Temperature Determines the Occurrence of CAM or C3 Photosynthesis in Pineapple Plantlets

Grown In vitro
Author(s): C. C. Nievola, J. E. Kraus, L. Freschi, B. M. Souza, H. Mercier
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 41, No. 6 (Nov. - Dec., 2005), pp.
832-837
Published by: Society for In Vitro Biology
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In Vitro Cell. Dev. Biol.-Plant 41:832-837, November-December 2005 DOI: 10.1079/IVP2005694
C 2005 Society for In Vitro Biology
1054-5476/05 $18.00+0.00

TEMPERATURE DETERMINES THE OCCURRENCE OF CAM OR C3 PHOTOSYNTHESIS

IN PINEAPPLE PLANTLETS GROWN IN VITRO

C. C. NIEVOLA,
J. E. KRAUS,L. FRESCHI,
B. M.SOUZA,ANDH. MERCIER*

Departamentode Botanica, Institutode Biociencias,Universidadede S&oPaulo, Caixa Postal 11461, 05422-970 Sdo Paulo, SP, Brazil

(Received23 September
2004;accepted20 May2005;editorP. Lakshmanan)

SUMMARY

Ananas comosus(L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperatureregimes
developed as CAM or as C3 plants. The plants used in this study were developed from the lateral buds of the nodal
etiolated stem explants culturedon Murashigeand Skoog mediumfor 3 mo. The cultureswere maintainedunder a 16-h
photoperiodfor differentthermoperiods.With 280C light/15?Cdarkthermoperiod,as comparedwith constant280C light
and dark,pineapple plants had a succulence index two times greater,and also a greaternocturnaltitratableacidity and
phosphoenolpyruvatecarboxylase(PEPCase)activity, indicating CAM-typephotosynthesis.The highest abscisic acid
(ABA)level occurredduringthe light period,8 h priorto maximumPEPCaseactivity,while the indole-3-aceticacid (IAA)
peak was found duringthe dark period, coinciding with the time of highest PEPCaseactivity. These plants were also
smallerwith thickerleaves and fewerroots,but had greaterdryweight.Theirleaves showedhistologicalcharacteristicsof
CAMplants, such as the presence of greaterquantitiesof chlorenchymaand hypoderm.In addition,their vascularsystem
was more conspicuous.In contrast,under constanttemperature(280Clight/dark)plants showedlittle succulence in the
leaves. Therewas no significantacid oscillationand diurnalvariationin PEPCaseactivityin these plants, suggestingthe
occurrenceof C3 photosynthesis.Also, no diurnalvariationin ABA and IAA contentswas observed.The results of this
studyclearlyindicatea role fortemperaturein determiningthe type of carbonfixationpathwayin in vitrogrownpineapple.
Evidence that ABA and IAA participatein CAMsignaling is provided.

Key words:Ananas comosus;Bromeliaceae;thermoperiod;phosphoenolpyruvatecarboxylase;abscisic acid; auxin; leaf

histology.

INTRODUCTION C3 to CAMshifts are stronglyenhancedwhen day/nighttemperature

Temperatureis the most importantfactor in the cultivation of
pineappleplants,which determinespineapple'sareas of cultivation
in differentpartsof the world(Bartholomewand Malkzieux,1994).
The optimalrange of temperaturesfor root and leaf growthis 29-
320C (Bartholomewand Kadzimin, 1977). Nevertheless, this
bromeliadis found in places with lower temperatures,demonstrat-
ing the environmentalplasticity of the plant (Bartholomewand
Malkzieux, 1994). Variations in temperatureinfluence rates of
growth and development, and also the internal and external
morphologicalstructureof Ananas comosusvar. SmoothCayenne
plants cultivated in the greenhouse (Bartholomewand Kadzimin,
1977).
The pineapple species studied have been shown to be
constitutive CAM plants (Borland and Griffiths, 1989; Medina
et al., 1994; Sayed, 2001). However,in other species, temperature
can determine CAM performance due to individual enzyme
characteristicsand tonoplastpermeability(Liittge,2004). Diurnal
changeswith lowernight-timeand higherdaytimetemperaturesare
favorablefor CAM (Liittge,2004). In the C3/CAMClusia minor,
*Authorto whomcorrespondenceshouldbe addressed:Emailhmercier@
usp.br
differencesare increased, while they are less sensitive to absolute
temperatures(Haag-Kerweret al., 1992).
Given the variety of environmentalfactors implicated in the
regulationof CAMexpression,substantialchanges in the contents
of endogenoushormoneshave always been observed (Taybiet al.,
2002). For instance, in Kalancho"blossfeldianaCAM plants an
increase in endogenouslevels of abscisic acid (ABA)preceded an
increase in phosphoenolpyruvatecarboxylase (PEPCase) activity
(Taybiet al., 1995).
In bromeliads, the use of in vitro culture for physiological
researchis rare.However,the techniquehas several advantagesfor
the controlof growthconditions,such as nutrients,temperature,etc.
In our laboratory,we have shown that bromeliadswith different
carbon fixing systems (CAM or C3) had their maximumnitrate
reductaseactivity at differenttimes of the 24 h cycle. For instance,
in the constitutiveCAMbromeliad,Tillandsiapohliana, cultivated
in vitro,leaf nitratereductionoccurredonly duringthe night, while
for the in vitrogrowthC3species, Vrieseafosteriana,the highestleaf
nitrate reductase activity was observed during the light period
(Nievola et al., 2001).
Little is knownaboutthe regulatoryaspects of carbonfixationin
pineapple. Adult, nonfloweringplants of cultivated pineapple are
considered to be constitutive CAM plants (Medina et al., 1994);

832
 CAM/C3PINEAPPLEPLANTS
FACULTATIVE
however, no data exist regarding photosynthesis in young
pineapples cultivated in vitro under controlled temperature. In this
study, we have investigated the role of temperature on the
carboxylation process in young pineapple plants cultivated in vitro.
We compared carbon fixation in plants grown at a constant
temperature of 280C with those grown under a thermoperiod of 28TC
during the light and 150C during the dark period. In addition, leaf
acidity levels, PEPCase activity, endogenous hormonal levels
(ABA and auxin), succulence index, and leaf histology were
compared between the two treatments.
MATERIALS AND METHODS
Pineapple sources. A. comosus(L.) Merr.var. SmoothCayenneplants
were obtained from a clone cultivated in vitro in the Laboratoryof Plant
Physiologyat the Universityof SIo Paulo, SP, Brazil. Parent plants were
cultivated in Murashigeand Skoog (1962) medium, pH 5.8, with agar
concentrationof 6 g 1-, 3% sucrose,and0.1 mg1-1 thiamin(growthmedium)
for 6 mo. The culture flasks were incubated at 25 ? 20C under 16h light
providedby cool-whitefluorescentlampsat 55 p.molm-2 s-1. Afterremoving
leaves, these plantsweretransferredto a new growthmediumand maintained
in the darkfor2 mo.to cause etiolation.Nodalexplantsof etiolatedplantswere
used forfurtherexperiments.A newplantwasdevelopedfromthelateralbudof
the nodal explant.
Two experimental conditions, defined as follows, were maintained:
(a) alternatingtemperatures(AT) of 28 and 150C duringthe light and dark
regime, respectively; (b) constant temperature(CT) of 280C maintained
duringbothlight and darkperiods.Each experimentaltreatmentconsistedof
10 replicates and each replicate consisted of 10 explantsinoculatedinto a
125 ml Erlenmeyerflask with 50ml of growth medium. The incubation
chambershad the photoperioddescribed above and temperaturevariation
was ? 0.20C. The cultures were maintainedfor 3 mo., after which both
treatmentswere analyzed.
All data presented here were obtained from a single of experiment.
However,the results of the second experimentconfirmedthose of the first
determination.
Growth analysis. Numberof leaves and roots, as well as shoot and
root lengths, were determined.Fresh and dry weights of shoots and roots
were also measured.For dry weight,the plant materialwas maintainedin a
dryingoven at 600Cfor 72 h. In all comparisons,n = 10 for bothtreatments.
Succulence index. The ratio between the quantity of water in the
leaves (freshweight/dryweight)and chlorophyllconcentrationprovidedthe
measure of leaf succulence (Kluge and Ting, 1978). Chlorophyllwas
measuredfollowingthe procedureof Arnon(1949). Fresh leaf tissue (0.5 g)
was homogenized with 5ml of cold 80% (v/v) aqueous acetone. The
homogenatewas filtered (Whatmanpaper, number1) and its solid residue
was washed five times with 4ml of cold 80% acetone. Chlorophyll
concentrationof the filtratewas measuredspectrophotometrically following
Hendryand Price (1993).
Amount of titratable acidity. The amountof titratableacidity was
analyzedfollowingOta and Yamamoto(1991) with some modifications.Leaf
samples (1 g) from each treatmentwere collected every 2 h for 24 h, after
which time samples were fragmentedand boiled in 30 ml distilled waterfor
5 min, followedby macerationfor 5 min in the remainingwater.Macerated
materialsof each samplewerefiltered(Whatmanpaper,number1). The final
volume of the filtratewas made up to 40 ml with waterand titratedto pH 7
with 0.02 N NaOH.The volumeof NaOHrequiredexpendedfor each sample
duringtitrationwas used to calculate acidity, expressedin p.Mof H+g of
fresh weight.
PEPCase extraction and assay. Leaves fromboth treatmentswere
harvestedevery2 h for24 h. Duringthe darkperiods,sampleswerecollected
under green light. Samples (0.5 g) were rinsed briefly with distilled water,
blottedwithtissue paper,placed in liquid nitrogenand storedat - 700Cuntil
used. All steps in the extractionorprocessingof leaf-proteinswerecarriedout
at 4?C.Extractionand assay of PEPCase(EC 4.1.1.31) followedthe method
described by Leport et al. (1996) with modifications.Leaf material was
homogenizedin 5 ml extractionbuffercontaining100 mMTris-HC1(pH8.0),
10% glycerol, (v/v), 1 mM EDTA, and 1 mM dithiothreitol(DTT). The
homogenatewas centrifugedfor 30 min at 11000 X g. The protein in the
833
homogenatewas precipitatedby the additionof ultrapure(NH4)2SO4 at 70%
saturationand kept at 40C overnight. After centrifugationfor 10min at
15 000 x g, the pelleted proteinsamplewas resuspendedin 2.5 ml extraction
bufferand desaltedagainstthe samebufferusinga 10 ml columnof Sephadex
G25. The desalted extract was used for the PEPCase assay and for the
determinationof soluble proteincontent(Bradford,1976). PEPCaseactivity
wasassayedin a reactioncontaining100 mMTris-HCl (pH8.0), 1 mMEDTA,
50 pM DTT, 10mM MgC12, 10 mM NaHCO3, 0.4 mM NADH, 3 mM
phosphoenolpyruvate (PEP), and 11.4 units of malate dehydrogenase.The
reaction was initiated by adding 800 p1lof the extract and the change in
absorbanceat 340 nm was measuredfor 2 min at 400C.Specific activitywas
definedas pmol NADHconsumedper mg of soluble proteinin the extract.
Leaf histology. Leavesfromthe fourthnode fromthe shoot apex were
used forthe histologicalanalysis.Samples(0.5 cm)fromthe middleportionof
the leaf laminawerefixedfor4 h at roomtemperaturein Karnovsky'ssolution
(Karnovsky,1965) modifiedwith the additionof glutaraldehyde/paraformal-
dehyde (4:1, v/v) in 0.1 M phosphatebuffer (pH 7.2). Fixed materialwas
dehydratedin a gradedethanolseries andembeddedin historesin.Transverse
sections(6 pm)weremountedon glass slides andstainedwith0.05%toluidine
blue O in 0.1 M phosphatebuffer,pH 6.8 (O'Brienet al., 1965).
Analysis of hormones. The levels of ABA and indole-3-acetic acid
(IAA) in the tissues were determined using high-performanceliquid
chromatography(HPLC) and an enzyme-linked immunosorbentassay
(ELISA)(Maldineyet al., 1986; Endreset al., 2002). For the analyses, leaf
samples(1 g) fromeach treatmentwerecollectedevery4 h for24 h, afterwhich
time sampleswere maceredwithliquid nitrogen,and 10 ml of cold 80% (v/v)
aqueousmethanol,which contained146 pM butylhydroxytoluene to prevent
oxidation,was added.Each maceratewas supplementedwithc. 100 000 dpm
of [3H]ABA(48Cimmol-1) and [3H]IAA (25Cimmol-1) as an internal
standard,then shaken for 60 h at 4OC.The extractswere filtered (0.45 and
0.2 pVm mesh),passedthrougha Sep-PackC18cartridge,and elutedwith80%
(v/v) aqueousmethanol.The resultingeluate was evaporatedundervacuum,
and then diluted with 200 p1 of 0.2% formic acid. HPLCwas used for the
separationof ABAandIAAfromthe pre-purifiedextracts.AuthenticABAand
IAA standardswere run with this system to establish their separationand
relative retentiontime. The hormoneswere eluted from the reverse-phase
HPLCcolumnC18 (PrepNova-PackHR C18,Waters?)with a 0.2% formic
acid (A)/methanol(B) gradientat a flowrate of 3 ml min- . Forthe gradient,
the followingprotocolwas used (%B):0-10min, 18%; 10-11min, 25%;
11- 65 min,33%;65-70 min,40%.Absorbanceof the effluentwasmonitored
at 270 nm.
IAA and ABA fractions were methylatedwith ethereal diazomethane
(Cohen, 1984) and analyzedwith polyclonalantibodiesagainst methylated
IAA, and monoclonalantibodiesagainstmethylatedABA, respectively.The
level of each hormonewas measuredfourtimes per sample,the resultsbeing
correctedfor recovery.Calculationswere madeby referenceto a calibration
curveestablishedon each microtitration plate witha fourth-orderpolynomial
regressionobtainedfromfourexperimentalstandardscurves(Maldineyet al.,
1986).
RESULTS
Growth patterns. After 3 mo. of in vitro culture, visible growth
differences were noticed in plants grown under different treatments
(Table 1, Fig. 1). AT plants were shorter than CT plants, yet dry weight
of the stem and root systems were greater in AT plants (Table 1). The
number of leaves did not vary; however, AT plants had thicker leaves
and fewer roots (Fig. 1A) as compared to CT plants (Fig. 1B).
Leaf structure and succulence index. Internal differences in
leaves among treatments were also clear (Fig. 2A, B). In both
treatments, the epidermis was one-layered with the adaxial cells being
larger and with a thicker cuticle than the abaxial cells. Differences
were in that leaves of AT plants had a mesophyll with a greater number
of cell layers, represented by chlorenchyma (Fig. 2A, B). A mechanical
hypoderm was only evident in AT leaves (Fig. 2A). The water-storing
parenchyma was similar in both treatments. The vascular system was
more conspicuous in AT plants than in CT plants (Fig. 2C, D). AT
plants had a greater succulence index (1.157) than CT plants (0.692).
834 NIEVOLAET AL.

TABLE1 AT plants showed greater IAA concentation during the dark

period (Table 2). The greatest level of this hormone in AT
GROWTHPARAMETERS(MEAN + SE) IN A. COMOSUSGROWN plants was found in the fourth hour of the dark period
IN VITROFOR 3 MO. UNDERALTERNATINGTEMPERATURE(AT)
(88.4 pmol g-l FW), coinciding with the highest PEPCase activity
(280CLIGHT/150CDARK)AND CONSTANTTEMPERATURE(CT)
TREATMENTS
(280CLIGHT/DARK) (Fig. 3B). In contrast, the IAA levels in CT plants showed no
significant diurnal variation.
Growthparameters AT CT

Plant height*(cm) 4.26 + 0.71 9.98 + 1.23 DiscussIoN

Numberof leaves 7.63 + 0.15 7.53 ? 0.25
Shootfresh weight*(g) 1.205 + 0.025 0.880 + 0.069 A. comosus and other species in this genus are normally
Shoot dry weight*(g) 0.108 ? 0.04 0.053 + 0.001 considered constitutive CAM plants (Medina et al., 1991, 1994:
Numberof roots 1.18 + 0.18 1.72 + 0.14
Martin, 1994; Zhu et al., 1997; Sayed, 2001). However, we
Root length (cm) 2.52 ? 0.39 1.8 ? 0.20
Root fresh weight (g) 0.10 + 0.001 0.08 + 0.001 demonstrate that plants from the same clone, cultivated in vitro, at
Root dry weight*(g) 0.019 + 0.0001 0.013 + 0.0001 constant temperature (280C, day and night) showed characteristics
of C3 photosynthesis. Plants grown in varying temperature (280C
*Significant,t-test, P < 0.05. light/150C dark) regimes, however, showed CAM photosynthesis.
While temperature has been shown to influence CO2 exchange in
adult pineapple grown in the greenhouse (Neales et al., 1980), this
Titratable acidity and PEPCase activity. AT plants showed is the first study to report the influence of temperature on carbon
greater titratable acidity during the dark period (Fig. 3A) than CT fixation in young pineapple plants.
ones. The greatest organic acids level in AT plants was found at the PEPCase activity and titratable acidity levels were both greater
end of the dark period (4 h). In the following hours of light, titratable in AT plants in the dark period. These two parameters are
acidity in AT plants decreased rapidly, reaching CT levels 6 h after considered important during the diurnal cycle and to determine
onset of illumination. Afterwards, titratable acidity in AT plants was whether a plant uses CAM (Chollet et al., 1996; Leport et al., 1996;
constant, similar to that in CT plants during daytime. Nimmo, 2000). Diurnal regulation of PEPCase in Crassula argentea
PEPCase activity was also greater in AT plants and its greatest (a CAM plant) is related to a declining nocturnal temperature, with
activity level was found in the fourth hour of the dark period a concomitant increase in the accumulation of malate in the
(Fig. 3B). Thereafter it declined constantly to low levels during the vacuoles (Chardot and Wedding, 1992). Temperature can influence
entire light period. In contrast, CT plants maintained constant cell membrane permeability, which is very important for organic
PEPCase activity during the dark and light periods. acid compartmentation (Friemert et al., 1988). In A. comosus grown
Hormonal analysis. Endogenous ABA levels in AT plants in vitro at 280C constant temperature (CT plants), it is likely that
varied during the diurnal cycle (Table 2). CT plants, on the other the capacity to accumulate acid in the vacuole decreases, following
hand, showed no significant diurnal changes in hormone content. the C3 pattern of photosynthesis. Increasing temperature can cause
The greatest ABA level in AT plants was found during the light a rapid decrease and disappearance of mRNA for the PEPCase
period (20.6pmolg-1 FW), occurring 8h prior to maximum kinase (Borland et al., 1999), resulting a reduction of PEPCase in
PEPCase activity (Fig. 3B). the active form. The increase in temperature is thought to be

FIG. 1. A. comosus morphologywhen grown in vitro for 3 mo. A, Alternatingtemperature(280C light/150C dark). B, Constant
temperature(280Clight/dark).For both experiments,a 16 h photoperiodwas maintained.Bars = 1 cm.
 CAM/C3PINEAPPLEPLANTS
FACULTATIVE 835

l?ljlll
WD8

A ri

4PI Aw

s .11 *
:,:-:WP

FIG.2. Leafhistologyof A. comosusgrownin vitrofor3 mo. Transversesections.A, C, Alternatingtemperature(280Clight/150Cdark).

B, D, Constanttemperature(280Clight/dark).(c, cuticle; ch, chlorenchyma;e, epidermis;h, mechanicalhypoderm;pf, pericycle fibers;
ph, phloem; s, stomata; vs, vascular system; xy, xylem; wp, water-storing parenchyma.) Bars = 100 [pm.

30
(A)
] TABLE2
--o-- AT
25- --- CT ABA AND IAA CONCENTRATIONDURINGA 24H CYCLEIN
A. COMOSUSGROWNIN VITROFOR 3 MO. UNDERALTERNATING
20 TEMPERATUREAT (280CLIGHT/150CDARK)AND CONSTANT
TEMPERATURECT (280CLIGHT/DARK)TREATMENTS
15 ABA IAA

10 Time (h) AT CT AT CT
-
-_ 20.00 6.8 ? 0.9 5.6 + 0.1 35.0 + 1.5 2.2 + 0.1
5- 24.00 12.9 ? 0.8 8.0 + 0.2 88.4 + 13.3 5.2 ? 0.5
04.00 13.1 + 1.2 9.0 + 0.2 33.5 + 1.4 3.9 + 0.3
0 08.00 16.9 + 1.0 6.3 + 0.2 5.0 + 1.2 1.1 + 0.2
20 22 24 2 4 6 8 10 12 14 16 18 20 12.00 18.3 + 0.4 5.8 + 0.4 6.8 + 1.7 2.0 + 0.2
Time (h) 16.00 20.6 ? 0.8 5.2 + 0.1 12.6 + 1.3 3.1 ? 0.2
(B)
Samples from dark periods are indicated in boldface. All data are in
? AT pmolg- FW. Values representmeans + SE, n = 4.
5 0.20 --- CT
Furthermore, at transcription level, CAM may be regulated
-on 0.15 through ABA signaling. External application of ABA has been
shown to induce CAM in Portulacaria afra (Ting, 1981), in
Mesembryanthemum crystallinum (Chu et al., 1990), and in
o 0.10
Kalanchoi" blossfeldiana (Taybi et al., 1995). The correlation
between ABA and induction of CAM is also supported by the
osmotic-stress induction of CAM in Peperomia camptotricha (Sipes
0.05
and Ting, 1985) and M. crystallinum (Thomas et al., 1992). Taybi
et al. (1995) reported an enhancement of PEPC activity in leaves
0.00 isolated from K. bloosfeldiana after a lag of 7 h from application of
20 22 24 2 4 6 8 10 12 14 16 18 20 external ABA via the petiole, whereas the controls remained
Time (h) unchanged. More recently, Taybi and Cushman (1999) provided
evidence in the facultative CAM plant M. crystallinum, that
FIG.3. Titratableacidity(A)and PEPCaseactivity(B) assaysduring24 h
in Ananas comosusgrownin vitro under alternatingtemperature(AT) and transcript abundance of Ppcl, a gene encoding for the CAM-specific
constanttemperature(CT)for 3 mo. The blackbar indicateshoursof dark. isoform of PEPCase, increased rapidly in response to exogenous
ABA treatment.
In the present work, we have demonstrated that pineapple plants
associated with an efflux of malate from the vacuoles (Friemert et al., grown in AT presented a gradual increase in the level of ABA
1988), and this cytosolic malate can be a negative feedback during the light period, with the highest accumulation at the end of
regulator (inhibitor) of PEPCase kinase expression (Nimmo, 2000). the day. In contrast, the highest auxin content in AT plants
836 NIEVOLAET AL.
occurred during the dark period. However, in CT plants, no diurnal
variation in ABA and IAA levels was observed. The highest
endogenous ABA level in AT plants was found 8 h prior to
maximum PEPCase activity, while the IAA peak coincided with the
time of its highest activity, thus suggesting that PEPCase
transcription in these plants could be controlled by ABA, and
enzyme phosphorylation may be mediated by IAA. Protein
phosphorylation is the key to the regulation of many proteins. In
the case of PEPCase, the phosphorylated form is considered the
active form (Nimmo, 2000). It has been shown that auxin stimulated
protein phosphorylation in maize embryonic axes treated for 3 h
with [32P] phosphate, increasing 49-62% in total 32P incorporation
(Perez et al., 1987). More recently, enhanced S6 ribosomal protein
phosphorylation on the 40S ribosomal subunit was found in IAA
treated maize axes. It was proposed that by this mechanism auxin
regulated the synthesis of specific proteins in maize tissues
(Beltriin-Pefia et al., 2002).
Our results suggest that temperature modulates the photosyn-
thetic enzyme machinery in C3/CAM plants by regulating the
expression of specific genes, and that ABA and IAA could be
involved in the transmission of the external signal to elicit gene
expression and PEPCase phosphorylation, converting this enzyme
into a catalytically active form during the dark period.
We also show morphological differences associated with the
growth pattern that agree with the biochemical assays. AT plants
were smaller than CT ones, yet the dry mass was greater, as was also
observed in a study of adult A. comosus plants cultivated in a
greenhouse, which showed that plants grown at 340C light/18?C
dark had greater dry mass than those grown at a constant 300C
(Bartholomew and Malhzieux, 1994). Roots in AT plants were fewer
when compared to CT plants. CAM plants present a reduced root/
shoot ratio (Osmond et al., 1999).
The succulence index (the ratio between the quantity of water and
chlorophyll) was also greater in AT plants. CAM plants have relatively
large vacuoles and large water to chlorophyll ratio (Kluge and Ting,
1978); this is the opposite of what was observed in C3 plants.
Histological analysis showed that leaves from AT plants had the
characteristics of CAM. Leaves were thicker in AT plants, as a
result of more chlorenchyma. The typical CAM photosynthetic cells
are large, thin walled, and with a narrow cytoplasm containing
chloroplasts around the large vacuole (Kluge and Ting, 1978; Fahn
and Cutler, 1992), as observed in AT leaves. According to Teeri
et al. (1981), there is the a correlation between the degree of leaf
thickness and the amount of titratable nocturnal increase in acidity.
Leaves in AT plants also presented a mechanical hypoderm, a
common structure in adult bromeliad leaves (Tomlinson, 1969). The
presence of mechanical hypoderm and schlerenchyma was needed
to support thicker leaves.
In summary, we demonstrated that in vitro grown pineapple
plants could function as CAM or C3 plants, depending upon the
temperature regimes used for culture. By unraveling the
temperature-dependent occurrence of CAM and C3 pathways, this
study established an experimental model for future physiological
studies of CAM regulation by temperature.
ACKNOWLEDGMENTS
We wish to thank Dr Bruno Sotta (Universityof Paris-VI) for kindly
donating the antibodies. We are also grateful to CNPq (process nos
301776/83 and 300732/93-7), FAPESP (03/02 677-6) and CAPESfor the
scholarshipawardedto C. C. N.
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