EFFECTS OF OZONE AND SULFUR DIOXIDE ON FOUR EPIPHYTIC BROMELIADS

kjfects of and sulfur on four epiphytic bromeliads. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 32, 25-32, 1992. Plants of Tillandsia balbisiana, T. paucifolia, T. recurvata and T. utriculata exposed to 0.15, 0.30 or 0.45 ppm 0 3 or to 0.30, 0.60 or 1.20 ppm S0 2 for 6 hr or sequentially to 0.30 ppm 0 3 (2 hr), 0.30 ppm 0 3 plus 0.60 ppm S0 2 (2 hr) and 0.60 ppm S0 2 (2 hr) did not exhibit visible injury. Fumigations also had no effect on foliar conductance or on ~H+ associated with Crassulacean acid metabolism. Characteristics responsible for plant tolerance to short exposures to these two gases probably included low stomata! conductance, the insulating indumentum of absorbing foliar hairs and inherently slow metabolism. Reasons for postulating that Tillandsia is a better indicator for technological metals and certain other pollutants than for brief exposures to 0 1 and S0 2 are discussed. We also conclude that epiphytic Tillandsia spp. offer advantages over lichens for air quality assessments under appropriate conditions. As vascular plants they exhibit sensitive, easily measured responses to stress and can be transplanted with ease to areas where neither they nor lichens occur naturally.


INTRODUCTION
exposures for injury and physiological effects from exposures to 0 3 and S0 2 • More broadly, we THE responses of two orchids with Crassulacean wished to determine whether epiphytic broacid metabolism (CAM) to brief exposures to 0 3 meliads could replace lichens for air quality and S0 2 were described in an earlier report. 191 assessment in Florida National Parks located in Four bromeliads from the same south Florida areas undergoing rapid urban and industrial forests received identical treatments. Although all development. Certain tropical regions, including six subjects grow on trees under similar conditions parts of extreme south Florida, support few suitboth bromeliads and orchids were tested because able lichens, but epiphytic bromeliads are abunthey absorb moisture and nutrients through foli-dant or can be transplanted to desired monitoring age or aerial roots, respectively, and may there-sites. Moreover, vascular plants provide sensitive fore differ in vulnerability to air contaminants. and easily quantified indicators of stress (e.g. Our immediate objective, as in the experiments stomata! conductance) that have no counterparts with tht> orchids. 19 ' was to t>stablish threshold in lower plants.

MATERIAL AND METHODS
Tillandsia balbisiana, T. paucifolia, T. recurvata and_ T. utriculata were collected in the Everglades Nat10nal Park and adjacent swamp forests injanuary 1985. Plants were maintained in a charcoalair-filtered greenhouse at 30-35°C day and about 25°C night temperatures, 70-75% relative ~mmidity, and natural illumination prior to, durmg and after exposures. These temperatures approximate those that prevail in south Florida excep~ when masses of colder air occasionally move mto the area during winter months.
Exposures to 0 3 and S0 2 were conducted at the University of California, Riverside in continuously stirred tank reactor (CTSR) exposure chambers 1.3 m in diameter and 1.36 m high covered with mil FEP Teflon film according to the procedures described in NYMAN et al.( 91 Three plants of each species were placed in a single chamber and exposed to 0.15, 0.3 or 0.45 ppm 03 for 6 hr or to 0.3, 0.6 or 1.2 ppm S0 2 for 6 h~ from 2100 to 0300 hr, an interval that began while sto~atal conductance was relatively high and contmued through a period of rapid acidification (Figs 1, 2). These exposures were repeated three times on different dates. Five additional plants of each species were subjected to sequential exposure to 0 3 and S0 2 • These subjects were first treated with 0 3 alone at 0.30 ppm for 2 hr, then to 0 3 at 0.30 ppm plus 0.6 ppm S0 2 for 2 hr, and finally to 0.60 ppm S0 2 for 2 hr. The exposures were replicated four times in four different chambers on the same date. Gas mixtures were selected to represent the midpoints of 03 and S0 2 concentrations applied when these agents were used alone. Stomata! conductance was measured with a LI-COR Steady-State Porometer (Model LI-1600, Licor, Inc., Lincoln, NB) equipped with a narrow (I cm -2 ) aperture. Two weeks before the experiments abaxial leaf surfaces of five T. utriculat.a plants were monitored consecutively at 3hr mtervals for 48 hr to determine conductance rhythms. Conductance was also recorded I hr prior to, at midpoint, immediately after and 12 hr ~ubsequent to each 6-hr fumigation period. Fohage of the other three species was too narrow to fit the porometer cuvette.
Tissue assayed for acid content was collected with a razor blade or cork borer at 3-hr intervals and frozen with dry ice. Samples were ground, extracted with boiling water and titrated to pH 7.5 with 0.01 N NaOH.
Immediately following exposure, 12, 24, 28 hr, 2, 12 weeks and 18 months later unsacrificed plant~ that had been returned to the greenhouse were mspected for visible injury. Foliar surfaces w:re also examined by scanning electron microscopy.

RESULTS
In the greenhouse all four bromeliads exhibited fluc_tuations in titratable acidity (~H+) that vaned somewhat in timing and intensity ( Fig.  2A,B), b~t were characteristic of CAM-type plants. Tzllandsia balbisiana, T. paucifolia and T. recurvata foliage began to acidify at about 2200 hr. Tillandsia utriculata reached this stage somewhat later. Acid accumulated by Tillandsia recurvata was utilized (decarboxylated) several hours before the other species exhausted reserves. Maximum acidity (H~.,) ranged between about 25 and 35 µeq/GFW among the four taxa.
A minimum number of plants were sacrificed for the acid determinations in order to preserve enough plants for the long term observations. Nevertheless, there was no evidence that any of the three concentrations of 0 3 influenced the   intensity of CAM in any of the tested bromeliads (Fig. 3). No dosage effect was apparent, i.e. acidification generally varied without regard to the severity of the treatment. Moreover, tt+ in the leaves of T. recurvata controls at 0700 hr was about three times that exhibited in the greenhouse. Acidification also occurred without discernible trends during exposures to S0 2 (Fig. 3). Treatment with 1.2 ppm S0 2 seemed to enhance acid accumulation by T. utriculata, but how much, if any, of this increase represented S0 2 dissolved in cell sap in this most severe of the treatments is not known. Equally inconsistent in their relationships to control values were the magnitudes of acid stores in plants exposed to combined 0 3 and S0 2 (Fig. 3). In this instance, tt+ in T. recurvata was well below that measured in controls. An explanation for at least part of the variability recorded in these runs is provided in the discussion.
Stomata! conductance in T. utriculata was greatest in the evening and early morning ( No injuries were apparent immediately after the fumigations nor did any necrosis or abnormal growth responses develop or flower abortion occur during the subsequent 18 months.

DISCUSSION
Tillandsia spp., particularly the xerophytic taxa including those tested in this study, possess characteristics that could increase sensitivity to air quality above that of most vascular plants. ( (Figs 4, 5). Tightly overlapping leafbases of T. utriculata form tanks in which falling debris and precipitation, ., ~ "' ., .,. the major sources of nutrient ions for this species, accumulate. The other three so-called atmospheric bromeliads lack shoot impoundments and obtain nutrients from rain and canopy runoff as it washes over foliage. l 5 l Critical to our decision to evaluate these four taxa is their dependence on aerial rather than terrestrial sources of nutrients. Two aspects of CAM probably influenced the responses of our subjects and those of the two orchids described earlierl 9 l to the tested gases. Crassulacean acid metabolism is part of the complex mechanism that allows these bromeliads and many other epiphytic and terrestrial xerophytes to achieve exceptional water use efficiency and to avoid severe desiccation during prolonged drought.11 41 This mechanism incorporates generally low stomata! conductances that peak at night and perhaps also accounted in our treatments for the absence of leaf damage from exposures to concentrations of 0 3 and S0 2 that injure some other plants. Further insulating the leafinterior were the thick-walled, dead cells comprising each of the trichome caps that form a continuous layer over the stomata (Figs 4, 5). In addition to these two physical features that slowed penetration of the fumigants, plant vulnerability may have been reduced by the inherently sluggish growth and metabolism associated with CAM.
Variations in flH+ that bore little if any relationship to the type or concentration of the fomigants used in these experiments are inherent to CAM plants. l 14 l Acid rhythms change in intensity depending on previous and immediate growing conditions (e.g. day and night temperature,   Two plant characteristics probably account for the sensitivity of bromeliads to some air pollutants: ( 1) dependence on ions drawn directly from the atmosphere rather than the ground, and (2) the relatively non-specific nature of the absorption mechanisms located in foliar trichomes. Effective nutrient scavenging is crucial to the epiphyte to maintain adequate nutrition in tree crown habitats, but these same mechanisms promote abnormal and even toxic accumulations ofrequired (e.g. Cu) and other substances if normal supplies are supplemented. l 5 l As a result, among vascular plants, xerophytic Tillandsia is an extraordinary indicator of contamination by technological metals and a variety of other agents that enter leaves via foliar trichomes. However, these epiphytes do not appear to be as sensitive as some other vegetation (e.g. certain crops) to short exposures to several gases that enter through stomata.