SummaryPrevious work from numerous laboratories has suggested that integration of Agrobacterium tumefaciens T-DNA into the plant genome occurs preferentially in promoter or transcriptionally active regions. However, all of these studies were conducted on plants recovered from selective conditions requiring the expression of transgenes. The conclusions of these studies may therefore have been biased because of the selection of transformants. In this study, we investigated T-DNA integration sites in the Arabidopsis genome by analyzing T-DNA/plant DNA junctions generated under non-selective conditions. We found a relatively high frequency of T-DNA insertions in heterochromatic regions, including centromeres, telomeres and rDNA repeats. These T-DNA insertion regions are disfavored under selective conditions. The frequency with which T-DNA insertions mapped to exon, intron, 5¢ upstream and 3¢ downstream regions closely resembled their respective proportions in the Arabidopsis genome. Transcriptional profiling indicated that expression levels of T-DNA pre-integration target sites recovered using selective conditions were significantly higher than those of random Arabidopsis sequences, whereas expression levels of genomic sequences targeted by T-DNA under non-selective conditions were similar to those of random Arabidopsis sequences. T-DNA target sites identified using non-selective conditions did not correlate with DNA methylation status, suggesting that T-DNA integration occurs without regard to DNA methylation. Our results indicate that T-DNA integration may occur more randomly than previously indicated, and that selection pressure may shift the recovery of T-DNA insertions into gene-rich or transcriptionally active regions of chromatin.

The majority of phosphorus (P) in seeds is found in phytic acid (InsP(6)) which accumulates as the mixed salt phytate. InsP(6) is generally considered to be an anti-nutrient and the development of low phytic acid (lpa) seed crops is of significant interest. We have employed a reverse genetics approach to examine the impact of disrupting genes involved in inositol phosphate metabolism on Arabidopsis seed InsP(6) levels. Our analysis revealed that knockout mutations in three genes (AtITPK1, AtITPK4, and AtMIK/At5g58730) reduced seed InsP(6) in addition to knockouts of four previously reported genes (AtIPK1, AtIPK2β, AtMRP5, and At5g60760). Seeds of these lpa mutants also exhibited reduced germination under various stress conditions. The greatest reduction in InsP(6) (>70%) was observed in atmrp5 seeds which were also among the least sensitive to the stresses examined. Expression analysis of the lpa genes revealed three distinct patterns in developing siliques consistent with their presumed roles. Disruption of each lpa gene resulted in changes in the expression in some of the other lpa genes indicating that transcription of lpa genes is modulated by other constituents of InsP(6) metabolism. While all the lpa genes represent possible targets for genetic engineering of low phytate seed crops, mutations in AtMRP5, AtMIK, and At5g60760 may be most successful for conventional approaches such as mutation breeding.

Rice (Oryza sativa L.) is sensitive to prolonged exposure to low temperature, which at the seedling stage can result in significant chilling injury and mortality. The objective of this study was to quantify physiological and biochemical changes in rice seedlings undergoing chilling stress and compare those changes with visual evaluation of tolerance. Seedlings from the cultivars M-202 (tolerant) and IR50 (sensitive) were subjected to 9°C for 14 days in a controlled environment chamber. Leaf tissues were harvested at various time points for determination of electrolyte leakage, proline, malondialdehyde, ascorbic acid and reduced glutathione. Significant differences between M-202 and IR50 were detected in electrolyte leakage, proline, and ascorbic acid starting at 7 days with IR50 exhibiting higher levels of these indicators. Most IR50 seedlings were dead at 14 days. A set of fifty rice accessions including M-202 and IR50 was evaluated at 10 days of cold treatment to examine the correlation of visual ratings with the physiological indices. Visual ratings were most highly correlated with electrolyte leakage and least correlated with proline content. Based on visual ratings and the physiological indices, we identified several cultivars that outperformed M-202 in cold tolerance while IR50 had the lowest tolerance of the cultivars tested.