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
9
12
15
Solar Time (h)
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
9
12
15
Solar Time (h)
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
Tropical Plant Biol.
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
Tropical Plant Biol.
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
Tropical Plant Biol.
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
<50 mm/month). Daylight lengths were about 12 h per day
(including skylight). Solar time for Phuket was −40 min
from Thailand Standard Time (GMT+7 h) on 31 Jan 2010
(http://www.powerfromthesun.net/CALCULATORS/
LocalToSolarTime.html [accessed 24/Jan/2010]). Leaves
were removed using scissors and kept in black cloth bags.
PAM measurements were made after incubating in the dark
for at least 10 min and no later than 40 min of collection.
Leaves for measurements of titratable acid were collected,
weighed and frozen before extraction of the acid.
PAR Irradiance in Phuket
SMARTS software (http://www.nrel.gov/rredc/smarts/
[Accessed 09/Nov/2009]) can be used to model solar
radiation reaching the earth’s surface at a given latitude
and solar angle (Gueymard 1995; Gueymard et al. 2002).
Using the SMARTS software and the NREL Solar
Position Calculator (SOLPOS) (http://www.nrel.gov/
midc/srrl_bms/ [accessed 09/Nov/2009]) it was possible
to calculate PAR irradiance (400–700 nm) at 15 min
intervals at the latitude of Phuket for any solar elevation
angle (corrected for refraction) over the period of a day for
a given date. Numerical integration methods could then be
used to calculate daily irradiances at chosen times of the
year.
Chlorophyll Determinations
Chlorophyll determinations are difficult to make on
CAM plants because the C4-dicarboxylic acids accumulated nocturnally in CAM plants cause denaturation of
chlorophylls into phaeophytins when attempts are made
to extract chlorophyll from leaves and stems of CAM
plants. However, the tissues of CAM plants like
pineapple (Chen et al. 2002) have minimal acidity if
collected in the late afternoon of daylight (15:00 to 18:00
Solar Time). Pineapple leaves were cut into small
rectangles (approx. 2×1 cm), measured with a ruler and
weighed to calculate projected surface area per g FW.
After cutting into thin slices, chlorophyll was extracted in
Mg carbonate-neutralized ethanol and assayed after clearing by centrifugation (Ritchie 2006). Chl a was calculated
as μg Chl a m−2 of projected leaf surface area and μg Chl
a g−1FW.
Modulation Fluorometry
Light saturation curve measurements were made using a
Junior PAM portable chlorophyll fluorometer (Gademann
Instruments GmbH, Wurzburg, Germany) fitted with a
1.5 mm diameter optic fiber and a blue diode (485±
40 nm) light source. PAM parameters (Effective Quantum Yield, ETR, qN, NPQ) were calculated using the
WINCONTROL software (v2.08 & v2.13) (Genty et al.
1989; van Kooten and Snel 1990) using the standard
settings for rapid light curves (absorptance factor = 0.84,
PSI/PSII allocation factor = 0.5) (Heinz Walz Gmbh,
Effeltrich, Germany) to calculate the Electron Transport
Rate (ETR) (Schreiber et al. 1995a, b; Rascher et al.
2000). Sets of PAM light curve measurements took about
88 s to complete with 10 s between actinic flashes of light
and each flash of light was 0.8 s duration. The flashes
were in order of increasing intensity but the steady-state
fluorescence yield (Fo) did not change by more than 10%
over the course of a run of ETR vs. E. Leaves were kept in
the dark 10 to 40 min before fluorometry measurements.
Only one light saturation experiment was run on each part
Tropical Plant Biol.
of a leaf to avoid confounding effects of multiple
experimental treatments and invalid estimates of Fo. The
measurements were made in the bright green central parts
of the leaves.
Titratable Acid
Titratable acid was measured in a freshweight basis (FW).
Leaves were taken on a 24 h cycle and frozen before
extraction. Acid was extracted in 30 ml of distilled water by
heating the leaves in a hot water bath (65°C) for 30 min.
After cooling, the free acid was titrated using 5 mol m−3
NaOH with phenolphthalein indicator (Mc Williams 1970;
Nievola et al. 2005; Griffiths et al. 2008; Ritchie and
Bunthawin 2010). Titratable acid was calculated as mol H+
g−1FW: this could be converted into mol H+ m−2 using the
surface area g−1FW data in Table 1. Estimates could then be
made of CO2 available from the nocturnally fixed reservoir
of dicarboxylic C4 acids on a leaf surface area basis
assuming one CO2 per 2H+ of titratable acid.
Analysis of PAM Experimental Results
determined by Björkman and Demmig (1987). These are
the standard settings used by the WINCONTROL software
of Walz PAM machines (Heinz Walz Gmbh, Effeltrich,
Germany).
Ritchie and Bunthawin (2010) presented a more convenient form of the waiting-in-line equation suitable for
modeling photosynthesis because it is easy to recognize
good starting values for the iterative determination of
ETRmax and Eopt from plots of ETR vs. Irradiance,
ETR ¼
ETRmax :E 1E=Eopt
:e
Eopt
ð2Þ
where, ETR is the Electron Transport Rate as mol m−2 s−1,
ETRmax is a scaling constant for the maximum height of
the curve, the Optimum Irradiance (Eopt) is a scaling
constant for the X-axis, and E is the Irradiance
(μmol m−2 s−1 PAR). At very low light intensities
photosynthesis is directly proportional to irradiance. The
maximum photosynthetic efficiency (Alpha,α) is the
initial slope of the curve at E=0 (α=ETRmax.e/Eopt).
Gross photosynthesis can be estimated from ETR based on
4 electrons moving through PSII for each O2 produced in
photosynthesis.
Yield
Quenching Analysis
The fluorescence yield was calculated using the WinControl
software (v2.08 & v2.13) as the Effective Quantum Yield
(Y or ΦPSII) as defined by Genty et al. (1989) and van
Kooten and Snel (1990).
Effective Quantum Yield
Effective Quantum Yield ranges from 0 up to 1 (maximum
usually no higher than ≈0.85). It is found experimentally
that if Y is plotted against irradiance (E) it follows a simple
exponential decay function of the form y=e-kx (Ritchie
2008; Ritchie and Bunthawin 2010).
Electron Transport Rate
The Electron Transport Rate (ETR) can be described by
the Waiting-in-Line equation (Ritchie 2008; Ritchie and
Bunthawin 2010). The Electron Transport Rate (ETR) is an
estimate of Pg and is defined as,
ETR ¼ Y E ðPSI=PSII allocation factorÞ
ðleaf absorptance factorÞ
ð1Þ
where, Y is the effective quantum yield, E is the irradiance
(mol m−2 s−1 PAR), the PSI/PSII allocation factor (0.5)
allows for about 50% of quanta being absorbed by PSII
(Melis 1989) and the leaf absorptance factor (0.84) is the
mean absorptance factor for a wide variety of plants
Equations for Non Photochemical Quenching are measures
of energy absorbed by the photosynthetic apparatus that is
not lost as fluorescence or used in photosynthetic electron
transport but lost as low-grade heat (Genty et al. 1989; van
Kooten and Snel 1990; Ralph and Gademann 2005) and are
calculated automatically by the WINCONTROL software.
The parameter qN requires measurements of Fo, which is
the fluorescence signal from the background measuring
light and valid values range only from 0 to 1. The
expression of non-photochemical quenching that does not
require a measure of Fo is given the symbol NPQ and
ranges from 0 to values greater than 1 (usually up to no
more than about 4). Both qN and NPQ can usually be
described by simple exponential saturation curves (Ralph
and Gademann 2005; Ritchie 2008; Ritchie and Bunthawin
2010) and qNmax and NPQmax can be calculated by nonlinear square curve fitting. The iterative least squares
routines used in the present study were implemented in
Microsoft EXCEL and fitted using the SOLVER Add-in
package. The EXCEL files are available upon request from
the senior author.
Acknowledgements This project was funded as part of an Endeavour
Executive Award (1324_2009) awarded to the author by the Department
of Education and Training, Commonwealth of Australia. The author
wishes to thank Prince Songkla University—Phuket for providing
facilities for the project and Prof Puwadon Butrat for his interest in the
project.
Tropical Plant Biol.
References
Björkman O, Demmig B (1987) Photon yield of O2 evolution and
chlorophyll fluorescence characteristics at 77K among vascular
plants of diverse origins. Planta 170:489–504
Broetto F, Duarte HM, Lüttge U (2007) Responses of chlorophyll
fluorescence parameters of the facultative halophyte and C3–
CAM intermediate species Mesembryanthemum crystallinum to
salinity and high irradiance stress. J Plant Physiol 164:904–912
Chen L-S, Lin Q, Nose A (2002) A comparative study on diurnal
changes in metabolite levels in the leaves of three crassulacean
acid metabolism (CAM) species, Ananas comosus, Kalanchoë
daigremontiana and K. pinnata. J Exp Bot 367:341–350
Cote FX, Andre M, Folliot M, Massimino D, Daquenet A (1989) CO2
and O2 exchanges in the CAM plant Ananas comosus (L.) Merr.
determination of total and malate-decarboxylation-dependent
C02-assimilation rates; study of light O2-uptake. Plant Physiol
89:61–68
Cushman JC, Borland AM (2002) Induction of crassulacean acid
metabolism by water limitation. Plant Cell Environ 25:297–312
Dodd AN, Borland AM, Haslam RP, Griffiths H, Maxwell K (2002)
Crassulacean acid metabolism: plastic fantastic. J Exp Bot
369:569–580
Duarte HM, Lüttge U (2007) Circadian rythmicity. In: Lüttge U (ed)
Clusia: a woody Neotropical genus of remarkable plasticity and
diversity, chapter 11, 2nd edn. Springer-Verlag, Berlin, pp 245–
256
Franco AC, Haag-Kerwer A, Herzog B, Grams TEE, Ball E, de
Mattos EA, Scarano FR, Barreto S, Garcia MA, Mantovani A,
Lüttge U (1996) The effect of light levels on daily patterns of
chlorophyll fluorescence and organic acid accumulation in the
tropical CAM tree Clusia hilariana. Trees 10:359–365
Franco AC, Herzog B, Hübner C, de Mattos EA, Scarano FR, Ball E,
Lüttge U (1999) Diurnal changes in chlorophyll a fluorescence,
CO2-exchange and organic acid decarboxylation in the tropical
CAM tree Clusia hilariana. Tree Physiol 19:635–644
Franklin LA, Badger MR (2001) A comparison of photosynthetic
electron transport rates in macroalgae measured by pulse
amplitude modulated chlorophyll fluorometry and mass spectroscopy. J Phycol 37:756–767
Genty B, Briantais JM, Baker NR (1989) The relationship between the
quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–
92
Griffiths H, Helliker B, Roberts A, Haslam RP, Girnus J, Robe WE,
Borland AM, Maxwell K (2002) Regulation of RUBISCO in
crassulacean acid metabolism plants: better late than never. Funct
Plant Biol 29:689–696
Griffiths H, Robe WE, Girnus J, Maxwell K (2008) Leaf succulence
determines the interplay between carboxylase systems and light
use during Crassulacean acid metabolism in Kalanchoë species. J
Exp Bot 59:1851–1861
Gueymard C (1995) SMARTS, A Simple Model of the Atmospheric
Radiative Transfer of Sunshine: algorithms and performance
assessment. Professional paper FSEC-PF-270-95, Florida Solar
Energy Center, 1679 Clearlake Rd., Cocoa, FL, USA 32922
Gueymard CA, Myers D, Emery K (2002) Proposed reference
irradiance spectra for solar energy systems testing. Sol Energy
73:443–467
Hartsock TL, Nobel PS (1976) Watering converts a CAM plant to
daytime CO2 uptake. Nature 262:574–576
Herppich WB, Midgley GF, Herppich M, Tüffers A, Veste M, von
Willert DJ (1998) Interactive effects of photon fluence rates and
drought on CAM-cycling in Delosperma tradescantioides
(Mesembryanthemaceae). Physiol Plant 102:148–154
Hew CS, Khoo SL (1980) Photosynthesis of young orchid seedlings.
New Phytol 86:349–357
Hew CS, Yong JWH (2004) The physiology of tropical orchids in
relation to the industry, 2nd edn. World Scientific, Singapore
Holt NE, Fleming GR, Niyogi NK (2004) Toward an understanding of
the mechanism of non-photochemical quenching in green plants.
Biochemistry 43:8281–8289
Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol 42:313–349
Lüttge U (2004) Ecophysiology of Crassulacean Acid Metabolism
(CAM). Ann Bot 93:629–652
Lüttge U (2007) Photosynthesis. In: Lüttge U (ed) Clusia: a woody
Neotropical genus of remarkable plasticity and diversity, chapter
8, 2nd edn. Springer-Verlag, Berlin, pp 135–186
Malda G, Backhaus RA, Martin C (1999) Alterations in growth and
crassulacean acid metabolism (CAM) activity of in vitro cultured
cactus. Plant Cell Tiss Org 58:1–9
Martin CE, Tüffers A, Herppich WB, von Willert DJ (1999)
Utilization and dissipation of absorbed light energy in the
epiphytic crassulacean acid metabolism Bromeliad Tillandsia
ionantha. Int J Plant Sci 160:307–313
Martyn AJ, Larkum AWD, McConchie R, Offord CA (2008) Photoinhibition and changes in pigments associated with bract
browning in waratahs (Telopea spp., Proteaceae). J Hortic Sci
Biotechnol 83:367–373
Maxwell K, Badger MR, Osmond CB (1998) A comparison of CO2
and O2 exchange patterns and the relationship with chlorophyll
fluorescence during photosynthesis in C3 and CAM plants. Aust
J Plant Physiol 25:45–52
Mc Williams EL (1970) Comparative rates of dark CO2 uptake and
acidification in the Bromeliaceae, Orchidaceae, and Euphorbiaceae. Bot Gaz 131:285–290
Medina E, Popp M, Olivares E, Janett H-P, Lüttge U (1993) Daily
fluctuations of titratable acidity, content of organic acids (malate
and citrate) and soluble sugars of varieties and wild relatives of
Ananas comosus L. growing under natural tropical conditions.
Plant Cell Environ 16:55–63
Melis A (1989) Spectroscopic methods in photosynthesis: photosystem stoichiometry and chlorophyll antenna size. Phil Trans R Soc
Lond B 323:397–409
Nievola CC, Kraus JE, Freschi L, Souza BM, Mercier H (2005)
Temperature determines the occurrence of CAM or C3 photosynthesis in pineapple plantlets grown in vitro. In Vitro Cell Dev
Biol-Plant 41:832–837
O’Neill PM, Shanahan JF, Schepers JS (2006) Use of chlorophyll
fluorescence assessments to differentiate corn hybrid response to
variable water conditions. Crop Sci 46:681–687
Osmond CB (1978) Crassulacean acid metabolism: curiosity in
context. Annu Rev Plant Physiol 29:379–414
Patel A, Ting IP (1987) Relationship between respiration and CAMCycling in Peperomia camptotricha. Plant Physiol 84:640–642
Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool to
assess photosynthetic activity. Aquat Bot 82:222–237
Rascher U, Liebig M, Lüttge U (2000) Evaluation of instant lightresponse curves of chlorophyll fluorescence parameters obtained
with a portable chlorophyll fluorometer on site in the field. Plant
Cell Environ 23:1398–1405
Ritchie RJ (2006) Consistent sets of spectrophotometric equations for
acetone, methanol and ethanol solvents. Photosynth Res 89:27–
41
Ritchie RJ (2008) Fitting light saturation curves measured using PAM
fluorometry. Photosynth Res 96:201–215
Ritchie RJ, Bunthawin S (2010) The use of PAM (Pulse Amplitude
Modulation) fluorometry to measure photosynthesis in a CAM
orchid, Dendrobium spp. (D. ‘Viravuth’ Pink). Int J Plant Sci
171:575–585
Tropical Plant Biol.
Schreiber U, Bilger W, Neubauer C (1995a) Chlorophyll fluorescence
as a non-intrusive indicator for rapid assessment of in vivo
photosynthesis. In: Schulze ED, Caldwell MM (eds) Ecophysiology of photosynthesis. Ecological studies, vol. 100. SpringerVerlag, Berlin, pp 49–70
Schreiber U, Endo T, Mi H-L, Asada K (1995b) Quenching analysis
of chlorophyll fluorescence by the saturation pulse method:
particular aspects relating to the study of eukaryotic algae and
cyanobacteria. Plant Cell Physiol 36:873–882
Silvera K, Santiago LS, Cushman JC, Winter K (2009) Crassulacean
acid metabolism and epiphytism linked to adaptive radiations in
the Orchidaceae. Plant Physiol 149:1838–1847
Sipes DL, Ting IP (1985) Crassulacean acid metabolism and
crassulacean acid metabolism modifications in Peperomia camptotricha. Plant Physiol 77:59–63
Slesak I, Karpinska B, Surówka E, Miszalski Z, Karpinski S (2003)
Redox changes in the chloroplast and hydrogen peroxide are
essential for regulation of C3–CAM transition and photooxidative stress responses in the facultative CAM plant Mesembryanthemum crystallinum L. Plant Cell Physiol 44:573–581
Taize L, Zeiger E (2002) Plant physiology. Sinauer Associates,
Sunderland
Ting IP (1985) Crassulacean acid metabolism. Annu Rev Plant
Physiol 36:595–622
van Kooten O, Snel JFH (1990) The use of chlorophyll fluorescence
nomenclature in plant stress physiology. Photosynth Res 25:147–
150
Vovides AP, Etherington JR, Dresser PQ, Groenhof A, Iglesias C,
Flores J, Lüttge U (2002) CAM-cycling in the cycad Dioon edule
Lindl. in its natural tropical deciduous forest habitat in central
Veracruz, Mexico. Bot J Linn Soc 138:155–162
White AJ, Critchley C (1999) Rapid light curves: a new fluorescence
method to assess the state of the photosynthetic apparatus.
Photosynth Res 59:63–72
Winter K, Holtum JAM (2002) How closely do the δ 13C values of
Crassulacean Acid Metabolism plants reflect the proportion of
CO2 fixed during day and night? Plant Physiol 129:1843–1851
Winter K, Garcia M, Holtum JAM (2008) On the nature of facultative
and constitutive CAM: environmental and developmental control
of CAM expression during early growth of Clusia, Kalanchoë,
and Opuntia. J Exp Bot 59:1829–1840
Zhu J, Goldstein G, Bartholomew DP (1999) Gas exchange and
carbon isotope composition of Ananas comosus in response to
elevated CO2 and temperature. Plant Cell Environ 22:999–1007