1003-B4

Radiation effects in the seedlings of some arbor species

Ioan Al. Creanga ¹

Abstract

The purpose of this study was to identify the putative influences of low intensity radiation on some biochemical parameters in young seedlings of forestry arbors. Three species from the plain area of Romanian country were taken for the experimental investigation: Quercus robur L., Populus nigra L., clone Sacrau-79 and Robinia pseudoacacia L. Since among external constraints frightening these species (and their ecosystems) the fluctuations of atmosphere radiation background may play a considerable role, the study was focused on the effects of small radiation doses in arbor seedlings during their early ontogenetic stages (up to six months old). Biochemical parameters considered for this research were: the content of chlorophyll a, chlorophyll b, total carotenes, the catalase activity and the content of nucleic acids. The exposure times were equal to: 1; 2; 6 and 12 hours. Assimilatory pigment levels were found mostly diminished after radiation exposure, depending also on the seedling age. Catalase activity was enhanced in younger seedlings (up to three months) while in older seedlings (six months) it was diminished proportionally to the exposure time. Average content of nucleic acids was generally diminished in exposed samples but an interesting positive correlation to the exposure time was revealed suggesting stimulatory effects of low intensity radiation upon the biosynthesis phenomena in the vegetal cells. The main issue of this preliminary investigation was the detection of net biochemical effects (statistically significant as Student t-test showed) for very small radiation doses, comparable to the environmental local fluctuation of the atmospheric background. Inhibitory but also stimulatory effects were revealed so that further investigation will be designed to clarify if low level ionizing radiation may actually induce changes in these arbor ecosystems.

Introduction

Situation of oak, poplar and black locust in Romania.

• Recolonisation of Europe by forest tree species after the last glaciation (well documented in the fossil pollen record, Petit et al., 1996) led to the spreading of oaks species to a larger area than in former periods. Now, the sessile and pedunculate oaks have a wide sympatric distribution over Europe from Spain to Russia and Romania and represent the largest economic resources among European broadleaf species. In Romania, especially pedunculate oak, Quercus robur L. is, at the moment, in serious decline, the surfaces with natural stands being decreased year after year, both because of natural factors and human action (Stanescu & Sofletea, 1995).
• Short history of species from genus Populus, presented by Wright (1965) shows that american black poplar (Populus deltoides Bartr) was introdus in Europe to the end of the XVIII th century, where hybridated rapidly with black European poplar (P. nigra L.) and natural hybrid that resulted have been quickly cultivated in small or large plantations. Black poplar (P. nigra), much convenient due to its high productivity, similar to that of P. alba, recorded a serious decline in Romania because of natural hybridations with other poplars but also because it was replaced in cultures with other species designed to restore natural biodiversity.

Poplar natural stands as well as poplar cultures (euramerican poplars) from Romanian plains are very important for economical and ecological reasons. Poplar species and clones provided by National Technical Norms for the utilization in Romanian production are: Populus × euramericana (Dode) Guinier, clones Robusta "Hârşova RO-16"; Robusta "Olteniţa RO-118"; Marilandica "Brăila RO-26"; Regenerata "Celei"; "Sacrau 79"; "I-45/51", "I-154", "I-214"; Populus deltoides Bartr, clone "Lux I-69/55"; Populus alba L., clone "Hemeiuş RO-724"; Populus × euramericana, clones "Triplo" (I-37/61); "Toropogritzki" - Russia (Benea si Filat, 1999).

Black locust is cultivated in Romania since 1750 when it is first mentioned in archives. The popularity of this species is related to its suitability for reforestation in degraded areas. Black locust is much appreciated as agroforestry species since it increases soil nitrogen level, improving its structure and fertility. Seedlings show positive response to fertilizer(...).

The importance of these species both for economical reasons and for ecological purposes motivated the choice of this study objective, i.e. the behavior of young seedlings after exposure to low intensity gamma radiation.

Material and method

Biological material was consistent with green tissue from seedling leaves, both for irradiated samples and controls. For every irradiation time and for every tree species five replays (seedlings with uniform genophond) were analyzed.

Radiation source. A laboratory Cobalt ⁶⁰ source with an activity of about 100 mCi was used to irradiate saplings. In order to do this, vessels were placed on the source and 24 hours after the irradiation the assimilatory pigments and catalase activity were determined.

Spectral device. A JASCO type spectrophotometer was used to read light extinction in 1 cm width quartz cells.

Spectrophotometric assay. Assimilatory pigment content was measured in 90% acetone extract (after maceration of crushed quantities of 0.02-0.03 g picked up from leaves according to standard procedure (Meyer-Bertenrath, modified by Stirban and Farcas (Stirban, 1987)). Small aliquots of CaCO₃ and MgCO₃ were added during extraction in order to avoid chlorophyll transformation in pheophytine. Acetone extract was filtered through paper filter and quantitatively transferred to coated bottles of 25 ml. Computation of chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenes pigment (c.p.) levels (mg pigment / 100 g green tissue) were performed by means of usual formulae:

Chl a=(9.784 E₆₆₂-0.99 E₆₄₄) 100v/w

Chl b=(21.426 E₆₄₄-4.65 E₆₆₂) 100v/w

carotene =[4.695 E_440.5-0.268 (5.134E₆₆₂+20.436E₆₄₄)] 100v/w

where:

E- light extinction at the wavelength , w-green tissue mass (g) and v-acetone extract (ml).

Average concentration of nucleic acids was evaluated spectrophotometrically with the same spectral device. Light extinction at 270 nm and 290 nm (E₂₇₀ and respectively E₂₉₀) were taken for quantitative evaluation of average content of nucleic acids (<AN>), in g/g, according to the next formula (Spirin method, Nuta and Busneag, 1981):

where: m (g) is the weighted amount of blood cells and v (ml) is the volume of perchloric acid used for nucleic acid extraction.

Iodometric assay. Catalase activity assay was carried out by means of iodometric titration (Artenie & Tanase, 1981). Catalase extraction was performed from sapling leaves using 0.1 M Na₂HPO₄ solution as extraction solvent. One catalase unit (C.U.) is the amount of enzyme that is capable to decompose in every minute 1 microMole of hydrogene peroxide (i.e. 0.034 mg).

Results and discussion

Statistical analysis. Student t-test (two tailed, pair type) was applied to evaluate reliability of modifications induced by radiation exposure in samples in comparison to the control. Regression curves were computed to reveal the dependence of every biochemical parameter to the exposure time as well as the relationship between pairs of biochemical parameters. Correlation coefficients were calculated by the less square method.

Experimental results and interpretation. Two lots of Q. robur L. seedlings, obtained from acorns provided by the same pedunculate oak stand, were analyzed: one at the age of 3 months and the other at the age of six months.

In 3 months old seedlings there is significant decrease of all assimilatory pigments to the enhance of the irradiation time (fig. 1) while for 6 months old seedlings (correlation coefficient over 0.90), linear increase of carotenes (for all exposure times) was noticed (fig. 2) together with polynomial variation of chlorophylls (correlation coefficients over 0.95). To explain this behavior the analysis of catalase activity was helpful as it represents a more sensitive indicator to the radiation action. The enzyme activity (fig. 3) of catalase is rapidly enhanced (to the exposure time enhancing) for 3 months seedlings while for 6 months old seedlings it is slightly diminished, in both cases linear approximation fitting experimental data with a correlation coefficient of about 0.9. Catalase having as unique substrate the hydrogen peroxide, which results in high concentration after water radiolysis, and living cell being able to adapt their biochemical functions (self adjusting phenomena known as feed-back reactions) if stress is not very strong, we might say that small radiation doses stimulate catalase biosynthesis. In the same time, radiations damage enzyme activity by destroying hydrogen bonds and other physical interactions that assure enzyme activity of a protein (either by direct or indirect action - by means of the radicals generated by water radiolysis).

So, in younger seedlings biosynthesis stimulation is stronger than molecule destruction while in older seedlings the balance is reversed. Further information could be obtained from nucleic acid assay. For shortest exposure time a significant decrease was obtained (fig. 4) while for longest exposure time a relevant enhance was obtained, sustaining the hypothesis that older seedlings (6 months old) are still able to recover the damages yielded by radiation action.

The study of Robinia pseudoacacia L. seedlings behavior after exposure to radiation was accomplished testing first 3 months old seedlings. Assimilatory pigment content is enhanced (fig.5) in all exposed samples (linear positive correlation) while catalase activity (fig. 6) is enhanced for the shortest and longest exposure times. Significant differences between control and exposed sample were obtained for: chlorophyll a - 1h and 6 h exposure time; chlorophyll b- 2h and 6 h exposure time; carotene: 1h, 2h and 12 h exposure time. Peroxidase activity (spectrophotometric assay) was supplementary assayed to get new data on radiation influence on enzyme activity: slight diminishing for the shortest exposure time followed by considerable amplification for the longest exposure time was obtained. Both catalase and peroxidase activities are significantly different in exposed samples in comparison to the control for 1h and 12 h exposure time. Stimulatory effect of low doses radiations upon biosynthesis mechanism (for both assimilatory pigment and enzymes) seems to be the dominating phenomenon revealed by these experimental data.

In 6 months old seedlings assimilatory pigment contents are diminished in samples (fig. 7) in comparison to the control, while catalase activity is exponentially degraded to the increase of the exposure time (fig. 8). This means that self-adjusting phenomena responsible to the recovering of losses produced by radiation action are not sufficiently activated to compensate damages caused by radiation at molecular level in chlorophylls and carotene as well as in catalase activity. Statistic analysis revealed smaller number of significant modifications than in previous case: chlorophyll a for 2h and 6 h exposure time, chlorophyll b for 2h and carotene for 6 h exposure time. Nucleic acid average content is also significantly decreased after radiation action confirming this interpretation. Linear correlation was revealed between the variation of these two biochemical parameters, correlation coefficient being equal to 0.8194. Both catalase and nucleic acid are considerably diminished by radiation in all exposed samples in comparison to the control.

In the case of black poplar P. nigra L. clone Sacrau-79, young plantlets were obtained from wood cuttings and they could be grown until the age of 3 months.

Analyzing the content of chlorophyll a, chlorophyll b and carotene, an enhance was noticed in all exposed samples in comparison to the control for the ratio chlorophyll a/chlorophyll b. As well, linear decrease was revealed in the ratio chlorophyll a/carotene to the exposure time increasing (correlation coefficient equal to 0.9632). The ratio chlorophyll b/ carotene was also significantly decreased in all samples in comparison to the control. The statistic analysis showed that the modifications induced by radiation are significant only for the next samples: chlorophyll a: 1 h and 12 h exposure time; chlorophyll b: 2h and 12 h exposure time; carotene: 1h and 6 h exposure time. The situation of catalase activity is described by an exponential enhance to the exposure time enhancing; statistic parameter t is over 0.05 only for the longest exposure time, so that correlation coefficient is also smaller than in other situations presented above (correlation coefficient equal to 0.69). Negative correlations were evidenced between catalase activity and pigment ratios (in the case of chlorophyll a/carotene correlation coefficient is equal to 0.7237).

CONCLUSION

More than the assimilatory pigment, enzyme activity, especially catalase activity was found to experience net modifications induced by low radiation doses in young arbor plantlets.
So:

(i) in oak seedings aged of 3 months, catalase activity enhanced rapidly (regression line slope equal to 130) to the exposure time increasing;
(ii) in oak seedlings 6 months old seedlings catalase activity diminished slowly (slope of regression line equal to 6.5) (Creanga et al, 2002);
(iii) in false acacia seedlings aged of 3 months a linear increasing of catalase activity was reported, peroxidase being also enhanced after exposure to gamma radiation (Artenie et al, 2002);
(iv) in black locust seedlings aged of 6 months a logarithmic decrease of catalase activity was revealed to the increasing of exposure time; (Creanga et al, 2002).

These are reasons to suppose that catalase represents a very sensitive indicator of radiation influence on young forestry plantlets. Never the less nucleic acid content was significantly changed after radiation exposure though more accurate method for the determination of such biochemical parameter is required. New tests should be accomplished to reveal deeper implications of radiation in biochemical processes from leaf cells and possibly to identify a suitable biotechnological tool in conducting the early ontogenetic stages of these forestry species. It is intended to follow the growth of seedlings exposed to low gamma radiation for several years in order to observe putative phenotypic changes in comparison to the control lots - possible causes of polymorphism.

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¹ Territorial Forestry Office-IASI, 28 Moara de Vant, 6600 Iasi, Romania, Europe,
e-mail: [email protected]