Relationships between Photosynthetically Active Radiation,Nocturnal Acid Accumulation, and CO2 Uptake for aCrassulacean Acid Metabolism Plant, Opuntiafcus-indica'
Received for publication June 15, 1982 and in revised form August 17, 1982
PARK S. NOBEL AND TERRY L. HARTSOCKDepartment of Biology and Laboratory of Biomedical and Environmental Sciences, University of Calfornia,Los Angeles, California 90024
ABSTRACT
The influences of photosynthetically active radiation (PAR) and waterstatus on nocturnal Crassulacean acid metabolism (CAM) were quantita-tively examined for a widely cultivated cactus, Opuntia ficus-indica (L.)Miller. When the total daily PAR was maintained at 10 moles photons persquare meter per day but the instantaneous PAR level varied, the rate ofnocturnal H+ accumulation (tissue acidification) became 90% saturatednear 700 micromoles per square meter per second, a PAR level typical forsimilar light saturation of C3 photosynthesis. The total nocturnal H'accumulation and CO2 uptake reached 90% of maximum for a total dailyPAR of about 22 moles per square meter per day. Light compensationoccurred near 0 moles per square meter per day for nocturnal H+ accu-mulation and 4 moles per square meter per day for CO2 uptake. Above atotal daily PAR of 36 moles per square meter per day or for an instanta-neous PAR of 1150 micromoles per square meter per second for more than6 hours, the nocturnal H+ accumulation actually decreased. This inhibition,which occurred at PAR levels just above those occurring in the field, wasaccompanied by a substantial decrease in chlorophyll content over a 1-week period.A minimum ratio of H+ accumulated to CO2 taken up of 2.5 averaged
over the night occurred for a total daily PAR of 31 moles per square meterper day under wet conditions. About 2 to 6 hours into the night under suchconditions, a minimum H+-to-CO2 ratio of 2.0 was observed. Under pro-gressively drier conditions, both nocturnal H+ accumulation and CO2uptake decreased, but the H+-tO-CO2 ratio increased. A ratio of two H+per CO2 is consistent with the H+ production accompanying the conversionof starch to malic acid, and it apparently occurs for 0. ficus-indica whenCAM CO2 uptake is strongly favored over respiratory activity.
Uptake of CO2 by CAM plants occurs primarily during thestomatal opening period at night (3, 8, 16). CO2 is incorporatedinto phosphoenolpyruvate leading to four-carbon organic acids,which are stored in the vacuoles of the chlorenchyma cells. Boththe CO2 uptake and the acid accumulation at night are influencedby the light regime and water status of the plants. For instance,field studies on cacti have shown that the net CO2 uptake or acidaccumulation at night is 909o saturated when the total daytimePAR incident on the stems is 20 to 24 mol m-2 d-' (10-12).Correlations between total PAR and acid accumulation are ex-
pected to be only approximations, inasmuch as the instantaneousPAR level should also affect the decarboxylation and C3 photo-
Supported by Department of Energy Contract DE-AM03-76-SFOOO 12.
71
synthetic processes occurring during the daytime, an aspect ofCAM that has not been fully investigated. Thus, one of theobjectives of the present study was to determine the effects ofvarious PAR levels on nocturnal CO2 uptake and acid accumu-lation under controlled conditions for a widely cultivated cactus,Opuntiaficus-indica.Another unresolved matter is the ratio of acid accumulated to
net CO2 uptake at night for cacti. The literature is -somewhatconfusing because acidity levels are often expressed per unit mass,while CO2 exchange and uptake of radioisotopes are generallyexpressed per unit area; also, the term 'acid' sometimes refers toH+ and sometimes to malic acid, from which two H's can disso-ciate (4, 6-8, 18, 24, 25, 27). For instance, Sale and Neales statethat the acid-to-CO2 ratio was 1.8 for pineapple, Ananas comosus(21). They refer to a previous publication for their method (7),where acidity levels are expressed as equivalents of H+ (one H+per acid), but they indicate that Nobel and Hartsock (14) had- anacid-to-CO2 ratio of 1.0 for Agave deserti, which means two H+per acid, since the latter authors argued that 2 mol of H+ wouldbe produced per mol of CO2 taken up. Szarek and Ting (25)presumed "a direct stoichiometric relationship between organicacid synthesis and CO2 uptake" for certain ecological considera-tions. However, the ratio might be variable in the field. In thepresent study, the influences of both PAR and water status on theH+-to-CO2 ratio were assessed in a quantitative fashion. The ratiowas found to vary with PAR level, time during the night, andplant water status.
MATERIALS AND METHODS
Opuntiaficus-indica (L.) Miller (Cactaceae) was obtained froma commercial plantation at Fillmore, CA (34°24'N, 1 18°53'W, 90m above sea level). Mature cladodes (flattened stems) were placedin loamy sand and maintained in environmental chambers withday/night air temperatures of 25°C/15°C. PAR between 400 and700 nm was provided for 12 h each day by 300-w cool-beamtungsten lamps and was measured with a Lambda Instruments LI-190S quantum sensor placed in the planes of the cladodes. ThePAR on each surface of the cladodes routinely averaged 560 ,umolm-2 s-1 (24 mol m-2 day-'), which is typical of the PAR onunshaded vertical surfaces during most of the year in the field (I 1,12). Other PAR levels were provided by adjusting the distancefrom the cladodes to the lamps. The RH averaged 40%7o during thedaytime and 60%o at night, and the CO2 content of the air averaged340 ,ul I-'. The plants were routinely watered each week with 1/10Hoagland solution no. 1 (2) so that the soil water potential in theroot zone was -0.2 ± 0.1 MPa (-2 ± 1 bars), as determined withWescor PT 51-05 soil thermocouple psychrometers.Net rates of CO2 exchange and water vapor loss were deter-
mined for attached cladodes placed in a modified Siemens null-point compensating closed-circuit flow system (14). Air tempera-tures, RH, PAR, and CO2 levels were the same as in the environ-mental chamber. The water vapor conductance was equated to thetranspiration rate divided by the water vapor concentration dropfrom leaf to air. To measure tissue acidity, two 1.14-cm2 samplesextending from the cladode surface to 1 mm interior to thechlorenchyma (about 4 mm overall) were removed at dawn ordusk with a cork borer, ground with sand in 30 ml distilled H20,and then titrated to an endpoint of pH 6.8 using 0.010 N NaOH(1; all acidity data refer to H+ amounts). Similar samples wereremoved for Chl extraction in 80o acetone/20% water (v/v).Actual cladode surface area was used in the expressions for PAR,CO2 exchange, acidity, and Chl. The transmittance of the epider-mis plus cuticle to PAR was 0.57 ± 0.03 for six samples, while thePAR transmittance through the chlorenchyma was less than 0.01,values that were used to calculate the photon absorption by thechlorenchyma. Tissue osmotic potentials were obtained by placinga small amount of macerated chlorenchyma in a Wescor C-52 leafchamber used in conjunction with a Wescor HR-33T microvolt-meter.
RESULTS
Both nocturnal CO2 uptake and water vapor conductance of 0.ficus-indica were considerably greater when the PAR was 560compared to 90o mol m-2 s-' throughout the daytime (Fig. 1). Atthe higher PAR, substantial stomatal opening (water vapor con-ductance >1 mm s-') and CO2 uptake occurred for most of thenight, while under the lower PAR the stomata never opened aswidely and net CO2 uptake occurred for only about 2 h (Fig. 1).At the lower PAR, the maximum rate of CO2 uptake was only 5%as large and the maximum water vapor conductance was 13% aslarge as for the higher PAR.To quantify the effect of PAR on CO2 uptake, the total CO2
uptake during the night was found by integrating the nighttimeCO2 flux densities for curves such as those in Figure IA. Also, thechange in tissue acidity accompanying the CO2 uptake was deter-mined. Both the total amount of CO2 taken up at night and the
12A
0 8
Cy~ ~ ~~Tm560o (h
NYE
0
o4-
0 ~ ~~90
FIG. 1. CO2 exchange (A) and water vapor conductance (B) of O.flcus-indica. Plants were maintained as in "Materials and Methods," except thatthe PAR at the surface of the cladodes for the 12-h daytimes was either560 y&mol m-2 s- (the usual condition) or 90 ,umol m-2 s- for 1 week(PAR values indicated in figure).
nocturnal H+ accumulation were 90%o light-saturated for a daytimePAR maintained for I week of 21 to 23 mol m-2 d-' (an instan-taneous value ofabout 500 ,umol m-2 s-' for 12 h; Fig. 2). However,the two responses differed considerably at low PAR. Specifically,the noctumal H+ accumulation was more or less directly propor-tional to PAR up to 15 mol m-2 d-', while very little CO2 uptakeoccurred until the total daily PAR was above about 4 mol m-2d-1, at which level the response to additional PAR was approxi-mately linear up to nearly 20 mol m-2 d-' (Fig. 2). Above about36 mol m-2 d-, both the nocturnal acid accumulation and CO2uptake were substantially reduced.To investigate the observed inhibition occurring at high PAR,
plants were exposed to 1,150 ,tmol m-2 s-' for various portions ofthe photoperiod (Fig. 3; 1,150 ,umol m-2 s- for 12 h each daycorresponds to 50 mol m-2 d-'). When the high PAR was presentfor up to 6 h, the nocturnal H+ accumulation increased propor-tionally. However, above about 6 h, the amount of acid accumu-lating actually decreased with increasing PAR. The decrease inacid accumulation was accompanied by a detectable bleaching orloss of Chl (Fig. 3).
Next, the effects on H+ accumulation were examined for variousinstantaneous levels of PAR but a constant and moderate totaldaily PAR (10.0 mol m-2 d-l). A background PAR of 40 ,tmolm- s-1 was maintained for 12 h each day to keep the photoperiodthe same. As the instantaneous level increased under these con-ditions, the total nocturnal H+ accumulation decreased (Fig. 4A),indicating that the daytime decarboxylation and/or C3 photosyn-thesis was becoming light-saturated. This is presented in a moreconventional way in Figure 4B. Both when the background PARof 40 ,umol m-2 s-' was ignored (upper curve) or when it wasassumed to produce a maximal effect (66 mmol H+ m-2; lowercurve), 90%o saturation of the rate of nocturnal H+ accumulationoccurred for an instantaneous PAR of about 700 ,umol m-2 s-'.Thus, acid accumulation at night could be used to assess thesaturation characteristics of the daytime photosynthetic behavior.
Instantoneous PAR ( mol m-2 s-')
E
E
a}00.
N0
0
N
E
EE0
0
E00
+I
0 10 20 30 40 50
Total daily PAR (mol rnX2 day-1)FIG. 2. Total nocturnal CO2 uptake or acid accumulation at various
levels of daytime PAR. Plants were maintained for I week under aparticular PAR, gas exchange was determined under that condition, andthen to insure uniform starting material the plants were returned for 2weeks under the usual PAR (560 ymol m-2 s-1 for 12 h each day, whichis 24 mol m-2 d-'). Samples for acid measurements were taken at thebeginning and end of the dark period from the same cladode used for gasexchange.
PAR, ACIDIFICATION, AND CO2 UPTAKE FOR A CAM PLANT
N
600-6 60 NyE
EE
400 - \ 4000
0~~~~~~~~~~~E h
0~~~~~~~~~~~(,200 - '00 :E
I
0 l0 2 4 6 8 10 12
Time at high PAR (h)
FIG. 3. Effect of the daytime period at a PAR of 1,150 ,umol m-2 S-1 onnocturnal acid accumulation. A background PAR of 40 ttmol m-2 s-' was
provided for 12 h each day to keep the photoperiod constant. The highPAR was presented for I week and was symmetrically distributed about12:00, e.g. 4 h at high PAR means 1, 150 ,umol m-2 s-' from 10:00 to 14:00.
N
CY
o
E
E -4
C._
CX
+
I
0
o
-9-a 2
E
u I
c
+ 0
-0-
0
400
500o
'00
100
o
40 - uncorrected
30 _ ,4 ^-~~<_
//corrected20 - ^g v"
10a I I
0 400 800
Instantaneous PAR ( . mol
FIG. 4. Influence of instantaneous PAR on nocttion (panel A) under a constant total daily PAR ofbackground PAR of 40 ,umol m-2 s-' was providewhich contributed 1.7 mol m-2 d-' to the total dailyinstantaneous PAR, which was supplied each day ftto the background PAR, contributed 8.3 mol m-2 4
PAR; it was presented for the appropriate time Idistributed about 12:00, e.g. the level of 576 ymol nfrom 10:00 to 14:00. In panel B, the data are expressaccumulation; this was calculated as the total acid acothe time at the indicated PAR ('uncorrected') ar
accumulated minus the maximum acid accumulatiorground PAR (66 mmol H+ m-2 for a PAR of 1.7divided by the time at the indicated PAR ('corrected
The relationship between nocturnal H+ accumulation and CO2uptake was also examined for stems subjected to various periodsof drought. As the drought period increased, the nocturnal sto-matal opening became less, and also there was less net CO2 uptake(Table I). Interestingly, nocturnal acid accumulation was reducedproportionally less than the net CO2 uptake. Hence, the ratio ofH+ accumulated to CO2 taken up progressively increased as thedrought period became longer, e.g. it went from 2.6 under wetconditions to 12.3 for severe drought (Table I).
Next, an attempt was made to identify the lowest value of theratio of nocturnal H+ accumulation to net CO2 uptake to helpunderstand the stoichiometry between these two quantities. Aspreviously indicated, this ratio was lower under wetter conditions(Table I). Also, the ratio became smaller as the daytime PAR wasraised to about 31 mol m-2 d-', where it was 2.5 (Fig. 2). To seewhether the ratio varied during the course of a night, aciditymeasurements were made every 2 h for a cladode whose gas
exchange was continuously monitored (Fig. 5). The rates of bothnet CO2 uptake and acid accumulation were lower at the beginningand especially at the end of the dark period. From 2 to 6 h intothe dark period, when stomata were open maximally (cf. Fig. 1),the H+-to-CO2 ratio was the lowest (Fig. 5). Since the ordinate onthe right in Figure 5 is numerically 2-fold greater than the ordinateon the left, the observed parallelism of the lines during this periodcorresponds to a ratio of 2.0. Thus, the minimum H+ accumulated/CO2 taken up was about 2.0.
DISCUSSION
The present studies with 0. ficus-indica indicate that the noc-turnal CO2 uptake and acid accumulation as well as their ratio areinfluenced by the daytime PAR. At a moderate total daily PAR(10 mol m-2 d'), both processes are 90%o light-saturated for aninstantaneous PAR of about 700 ,umol m-2 s-' (Fig. 2). Such asaturation level, which is 35% of full sunlight, commonly occursfor C3 photosynthesis, which prevails during the daytime for cacti(3). However, the PAR level on a cladode constantly varies in thefield due to the sun's trajectory. For randomly oriented unshadedvertical surfaces at 340 north latitude, the PAR annually exceeds700 ,umol m-2 s-' for about 25% of the daytime (11). Sinceinstantaneous PAR levels above about 700 ,Lmol m2 s' are not
efficiently used, 90%1o light saturation should require a somewhat0 higher total daily PAR in the field than for a square wave pattern
in the laboratory. This was indeed observed, since the PARleading to 90%o saturation of nocturnal acid accumulation was 24mol m-2 d-' for normal diurnal variation in the field (13) and 21mol m-2 d-' for the square-wave pattern used in the laboratory(Fig. 2) for 0. ficus-indica. Although useful as a field index ofCAM activity (11, 12), the total incident daytime PAR is only an
approximate indicator of nighttime metabolism (Fig. 4A).Below a PAR of 4 mol m-2 d-1, essentially no net nocturnal
1200 1600 CO2 uptake occurred for O. ficus-indica (Fig. 2). This is consistentwith field studies on Ferocactus acanthodes, where net nighttime
m~2 Sg1 ) CO2 uptake did not become positive until the total daily PAR alsournal acid accumula- exceeded 4 mol m-2 d-' (10). Such PAR responses have important10.0 mol m-2 d-'. A ecological implications for cacti. For instance, light may be limit-
d for 12 h each day, ing for Carnegiea gigantea on certain north-facing slopes (9), justf PAR. The indicated as it is for Agave deserti (29?. Also, since the total daily PARor I week in addition averages about 20 mol m-2 d- over the year for vertical unshadedd-l to the total daily surfaces on clear days at various latitudes (11, 12), 4 mol m-2 d-'period symmetrically would correspond to an 80%1o reduction in PAR. Approximatelyn-2 s-i was presented the same PAR reduction due to shading prevailed when 0. echios;ed as the rate of acid no longer occurred along a transect in the Galapagos Islandscumulated divided by where the height of the surrounding vegetation varied (12), sug-nd as the total acid gesting that this species was then below light compensation. Duea caused by the back- to light compensation near a PAR of 4 mol m-2 d'1 for net CO2mol m-2 d-', Fig. 2) uptake at night but at 0 mol m-2 d-' for nocturnal H+ accumula-i'). tion (Fig. 2), a given increase in PAR in the range of 4 to 20 mol
Table 1. Drought Effects on Nocturnal CO2 Uptake and Acid AccumulationDiurnal patterns of gas exchange and tissue acidity levels were determined at various times after watering was
ceased. The PAR was 560 LMol m-2 s-' for 12 h each day (24 mol m-2 d-').
Time Tissue Maximum Net Nocturnalsince Osmotic Nocturnal NoctualH+ H Accumulated
Watering Potential Water Vapor C2Utk cuuain C2TknUConductance Uptak Accumulation CO2 Taken Up
Time in dark (h)FIG. 5. Changes in tissue acid level and net CO2 uptake during the
course of a night. A well-watered plant was maintained for I week at an
approximately saturating PAR of 720 ,umol m 2 s-' for the usual 12-hdaytime period (31 mol m-2 d-'). CO2 uptake was measured continuously(cf. Fig. 1) and tissue acid level was determined at 2-h intervals throughoutthe night.
m-2 d-I can have a proportionally much greater effect on CO2uptake than on acid accumulation.
0. ficus-indica studied here in the laboratory had the greatestnocturnal H' accumulation so far reported for cacti, 860 mmolm-2. The highest previous values were for field studies, viz. 760mmol m-2 for 0. chlorotica (11), 700 mmol m-2 for 0. inermis(18), and 630 to 670 mmol m2 for 0. ficus-indica (13, 22). Themaximum nocturnal CO2 uptake for O.ficus-indica was 340 mmolm2 (Fig. 2), which corresponds to 7.9 ,umol m2 s-' averaged overthe whole night-instantaneous values for net CO2 uptake wereas high as 12.2 itmol m2 s-'. By way of companson, the highestreported nocturnal values are 3.9 t,mol m-2 s-' for pineapple (21;when expressed per actual total surface area, as is done for 0.
ficus-indica, the pineapple value is only 2.0 ,umol m 2 s-I averagedover both leaf surfaces) and 1.8 smol m-2 S-' for Agave americana(15; expressed per actual total surface area).When 0. ficus-indica was subjected to progressively greater
periods of drought, the degree of nocturnal stomatal openingcontinually declined, nocturnal CO2 uptake became less, and sodid nocturnal acid accumulation. However, the ratio of H+ accu-mulated to net CO2 uptake steadily increased (Table I). Similarly,the ratio for 0. inermis in the field was 2.4 under wet conditionsand 4.2 under dry conditions (18). Comparable results were ob-tained for 0. basilaris, where some diurnal changes in acidityoccurred even when the stomates remained closed in response towater stress, thus preventing net CO2 uptake (24, 25). Further, a
plant of 0. bigelovii subjected to drought for 3 years had asubstantial diurnal fluctuation in tissue acidity, although no sto-matal opening (26). Thus, although convenient to measure, diurnalchanges in tissue acidity do not stoichiometrically indicate the netCO2 uptake by a CAM plant.The slope of the PAR response curves (Fig. 2) can indicate the
photon requirement for the nocturnal CAM processes. For thesteepest part of the curve, 45 incident photons were required perCO2 taken up. Since the transmittance of the epidermis and cuticlefor PAR was 0.57 and the transmittance through the chlorenchymawas 0.01, 25 photons absorbed by the chlorenchyma were requiredper CO2 taken up at night. This represents a higher photonrequirement than found for agricultural plants, where the valuesusually range from 11 to 22 for daytime CO2 uptake (20, 23). Thereported photon requirement is also high for other CAM plants,e.g. 46 absorbed photons are required per CO2 fixed at night forA. deserti (14) and 68 for F. acanthodes in the field (10). Using aratio of 2H+ accumulated at night per CO2 taken up, a 56%absorbance of PAR by the chlorenchyma, and the initial slope ofthe PAR curves, the quantum requirement is 22 absorbed photonsper CO2 fixed at night for the cacti 0. chlorotica ( 1) and Tricho-cereus chilensis (12), which is similar to the higher quantumrequirements for agricultural plants (20, 23). Moreover, for theCAM plant Sedumpraealtum, the quantum requirement measuredduring the daytime was 16 absorbed photons per CO2 at 2 to 4 hinto the photoperiod when the stomata were closed, although thevalue rose toward the end of the daytime (23).The decrease in nocturnal CO2 uptake and acid accumulation
at the higher PAR levels may represent a form of photoinhibition,where the light-harvesting reactions and hence CO2 assimilationability of the chloroplasts are inhibited ( 17, 19). Also, the decreasein Chl per unit cladode area at the higher total daily PAR (cf. Fig.3) suggests that photooxidation has occurred. Even though the 0.ficus-indica used here had been maintained in the laboratory at560 ,umol m-2 s-1 for 12-h days (a total PAR of 24 mol m-2 d-l)for 4 to 6 months before use, it is of interest to extrapolate thepresent results to the field situation. Specifically, the total dailyPAR in the field for vertical cladodes could reach but would notexceed 38 mol m-2 d-' for various latitudes and seasons (11-13).Also, an instantaneous rate of 1,150 ,umol m-2 s-1 would not beexceeded for 6 h. Inhibition for 0. ficus-indica did not set in untilthese two values were exceeded (Figs. 2 and 3), suggesting thatsuch damage would generally not occur for cacti in the field.However, incident PAR can approach these limits in the field,suggesting that photoinhibition and/or photooxidation might oc-cur if other circumstances made the cacti more sensitive to suchreactions.Although the ratio of H' accumulated at night to CO2 taken up
varies considerably, it appears to have a minimum value near 2.0(Fig. 5). Conditions that restrict stomatal opening, such as drought(Table I), lower daytime PAR (Fig. 1), or even the beginning aswell as the end of the dark period (Figs. 1 and 5), lead to a higherratio for 0. ficus-indica. Likewise for Spanish moss, Tillandsiausneoides, the minimum value of 2.0 occurred following clear
PAR, ACIDIFICATION, AND CO2 UPTAKE FOR A CAM PLANT
days, while a value of 4.3 occurred after cloudy days (5). Underconditions that decrease stomatal opening, respiratory release ofCO2 may be responsible for a higher fraction of the CO2 incor-porated into organic acids, which in turn would lead to a higherratio of H+ accumulated to CO2 taken up. When respiration canbe ignored, then the overall net reaction for carbon involves starchor another glucan being converted to an organic acid such asmalic acid, e.g.
1/2 starch monomer + CO2 + ½ H20 --*malic acid
Malic acid has a pKi of 3.4 and a pK2 of 5.1 at 25°C (28), and soa titration to pH 6.8 would require essentially two OH per CO2.Hence, under conditions where respiration is low compared toCAM uptake of C02, such as 2 to 6 h into the dark period fornondroughted cacti exposed to fairly high daytime PAR levels,the H+ accumulated per CO2 taken up is predicted to be two. Thisis in good agreement with the lowest ratio of nocturnal H+accumulated to CO2 taken up found for 0. ficus-indica, 2.0.
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