Báo cáo lâm nghiệp: "Apoplast: a sensitive site for assessing some biochemical in Norway spruce needles"

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Apoplast: a sensitive site effects of for assessing some biochemical 32 O or SO in Norway spruce needles H. G. F.J. Castillo Greppin Ogier, Biochemistry and Physiology, University of Geneva, CH-1211, Geneva 4, Laboratory of Plant Switzerland (IWF) and in the residual cell material Introduction (RCM). The plants treated in summer remained 12 wk longer in the chambers in The study of the cell wall-plasma mem- order to assess any visible injury caused brane interphase is of great importance for by 0 in autumn. 3 the understanding of gaseous air pollu- tants and leaf cell interactions. In the apo- plast liquid phase, the pollutants are solu- bilized and they can generate oxidative Materials and Methods products (Tingey and Taylor, 1982). For example, 0 or S0 could lead to H 32 0 2 Two groups of 20 clone saplings (4 yr old graft- production (Tingey and Taylor, 1982; Khan ed P. abies) were selected from the nursery of and Malhotra, 1982). In order to protect the Swiss FedEaral Institute of Forestry Re- the plasma membrane and the compo- search (Birmensdorf, CH), one group for each experiment. Prior to fumigation, the plants were nents of the extracellular matrix, cells are distributed randomly into 4 semi-open top believed to dispose of oxidant-scavenging chambers (5 individuals per chamber). Current mechanisms. One of the enzymatic sys- year old needles and 1 yr old needles were tems which could play a protective role analyzed in samples harvested at the end of the against oxidative stresses includes per- fumigation period. The experimental approach is shown in Table I. oxidases (Castillo and Greppin, 1988). The IWF was obtained after infiltration of Peroxidase activity, with guaiacol as the buffer (40 mM, pH 4.5), 0.1 M KCI, phosphate electron donor, and protein content were 3 pM EDTA, and centrifugation (10 000 x g, measured in Norway spruce needles 4°C, 10 min) according to Castillo et al. (1987). The RCM extract was obtained from 0.5 g of (Picea abies (L.) Karst) after fumigation the remaining needles, which were ground (24 h/d) in semi-open top chambers, for under liquid nitrogen, in the presence of PVP 12 wk in summer with 0 or for 10 wk in 3 (0.5 g), then solubilized with 3 ml of phosphate winter with S0 These parameters were . 2 buffer (66 mWl, pH 7), and centrifuged 10 (10 000 x g, 4°C, min). followed in the intercellular washing fluid
Peroxidase activity was assayed by mea- This enzyme activity was respectively). suring the oxidation of guaiacol at 470 nm. not affected in the IWF by S0 treatment. 2 This activity was carried out using phosphate The only noticeable change in the RCM buffer (66 mM, pH 6.1), 16 mM guaiacol, increase in 1 yr old needles of the was an 3.3 mM H and 0-10 ul of enzyme extract. 0 2 ambient air-treated plants (124% Protein contents were determined according to summer Bradford (1976) using a Bio-Rad protein assay of the pooled values of the plants exposed (0-20,ul of enzyme extract). to controlled conditions). The protein content (Fig. 2) in the IWF Statistics. The only environmental factor dif- of young needles was 1.3-2.3 times great- fering between groups was the air composition er after low and high ozone exposure, and within the chambers. Considering this factor, 1.6-1.7 times greater after low and high plants fumigated with either filtered air (fa) or fa plus added pollutants were under controlled S0 concentrations, respectively, 2 as com- conditions (a); whereas in ambient air cham- to the control values. On the other pared bers, the fumigation conditions were uncon- the protein content of the RCM was hand, trolled (b). In both experiments, the data of the only affected by the high ozone exposure (a) plant groups were tested by analysis of vari- ance. Means which were significantly different lower. In the 1 yr old needles, the and was were identified using a t-test. The data of the only change observed was an increase of groups which were statistically equivalent were the protein content in the IWF after high pooled. Then, those of the (b) plant groups ozone exposure. were compared to the pooled or unpooled ones of the (a) plant groups using a t-test. For these No visible damage could be noted when analyses, we chose P
The long lasting period of high 0 3 Discussion and Conclusion concentration (200 !g 0 12 wk in , /m 3 summer + 12 wk in autumn) together with This type of experiment does not allow us subzero temperatures during autumn to know the actual leaf could be responsible for the drop of the pollutant uptake, which is controlled in part by the thickness needles (Brown et aL, 1987; Barnes and of the boundary layer, the opening of the Davison, 1988). These authors have stomata and the transpiration rate of the reported that 1 yr old needles from 3 out cells. However, according to the marked of 10 and 3 out of 8 clones were sensitive differences of responses between young to frost injuries due to long-term 0 3 fumigations (> 200 P 0 Despite the g3 ). M / and old needles, one can assume that young needles take up more pollutants fact that in our case both current and 1 yr than old ones (Tingey and Taylor, 1982). old needles were injured, their fall after
The decreased peroxidase activity in the a high 0 concentration indi- 3 exposure to cates the sensitivity of our clone to this IWF after high 0 exposure could result 3 from either altered enzyme secretion or pollutant. Moreover, this sensitivity is prob- direct enzyme denaturation by 0 or its ably revealed by frost events in late 3 by-products. In an earlier study (Castillo autumn. et al., 1987), an increase of extracellular The enhancement of the protein con- peroxidase activity in needles of Picea tents in the IWF promoted by both pol- abies saplings fumigated with 300 Jig lutants in current-year needles and by 03 0 7 h/d for 4 wk was observed. The , M / 3 alone in 1 yr old needles, could be attribut- apparent contradictory response of extra- ed to the alteration of protein secretion. cellular peroxidase between both experi- Whether this change is a consequence of ments is probably due to different experi- an increased secretion or leakage of mental conditions. In the previous paper stored or newly synthesized proteins is (Castillo et al., 1987), the experiment was currently under investigation. carried out with a heterogeneous popula-
tion of saplings and the total dose for that References short-term 0 fumigation was 30 ppm/h. In 3 this report, the data were obtained from Barnes J.D. & Davison A.W. (1988) The influ- grafted saplings originating from the same of ozone on the winter hardiness of Nor- ence clone and the total dose for this long-term way spruce (Picea abies (L.) Karst.). New Phy- 0 fumigation was 200 ppm/h. Apparently, 3 tol. 108, 159-16E! extracellular peroxidase responds in a dif- Bradford M. (1976) A rapid and sensitive ferent way depending upon the level and method for the quantitative determination of microgram quantities of protein utilizing the length of pollutant exposure and/or on the principle of protein-dye binding. Anal. Bio- genetic characteristics of the plant mate- chem. 72, 248-2.54 rial. Brown K.A., Rot>erts TM. & Blank L.W. (1987) In the case of high 0 exposure, the 3 Interaction between ozone and cold sensitivity decreased extracellular enzyme activity in Norway spruce: a factor contributing to the forest decline in central Europe? New PhytoL and the increased protein content in the 105, 149-155 IWF of young needles could be explained Castillo F.J. & Greppin H. (1988) Extracellular by the high 0 concentration applied 3 ascorbic acid and enzyme activities related to (200 ,ug 0 24 hid, for 12 wk), which is , M / 3 ascorbic acid metabolism in Sedum album probably above the threshold value that leaves after ozone exposure. Environ. Exp. the plant can tolerate without disruption of Bot. 28, 231-238 homeostasis. Castillo F.J., Miller P.R. & Greppin H. (1987) Extracellular biochemical markers of photo- Based on these observations, it ap- chemical oxidant air pollution damage to Nor- pears that the apoplast of Norway spruce s way spruce. Experientia 43, 111-115 needles is a sensitive site for the detection Khan A.A. & M:alhotra S.S. (1982) Peroxidase of stresses induced by gaseous pollutants. activity as an indicator of S0 injury in jack pine 2 and white birch. Biochem. Physiol. Pflanz. 177, 643-650 Tingey D.T. & T G.E. (1982) Variation in ayior Acknowledgments plant response to ozone: a conceptual model of physiological events. In: Effects of Gaseous Air Pollution in Agriculture and Horticulture. Butter- This work was supported by Grant Number 4.849.0.85.14 from the Swiss FNRS. worths, London, pp. 111-138