Jan-Elo Jørgensen

SHOREmap: simultaneous mapping and mutation identification by deep sequencing

Supplementary Figure 1 Method workflow Supplementary Figure 2 Visual output from SHOREmap DENOVO Supplementary Table 1 Top 10 ranked mutations from the SHOREmap ANNOTATE output Supplementary Table 2 Command line programs, parameters and run time used for the computational analysis of Illumina data Supplementary Table 3 Identification of additional AT4G35090 mutant alleles Supplementary Table 4 Output of SHOREmap ANNOTATE using the interval based on SHOREmap DENOVO data Supplementary Note Mapping large deletions, QTLs and dominant or recessive lethal mutations. Supplementary Methods Lab protocols and computational algorithms Supplementary Data SHORE and SHOREmap example files

Hairy roots — a short cut to transgenic root nodules

To facilitate molecular studies of symbiotic nitrogen fixation a procedure for rapid production of transgenic root nodules was established on the legumeLotus corniculatus (Birdsfoot trefoil). Regeneration of transgenic plants is not required as transgenic nodules are formed onAgrobacterium rhizogenes incited roots inoculated withRhizobium. Easy identification of transformed roots is possible using a set ofA. rhizogenes acceptor strains carrying assayable marker genes such as chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or luciferase (LUC) under control of the cauliflower mosaic virus (CaMV) 35S promoter. Counterselection ofA. rhizogenes after infection of plants was improved using an auxotrophy marker.

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters

SummaryThe qualitative and quantitative contributions of four separate cis-acting DNA elements controlling the root nodule-specific soybean leghemoglobin lbc3 gene were analyzed in transgenic Lotus corniculatus plants. Expression from internal deletions in the 5′ region between positions −49 and −1956 was monitored from a CAT reporter gene. The strong positive element (SPE; −1090, −947) responsible for high-level expression was demonstrated to be an organ-specific element by deleting proximal nodule-specific control elements. Deletion of the downstream qualitative organ-specific element (OSE; −139, −102) containing the putative nodulin consensus sequences 5′AAAGAT and 5′CTCTT resulted in a low expression level. Efficient SPE enhancement is therefore dependent on the organ-specific element, which by itself does not enhance expression. This quantitative effect of the immediate upstream region carrying the consensus sequences was also found in hybrid promoter studies using the soybean nodulin N23 gene promoter, suggesting the involvement of these motifs in a regulatory mechanism for nodulin genes. Deletion of the lbc3negative element (NE, −102, t-49) linking the SPE and OSE onto the TATA box did not lead to unregulated expression. These results indicate that interaction between positive, negative and neutral qualitative elements controls lbc3 expression. Binding of the nuclear protein NAT2 at the lbc3 weak positive element (WPE; −230, −170) is probably not directly required for this mechanism.

Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis

Catalase directly interacts with and detoxifies reactive oxygen species. This work identifies catalase-deficient mutants in a screen for suppression of cell death and finds that promotion of cell death associated with the plant hypersensitive response requires catalase, suggesting that catalase could act as a direct molecular link between reactive oxygen species and cell death signaling. Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.

Identification of Trans-Acting Factors Regulating Nodulin Gene Expression

Functional studies of nodulin gene promoters in transgenic legumes have identified a number of cis-regulatory elements important for nodule specific expression. One example is the soybean leghaemoglobin (lb) c3 promoter which were investigated in details in transgenic Lotus corniculatus plants. By analysing 5’ promoter deletions and hybrid promoters fused to the GUS and CAT reporter genes the following elements were identified in the lbc3 promoter; A strong positive element, a weak positive element, an organ specific element and a negative element (Figure 1, Stougaard et al. 1990).