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Planto 9 Springer-Verlag 1993

Environmental effects on circadian rhythms in photosynthesis and stomatal opening* Timothy L. Hennessey 1'2, Arthur L. Freeden 1, and Christopher B. Field 1'** i Department of Plant Biology,Carnegie Institution of Washington, Stanford, CA 94305, USA 2 Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA Received 18 May; accepted 4 August 1992

Abstract. Persistent circadian rhythms in photosynthesis and stomatal opening occurred in bean (Phaseolus vul9aris L.) plants transferred from a natural photoperiod to a variety of constant conditions. Photosynthesis, measured as carbon assimilation, and stomatal opening, as conductance to water vapor, oscillated with a freerunning period close to 24 h under constant moderate light, as well as under light-limiting and CO2-1imiting conditions. The rhythms damped under constant conditions conducive to high photosynthetic rates, as did rates of carbon assimilation and stomatal conductance, and this damping correlated with the accumulation of carbohydrate. No rhythm in respiration occurred in plants transferred to constant darkness, and the rhythm in stomatal opening damped rapidly in constant darkness. Damping of rhythms also occurred in leaflets exposed to constant light and CO2-free air, demonstrating that active photosynthesis and not simply light was necessary for sustained expression of these rhythms. Key words: Circadian rhythm - Phaseolus- Photosynthesis - Stomate

Introduction Circadian rhythms in photosynthesis and stomatal opening occur in a broad variety of vascular plants (ChiaLooi and Cumming 1972; Pallas et al. 1974; Deitzer and Frosch 1990). In bean plants (Phaseolus vulgaris L.) these rhythms are closely coupled and have a period of approx. 24.5 h under constant conditions (Hennessey and Field * This is CIWDPB Publication No. 1142 ** To whom correspondence should be addressed; FAX: 1 (415) 325 6857 Abbreviations: Ci=intercellular CO2 partial pressure (Pa); PFD=photon flux density; Rubisco=ribulose-l,5-bisphosphate carboxylase-oxygenase; RuBP= ribulose-1,5-bisphosphate

1991, 1992). The rhythm in photosynthesis, although synchronized with the rhythm in stomatal conductance, is not entirely the result of stomatal effects on CO2 diffusion because a rhythm in photosynthesis occurs even when the intercellular CO/level is held constant (Hennessey and Field 1991). The basis of the non-stomatal component in the photosynthetic rhythm of vascular plants is unclear. Processes that might individually or collectively contribute to a photosynthetic rhythm occur at many levels and feedback between these processes can occur. In algae, circadian regulation of PSII activity indicates that the rhythm in photosynthesis is correlated with rhythms in light harvesting (Samuelsson et al. 1983). In vascular plants, rhythms in whole-chain electron flow (Lonergan 1981), and the level of ribulose- 1,5-bisphosphate (RuBP) (Freeden et al. 1991) are also consistent with rhythms in light-harvesting efficiency. No rhythms have been substantiated in the activity of ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco), the central enzyme in the dark reactions, although a rhythm has been reported in the transcription of the small subunit of this enzyme's gene (Spiller et al. 1987; Giuliano et al. 1988). In addition to the biochemical approaches discussed above, control of the rhythm in photosynthesis can be analyzed in intact leaves using gas-exchange techniques. An advantage of this approach is that repeated measurements can be made on a plant in vivo under a variety of conditions. One objective of this study was to use gasexchange techniques to examine whether rhythms in photosynthesis occurred under both light-limiting and CO2-1imiting conditions. A second objective of this study was to establish the range of conditions under which rhythms in stomatal conductance and carbon assimilation occur. Under constant conditions with moderate light intensities and CO2 levels comparable to those in the growth environment, the amplitude of the rhythms damps slowly with little change in the average rates of photosynthesis and stomatal conductance (Hennessey and Field 1991). Under other conditions, however, expression of the rhythms

370 m a y be a l t e r e d b y f e e d b a c k effects f r o m the a c c u m u l a t i o n o r d e p l e t i o n o f c a r b o h y d r a t e . A l s o , r h y t h m s in respiration have been r e p o r t e d in v a s c u l a r p l a n t s ( C h i a - L o o i a n d C u m m i n g 1972; Pallas et al. 1974) a n d m a y c o n t r i b ute to the overall r h y t h m in c a r b o n a s s i m i l a t i o n . R e l a t e d to the issue o f c a r b o n e x c h a n g e u n d e r r e s p i r a t o r y cond i t i o n s is the e x p r e s s i o n o f s t o m a t a l r h y t h m s in either d a r k n e s s o r in light with CO2-free air. M o s t studies o f s t o m a t a l r h y t h m s r e p o r t r a p i d d a m p i n g in c o n s t a n t d a r k n e s s ( M a r t i n a n d M e i d n e r 1972; H e a t h 1984), p o s sibly d u e to d e p l e t i o n o f c a r b o h y d r a t e reserves. T h e effects o f d a r k n e s s can be s e p a r a t e d f r o m the effects o f c a r b o h y d r a t e d e p l e t i o n b y e x a m i n i n g r h y t h m s in cons t a n t light in a CO2-free e n v i r o n m e n t .

Material and methods Bean (Phaseolus vulyaris L., cv. Blue Lake Bush 274) plants were grown individually in 1-1 pots containing a 1:1 (v/v) mixture of vermiculite and perlite. Plants were grown in either a growth chamber at 500gmol.m - 2 - s -~ photon flux density (PFD) (12h light:12 h dark cycle) under combined fluorescent-incandescent lighting or in a greenhouse under natural light with a photoperiod of approx. 12 h and a maximum PFD of approx. 1600 lamol " m - 2 . s 1. Temperatures in the growth chambers were held constant at 28 ~ C; in the greenhouse, temperatures were maintained at 28~ (day) and 20~ (night). Plants were frequently irrigated with a complete nutrient solution (Hennessey and Field 1991). Constant-light experiments were initiated at the beginning of the photoperiod after a normal dark cycle, with hour 0 corresponding to dawn. Time periods corresponding to the entraining light and dark periods are referred to as subjective day and subjective night, respectively. The gas-exchange system used in this study and its components have been described elsewhere (Field et al. 1982, 1989; Hennessey 1991). The gas-exchange chamber was approx. 300 cm 3 in volume and the air flow was approx. 2 1 9rain- t. Environmental conditions within the gas-exchange chamber, such as temperature, relative humidity, CO2 level and PFD could all be simultaneously controlled and held constant. Computerized control of the system made it possible to control the intercellular CO2 partial pressure (C~) by adjusting the ambient CO2 partial pressure. Photosynthesis and respiration were determined from the net exchange of carbon dioxide, and stomatal opening was measured as conductance to water vapor. In experiments with CO2-free air, the only source of CO 2 was respiration by the leaflet in the chamber. Physiological and environmental variables were monitored every minute and variables under computer control were adjusted as frequently as every minute to achieve targeted values. Data in the figures correspond to individual readings recorded to computer disk every 12 min. The remainder of the plant was exposed to a similar PFD as the assay leaflet and to atmospheric partial pressures of CO2 (approx. 35 Pa). The center leaflet of the uppermost fully expanded trifoliate was selected for gas exchange and the entire plant was illuminated at approximately the same PFD as the assay leaflet. In all gasexchange experiments, leaflet temperatures were maintained at 28 ~ C and the vapor-pressure difference between the leaflet and air was maintained at 1 kPa. The light source during gas-exchange experiments was a 1000-W high-intensity multi-vapor lamp attenuated with neutral filters. Carbohydrate analysis was carried out according to Fader and Kolter (1984) after extraction of 3.83 cm 2 of leaf material (middle leaflet of the second trifoliate, 8-9 d after emergence) in 2 cm 3 of 80% ethanol in water. The extract was centrifuged for 10 min at 10000.g and the supernatant removed for sucrose and glucose analysis. The pellet was dried under a stream of N2 gas, 3 cm 3 of H20 was added, and all samples were capped and autoclaved for

T.L. Hennessey et al. : Circadian rhythms in photosynthesis I h at 105 ~ C. Samples were cooled and 1.0 cm 3 of 300 mol - m -3 acetate buffer (pH 4.5) containing 5 units of amyloglucosidase (Sigma A 7420; Sigma Chemical Co., St. Louis, Mo., USA) was added for a 24-h incubation on a rotating platform at 45 ~ C. Tubes were centrifuged for 5 min at 10000 9g and glucose was analyzed in the supernatant by a glucose-oxidase procedure (Sigma diagnostic kit No. 510A).

Results P h o t o s y n t h e s i s a n d s t o m a t a l c o n d u c t a n c e oscillated with a p e r i o d close to 24 h in Phaseolus vulgaris p l a n t s transferred f r o m a n a t u r a l p h o t o p e r i o d to c o n s t a n t m o d e r a t e i l l u m i n a t i o n (Fig. 1). T h e i n t e r c e l l u l a r CO2 level in this e x p e r i m e n t , 28 Pa, was c o m p a r a b l e to t h a t e x p e r i e n c e d b y P. vulgaris u n d e r the s a m e i r r a d i a n c e a t n o r m a l CO2 levels ( H e n n e s s e y a n d Field 1991). A s we c o n c l u d e d earlier ( H e n n e s s e y a n d F i e l d 1991), the e x p r e s s i o n o f a r h y t h m in p h o t o s y n t h e s i s at c o n s t a n t C~ d e m o n s t r a t e d t h a t this r h y t h m was n o t entirely the result o f s t o m a t a l effects o n the diffusion o f CO2. A v a r i e t y o f processes, a c t i n g i n d e p e n d e n t l y o r collectively, could c o n t r i b u t e to the n o n - s t o m a t a l c o m p o n e n t o f the r h y t h m in c a r b o n assimilation. T o investigate the role different processes p l a y e d in the p h o t o s y n t h e t i c r h y t h m , we e x p o s e d leaflets to a variety o f limiting conditions. U n d e r c o n d i t i o n s o f low light a n d high CO2, for e x a m p l e , p h o t o s y n t h e s i s s h o u l d be limited b y the availability o f energy f r o m light to r e g e n e r a t e R u B P . C o n versely, u n d e r c o n d i t i o n s o f high light a n d low CO2, the rate o f p h o t o s y n t h e s i s s h o u l d be limited p r i m a r i l y by the rate o f the d a r k r e a c t i o n s t h a t c o n s u m e R u B P a n d r e d u c e carbon. U n d e r c o n d i t i o n s o f low light a n d high CO2, a r h y t h m in p h o t o s y n t h e s i s was still expressed a n d this r h y t h m r e m a i n e d s y n c h r o n i z e d with the r h y t h m in s t o m a t a l cond u c t a n c e (Fig. 2). T h e rate o f p h o t o s y n t h e s i s was l o w e r in Fig. 2 t h a n in Fig. 1, reflecting l i m i t a t i o n o f p h o t o s y n thesis b y low light, b u t the e x p r e s s i o n o f the c i r c a d i a n r h y t h m was u n c h a n g e d . W h e n p l a n t s were e x p o s e d to high light a n d low CO2 levels, c o n d i t i o n s u n d e r which the d a r k r e a c t i o n s s h o u l d limit p h o t o s y n t h e s i s , a r h y t h m in c a r b o n a s s i m i l a t i o n also o c c u r r e d (Fig. 3). R h y t h m s in p h o t o s y n t h e s i s a n d s t o m a t a l c o n d u c t a n c e r e m a i n e d sync h r o n i z e d , a l t h o u g h the p e r i o d s u n d e r high i r r a d i a n c e (Fig. 3) a p p e a r e d slightly l o n g e r t h a n those u n d e r l o w e r i r r a d i a n c e s (Figs. 1, 2). A related objective o f this s t u d y was to d e t e r m i n e the r a n g e o f c o n d i t i o n s u n d e r which r h y t h m s in p h o t o s y n thesis a n d s t o m a t a l c o n d u c t a n c e occurred. F i g u r e s 1-3 i n d i c a t e d that, u n d e r a v a r i e t y o f light a n d CO2 conditions, these processes oscillated on a c i r c a d i a n basis. In each o f these cases, however, the r a t e o f p h o t o s y n t h e s i s was limited by either the a v a i l a b i l i t y o f light (Fig. 2), CO2 (Fig. 3), o r p o s s i b l y b o t h (Fig. 1). In the f o l l o w i n g experiments, we e x a m i n e d expression o f p h o t o s y n t h e t i c a n d s t o m a t a l r h y t h m s u n d e r c o n d i t i o n s t h a t p r o d u c e d either high o r negative rates o f c a r b o n a s s i m i l a t i o n . W h e n Ca was held at 3 5 P a a n d light was increased to 500 g m o l . m 2. s - 1 P F D , the rate o f c a r b o n assimilation was initially m u c h higher (Fig. 4) t h a n t h a t o b s e r v e d

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