Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure
Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. (Konno 2011 Gaupels and Ghirardo 2013 Exudates from the EFP contain toxic cucurbitacin steroids alkaloids and terpenoids as a preformed barrier against invaders (Konno 2011 Moreover metabolomic and proteomic approaches revealed that leaf damage triggered SWR in the EFP of (pumpkin) amongst others by JA and redox signaling (Gaupels et al. 2012 In the present study we aimed at further exploring signal transduction induced in the EFP during systemic wound responses. We were particularly interested in alterations of the antioxidant system as a hint toward induced redox changes and in signaling by NO-mediated protein modifications and cGMP. The observed damage responses might be transmitted over long distances via the phloem or could be part of EFP-internal defense mechanisms triggered by systemic messengers. Materials and methods Plant treatment and sampling Leaf edges of 4-5 week-old pumpkin plants (cv. Gele Centenaar) grown under green-house conditions were crushed between the lids of two 50 ml Dihydrocapsaicin polypropylene reaction tubes. Control plants were left untreated. Phloem sap was collected as described earlier (Gaupels et al. 2012 Petioles and stems were cut using Dihydrocapsaicin a razor blade and the basal side of the cut was immediately blotted with tissue paper. The exuding phloem sap was subsequently collected by a micropipette and mixed with an equal volume of phloem buffer (50 mMTris/HCl pH 7.8 0.1% β-mercaptoethanol; Dihydrocapsaicin McEuen and Hill 1982 Pumpkin leaf extracts were prepared by grinding 0.5 g leaf material in liquid N2 addition of 3 ml homogenization buffer (50 mM TrisCl pH 7.8 1 mM EDTA 7.5% [w/v] soluble polyvinylpyrrolidone 2 mM ascorbate) and subsequent centrifugation. The supernatant was used for APX measurements. Measurements of antioxidant enzymes glutathione and ascorbate All enzyme measurements were done with an Ultrospec 3100 Pro photometer (GE Healthcare Life Sciences) following previously published protocols (Harrach et al. 2008 APX activity was measured in 36 μl phloem sample (phloem exudate plus phloem buffer) or 50 μl leaf extract while 10 and 32 μl aliquots of phloem samples were used for determination of DHAR and GR activities respectively. For the glutathione and ascorbate measurements 10 μl phloem exudate was added to 90 μl of 5% meta-phosphoric acid. Samples were incubated for 10 min at RT and centrifuged for 30 min at 14000 rpm. The supernatant was stored at ?20°C until further analysis. Immediately before the measurements samples were neutralized by adding 25 μl 1 M triethanolamine. Glutathione was measured in 5 GABPB2 μl neutralized extract using the Amplite? Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit (AAT Bioquest) following the manufacturer’s instructions. For ascorbate measurements a colorimetric protocol was used (Harrach et al. 2008 Five microliter neutralized phloem extract was mixed with 150 μl 150 mM NaPO4 (pH 7.4) and 150 μl H2Odest to determine reduced ascorbate. For the measurement of total ascorbate neutralized extract was mixed with 150 μl 150 mM NaPO4 and 75 μl 10 mM dithiothreitol. After 10 min incubation at RT 75 μl 0.5% N-ethylmaleimide was added to the sample. The reaction protocol is the same for both reduced and Dihydrocapsaicin total ascorbate. The sample was combined with 300 μl 10% (w/v) trichloroacetic acid 300 μl 44% (v/v) phosphoric acid 300 μl 4% (w/v) bipyridyl (in 70% EtOH) and 150 μl 2% (w/v) FeCl3. After 1 h incubation at 37°C the absorption of the sample was measured at 525 nm. Thiobarbituric acid reactive substances determination Proteins were removed from 100 μl phloem exudate by adding 200 μl ice-cold trichloroacetate incubation for 15 min on ice and subsequent centrifugation. Two hundred fifty microliters of the supernatant was used for determining the content of thiobarbituric acid reactive substances (TBARS) according to Hodges et al. (1999). Determination of the total S-nitrosothiol content The total S-nitrosothiol content of phloem sap was analyzed by a Nitric Oxide Analyzer (Siever’s NOA 280i GE Power and Water Analytix). Seventy five microliters phloem exudate was treated for 10 min at RT with 19 μl 5% sulfanilamide (w/v in 1 M HCl) in order to scavenge nitrite. The sample was then injected into the NOA reaction vessel which contained a reducing triiodide.