The regulation of Crassulacean acid metabolism (CAM) in the fern Pyrrosia piloselloides (L.) Price was investigated in Singapore on two epiphytic populations acclimated to sun and shade conditions. The shade fronds were less succulent and had a higher chlorophyll content although the chlorophyll a:b ratio was lower and light compensation points and dark-respiration rates were reduced. Dawn-dusk variations in titratable acidity and carbohydrate pools were two to three times greater in fronds acclimated to high photosynthetically active radiation (PAR), although water deficits were also higher than in shade fronds. External and internal CO2 supply to attached fronds of the fern was varied so as to regulate the magnitude of CAM activity. A significant proportion of titratable acidity was derived from the refixation of respiratory CO2 (27% and 35% recycling for sun and shade populations, respectively), as measured directly under CO2-free conditions. Starch was shown to be the storage carbodydrate for CAM in Pyrrosia, with a stoichiometric reduction of "C3-skeleton" units in proportion to malic-acid accumulation. Measurements of photosynthetic O2 evolution under saturating CO2 were used to compare the light responses of sun and shade fronds for each CO2 supply regime, and also following the imposition of a photoinhibitory PAR treatment (1600 μmol·m(-2)·s(-1) for 3 h). Apparent quantum yield declined following the high-PAR treatment for sun- and shade-adapted plants, although for sun fronds CAM activity derived from respiratory CO2 prevented any further reduction in photosynthetic efficiency. Recycling of respiratory CO2 by shade plants could only partly prevent photoinhibitory damage. These observations provide experimental evidence that respiratory CO2 recycling, ubiquitous in CAM plants, may have developed so as to alleviate photoinhibition.

Photosynthetic carbon assimilation in the roots of a shootless orchid Chiloschista usneoides (DON) LDL involves the synthesis and accumulation of malic acid from CO2 in darkness. Malic acid is consumed in the light.The roots do not possess stomata or any means of diurnally regulating the diffusive conductance of the pathway between the internal gas phase ofthe plant and the atmosphere. Regulation of internal CO2 concentration near to atmospheric levels avoids a large net loss of CO2 to the atmosphere during malic acid consumption in the light.The water-absorbing function of the velamen conflicts with the photosynthetic function of the roots. Plants with water-saturated velamina do not acquire CO2 from the atmosphere at night.Photosynthesis in many epiphytic orchids involves the nocturnal acquisition of CO2 and accumulation of malic acid followed the next day by the release of CO2 from malic acid and its utilization in the reductive pentose phosphate pathway. Stomatal resistance is high at night and low during the day. Clearly these plants exhibit CAM (1).Another commonly observed feature of such orchids is the possession ofChl-containing aerial roots and it has recently been shown that these roots, like leaves, exhibit the diurnal fluctuation in acid content and CO2 exchange pattern characteristic ofCAM (6). The aim of the present work is to extend information on the photosynthetic metabolism of orchid roots.The experimental material used in this study is Chiloschista usneoides, a member of the group known as shootless orchids which has been eloquently described by Benzing et al. (2) CO2 Exchange. The CO2 concentration in air which had passed at a rate of 300 ml-' min-' through a glass cuvette maintained at 25°C containing a whole plant was estimated by open-system, differential analysis using a Beckman model 865 IR gas analyzer. Water vapor was removed from the usually saturated gas stream by absorption in silica gel prior to analysis. Calibration of the system utilized standard CO2 mixtures. Plants were illuminated by a 700 w Philips mercury lamp and PAR was measured using a LI-COR quantum meter with a LI-190 SB sensor. Experiments were repeated at least four times.Products of Dark '4CO2 Fixation. Radioactive CO2 (50 ACi in 1 Mmol) generated from NaHCO3 by the addition of lactic acid was injected through a serum bottle stopper into darkened 50-ml flasks containing intact plants. After incubation at 25°C for 14 h, the plants were killed and extracted in boiling ethyl alcohol.The ethanol extract was analyzed by paper chromatography and autoradiography using propan-1 -ol, ammonia, water, and phenol; water as solvents. The identity ofradioactive compounds from two experiments was confirmed by co-chromatography and relative amounts of radioactivity measured by Geiger Muller counting of the chromatograms.Internal CO2 Concentration. Using a microsyringe, I0-js1 samples of gas were removed from the internal atmosphere of plants under the conditions detailed in the results section and analyzed by GC on Porapak...

SUMMARYThe stomata of Arachnis cv. Maggie Oei, Aranda cv. Deborah, Arundina graminifolia, Bromheadia finlay so niana, Cattleya bowringiana X C. forhesii and Spathoglottis plicata (Orchidaceae) occur only on the lower epidermis of the leaves and are located within hyperstomatic chambers formed by cuticular ledges extending from the guard cells. Arachnis, Aranda and Cattleya have thick leaves which exhibit Crassulacean acid metabolism, and their stomata open when acidity levels are lowest, or shortly thereafter. Aranda and Arachnis require higher light intensities for sufficient deacidification to permit stomatal opening than Cattleya. Stomata of the thin-leaved Arundina, Bromheadia and Spathoglottis open during the day. The stomatal rhythms, morphology and distribution, as well as the pathways of carbon fixation and light requirements for deacidification, reflect the natural habitat of each species or the parents of the three hybrids.

The labeling patterns in malic acid from dark (13)CO2 fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12-14 h dark (13)CO2 fixation the ratio of [4-(13)C] to [1-(13)C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The (13)C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87-89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following (13)CO2 fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [(13)C] malic acid ((13)CO2 or [1-(13)C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to (13)CO2 in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284-291). In the shortest times, only singly-labeled [4-(13)C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.

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