Photosynthesis in Pineapple (Ananas comosus comosus [L.] Merr ...

Tropical Plant Biol. DOI 10.1007/s12042-010-9057-y

Photosynthesis in Pineapple (Ananas comosus comosus [L.] Merr) Measured Using PAM (Pulse Amplitude Modulation) Fluorometry Raymond James Ritchie & Sakshin Bunthawin

Received: 10 June 2010 / Accepted: 16 September 2010 # Springer Science+Business Media, LLC 2010

Abstract PAM (Pulse Amplitude Modulation) fluorometer techniques directly measure the light reactions of photosynthesis that are otherwise difficult to estimate in CAM (Crassulacean Acid metabolism) plants such as pineapple (Ananas comosus comosus cv. Phuket). PAM machines calculate photosynthesis as the Electron Transport Rate (ETR) through PSII (4 electrons per O2 produced) as mol m−2 s−1. P vs. E curves fitted the waiting-in-line function (an equation of the form ETR ¼ ðETRmax E=Eopt Þ:e1E=Eopt ) allowing half-saturating and optimal irradiances (Eopt) to be estimated. Effective Quantum Yield (Ymax), Electron Transport Rate (ETRmax) and the NonPhotochemical Quenching parameter, NPQmax all vary on a diurnal cycle but the parameter qNmax does not show a systematic variation over a diurnal period. Phuket pineapple is a “sun plant” with Optimum Irradiance (Eopt) from 755 to 1,130 μmol m−2 s−1 (400–700 nm) PAR but photosynthetic capacity is very low in the late afternoon even though light conditions are favourable for rapid photosynthesis. Total CO2 fixed nocturnally as C4-dicarboxylic acids by leaves of the Phuket pineapple was only ≈0.14 gC m−2 d−1 (0.012 mol C m−2 d−1). Titratable acid of leaves was depleted about 3 pm (15:00) and shows a classical CAM diurnal cycle. The Phuket pineapple variety only stored enough CO2 as C4 acids to account for only about 2.5% of photosynthesis (Pg) estimated using the PAM machine

(≈5.6 gC m−2 d−1). Phuket pineapples are classifiable as CAM-Cycling plants but nocturnal fixation of CO2 is so low compared to the more familiar Smooth Cayenne variety that it probably recycles only a small proportion of the respiratory CO2 produced in leaves at night and so even CAM-cycling is only of minor importance to the carbon economy of the plant. Unlike the Smooth Cayenne pineapple variety, which fixes large amounts of CO2 nocturnally, the Phuket pineapple is for practical purposes a C3 plant. Keywords Pineapple . Cultivar Phuket . CAM photosynthesis . Carbon fixation . Diurnal cycle . Gross photosynthesis . PAM fluorometry . PAR . Primary productivity Abbreviations α Photosynthetic efficiency E Irradiance (mol m−2 s−1) PAR Eopt Optimum irradiance for maximum photosynthesis ETR Electron transport rate PAM Pulse Amplitude Modulation fluorometry PAR Photosynthetically Active Radiation (400–700 nm) (sometimes alternatively termed Photosynthetic Photon Flux Density PPFD) Pg Gross photosynthesis PSI Photosystem I PSII Photosystem II

Communicated by: Paul Moore R. J. Ritchie (*) : S. Bunthawin Biotechnology of Electromechanics Research Unit, Faculty of Technology and Environment, Prince of Songkla University, Phuket 83120, Thailand e-mail: [email protected] S. Bunthawin e-mail: [email protected]

Introduction PAM machines can perform measurements of the light reactions of photosynthesis very quickly (Krause and Weis 1991; Schreiber et al. 1995a, b; White and Critchley 1999; Rascher et al. 2000; Franklin and Badger 2001; Ralph and

Tropical Plant Biol.

Gademann 2005; Lüttge 2007; Ritchie 2008; Ritchie and Bunthawin 2010): experiments that can take 4 to 6 h or days using oxygen electrode or gas exchange apparatus such as Infrared Gas Analyzers (IRGA) (Cote et al. 1989) can be done with a PAM machine in 2 to 3 min. PAM machines are very useful in making comparative studies of the effects of environmental stress on plants and for rapid screening of the physiological condition of plants, particularly in field situations (Franco et al. 1996, 1999; Rascher et al. 2000; Martyn et al. 2008). However, without independent respiratory information Net Photosynthesis (Pn) cannot be estimated. Additional oxygen electrode, 14C or IRGA measurements are necessary to make quantitative estimates of net photosynthesis from PAM data. CAM (Crassulacean Acid Metabolism) plants close their stomates during at least some of daylight, creating a sealed compartment in the stems and leaves precluding measuring photosynthesis by CO2 gas exchange-based methods. Mature leaves or photosynthetic stems of constitutive or obligate CAM plants always exhibit significant dark fixation of CO2 whereas facultative (C3/CAM intermediates) CAM species are plants which express CAM metabolism only under certain environmental conditions or seasons of the year (Osmond 1978; Taize and Zeiger 2002; Lüttge 2004, 2007; Winter et al. 2008). PAM techniques provide valuable information on the light reactions of photosynthesis of CAM plants (Maxwell et al. 1998; Ritchie and Bunthawin 2010) which are not readily available using other methods based on gas exchange such as IRGA, 14C labeling or oxygen electrode methods because CAM plants close their stomata during the day. The study of Cote et al. (1989) on pineapple (Ananas comosus comosus [L.] Merr, Bromeliaceae, cv. Smooth Cayenne) is a rare example of where CO2 fixation and O2 production were simultaneously measured. PAM and other related fluorescence methods have been extensively used on facultative CAM species of Clusia (Franco et al. 1996, 1999; Lüttge 2004, 2007) and a few other facultative CAM plants such as Delosperma tradescantioides (Herppich et al. 1998), Mesembryanthemum crystallinum (Slesak et al. 2003; Broetto et al. 2007) and the obligate CAM species Kalanchoë daigremontiana and Hoya camosa (Maxwell et al. 1998); Kalanchoë daigremontiana and K. pinnata (Griffiths et al. 2008) and Dendrobium spp. cv. ‘Virathuth’ (Ritchie and Bunthawin 2010). Maxwell et al. (1998) found a good quantitative relationship between CO2, O2 and PAM estimates of photosynthesis in Kalanchoë daigremontiana and Hoya camosa. CAM plants can be difficult systems for photosynthetic studies, in particular dealing with the issue of the proportion of carbon fixed by the plants nocturnally compared to direct C3 fixation during daylight (Osmond 1978; Ting 1985; Cushman and Borland 2002; Dodd et al.

2002; Winter and Holtum 2002; Lüttge 2004, 2007; Winter et al. 2008). Gas exchange methods for estimating photosynthesis require both day and night measurements in obligate CAM plants. Misleading experimental artifacts are common. In vitro measurements of photosynthesis in protoplasts or cell suspensions obtained by enzymatic digestion of leaves of CAM plants generally exhibit little or no CAM activity. Very young leaves of CAM plants or experimentally convenient seedlings or small plantlets also typically exhibit little or no detectable CAM activity even in supposedly obligate CAM species (Hew and Khoo 1980; Dodd et al. 2002; Winter et al. 2008) including Smooth Cayenne pineapple (Cote et al. 1989; Nievola et al. 2005). Callus cultures typically behave as C3 plants with little or no CAM activity, for example in cacti (Malda et al. 1999) and Smooth Cayenne pineapple (Nievola et al. 2005). There is an intimate relationship between CAM physiology and water stress of CAM plants (Cushman and Borland 2002). Some even supposedly obligate CAM plants completely switch to C3 photosynthesis if heavily watered (Agave deserti, Hartsock and Nobel 1976); other obligate CAM species switch to a form of CAM called CAMCycling (sometimes dismissively called Weak CAM) where stomates are closed at night and nocturnal respiration of the plant is at least partly recovered by the plant as C4 acids which are used as a partial source of CO2 the next day (Ting 1985; Sipes and Ting 1985; Patel and Ting 1987; Vovides et al. 2002; Lüttge 2004, 2007; Ritchie and Bunthawin 2010). Cote et al. (1989), working on heavily watered (6 times /day) small Smooth Cayenne pineapple plants under saturating humidity, found limited C4 fixation at night: most carbon was fixed in daylight directly from the atmosphere. Their results are consistent with their plants being in CAM-Cycling mode. Facultative CAM species (by definition) adjust the degree of nocturnal fixation of C4 acids depending on the environmental conditions. Pineapple is the most important food crop, which is a CAM plant (Taize and Zeiger 2002) but under commercially cultivated conditions may fix most CO2 directly from the atmosphere during daylight (Ting 1985; Zhu et al. 1999; Winter and Holtum 2002). PAM machines are excellent for screening plants for signs of stress (O’Neill et al. 2006) and so PAM techniques are potentially very valuable tools for studies of stress physiology of cultivated pineapple (Chen et al. 2002). Cacti, Bromeliads and Orchids are also major components of the globally important ornamental plant industry (Hew and Yong 2004). CAM plants are often propagated using cell and tissue culture techniques and the critical steps are acclimating plants from the culture room to greenhouses and finally to the open air. PAM technology offers an accessible routine technique for monitoring plants during this critical step of horticultural production.

Tropical Plant Biol. Yieldmax vs. Time (h)

1

Results

0.9 Yield (max)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

3

9 12 15 Solar Time (h)

18

21

24

least squares methods based on 9 different light intensities and 12 leaves giving 108 data points (r=0.9101, p≪0.001). The ETRmax was 65.51±2.27 μmol m−2 s−1 and the Optimum Irradiance (Eopt) was 755±42 μmol m−2 s−1 PAR or about 40% of full sunlight at the season of the year when the study was done in Phuket (Fig. 1). The photosynthetic efficiency (α) was 0.2359±0.0154, which is a typical value found for vascular plants, including the CAM orchid Denbrobium spp cv. ‘Viravuth’ (Ritchie 2008; Ritchie and Pineapple Exponential Waiting-in-Line Fit at 15:00 h

Diurnal Irradiance for Phuket (7 53' N) Light Curve SS Light Curve SE Light Curve WS 31 Jan 2010

80 70

ETR (µmol m -2s-1 )

2000

6

Fig. 2 Photosynthetic Yield (Ymax) of pineapple leaves collected over the course of a day. Daylight length at the time (Jan 2009) was approximately 12 h/day. Ymax was estimated using non-linear leastsquares fitting of an exponential decay function (Ritchie and Bunthawin 2010). Data are based on 12 replicates of light curves with 9 different irradiances for all the times (n=12×9=108). Yield estimates are means and error bars are ±95% confidence limits

o

2500 PAR Irradiance (μmol m -2 s-1)

0.8

Yield max

Figure 1 shows diurnal light curves (Global Irradiance: direct irradiance + diffuse irradiance) for Phuket (Lat. 7°53′ N, Long. 98°24′E), Thailand at the summer solstice, spring equinox and the winter solstice. The present study was conducted in January–February 2010, which is during the monsoonal dry season. Daily maximum irradiance on the 31 January 2010 was about 1,978 μmol m−2 s−1 (400– 700 nm) PAR. Days were typically clear with about 12 h of sunshine per day. The average daily irradiance for January– February was 51 mol m−2 d−1 (400–700 nm). There is little seasonal variation in clear-sky irradiance over the year at Phuket (Fig. 1) but during the wet season it is typically overcast for much of the day, leading to much lower daily irradiances. PAM measurements of photosynthetic parameters in pineapple show a pronounced diurnal behavior. Circadian cycling of metabolism is an essential component of CAM physiology (Duarte and Lüttge 2007). Maximum Photosynthetic yield (Ymax) by leaves sampled during the nighttime was only about 0.2–0.3, rose to about 0.7 at midday but in the afternoon, well before the sun had set, there was a significant decline in yield (Fig. 2). Correlation coefficients for fits to plots of Yield vs. Irradiance curves were all very high, for example for leaves collected at 15:00 Solar Time was r=0.9866 (p≪0.001). The exponential constant (ky) values are tabulated in Table 2. Figure 3 is a graph of ETR vs. Irradiance up to sunlight irradiances (≈1,900 μmol m−2 s−1 PAR for Phuket in January–February) for pineapple leaves collected at solar mid-afternoon (15:00). Equation 2 was fitted by iterative

1500

1000

500

60 50 40 30 20

Electron Transport Rate

10

Least Squares Fit

0

0 0

3

6


18

21

24

Fig. 1 Diurnal light curves (Global Irradiance: direct irradiance + diffuse irradiance) for Phuket (Lat. 7°53′N, Long. 98°24′E), Thailand at the summer solstice, spring equinox, the winter solstice and 31 Jan 2010. Fifteen (15) minute time intervals were chosen as suitable for the purposes of the present study and the refractive effects of the atmosphere were allowed for. The maximum PAR irradiance on 31 January 2010 was 1,978 μmol m−2 s−1 (400–700 nm). The average daily irradiance for January–February was 51 mol m−2 d−1 PAR

0

500

1000 1500 PAR Irradiance (µmol m-2 s-1)

2000

Fig. 3 Plot of ETR vs. Irradiance of pineapple leaves collected at 15:00 solar time. The Waiting-in-Line equation (Eq. 2) was fitted using non-linear least-squares fitting as described by Ritchie and Bunthawin (2010). The ETRmax was 65.51±2.268 μmol m−2 s−1 (mean ±95% confidence limit) and the Optimum Irradiance (Eopt) was 754.9±41.8 μmol m−2 s−1 PAR (mean ±95% confidence limit) based upon 12 replicate light curves with 9 different irradiances on each leaf (n=108)

Tropical Plant Biol. Table 1 Chlorophyll and leaf data on Phuket pineapple leaves Chl a (FW basis) Chl a (Leaf Surface Area basis) Leaf Surface Area Succulence Index

−1

110±18.11 μg Chl a g FW (n=24) 97.0±15.9 mg Chl a m−2 1.137 (±0.0477)×10−3 m2 g−1FW (n=36) 892.7 (±37.8) g FW m−2 (n=36)

Bunthawin 2010). The approximate maximum Gross Photosynthetic rate (Pg) would be 16.38±0.57 μmol m−2 s−1 based on one O2 is produced per 4e− passing through PSII (Maxwell et al. 1998). On a chlorophyll a basis (Table 1), the ETRmax was 2,432±407 μmol mg Chl a−1 h−1 or Pg ≈ 608±102 μmol O2 mg Chl a−1 h−1. The Optimum Irradiance values (Eopt), determined using least squares fitting, are tabulated in Table 2. For leaves collected at most times over a 24 h period the Optimum Irradiance (Eopt) value was about 700 μmol m−2 s−1 PAR with the notable exception of leaves collected in the afternoon (15:00 to 18:00 solar time), which had very low Eopt values and suppressed ETRmax. Phuket pineapple leaves taken at solar midnight had low fluorescence yield values (Fig. 2). Leaves were sampled at midnight on two occasions and so overall photosynthetic parameters could be calculated based on 18 irradiance levels and 24 leaves giving a total sample size of n=216. The ETRmax was a very low value of 18.27±1.76 μmol m−2 s−1 compared to ETR measurements made during the middle of the day (see above). However, the Optimum Irradiance (Eopt) was 662±120 μmol m−2 s−1 PAR which was not significantly different to the optimum irradiance found for leaves sampled at solar midday above. The photosynthetic efficiency (α) was a very low value of 0.0750±0.0153. The approximate Gross Photosynthetic rate (Pg) was 4.57± 0.439 μmol O2 m−2 s−1. On a chlorophyll a basis (Table 1) the ETRmax was 678±129 μmol mg Chl a−1 h−1 or Pg ≈170± 32 μmol mg Chl a−1 h−1. Figures 4 and 5 show ETR vs. Solar time and Photosynthetic Efficiency (α) vs. Solar Time. Both ETR

and Photosynthetic Efficiency (α) show a strong diurnal cycle with low values at night followed by increasing ETRmax and α during the morning, reaching a peak at about solar midday followed by a sharp decrease to values similar to those found in the dark in the afternoon. Note that this occurred well before the sun had set. Figure 6 shows that the maxima of the two parameters used to express Non-Photochemical Quenching (expressed as qN and NPQ) vary in a different way over the diurnal cycle. Maximum qN varies little over time and averaged about 0.6 to 1. NPQmax was about 2.5 in leaves sampled during the night. During the light period NPQmax and qNmax were both about 1 in the mornings and late afternoons but NPQmax rose to about 2 during the middle of the day. The exponential constants for qN and NPQ determined as described by Ritchie and Bunthawin (2010) are tabulated in Table 2. The two exponentials of the nonphotochemical quenching parameters are not similar in magnitude and are not obviously related to the ky of photosynthetic yield (Table 2). Figure 7 shows that pineapple has a clear diurnal cycle of titratable acid in its leaf tissues typical of a CAM plant. As found previously in Pineapple (Ananas comosus comosus cv. Smooth Cayenne) (adult plants, Chen et al. 2002; cultured plantlets, Nievola et al. 2005) and Kalanchoë daigremontiana and K. pinnata, accumulation of titratable acid and its depletion during daylight were approximately linear (Chen et al. 2002; Nievola et al. 2005; Griffiths et al. 2008). In the present study, there was a delay of several hours before the titratable acid of the leaves started to decrease in daylight. After about 9:00 solar time, the titratable H+ in the plant tissue rapidly depleted to a minimum in the late afternoon (Solar Time 15:00 to 18:00). The minimum titratable acid in Phuket pineapple leaves in the present study was 5.600±0.577 μmol g−1 FW (n=16) at 18:00. The acid content of leaves sampled at 6:00 solar time was 31.93±2.66 μmol g−1 FW (n=16). The net accumulation of acid during the night period was therefore

Table 2 Fitted exponential coefficients for Phuket pineapple. Data presented as means ±95% confidence limits are based upon 12 replicates (n= 108 data points) Solar time h

Yield (ky)

0:20 6:00 9:00 10:30 12:00 15:00 18:00 21:00 24:00

0.002647±0.000480 0.002133±0.000194 0.001512±0.0000885 0.001362±0.000125 0.001368±0.000137 0.001571±0.0000709 0.004975±0.00102 0.002695±0.000500 0.004601±0.000664

Optimum irradiance (Eopt) 536±114 1,130±419 804.5±36.4 711.4±52.8 756±75.4 754.9±41.8 198±27.9 547±116 906±300

qN (kqN)

NPQ (kNPQ)

0.006092±0.000887 0.002365±0.000347 0.002250±0.000247 0.005744±0.000826 0.00436±0.000754 0.001667±0.000147 0.003778±0.000814 0.003628±0.000525 0.004514±0.000621

0.003202±0.000536 0.001273±0.000313 0.001141±0.000238 0.002990±0.000738 0.001769±0.000600 0.0009918±0.000142 0.002390±0.000612 0.001346±0.000475 0.002139±0.000501

Tropical Plant Biol. P (ETR)max vs. Time (h)

90

ETR (max)

80

-2

-1

ETRmax (μmol m s )

Non-Photochemical Quenching vs. Time (h)

4.5

70 60 50 40 30 20 10

Non-Photochemical Quenching (qN & NPQ)

100

qN (max) NPQ (max)

4 3.5 3 2.5 2 1.5 1 0.5 0

0

0

0

3

6

9

12

15

18

21

3

6

9

24

12

15

18

21

24

Solar Time (h)

Solar Time (h)

Fig. 4 Maximum ETR (ETRmax) of pineapple leaves collected over the course of a day. ETRmax was estimated using non-linear leastsquares fitting of Eq. 2. Data presented as means ±95% confidence limits are based upon 12 replicates (n=108 data points)

31.93 (±2.66)–5.600 (±0.577)=26.33±2.61 μmol g−1 FW or 23.16 (±2.49)×10−3 mol m−2 (from Table 1). Since the dicarboxylic acids accumulated by CAM plants have two titratable H+ per CO2 fixed, then the overnight CO2 fixation of pineapple leaves would have been 11.58 (±1.25)× 10−3 mol CO2 m−2d−1 or 0.139±0.0149 gC m−2d−1. These values are much lower than found previously in adult Smooth Cayenne pineapple plants (≈3 to 5 gC m−2 d−1; Zhu et al. 1999; Chen et al. 2002) and the wild and noncommercial subsistence-farming pineapple varieties found in South America (Medina et al. 1993). Adult leaves of Phuket pineapple used in the present study store about the

Fig. 6 Non-Photochemical Quenching calculations on pineapple leaves collected over the course of a day. The two expressions for non-photochemical quenching (qN and NPQ) estimated using nonlinear least-squares fitting (Ritchie and Bunthawin 2010). Data presented as means ±95% confidence limits are based upon 12 replicates (n=108 data points)

same amount of CO2 as dicarboxylic acids nocturnally as found in Dendrobium ‘Viravuth’ (Ritchie and Bunthawin 2010) and Smooth Cayenne pineapple plantlets grown in culture (Nievola et al. 2005) and Smooth Cayenne plantlets under saturated humidity conditions (Cote et al. 1989). They also agree with values for C4 fixation in the facultative CAM species Delosperma tradescantioides under well-watered conditions (Herppich et al. 1998).

Pineapple Titratable Acid vs. Solar Time

40

Alpha ( )

0.3 0.25 0.2 0.15

Titratable H + (μmol g -1 FW)

0.35 Photosynthetic Efficiency (8)

35

Photosynthetic Efficiency (8) vs. Time (h)

0.4

30 Titratable Acidity

25 20 15 10 5

0.1

0

0.05

0

3

6

9

12

15

18

21

24

Solar Time (h)

0

0

3

6


18

21

24

Fig. 5 Photosynthetic Efficiency (α) of pineapple leaves collected over the course of a day. The photosynthetic efficiency was calculated from kw and Pmax estimated using non-linear least-squares fitting of Eq. 2. Data presented as means ±95% confidence limits are based upon 12 replicates (n=108 data points)

Fig. 7 Titratable acid of pineapple leaves collected over the course of a day. Data are means based on 6 replicates and error bars are ±95% confidence limits except for the measurements at solar time 6:00 and 18:00 which are based on 16 replicates. Minimum acidity was in the later afternoon (≈1 μmol g−1FW) (15:00 to 18:00), rose to ≈18 μmol g−1FW at dawn and steadily decreased during the day back to the late-afternoon minimum


Taking the irradiance data (Fig. 1, 31 January 2010) and estimates of ETRmax and Eopt taken during the course of the day (Fig. 4 and Table 2) it is possible to calculate Pg of pineapple leaves over the course of a day using Eq. 2. The results were then integrated using the trapezium rule to estimate cumulative and total daily Pg (Fig. 8). For comparison, the CO2 reservoir as C4 acids is also shown. The C4 reservoir is clearly insufficient to account for total daily photosynthesis. Total Pg increased rapidly during the morning but leveled off during the middle of the day because of photoinhibition during the middle of the day, followed by resumption of high photosynthesis in the afternoon. The PAM data gives an estimation of total daily photosynthesis of about 5.6 g m−2 d−1 of which only about 0.139 gC m−2 d−1(or ≈2.5%) the total would be derived from nocturnal fixation of CO2.

Discussion Phuket pineapple plants were shown to fix about 5.6 gC m−2 d−1. This estimate of Pg is similar to values found by Zhu et al. (1999) and Chen et al. (2002) on the Smooth Cayenne variety but more than double the value of about 1.8 gC m−2 d−1 found on the heavily watered immature plantlets used by Cote et al. (1989). We found, in contrast to the studies by Zhu et al. (1999) and Chen et al. (2002), that only a very small proportion of total carbon was fixed nocturnally (0.139±0.0149 gC m−2d−1 or ≈2.5%). In the present study, nocturnal fixation of CO2 as C4 acids accounts for such a low proportion of total photosynthesis Total Daily Gross Photosynthesis for Pineapple

6000

Total Cummulative Pg

5000

Reserves of CO2 as C4 Acids

ΣPg (mg C m-2 )

Approx. Dark Period

4000 3000 2000 1000 0 0

3

6

9

12

15

18

21

24

Solar Time (h)

Fig. 8 Estimated total Gross Photosynthesis (Pg) of pineapple leaves over the course of a day based upon ETRmax determinations (Fig. 4) and the Eopt data tabulated in Table 2 inserted into Eq. 2 and using irradiances values shown in Fig. 1. For comparison, the CO2 reservoir as C4 acids is also shown. The PAM data gives an estimation of total daily photosynthesis of ≈5.6 g m−2 d−1. This is much greater than what was available from the reservoir of nocturnal fixation of CO2 as C4 acids

that the Phuket pineapples could not be fixing a large proportion of nocturnal respiratory CO2 and so CAMCycling contributes very little to the carbon economy of the plant. In practical terms they were behaving as C3 plants. The Phuket pineapple plants used in the present study were growing during the dry season when they would be expected to show the heaviest seasonal dependence on CAM physiology but were regularly watered and so were not water-stressed or CAM Idling (Osmond 1978; Herppich et al. 1998; Zhu et al. 1999; Chen et al. 2002; Dodd et al. 2002; Lüttge 2004; Winter et al. 2008; Silvera et al. 2009). Obligate CAM species grown under field conditions typically have δ13C/12C ratios intermediate between C3 and C4 plants: about 70–80% of carbon fixed by the plants is fixed nocturnally by C4 biochemistry (Winter and Holtum 2002). Zhu et al. (1999) found using gas exchange that about 70–84% of carbon was fixed nocturnally in Smooth Cayenne pineapples. This conclusion was confirmed by the δ13C/12C ratios found in their study because they are consistent with the relationship between stable carbon ratios and C3/C4 carbon fixation later found by (Winter and Holtum 2002). Pineapples show most qualitative aspects of the classical CAM diurnal cycle of fixation of carbon: CO2 is fixed as C4 acids nocturnally (Phase I as defined by Osmond 1978), followed by mobilization during the day (Phase III) (Fig. 7), however different studies disagree on the importance of Phase II (early daylight atmospheric uptake of CO2) and Phase IV (late afternoon C3 photosynthesis using CO2 directly from the atmosphere). The importance of direct fixation by C3 photosynthesis in pineapples varies greatly (compare, Cote et al. 1989; Zhu et al. 1999; Chen et al. 2002 to Figs. 7 and 8 in the present study). Chen et al. (2002) in their study of Smooth Cayenne pineapple and Kalanchoë daigremontiana and K. pinnata found that they accumulated 120–150 μmol g−1 FW of malate (240– 300 μmol g−1 FW titratable acid or about 13-times that found in the present study) in their leaves at night as virtually their sole source of CO2 for photosynthesis. Under their experimental conditions, Smooth Cayenne pineapples had essentially no CAM Phases II & IV but had daily carbon fixation rates of ≈5 gC m−2 d−1. Zhu et al. (1999) measured more modest production of about 0.3 mol m−2 d−1 or 3.6 gC m−2 d−1 of which 69 to 84% was based on nocturnal fixation of carbon as C4 compounds. Smooth Cayenne pineapple plantlets grown in culture show very little diurnal accumulation and depletion of titratable acid if grown under constant temperature (28°C, Light:16 h/Dark: 8 h) but substantial CAM characteristics if grown under variable temperature (Light 25°C,16 h/Dark 15°C,8 h) (Fig. 3, Nievola et al. 2005). In the present study, Fig. 7 shows that there was a long delay of about 3 h after dawn before the nocturnally fixed C4 acid content of the leaves started to


decline. This is consistent with the Phuket variety of pineapples having a predominant CAM Phase II of early morning C3 fixation directly from the atmosphere. Phase IV (Solar time 15:00 to 18:00) is probably not important judging from the poor photosynthetic performance by the pineapples late in the day (Figs. 4 and 8, Table 2). In our previous study (Ritchie and Bunthawin 2010) we found that the orchid Dendrobium spp. cv. ‘Virathuth’ was another obligate CAM species that nevertheless fixed most of its CO2 during the day from the atmosphere. Thus the Phuket pineapple and Dendrobium ‘Virathuth’ are obligate CAM species that fix most of their carbon by conventional C3 photosynthesis if conditions permit but nevertheless show the typical CAM cycle of some nocturnal fixation of CO2 as C4 acids followed by mobilization of CO2 during the day. Nocturnal fixation of carbon is only on a small scale and so these plants are best regarded as CAM-Cycling plants. Other examples of CAM-Cycling plants are Peperomia (Sipes and Ting 1985; Patel and Ting 1987) and the cycad Dioon (Vovides et al. 2002). Similarly, Herppich et al. (1998) working on a facultative CAM plant, Delosperma tradescantioides, concluded that only about 24% of total carbon fixed was derived from CO2 fixed as C4 acids at night. Photosynthetic light saturation curves for pineapple show that it is a “sun plant” because the Optimum Irradiance (Eopt) is about 700 to 1,100 μmol m−2 s−1 PAR (Fig. 3 and Table 2) (Herppich et al. 1998; Martin et al. 1999). Figure 8 is a plot of the cumulative Pg of Phuket pineapple. Since irradiance reached over 1,900 μmol m−2 s−1 at midday in Phuket during the time of the study, then Eq. 2 predicts substantial photoinhibition of photosynthesis during the middle of the day (≈50%) but favorable circumstances for high photosynthesis in the morning and afternoon. On the contrary, Figs. 4 and 8 show that there was a steep decline in photosynthesis in the late afternoon, corresponding to the time when the internal reservoir of C4 acids were completely depleted (Fig. 7). This result is consistent with hydrogen peroxide photodamage of the photosystems occurring in the late afternoon as documented by (Slesak et al. 2003) in Mesembryanthemum crystallinum under conditions of high light, combined with lack of CO2 substrate for the Calvin cycle. Similar effects can occur in C3 plants such as Telopea when stomates are closed in the middle of the day due to water stress (Martyn et al. 2008). Very high oxygen tensions build up within the leaves of Clusia when in CAM mode resulting in high photorespiration (Lüttge 2007). Similar inferences can be drawn about the oxidative environment prevailing inside leaves of pineapple based upon the diurnal pattern of PAM parameters in pineapple. Routine pre-dawn measurements of PAM parameters (Martyn et al. 2008) can therefore give misleading information about photosynthetic performance

in daylight, particularly in the afternoon. Figures 2, 4 & 5 and the Eopt data in Table 2 show that the maximum effective quantum yield (Ymax), Electron Transport Rate (ETRmax), Optimum Irradiance (Eopt) and NPQmax all vary on a diurnal cycle as found previously for Dendrobium (Ritchie and Bunthawin 2010). Effective quantum yield (Y) and ETR are both related to the flow of electrons through PSII to PSI eventually to form NADPH2 which is used to fix CO2. Ymax and ETRmax of the light reactions of pineapple were much lower during the night-time (Fig. 2). Non-Photochemical Quenching expresses the amount of light energy absorbed by PSII but not used for photochemistry and is lost as low-grade heat (Schreiber et al. 1995a, b; Martin et al. 1999; Holt et al. 2004; Ralph and Gademann 2005). The qNmax parameter does not show an obvious systematic variation over a diurnal cycle (Fig. 6). NPQmax increases greatly at night when photosynthesis does not normally occur (Fig. 6) and RUBISCO in CAM plants is known to be partially deactivated (Ficus belgica Fig. 8 in Griffiths et al. 2002). It can be concluded that NPQ is a better measure than qN of conversion of energy absorbed as 400–700 nm PAR into waste heat. The high NPQ values during the dark period indicate that if plants are exposed to light at night the photosynthetic apparatus disperses absorbed light energy as heat rather than generating a proton motive force, in other words the photosynthetic electron transport chain is uncoupled. The peaks of NPQ found in Phuket pineapple during the middle of the day (Fig. 6) probably indicates photooxidative stress during the heat of the day when the stomates are closed and no CO2 is available inside the leaves. A lot of synthesis and repair of PSII and PSI and antennae proteins and RUBISCO would be going on during the night in pineapples, hence the aberrant NPQ values found at night (Fig. 6). It is known that the level of expression of CAM physiology in pineapples depends upon the watering regime under which they are kept. Well-watered plants accumulate lesser amounts of C4 acids at night (Ting 1985; Cote et al. 1989; Medina et al. 1993; Zhu et al. 1999; Cushman and Borland 2002; Winter and Holtum 2002). With the exception of the work of Medina et al. (1993) most published work is on the Smooth Cayenne pineapple variety. The question naturally arises whether the very low nocturnal accumulation of C4 acids in the Phuket variety found in the present study represents a varietal difference or simply that the plants were well-watered. One of the authors (RJR) had several Smooth Cayenne plants growing as ornamentals in a suburban garden in Sydney, Australia (≈34 °S). Leaves were taken from these plants on the autumn equinox (22 March 2010) at dawn and dusk and assayed for titratable acid. The leaves taken at dawn had very high acid levels (257±19 μmol H+ g−1 FW, n=8) and at dusk fell to 42±3 μmol H+ g−1 FW (n=8) and so nocturnal accumulation


of acid was about 215±19 μmol H+ g−1 FW or more than 8 times that found in Phuket pineapples. Such values are similar to previously published values for the Smooth Cayenne variety (Zhu et al. 1999; Chen et al. 2002) and so the very low level of CAM expression in Phuket pineapples is a varietal difference. Phuket pineapple fruits are noted for their very low acidity and sweet taste. It also puts into context the wide variation in nocturnal C4 acid accumulation found in wild and subsistence varieties of pineapple (Medina et al. 1993). We found that Phuket pineapples fixed enough CO2 directly by C3 photosynthesis (CAM Phase II) to account for nearly all daily photosynthesis. Previous studies on adult plants of the Smooth Cayenne pineapple variety found similar overall carbon fixation rates as the present study but Phase II was of only minor importance and most CO2 was fixed nocturnally as C4 acids (Zhu et al. 1999; Chen et al. 2002). There are large differences in the degree of expression of CAM in different varieties of pineapple. This information is likely to be important for understanding water-use efficiency and productivity of different varieties of pineapples under cultivation.

Methods Experimental Materials Pineapple plants (Ananas comosus comosus [L.] Merr) cv. Phuket were grown in pots in a sunny location on the Prince Songkla University Phuket campus, Phuket Province, Thailand (Lat. 7°53′N, Long. 98°24′E) in December– February 2010. The Phuket pineapple plants were grown from crowns of fruits purchased locally and kept in a shadecloth green house (≈80% shade) for about 2 weeks before being moved into full sunlight. Plants were watered daily. Phuket has a monsoon climate and the experimental period was during the dry season (precipitation December to March