New insights into phloem unloading and expression of sucrose transporters in vegetative sinks of the parasitic plant Phelipanche ramosa L. (Pomel)

The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in concordance with the PrSUT3 mRNA accumulation in multiple sink areas including apical meristem, scale-leaf primordia, immature vasculature and even storage parenchyma. However, the PrSUT3 transcripts did not accumulate in the mature phloem cells. The transient expression experiments in Arabidopsis protoplasts suggested a tonoplast localization of PrSUT3, for which nevertheless the involvement in intracellular sucrose transport needs clarification

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8\u27-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2A, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

<p>Abstract</p> <p>Background</p> <p>Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in <it>Nicotiana benthamiana </it>was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system.</p> <p>Results</p> <p>A <it>LOX </it>gene which is expressed after treatment of the viral vectors was isolated from <it>Nicotiana benthamiana</it>. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as <it>Nb-9-LOX</it>. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml^-1cell cultures) which can oxygenate linoleic acid resulting in high yields (18 μmol ml^-1cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C₉-aldehydes (3.3 μmol mg^-1crude protein). The yield of C₉-aldehydes from linoleic acid was 64%.</p> <p>Conclusion</p> <p>The yeast expressed Nb-9-LOX can be used to produce C₉-aldehydes on a large scale in combination with a <it>HPL </it>gene with 9-HPL function, or to effectively produce 9-hydroxy-10(<it>E</it>),12(<it>Z</it>)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent.</p

Characterisation of resistance to branched broomrape, Phelipanche ramosa, in winter oilseed rape

a b s t r a c t Over the past decade, Phelipanche ramosa, a weedy parasitic plant (broomrape), has been increasingly infesting winter oilseed rape (WOSR) fields in France. Elite WOSR lines have shown different responses in P. ramosa infested fields, suggesting that genetic variability might be available for breeding programmes targeting broomrape resistance. Ten WOSR genotypes selected for their contrasting response in field experiments were analysed using mini-rhizotron and greenhouse co-culture experiments to determine the components of resistance to broomrape. Partial resistance was revealed at three developmental stages of the parasitic plant. First, at the germination stage, parasite attachment to the roots of some WOSR lines was limited and associated with a low rate of parasite seed germination. This mechanism of parasite avoidance could nevertheless be suppressed under high infestation in mini-rhizotron and greenhouse conditions. Second, at the root attachment stage, limited parasite attachment was observed in mini-rhizotron conditions under low and high infestation, and in greenhouse conditions. Third, after successful parasite attachment, some WOSR genotypes retarded and even disturbed the growth of tubercles, minimising and delaying parasite emergence from the soil. Although the exact mechanisms limiting parasite attachment and tubercle development require further investigation, our findings suggest that, by cumulating various resistance traits in new genotypes to enhance effectiveness and potential durability of resistance, breeding could be a promising control strategy in WOSR