Photosynthetic Response of Yellow-Poplar Seedlings to the Antioxidant Chemical Ethylenediurea

Materials and Methods

Starting in mid-May, 50 two-yr-old seedlings of yellow-poplar were planted in 2-L plastic containers filled with an artificial potting media (Terra Lite 500). The seedlings were placed in the greenhouse under natural photoperiods for 8 wks prior to initiation of the study. During this time the plants were watered thoroughly twice each week and fertilized biweekly with 500 ml of a commerical fertilizer solution containing 200 ppm each of nitrogen, phosphoric acid and soluble potash as 20.ON-8.6P-16.6K. Temperature and relative humidity conditions in the greenhouse were 26±5 C and 72±12%, respectively. Toward the end of July, 36 seedlings of uniform size were selected and 12 were randomly assigned to each of the following treatments: 1) stem injection with 5 ml EDU at a concentration of 500 ppm; 2) soil drench with 250 ml EDU, also at a concentration of 500 ppm; 3) no EDU treatment. These applications of EDU had been used successfully in an earlier investigation on the effectiveness of the chemical in modifying the sensitivity of woody plants to O₃ fumigation (16).

Following the assignment of EDU treatments, but prior to application of the chemical (day 0), Pn was measured on a single leaf from each seedling by a transient measuring technique (Li-Cor 6000 Portable Photosynthesis System). Photosynthetically active radiation, 410-428µE s⁻¹ m⁻², was derived from a high pressure sodium lamp filtered through 6 cm of water. Pn was calculated as CO₂ uptake per unit leaf area based on average leaf area determinations made at day 0 and again at the end of the experiment. The leaf used for Pn determinations had a plastochron index value of 5, and leaf area was recorded from a photocopy of the leaf surface measured with an area meter (Hayashi Denko AAM-5). After the measurement at day 0, Pn was remeasured on the same leaf at day 2, 4, and 7. Following the 7 day measurement, 4 seedlings in each treatment (EDU injection; EDU drench; no EDU) were fumigated for 3 hr in a continuously stirred tank reactor system (12) with one of the following O₃ concentrations: 0, 0.35 or 0.95 ppm (0, 693 or 1881 µg m⁻³). After O₃ fumigation the seedlings were returned to the greenhouse for 2 days prior to remeasuring Pn on the same leaf as previously described. At this time, the degree of O₃ injury for each seedling was rated subjectively on a scale ranging from 0-10. Seedlings exhibiting no visible foliar injury were rated 0. For those plants showing chlorotic areas between the veins, a rating of 0.5 was scored. Small interveinal necrotic spots rated at 1.0. For ratings of 2 to 10, 20% to 100% of the sensitive leaves, respectively, exhibited bifacial necrosis.

Results and Discussion

The results of this study show that there was no significant change in Pn for yellow-poplar seedlings treated with EDU (either injection or drench) over the 7 day period following application of the chemical (Table 1). These data suggest that EDU treatment per se has no appreciable effect on Pn, and are in general agreement with the results reported by Bennett et al (2) for soil-applied EDU on bean seedlings. While EDU had no significant influence on Pn, seedlings without EDU showed a substantial decrease in Pn rate between day 4 and day 7 (Table 1). The reason for this decline is not clear, but may be related to the large increase in leaf area noted for those plants without EDU (data not shown). While leaf area increased on average of 26% for seedlings treated with EDU, the corresponding increase for untreated plants was substantially greater (41 %).

Measurements of leaf stomatal conductance, taken simutaneously with determination of Pn, showed that stomatal opening in all treatments increased gradually over the first 4 days (Table 2). However, between day 4 and day 7 there was a significant decline in stomatal conductance in the EDU treated plants. These data correspond with the trends noted for Pn in Table 1, and suggest that CO₂ assimilation in this study was probably influenced more by stomatal opening than by the direct effect of EDU on the photosynthetic mechanism per se. As a general observation, stomatal conductance of EDU-treated plants (either injection or drench) tended to be somewhat greater than corresponding plants without EDU, although the values are only significant for the EDU injection treatment on day 4 (Table 2).

The data on Pn of EDU-treated seedlings following fumigation with O₃ are found in Fig. 1. Two days after fumigation, Pn of those seedlings without EDU showed a reduction of 32%. These results were not unexpected, and illustrate the potential harmful effect of O₃ on physiological activity of fumigated plants (1,4,17). For seedlings treated with EDU drench, Pn decreased 20% at 0.35 ppm O₃ and an additional 7% at 0.95 ppm O₃. Thus, EDU treatment afforded some degree of protection from O₃ in terms of moderating the extend of Pn decline. With the EDU injection treatment Pn actually increased at O₃ concentrations of 0.35 and 0.95 ppm when compared to the values for unfumigated seedllings (Fig. 1). These data may be somewhat misleading and difficult to interpret because of the confounding influence of wounding during the injection process, although the metabolic after-effects of wounding are normally quite short-lived (3).

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Fig. 1.

Net photosynthesis of 2-yr-old containerized yellow-poplar seedlings 2 days after fumigation with 0₃. Each value represents the mean of 5 consecutive measurements made on individual leaves (plastochron Index = 5) from each of 4 seedlings. Injection treatments made with 5 ml EDU (500 ppm) per seedling; drench treatments with 250 ml (500 ppm) per container. Ozone fumigations were for 3 hr at concentrations of 0, 0.35 and 0.95 ppm (0, 693 and 1881 µg m⁻³).

Stomatal conductance measurements taken 2 days after fumigation with O₃ show an increase in stomatal opening for EDU-injected seedlings at both O₃ levels used in this study (Fig. 2). These data correspond with the trends for Pn of EDU-injected seedlings noted earlier (Fig. 1), and show that increased CO₂ exchange is probably related to enhanced stomatal opening. However, it is still not possible from these results to distinguish between the effects of EDU on stomatal behavior and those associated with the wounding phenomenon. Yellow-poplar seedlings receiving no EDU treatment exhibited an unusual stomatal response to O₃ fumigation (Fig. 2). At 0.35 ppm O₃, stomatal conductance declined 55% as might be expected, but at 0.95 ppm O₃ conductance was substantially greater than in the absence of the pollutant. These data may be explained on the basis that at high pollutant concentrations guard cell integrity and photosynthetic capacity are functionally damaged. Thus, even though stomatal conductance increases (Fig. 2), CO₂ assimilation remains depressed (Fig. 1).

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Fig. 2.

Stomatal conductivity of 2-yr-old containerized yellow-poplar seedlings 2 days after fumigation with O₃. Refer to legend for Fig. 1.

The degree of foliar injury for seedlings fumigated with O₃ is recorded in Table 3. As previously reported (16), stem injection of EDU seems to be an effective means of reducing the sensitivity of many woody plants to O₃ damage. In this study, stem injection of EDU reduced foliar injury 69% at 0.35 ppm O₃ and 32% at 0.95 ppm O₃ when compared to seedlings receiving no EDU. Based on the degree of damage, it is reasonable to anticipate that Pn would decrease as foliar injury increases. There was a significant increase in foliar injury for both EDU treatments as the fumigation level increased from 0.35 to 0.95 ppm O₃ (Table 3). Correspondingly, a decrease in Pn was noted for these same treatments (Fig. 1). However, for seedlings without EDU, as foliar injury increased from 14% to 50%, Pn remained unchanged. These results are unexpected and may have resulted from the fact that folilar injury ratings were recorded for entire seedlings, while Pn measurements were made on a single leaf from each seedling. Thus, there may not be the strong correlation between foliar injury and Pn which normally would be expected in a study of this type.

The results of this investigation show that EDU treatment, while affording protection from O₃-induced foliar injury in yellow-poplar seedlings, does not adversely affect Pn. These data would suggest that the chemical could probably be used safely for protecting high value urban trees from air pollution injury without the risk of altering normal physiological activity. This is an important consideration for arborists who may contemplate use of EDU in the urban landscape.