high levels of Cd (1 mg per kg dw) and Mo (32.8 mg per kg dw), respectively. Pb, As and Cu concentrations were low in the plant leaves from all sites. The low Cu level in important fodder plant species (Cynodon aethiopicus, Acacia tortilis and Opuntia ficus-indicus) implies potential deficiency in grazing and browsing animals. Compared to the Canadian environmental quality guideline and maximum allowable concentration in agricultural soils, the total soil trace element concentrations at the studied sites are safe for agricultural crop production. Enrichment factor was high for Zn in soils around Lakes Ziway and Awassa, resulting in moderate to high transfer of Zn to the studied plants. A six step sequential extraction procedure on the soils revealed a relatively high mobility of Cd, Se and Mn. Strong association of most trace elements with the redox sensitive fraction and mineral lattice was also confirmed by partial redundancy analysis. TF (mg per kg dw plants/mg per kg dw soil) values based on the total (TF(total)) and mobile fractions (TF(mobile)) of soil trace element concentrations varied widely among elements and plant species, with the averaged TF(total) and TF(mobile) values ranging from 0.01-2 and 1-60, respectively. Considering the mobile fraction in soils should be available to plants, TF(mobile) values could reflect trace elements transfer to plants in the most realistic way. However, the present study indicates that TF(total) values also reflect the transfer of elements such as Mn, Cd and Se to plants more realistically than TF(mobile) values did.

The FRO2 ferric reductase is required for glycine betaine's effect on chilling tolerance in Arabidopsis roots.

FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.

ROS Signaling Pathways in Chilling Stress.

It was known that application of glycine betaine (GB) to plants could improve tolerance to stress caused by chilling, frost, salt, drought and high light intensities, and that this effect was accompanied by gene expression changes, but whether the gene expression changes were implicated in GB's effect and which genes were involved has been unclear. In the fourth issue of Physiologia Plantarum, we identified genes upregulated by GB that are involved in reactive oxygen species (ROS) metabolism and membrane trafficking processes. Direct evidence was provided for a role for a membrane trafficking protein (RabA4c) in GB's effect on ROS accumulation during chilling. In this Addendum, we discuss our findings that chilling stress is so closely linked with ROS accumulation. Chilling elevates ROS levels and results in inhibited root growth upon transfer of plants back to normal growing conditions. During the 2-4 day recovery period, ROS levels decline in root tips and in leaves. If ROS accumulation in response to chilling is blocked by pretreatment with GB, optimal root growth begins as soon as plants are transferred back to normal growing conditions without a recovery period, suggesting that chilling stress involves a ROS signaling pathway.