An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

BACKGROUND: It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. METHODS: Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. RESULTS: The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41 µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100 mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1 pg/µl while root gDNA remained amplifiable at 10 pg/µl. Genomic DNA obtained was also suitable for sequencing. CONCLUSION: This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Luminescence imaging of leaf damage induced by lipid peroxidation products and its modulation by ß-cyclocitral.

Lipid peroxidation is a primary event associated with oxidative stress in plants. This phenomenon secondarily generates bioactive and/or toxic compounds such as reactive carbonyl species (RCS), phytoprostanes, and phytofurans, as confirmed here in Arabidopsis plants exposed to photo-oxidative stress conditions. We analyzed the effects of exogenous applications of secondary lipid oxidation products on Arabidopsis plants by luminescence techniques. Oxidative damage to attached leaves was measured by autoluminescence imaging, using a highly sensitive CCD camera, and the activity of the detoxification pathway, dependent on the transcription regulator SCARECROW-LIKE 14 (SCL14), was monitored with a bioluminescent line expressing the firefly LUCIFERASE (LUC) gene under the control of the ALKENAL REDUCTASE (AER) gene promoter. We identified 4-hydroxynonenal (HNE), and to a lesser extent 4-hydroxyhexenal (HHE), as highly reactive compounds that are harmful to leaves and can trigger AER gene expression, contrary to other RCS (pentenal, hexenal) and to isoprostanoids. Although the levels of HNE and other RCS were enhanced in the SCL14-deficient mutant (scl14), exogenously applied HNE was similarly damaging to this mutant, its wild-type parent and a SCL14-overexpressing transgenic line (OE:SCL14). However, strongly boosting the SCL14 detoxification pathway and AER expression by a pre-treatment of OE:SCL14 with the signaling apocarotenoid ß-cyclocitral canceled the damaging effects of HNE. Conversely, in the scl14 mutant, the effects of ß-cyclocitral and HNE were additive, leading to enhanced leaf damage. These results indicate that the cellular detoxification pathway induced by the low-toxicity ß-cyclocitral targets highly toxic compounds produced during lipid peroxidation, reminiscent of a safener-type mode of action.

Asparagine accumulation in chicory storage roots is controlled by translocation and feedback regulation of asparagine biosynthesis in leaves.

The presence of acrylamide (AA), a potentially carcinogenic and neurotoxic compound, in food has become a major concern for public health. AA in plant-derived food mainly arises from the reaction of the amino acid asparagine (Asn) and reducing sugars during processing of foodstuffs at high temperature. Using a selection of genotypes from the chicory (Cichorium intybus L.) germplasm, we performed Asn measurements in storage roots and leaves to identify genotypes contrasting for Asn accumulation. We combined molecular analysis and grafting experiments to show that leaf to root translocation controls Asn biosynthesis and accumulation in chicory storage roots. We could demonstrate that Asn accumulation in storage roots depends on Asn biosynthesis and transport from the leaf, and that a negative feedback loop by Asn on CiASN1 expression impacts Asn biosynthesis in leaves. Our results provide a new model for Asn biosynthesis in root crop species and highlight the importance of characterizing and manipulating Asn transport to reduce AA content in processed plant-based foodstuffs.

Singlet Oxygen-Induced Cell Death in Arabidopsis under High-Light Stress Is Controlled by OXI1 Kinase.

Studies of the singlet oxygen ((1)O2)-overproducing flu and chlorina1 (ch1) mutants of Arabidopsis (Arabidopsis thaliana) have shown that (1)O2-induced changes in gene expression can lead to either programmed cell death (PCD) or acclimation. A transcriptomic analysis of the ch1 mutant has allowed the identification of genes whose expression is specifically affected by each phenomenon. One such gene is OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) encoding an AGC kinase that was noticeably induced by excess light energy and (1)O2 stress conditions leading to cell death. Photo-induced oxidative damage and cell death were drastically reduced in the OXI1 null mutant (oxi1) and in the double mutant ch1*oxi1 compared with the wild type and the ch1 single mutant, respectively. This occurred without any changes in the production rate of (1)O2 but was cancelled by exogenous applications of the phytohormone jasmonate. OXI1-mediated (1)O2 signaling appeared to operate through a different pathway from the previously characterized OXI1-dependent response to pathogens and H2O2 and was found to be independent of the EXECUTER proteins. In high-light-stressed plants, the oxi1 mutation was associated with reduced jasmonate levels and with the up-regulation of genes encoding negative regulators of jasmonate signaling and PCD. Our results show that OXI1 is a new regulator of (1)O2-induced PCD, likely acting upstream of jasmonate.

METHYLENE BLUE SENSITIVITY 1 (MBS1) is required for acclimation of Arabidopsis to singlet oxygen and acts downstream of ß-cyclocitral.

Singlet oxygen (1 O2 ) signalling in plants is essential to trigger both acclimatory mechanisms and programmed cell death under high light stress. However, because of its chemical features, 1 O2 requires mediators, and the players involved in this pathway are largely unknown. The ß-carotene oxidation product, ß-cyclocitral, is one such mediator. Produced in the chloroplast, ß-cyclocitral induces changes in nuclear gene expression leading to photoacclimation. Recently, the METHYLENE BLUE SENSITIVITY protein MBS has been identified as a key player in 1 O2 signalling leading to tolerance to high light. Here, we provide evidence that MBS1 is essential for acclimation to 1 O2 and cross-talks with ß-cyclocitral to mediate transfer of the 1 O2 signal to the nucleus, leading to photoacclimation. The presented results position MBS1 downstream of ß-cyclocitral in 1 O2 signalling and suggest an additional role for MBS1 in the regulation of plant growth and development under chronic 1 O2 production.

Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.

Identification of potato varieties suitable for cold storage and reconditioning: A safer alternative to anti-sprouting chemicals for potato sprouting control.

Low temperature storage as an alternative to anti-sprouting chemicals in potato storage may induce reducing sugars (RS) accumulation (i.e. glucose and fructose) in potato tubers. This phenomenon is called "cold induced sweetening" (CIS) and occurs in certain varieties. CIS leads to a decrease in the organoleptic qualities and darkening of processed potato and the accumulation of toxic molecules such as acrylamide. To identify potato varieties suitable for storage at low temperatures, we screened six commercial processing varieties: Lady Claire (LC), Verdi, Kiebitz (KB), Pirol, Agria and Markies for their CIS characteristics and sprout-forming potential after storage at 4 °C and 8 °C. Our findings reveal that 4 °C storage allows for efficient sprout reduction in all six tested varieties for up to 4.5 months of storage. Three CIS-resistant varieties, namely Verdi, Lady Claire and Kiebitz, were identified as able to be stored for up to four months at 4 °C with limited increase in glucose content. Conversely, Pirol, Agria and Markies showed an increase in glucose content with a decrease in storage temperature and can be considered as CIS-susceptible varieties. After processing into crisps, the CIS-susceptible varieties displayed poor crisp color quality (brown to black color crisps) after storage for two months at 4 °C compared to the storage at 8 °C, whereas the CIS-resistant varieties had good crisp color quality (pale yellow color crisps) after storage at both 4 and 8 °C. Interestingly, the trends of total RS and/or glucose content in the CIS-resistant and in the CIS-susceptible varieties were correlated with the trends in Vacuolar Invertase (VInv) gene expression for most varieties, as well as with the trends in acrylamide content after processing. In addition, reconditioning of Markies variety after storage at 4 °C by gradually increasing the temperature to 15 °C resulted in a significant decrease of VInv transcript levels (reduction of 80 %), acrylamide content (reduction of 75 %) and glucose content when compared to a storage at 4 °C without reconditioning. Those results demonstrate that the reconditioning technique is a key factor for a sustainable potato storage and for improving the quality of processed potatoes.

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required.