Research Article Allocation of Secondary...

Research ArticleAllocation of Secondary Metabolites Photosynthetic Capacityand Antioxidant Activity of Kacip Fatimah (Labisia pumilaBenth) in Response to CO2 and Light Intensity

Mohd Hafiz Ibrahim1 Hawa Z E Jaafar2 Ehsan Karimi2 and Ali Ghasemzadeh2

1 Department of Biology Faculty of Science Universiti Putra Malaysia 43400 Serdang Selangor Malaysia2 Department of Crop Science Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Mohd Hafiz Ibrahim mhafizphdyahoocom

Received 4 July 2013 Accepted 26 December 2013 Published 10 February 2014

Academic Editors H A Alegria and R Michalski

Copyright copy 2014 Mohd Hafiz Ibrahim et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A split plot 3 by 4 experiment was designed to investigate and distinguish the relationships among production of secondarymetabolites soluble sugar phenylalanine ammonia lyase (PAL EC 4315) activity leaf gas exchange chlorophyll contentantioxidant activity (DPPH) and lipid peroxidation under three levels of CO

2(400 800 and 1200120583molmol) and four levels of

light intensity (225 500 625 and 900 120583molm2s) over 15 weeks in Labisia pumila The production of plant secondary metabolitessugar chlorophyll content antioxidant activity and malondialdehyde content was influenced by the interactions between CO

2and

irradiance The highest accumulation of secondary metabolites sugar maliondialdehyde and DPPH activity was observed underCO2at 1200120583molmol + light intensity at 225120583molm2s Meanwhile at 400 120583molmol CO

2+ 900 120583molm2s light intensity the

production of chlorophyll and maliondialdehyde content was the highest As CO2levels increased from 400 to 1200 120583molmol

the photosynthesis stomatal conductance 119891V119891119898 (maximum efficiency of photosystem II) and PAL activity were enhancedThe production of secondary metabolites displayed a significant negative relationship with maliondialdehyde indicating loweredoxidative stress under high CO

2and low irradiance improved the production of plant secondary metabolites that simultaneously

enhanced the antioxidant activity (DPPH) thus improving the medicinal value of Labisia pumila under this condition

1 Introduction

Labisia pumila locally known as Kacip Fatimah is a herba-ceous plant belonging to the family Myrsinaceae It is widelyscattered in the lowland and hilly rain forests of MalaysiaThailand IndochinaThe Philippines andNewGuinea [1ndash3]Three varieties of L pumila are identified in Malaysia clas-sified as L pumila var alata L pumila var pumila and Lpumila var lanceolata [2] It is usually used as a tonic drinkamong females and its indication for womenrsquos health may berelated to its phytoestrogen effects and having similar chem-ical structure to estrogen [4] The varieties most universallyutilized by the traditional healers are L pumila var alata andL pumila var pumila Kacip Fatimah has been generally usedby generations of Malay women by boiling it in water It isbelieved that the decoction drink can induce and facilitate

childbirth as well as being a postpartum medication to helpcontract the birth channel to tone the abdominal musclesand to regain body strength [5 6]The other uses of this herbinclude treatments for dysentery dysmenorrheal flatulenceand gonorrhea [7ndash10]

Recently it was found thatLabisia pumila extract containsflavanoids phenolics and various bioactive volatile com-pounds [11] These compounds include demethylbelamcan-daquinone B fatimahol dexyloprimulanin epoxyoleananeglycosides alkenated phenolics cerebroside glycerogalac-tolipids and lipids [12] These compounds have been iden-tified as natural antioxidants that reduce oxidative damageto the human body [13ndash15] The concentration of plantsecondary metabolites was found to be influenced by envi-ronmental conditions such as light intensity CO

2levels tem-

perature fertilization and biotic and abiotic factors which

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 360290 13 pageshttpdxdoiorg1011552014360290

2 The Scientific World Journal

can change the concentration of these active constituents[16 17] Changes in the accumulation pattern of secondarymetabolites due to environmental factors are however theresult of changes in both the rate of synthesis and therate of catabolism [18 19] The activity of L-phenylalanineammonia lyase (PAL EC 4315) determines the extent towhich phenylalanine is withdrawn from primarymetabolismand enters the general phenylpropanoid pathway [17] Latelyit was found that the enrichment of L pumila with highlevels of CO

2increased the secondary metabolite production

(phenolics and flavonoids) of this plant [20ndash25] Similarresults were observed in ginger (Zingiber officianale) [24]

Atmospheric CO2concentration has increased by about

a third over preindustrial levels and is continuing to increaseat around 04 per year By the year 2100 the atmosphericpartial pressure ofCO

2is projected to be 700 parts permillion

(ppm) or higher [26] Increased CO2concentration in the

air promotes growth and accelerates physiologic processesin young trees because it causes an increase in internalcarbon availability and changes partitioning of assimilatesamong plant parts (roots stems and leaves) and primaryand secondary metabolites [27] Plant responses to CO

2will

likely be influenced by other environmental factors [28]including nutrient and water availability [29 30] and climatechange [31] Irradiance also directly affects plant growth andphytochemistry [31] and is expected to mediate effects ofelevatedCO

2onplants Both irradiance andCO

2are required

resources for photosynthesis and their levels will interactivelyaffect primary and secondary plant metabolism [31] How-ever studies on interactive effects between irradiance andCO2levels on phytochemistry of herbal plants were limited

Recently researchers have reported that environmental fac-tors such as irradiance and CO

2concentration can signifi-

cantly alter secondary metabolite synthesis and productionin plants [32ndash36] The synthesis of medicinal componentsin the plant is affected by CO

2and light intensity with

changes observed in plant morphology and physiologicalcharacteristics [37 38] Both factors were known to adjust notonly plant growth and development but also the biosynthesisof primary and secondary metabolites [39]

Two theories that best describe plant phyto-chemicalresponses to elevated CO

2and different irradiance have

receivedmuch attentionThe carbonnutrient balance (CNB)theory [40 41] proposes that allocation to storage andsecondary metabolites is determined by the balance betweenthe availability of carbon and nutrients such that carbonin excess of growth demands is allocated to carbon-basedsecondary metabolites or storage compounds The growth-differentiation balance (GDB) theory [42] is premised upongeneral patterns for relative responses of net assimilationand growth to resource availability (eg water nutrientsand light) such that conditions limiting growth more thanassimilation allow resources to be available for differentiationprocesses such as secondary metabolism

There have been several studies that investigated theeffects of elevated CO

2on plant primary and secondary

metabolites [25 43ndash45] but only a few covered the responsesof secondary metabolites under increasing CO

2and irradi-

ance Although secondarymetabolites constitute a significant

sink for assimilated carbon to date the picture is unclear asto how these compounds respond to different levels of CO

2

and irradiance However the interaction of CO2enrichment

with different irradiance levels is expected to enhance themedicinal value of this plant Hence the objective of thisstudy was to examine the effects of different CO

2and irra-

diance levels on the secondary metabolites (total flavonoidsphenolics anthocyanins and ascorbic acid) soluble sugarsleaf gas exchange characteristics phenylalanine ammonialyase (PAL) activity lipid peroxidation and antioxidantactivity (DPPH) of L pumila seedlingsThe study also aims toinvestigate at which CO

2and irradiance levels the production

of secondary metabolites was enhanced The relationshipsbetween all these parameters were also investigated

2 Materials and Methods

21 Experimental Location Plant Materials and TreatmentsThe experiment was carried out in the glasshouse complexat Field 2 Faculty of Agriculture Universiti Putra Malaysia(longitude 101∘ 441015840N and latitude 2∘ 581015840 S 68m above sealevel) with a mean atmospheric pressure of 1013 kPa Three-month-old L pumila var alata seedlings were left for amonthin the nursery to acclimatize until they were ready for thetreatments CO

2enrichment treatments were initiated when

the seedlings reached four months of age and when plantswere exposed to 400 800 or 1200120583molmol CO

2 Labisia

pumila plants were grown under four levels of shade (020 40 or 60 shade) using black net The average lightintensity passing through each of the 0 20 40 and 60shade treatments was 900 625 500 and 225120583molm2srespectively The photosynthetic photon flux density wasmeasured using LICOR-1412 quantum sensors When theseedlings had reached 4 months of age they were fertilizedwith NPK Blue Special at 15 g per plant The seedlings wereplanted in a soilless medium containing coco-peat burntpaddy husk and well composted chicken manure in a 5 5 1(vv) ratio in 25 cm diameter polyethylene bags Day andnight temperatures in the glasshouse were maintained at27ndash30∘C and 18ndash21∘C respectively The relative humiditywas maintained between 50 and 60 All seedlings wereirrigated using overhead mist irrigation which was givenfour times a day or when necessary Each irrigation sessionlasted for 7min The factorial experiment was arranged ina split plot using a randomized complete block design withthe three CO

2levels (400 800 and 1200120583molmol) as the

main plots and four levels of light intensity (900 625 500 and225120583molm2s) as the subplots replicated three times Eachexperimental unit consisted of seven seedlings with a total of252 seedlings in the experiment All plants were harvested atthe end of 15 weeks of treatments

22 Growth Chamber Microclimate and CO2Enrichment

The seedlings were raised in specially constructed growthchambers receiving 12 h photoperiod and average photosyn-thetic photon flux density of 330 120583molm2s Day and nighttemperatures were recorded at 30 plusmn 10∘C and 20 plusmn 15∘C

The Scientific World Journal 3

respectively with a relative humidity of about 70 to 80Vapor pressure deficit ranged from 111 to 232 kPa Carbondioxide at 998 purity was supplied from a high-pressureCO2cylinder and injected through a pressure regulator

into the fully sealed 2 times 3m growth houses at 2 h dailyand applied continuously from 0800 to 1000 am The CO

2

concentrations of the different treatments were measuredusing Air Sense CO

2sensors designated to each chamber

during CO2exposition period Plants were watered three to

four times a day at 5min per session to ensure normal growthof plants using drip irrigation with emitter capacity of 2 Lh[46]

23 Total Phenolics and Flavonoids Quantification Theextraction and quantification of total phenolics andflavonoids followed the method described by Ibrahimet al [47] An amount of ground tissue samples (01 g) wasextracted with 80 ethanol (10mL) on an orbital shaker for120 minutes at 50∘C The mixture was subsequently filtered(Whatman Number 1) and the filtrate was used for thequantification of total phenolics and total flavonoids Folin-Ciocalteu reagent (diluted 10-fold) was used to determinethe total phenolics content of leaf samples Two hundred120583Lof the sample extract was mixed with Follin-Ciocalteaureagent (15mL) and allowed to stand at 22∘C for 5 minutesbefore adding NaNO

3solution (15mL 60 g Lminus1) After two

hours at 22∘C absorbance was measured at 725 nm Theresults are expressed as mg gminus1 gallic acid equivalent (mgGAE gminus1 dry sample) For total flavonoids determinationa sample (1mL) was mixed with NaNO

3(03mL) in a test

tube covered with aluminium foil and left for 5 minutesThen 10 AlCl

3(03mL) was added followed by addition of

1M NaOH (2mL) The absorbance was measured at 510 nmusing a spectrophotometer with rutin as a standard (resultsexpressed as mgg rutin dry sample)

24 Anthocyanin Content Anthocyanin content was deter-mined according to Bharti and Khurana [48] Fresh leaves(1 g) were added in 10mL acidic methanol (1 vv HCl)and incubated overnight Anthocyanin was partitioned fromchlorophyll with 10mL chloroform followed by addition of9mL of double deionised water The test tubes containingthe samples were shaken gently and the mixture was allowedto settle The absorbance was read at 505 nm Petunidin wasused as a standard Anthocyanin content was recorded asmgg petunidin fresh weight

25 Ascorbic Acid Content The ascorbic acid content wasmeasured using the modified method of Davies and Mas-ten [49] Fresh leaf samples (1 g) were extracted in 1 ofphosphate-citrate buffer (pH 35) using a chilled pestle andmortar The homogenate was filtered The filtrate was addedto 1mL of 17mM 26-dichloroindophenol (26-DCPIP) ina 3mL cuvette The absorbance at 520 nm was read within10min of mixing the reagentsThe extraction buffer was usedas a blank L-Ascorbic acid was used as a standard Ascorbicacid was recorded as mgg L-ascorbic acid fresh leaves

26 Total Soluble Sugar Determination Total soluble sugarwas measured spectrophotometrically using the method ofIbrahim and Jaafar [50] Samples (05 g) were placed in 15mLconical tubes and distilled water was added to make upthe volume to 10mL The mixture was then vortexed andlater incubated for 10min Anthrone reagent was preparedby dissolving anthrone (Sigma Aldrich St Louis MO USA01 g) in 95 sulphuric acid (Fisher Scientific USA 50mL)Sucrose was used as a standard stock solution to prepare thestandard curve for the quantification of sucrose in the sampleThe mixed sample of ground dry sample and distilled waterwas centrifuged at a speed of 3400 rpm for 10min and thenfiltered to get the supernatant A sample (4mL) was mixedwith anthrone reagent (8mL) and then placed in a waterbath set at 100∘C for 5min before the sample was measuredat an absorbance of 620 nm using a spectrophotometer(model UV160U Shimadzu Scientific Kyoto Japan) Thetotal soluble sugar in the sample was expressed as mggsucrose dry sample

27 Phenylalanine Ammonia Lyase (PAL) Activity Phenylala-nine ammonia lyase (PAL) activity was measured using themethod described by Martinez-Tellez and Lafuente [51] Theenzyme activity was determined spectrophotometrically bymeasuring the production of trans-cinnamic acid from L-phenylalanine Enzyme extract (10 120583L) was incubated at 40∘Cwith 121mM L-phenylalanine (90120583L Sigma) prepared in50mM Tris-HCl (pH 85) After 15min of reaction trans-cinnamic acid yield was estimated by measuring increase inthe absorbance at 290 nm The standard curve was preparedusing trans-cinnamic acid as standard (Sigma) and thePAL activity was expressed as nM trans-cinnamic acid120583gproteinh

28 Leaf Gas Exchange Measurement Themeasurement wasobtained using a closed infrared gas analyzer (LICOR 6400Portable Photosynthesis System IRGA Licor Inc NebraskaUSA) Prior to use the instrument was warmed for 30minutes and calibrated with the ZERO IRGA mode Twosteps are required in the calibration process first the initialzeroing process for the built-in flowmeter and second zeroingprocess for the infrared gas analyzer The measurementsused optimal conditions set at 400 120583molmolminus1 CO

230∘C

cuvette temperature 60 relative humidity with air flowrate set at 500 cm3minminus1 and modified cuvette conditionof 800120583molmminus2 sminus1 photosynthetically photon flux density(PPFD) The measurements of gas exchange were carried outbetween 0900 and 1100 am using fully expanded youngleaves numbered three and four from the plant apex to recordnet photosynthesis rate (119860)The operationwas automatic andthe data were stored in the LI-6400 console and analyzedusing ldquoPhotosyn Assistantrdquo software (Version 3 Lincoln IncUSA) Several precautions were taken to avoid errors duringmeasurement Leaf surfaces were cleaned and dried usingtissue paper before being enclosed in the leaf cuvette Thelight response curve was produced following proceduresdescribed in Ibrahim and Jaafar [6] to generate the apparentquantum yield and dark respiration rate


29 Maximum Quantum Efficiency of Photosystem II (119891V119891119898)Measurements of chlorophyll fluorescence were recordedon fully expanded second leaves Leaves were darkened for15 minutes by attaching light-exclusion clips to the centralregion of the leaf surface Chlorophyll fluorescence wasmeasured using a portable chlorophyll fluorescence meter(Handy PEA Hansatech Instruments Ltd Kings Lynn UK)Measurements were recorded for up to 5 seconds [52] Thefluorescence responses were induced by emitting diodesMeasurements of 119891

0(initial fluorescence) 119891

119898(maximum

fluorescence) and 119891V (variable fluorescence) were obtainedThe 119891V values were derived as the differences between 119891119898 and1198910

210 DPPHRadical Scavenging Assay TheDPPH free radicalscavenging activity of each sample was determined accordingto the method described by Joyeux et al [53] A solutionof 01mM DPPH in methanol was prepared The initialabsorbance of the DPPH in methanol was measured at515 nm An aliquot (40 120583L) of an extract was added to 3mLof methanolic DPPH solution The change in absorbance at515 nm was measured after 30min The antiradical activity(AA) was determined using the following formula

AA

=100 minus [(Abs sample minus Abs empty sample)] times 100

Abs control

(1)

The optic density of the samples the control and the emptysamples were measured in comparison with methanol Thesynthetic antioxidant BHT (butylhydroxytoluene) and 120572-tocopherol were used as positive controls

211 Malondialdehyde (MDA) Determination Lipid peroxi-dation of plant parts was estimated by the level of malon-dialdehyde (MDA) production using the thiobarbituric acid(TBA) method as described by Alexieva et al [54] One gramof ground (025mm) plant sample was homogenized with amortar and pestle in 05 trichloracetic acid (TCA 1mL)The homogenate was centrifuged at 9000 rpm for 20minThe supernatant (05mL) was mixed with 20 TCA (25mL)containing 05 TBA and heated in a boiling water bathfor 30min and allowed to cool in an ice bath quickly Thesupernatant was centrifuged at 9000 rpm for 10min and theresulting supernatant was used for determination of MDAcontent Absorbance values at 532 nm were recorded

212 Statistical Analysis Data were analyzed using the anal-ysis of variance procedure in SAS version 17 Means separa-tion test between treatments was performed using Duncanmultiple range testThe standard error of differences betweenmeans was calculated with the assumption that data werenormally distributed and equally replicated [55 56]

3 Results and Discussion

31 Total Phenolics and Flavonoids Accumulation of totalphenolics and flavonoids in L pumila was influenced by

the interaction effect between CO2and irradiance (119875 le

001 Table 1) Generally total phenolics and flavonoidswas observed to be the highest at 1200120583molmol CO

2+

225120583molm2s (325mg gallic acidg dry weight) and wasthe lowest at 400120583molmol CO

2+ 900120583molm2s (092mg

gallic acidg dry weight) Total flavonoids content followedthe same trend as total phenolics where the highest totalflavonoids content was observed at 1200 120583molmol CO

2+

225120583molm2s irradiance which registered 212mg rutingdry The lowest weight was recorded at 400120583molmol CO

2

+ 900120583molm2s irradiance which contained only 046mgruting dry weight The present results are in contrast witha previous study by McDonald et al [57] who found thatthe highest bioactive compound was accumulated underelevated CO

2and high light conditions in Populus tremu-

loides Betula papyrifera and Acer saccharum In the presentstudy the enrichment with high CO

2(1200120583molmol) under

low irradiance (225120583molm2s) was found to increase theproduction of L pumila secondary metabolites Enhancedproduction of secondary metabolites under high levels ofCO2is usually reported [47ndash49] The present results indicate

that enrichment of L pumilawith high CO2(1200120583molmol)

with 225120583molm2s irradiance can upregulate the produc-tion of total phenolics and flavonoids in the plant Theincreased production of secondary metabolites under highCO2and low irradiance is attributed to the enhanced avail-

ability of soluble carbohydrates under this condition thatupregulates the production of total phenolics and flavonoids[58] Furthermore the phenolics and flavonoids contentshave high significant positive correlationships with solublecarbohydrates (total phenolics 1199032 = 0901 119875 le 005 totalflavonoids 1199032 = 0783 119875 le 005 Table 2) which indicatesthat the up-regulation of secondary metabolites in L pumilaunder this condition might be contributed by the increase insoluble carbohydrate content The increase in carbohydratecontentmight increase the production ofL pumila secondarymetabolites due to increase in substrates to the shikimicacid pathway [59] The high total phenolics and flavonoidscontent under high CO

2and low irradiance in the plant has

been shown to have anticancer properties and also to haveapplications for the use as antibiotics antidiarrhea antiulcerand anti-inflammatory agents as well as in the treatment ofdiseases such as hypertension vascular fragility allergies andhyperchlolesterolemia [60 61] The results imply that CO

2

enrichment under low irradiance can enhance the medicinalproperties of this herb

32 Soluble Carbohydrates The accumulation of soluble car-bohydrates in L pumila followed the same trends with totalphenolics and flavonoids The soluble sugar was influencedby the interaction between CO

2and irradiance (119875 le 005

Table 3) The soluble sugar content was found to be higheras CO

2levels increased from 400 to 1200120583molmol and

as the irradiance decreased from 900 to 225120583molm2swith the highest accumulation of soluble sugar observedat 1200120583molmol CO

2+ 225120583molm2s irradiance which

registered 4071mgg sucrose dry weight and it was the


Table 1 Interaction effects of CO2 levels and irradiance on total phenolics and total flavonoids of Labisia pumila

CO2 levels (120583molmol) Irradiances (120583molm2s) Total phenolics(mg gallic acidg dry weight)

Total flavonoids(mg ruting dry weight)

400

225 271 plusmn 001c 154 plusmn 021c

500 214 plusmn 003e 123 plusmn 011e

675 164 plusmn 004g 098 plusmn 091g

900 092 plusmn 021j 046 plusmn 021j

800

225 301 plusmn 021b 176 plusmn 003b

500 256 plusmn 003d 142 plusmn 001d

675 172 plusmn 001f 107 plusmn 015f

900 136 plusmn 011i 062 plusmn 002i

1200

225 325 plusmn 023a 212 plusmn 004a

500 277 plusmn 014d 150 plusmn 005de

675 186 plusmn 024f 111 plusmn 007fg

900 147 plusmn 021h 086 plusmn 002h

All analyses are expressed as means plusmn standard error of mean (SEM)119873 = 21 Means not sharing a common letter within a column were significantly differentat 119875 le 005

Table 2 Pearsonrsquos correlation parameters

Characteristics 1 2 3 4 5 6 7 8 9 10 11 12(1) Phenolics 1000(2) Flavonoids 0899lowast 1000(3) Sugar 0901lowast 0789lowast 1000(4) Anthocyanin 0879lowast 0802lowast 0567 1000(5) Ascorbic Acid 0872lowast 0823lowast 0887lowast 0776lowast 1000(6) PAL activity 0921lowast 0895lowast 0665 0723lowast 0712lowast 1000(7) Chlorophyll minus0892lowast minus0899lowast minus0234 minus0567 minus0446 minus0032 1000(8) Photosynthesis 0987lowast 0786lowast 0722lowast 0668lowast 0056 0456 minus0445 1000(9) S conductance 0932lowast 0821lowast 0678 0556 0034 0332 0223 0987lowast 1000(10) 119891V119891119898 0887lowast 0546 0801lowast 0505 0021 0303 0445 0887lowast 0666lowast 1000(11) MDA minus0876lowast minus0821lowast 0045 0003 0042 minus0789lowast 0567lowast minus0654lowast 0324lowast 0012 1000(12) DPPH 0923lowast 0887lowast 0213 0887lowast 0104 0901lowast minus0445 0901lowast 0023 0344 minus0455lowast 1000Note 119891V119891119898 maximum efficiency of photosystem II PAL phenyl alanine lyase activity DPPH 11-diphenyl-2-picryl-hydrazyl (DPPH) assay lowastSignificant at119875 le 005

Table 3 Interaction effects of CO2 levels and irradiance on leaf total phenolics and total flavonoids production in Labisia pumila

CO2 levels (120583molmol) Irradiances (120583molm2s) Soluble sugar(mg sucroseg dry weight)

Anthocyanin(mg petuniding dry weight)

Ascorbic acid(mgg dry weight)

400

225 32110 plusmn 2110c 0860 plusmn 0211b 0064 plusmn 0001c

500 26220 plusmn 0340f 0701 plusmn 0011c 0050 plusmn 0003e

675 23100 plusmn 0231i 0610 plusmn 0231e 0041 plusmn 0004g

900 18341 plusmn 1241l 0371 plusmn 005h 0024 plusmn 0003j

800

225 35210 plusmn 2152b 0881 plusmn 0041a 0065 plusmn 0002b

500 28910 plusmn 0452e 0750 plusmn 0012b 0053 plusmn 0006d

675 24271 plusmn 2441h 0640 plusmn 0060d 0043 plusmn 0001f

900 19722 plusmn 3212k 0400 plusmn 0073g 0030 plusmn 0003i

1200

225 40712 plusmn 2561a 0920 plusmn 0020a 0071 plusmn 0002a

500 30721 plusmn 2110d 0790 plusmn 0012b 0059 plusmn 0001d

675 26110 plusmn 0450g 0671 plusmn 0053d 0047 plusmn 0004f

900 21211 plusmn 3212j 0480 plusmn 0031f 0036 plusmn 0003h

All results are expressed as means plusmn standard error of mean (SEM)119873 = 21 Means not sharing a common letter within a column were significantly differentat 119875 le 005


lowest at 400 120583molmolCO2+900120583molm2s irradiance that

only registered 1834mgg sucrose dry weight The presentresults suggest that at high levels of CO

2(1200120583molmol)

under low irradiance (225120583molm2s) the production offlavonoids soluble sugars was enhanced Ghasemzadeh etal [24] described the accumulation of carbohydrates as asignal of an increase in production of secondary metabolitesthat enhanced the medicinal quality of plants The extracarbohydrates accumulated in L pumila seedlings in thepresent study might be channeled for the production ofsecondary metabolites (total phenolics and flavonoids) [62ndash68] Carbohydrates are the basic compounds required toproduce phenolic compounds through the shikimic acidpathway where extra carbohydrates derived from glycolysisand the penthose phosphate pathway are converted intoaromatic amino acids [69]The upregulation in carbon-basedsecondary metabolites (CBSM) in the present study mightbe related to the balance between carbohydrate source andsink as the greater the source-sink ratio the greater theproduction of secondary metabolites that might occur [70]The present findings are in agreement with Guo et al [71]who found that an increase in sucrose content correspondedwith the enhanced production of ascorbic acid glucosino-lates sulforaphane anthocyanins and total phenolics andincreased the antioxidative activities in broccoli sprouts Thecurrent results indicate that the exposure of L pumila to highCO2under low irradiance can enhance the health promoting

effects of this plant

33 Anthocyanin Content In the present study anthocyanincontent was found to be influenced by the interaction effectsbetween CO

2and irradiance levels (119875 le 005 Table 3)

The accumulation of anthocyanin exhibited a similar patternwith total phenolics flavonoids and soluble sugars In theleaves the imposition of 225120583molm2s irradiance increasedanthocyanin production to a maximum at 1200120583molmol(097mgg fresh weight) whilst the lowest anthocyanincontent of 069mgg fresh weight was recorded at ambientCO2levels (400 120583molmol) under 900120583molm2s irradi-

ance The same patterns that were observed for total phe-nolics flavonoid and soluble sugar accumulation suggestthat enhancement of secondary metabolites production inL pumila was optimized under the highest level of CO

2

(1200120583molmol) under low irradiance (225120583molm2s) Theincrease in anthocyanins under high CO

2was also observed

in Populus euramericana [72] and strawberry [73] Antho-cyanins are naturally occurring phenolic compounds respon-sible for the color of many flowers fruits and berries [63]They are the most important group of water soluble pigmentsin plants and they have beneficial health effects as antiox-idant and anti-inflammatory agents [74 75] Tamura andYamagami [76] reported that anthocyanins possess positivetherapeutic value mainly associated with their antioxidantactivity Improvement in the anthocyanin content in thecurrent study with elevated CO

2and low irradiance suggests

a possible mechanism for increasing the medicinal quality ofL pumila

34 Ascorbic Acid Content Ascorbic acid was found to beinfluenced by the interaction effects of elevated CO

2and

irradiance levels (119875 le 005 Table 3) The imposition of1200120583molmol CO

2+ 225 120583molm2s irradiance significantly

increased the ascorbic acid content compared to the othertreatments At the end of 15 weeks the ascorbic acid con-tent with 800120583molmol CO

2+ 225120583molm2s irradiance

400 120583molmol CO2+ 225120583molm2s 1200120583molmol CO

2

+ 500120583molm2s 800120583molmol CO2+ 500120583molm2s

400 120583molmol CO2+ 500 120583molm2s 1200120583molmol CO

2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2

+ 900120583molm2s 800120583molmol CO2+ 900120583molm2s and

400 120583molmol CO2+ 900120583molm2s was 00065 00064

00059 00053 00050 00047 00043 00041 00036 00030and 00024mgg respectively compared to 00071mgg with1200120583molmol CO

2+ 225120583molm2s irradiance There were

no previous reports on the combined impact of elevatedCO2and irradiance on the accumulation of ascorbic acid

However previous research had shown that exposure toelevated CO

2can enhance the production of ascorbic acid in

Capsicum anuum [77] tomato [78] and potato [79] Ascorbicacid had a significant positive correlation with soluble sugar(1199032 = 0887 119875 le 005) which indicated that the increasein production of ascorbic acid under high CO

2and low

irradiance can be attributed to the increased carbohydratecontent that upregulated the production of this componentThe increase in ascorbic acid content in L pumila seedlingsis attributed to the high production of TNC under highCO2and low irradiance TNC (d-glucose) is a precursor for

ascorbic biosynthesis in plants and the more the availabilityof TNC was the more the ascorbic acid would be producedin the L-galactose pathway [80 81]

35 Phenylalanine Ammonia Lyase (PAL) Activity Thephenyl alanine ammonia lyase (PAL) activity was influencedby interaction effects between CO

2and irradiance levels

(119875 le 005 Figure 1) In general it was observed thatincrease in light intensity reduced the activity of this enzymein Labisia pumila The highest PAL activity was found tobe under 1200120583molmol CO


that recorded 4721 nM transcinnamic mgproteinhour andthe lowest was at 400 120583molmol CO

2+ 900120583molm2s

irradiance which only registered 1571 nM transcinnamicmgproteinhour The increase in secondary metabolitescontent in the present study is attributed to the increase inPAL activity under high CO

2and low irradiance Correlation

analysis revealed that PAL activity had a significant positivecorrelation with total phenolics (1199032 = 0921 119875 le 005) andtotal flavonoids (1199032 = 0895119875 le 005) suggesting an increasein secondary metabolites production with increasing PALactivityThe increase in PAL activity under high CO

2and low

irradiance might be due to the reduction in nitrogen contentunder high CO

2and low irradiance that enhanced growth

and reduced the nitrogen pool in the plant this restrictedthe protein production and thus more phenylalanine wasavailable for secondary metabolites production [82 83]


60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900

Light intensity (120583molm2s)400

800

1200

Figure 1 Interaction effects of CO2and irradiance on PAL activity

of Labisia pumila 119873 = 21 Bars represent standard error ofdifferences between means (SEM)

35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)

Irradiances (120583molm2s)

400

800

1200

225 500 675 900

Figure 2 Interaction effects of CO2and irradiance on total

chlorophyll content of Labisia pumila 119873 = 21 Bars representstandard error of differences between means (SEM)

36 Total Chlorophyll Content Similar to PAL activity thechlorophyll content was also influenced by the interactionbetween CO

2and irradiance levels (119875 le 001 Figure 2) The

chlorophyll content was found to be the highest at ambi-ent CO

2under 225120583molm2s irradiance which registered

3071mgg fresh weight and the lowest at 1200120583molmol CO2

+ 900120583molm2s irradiance which recorded only 1421mggfresh weight The decrease in chlorophyll content withincreasing CO

2levels has been reported by Porteaus et al

[84] in wheat The correlations revealed (Table 2) that totalchlorophyll were significantly (119875 le 005) and negativelyrelated to total phenolics (1199032 = minus0892) flavonoids (1199032 =minus0899) anthocyanins (1199032 = minus0867) and ascorbic acid

(1199032 = minus0846) Competition between secondary metabolitesand chlorophyll content fitted well with the prediction ofthe protein competition model (PCM) and indicated thatthe secondary metabolites content was controlled by thecompetition between protein and secondary metabolitesbiosynthesis pathway and its metabolites regulation [85]The negative relationship between the secondary metabolitesand chlorophyll was a sign of gradual switch of investmentfrom protein to polyphenols production [86] The presentresults indicate that under high CO

2and low irradiance the

production of total chlorophyll content was downregulated inL pumila that also indicated the upregulation on productionof secondary metabolites

37 Leaf Gas Exchange Properties The photosynthesis ratestomatal conductance and maximum efficiency of photosy-stem II (119891V119891119898 ratio) were influenced by CO

2levels

(119875 le 005 Figure 3) No irradiance and interaction effectswere observed Leaf net photosynthesis stomatal conduc-tance and119891V119891119898 rate increasedwith increasingCO2 from400to 1200120583molmol The highest net photosynthesis was obta-ined in L pumila exposed to 1200120583molmol CO

2(1145

120583molm2s) followed by 800120583molmol (866 120583molm2s)and 400 120583molmol (444 120583molm2s Figure 3(a)) A similartrend was observed for stomatal conductance where thestomatal conductance for 1200 800 and 400120583molmolCO2was 3015 2512 and 1240mmolm2s respectively

(Figure 3(b)) The 119891V119891119898 ratio was 16 and 25 higher intreatments with 800 and 1200120583molmol CO

2 respectively

compared to the ambient CO2level (Figure 3(c)) These

findings exhibit the importance of CO2in further enhancing

properties of leaf gas exchange and photosystem II efficiencyof L pumila plants The increase in photosynthesis andphotosystem II efficiency in the present work could havestimulated the production of plant secondary metabolites assuggested by the positive correlation coefficients in Table 2between photosynthesis and secondary metabolites of totalphenolics (1199032 = 0987 119875 le 005) and flavonoids (1199032 = 0786119875 le 005) and also between 119891V119891119898 and total phenolics (1199032 =0887 119875 le 005) and flavonoids (1199032 = 0846 119875 le 005) Thusan increase in photosynthetic and photochemical efficiencyhad increased the shikimic acid pathway and enhanced theproduction of plant secondary metabolites and this was inturn due to an increase in the concentration of soluble sugars[87 88] Some studies had reported that when production ofsecondary metabolites increased the photosynthesis woulddecrease due to feedback control of the secondarymetabolitesproduction [89ndash91] however such an effect was not observedin the present study

38 DPPH Radical Scavenging Activity DPPH activity wasinfluenced by the interaction between elevated CO

2and

irradiance levels (119875 le 005 Figure 4) The highest DPPHantioxidant activity was recorded with 1200120583molmol CO

2

followed by 800120583molmol CO2 and it was lowest in the

treatment with 400 120583molmol CO2 As the level of irradiance

was reduced from 900 to 225 120583molm2s the DPPH activitywas increased At 320 120583gmL the DPPH antioxidant activity


400 800 1200

14

12

10

8

6

4

2

0

CO2 levels (120583molmol)

Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)

Figure 3 Impact of elevated CO2on (a) net photosynthesis (b) stomatal conductance and (c)maximum efficiency of photosystem II (119891V119891119898)

in Labisia pumila119873 = 84 Bars represent standard errors of differences between means (SEM)

recorded the highest inhibition value when exposed to1200120583molmol CO

2+ 225120583molm2s irradiance (6544)

followed by the 800 120583molmol + 225120583molm2s (6321)400 120583molmol + 225120583molm2s (6021) 1200 120583molmol+ 500 120583molm2s (5721) 800 120583molmol + 500120583molm2s(5623) 400 120583molmol + 500120583molm2s (5211)1200120583molmol + 625120583molm2s (4921) 800 120583molmol +625 120583molm2s (4832) 400 120583molmol + 625120583molm2s(4527) 1200120583molmol + 900120583molm2s (4021) 800120583molmol + 900120583molm2s (3921) and the least in the400 120583molmol CO


treatment The results of the current work also showed thatthe DPPH radical scavenging ability of the plant extracts at320 120583gmL was less than those of the reference antioxidantsbutylated hydroxytoluene (BHT 6641) and 120572-tocopherol(8038) This study showed that L pumila methanolicextract had good free radical scavenging activity and hence

it can be used as a radical scavenging agent acting possibly asa primary antioxidantThe results also imply that exposure tohigh CO

2concentrations under low irradiance could signi-

ficantly enhance the DPPH radical scavenging activity of themedicinal plant It must be noted that the DPPH assayprincipally measures the activity of water-soluble antiox-idants [92] Results of the current work suggest that high CO

2

supply under low irradiance is advantageous to L pumila inthe improvement of antioxidant activity of the water-solubleantioxidants In our previous study besides phenolics andflavonoid compounds other water-soluble antioxidantsof the extracts such as ascorbic acid and anthocyaninswere suggested to exert additive effects on DPPH radicalscavenging activity This was evident from the current resultswhere DPPH had a significant positive correlationship withtotal phenolics (1199032 = 0923 119875 le 005) total flavonoid(1199032 = 0887 119875 le 005) anthocyanin (1199032 = 0887 119875 le 005)and ascorbic acid (1199032 = 0904 119875 le 005) The results imply


30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)

Irradiance (120583molm2s)

400

800

1200

225 500 675 900

Figure 4 Interaction effect of CO2and irradiance on DPPH

scavenging activity of Labisia pumila BHT and 120572-tocopherol wereused as controls and valued at 6641 and 8041 respectively119873 = 21Bars represent standard errors of differences betweenmeans (SEM)

6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900

Figure 5 Interaction effects of CO2levels and irradiance on lipid

peroxidation DPPH of Labisia Pumila 119873 = 21 Bars representstandard errors of differences between means (SEM)

that the increase in secondary metabolites production underhigh CO

2and low irradiance increases the activity of DPPH

39 Lipid Peroxidation Activity Malondialdehyde (MDA)is a highly reactive three carbon dialdehyde by-product ofpolyunsaturated fatty acid peroxidation and arachidonic acidmetabolismThe production of MDAwas influenced by CO

2

levels imposed onto irradiances (119875 le 005 Figure 5) It wasobserved that as the level of CO

2was enhanced from 400 to

1200120583molmol CO2the production of MDA was decreased

TheMDA content was also found to decrease with increasingirradiance (from 900 to 225 120583molm2s)This suggests that as

CO2levels and irradiance were reduced the oxidative stress

in L pumila cells and tissues was decreased thus implyingoccurrence of lipid peroxidation in L pumila under low CO

2

and high irradiance Yu et al [93] working on P subcordi-formis had also observed that the reduction in MDA at highconcentrations of CO

2was due to reduced photorespiration

that simultaneously reduced photoreduction of dioxygenIn this case fewer electrons were transported to dioxygenduring photosynthesis and in effect reducing the potentialdamage of active oxygen to the membrane system (MDA)The MDA production had established significant negativecorrelations with total phenolics (1199032 = 0876 119875 le 005)and total flavonoids (1199032 = 0821 119875 le 005) indicating thatreduction in MDA might be involved in the upregulation ofsecondary metabolite production under high CO

2and low

irradiance in L pumila (Table 2) The formation of MDAwas considered as a measure of lipid peroxidation that wasinduced by a high stress level [92] MDA a decompositionproduct of polyunsaturated fatty acid hydroperoxides hasbeen utilized very often as a suitable biomarker for oxidativestress Usually the increase in lipid peroxidation is simulta-neously accompanied by an increase in hydrogen peroxidelevels but in the present study hydrogen peroxide contentwasnot measured Hydrogen peroxide may function as a signalfor the induction of plant defense systems and this couldenhance secondary metabolite production [94] Hence theresults of the present study suggest that oxidative stress wasreduced and was not a prerequisite for secondary metabolitesynthesis in L pumila under high CO

2and low irradiance

4 Conclusions

The present work has demonstrated that high levels of CO2

and low irradiance are able to change the synthesis and alloca-tion of secondary metabolites in Labisia pumila The produc-tion of total phenolics flavonoids anthocyanins and ascorbicacid was enhanced as CO

2levels increased from 400 to

1200120583molmol CO2and irradiance was reduced from 900 to

225120583molm2s The increase in the production of secondarymetabolites was followed by an increase in the soluble sugarcontent The DPPH antioxidant activity was found to be thehighest at 1200 120583molmol CO


and the lowest under 400 120583molmol CO2+ 900120583molm2s

irradiance Elevated CO2significantly improved the leaf gas

exchange properties of Labisia pumila The high productionof secondary metabolites under high CO

2is attributed to

increased PAL activity under these conditions MDA had asignificant negative correlation with production of secondarymetabolites indicating that reduction in lipid peroxidationunder high CO

2and low irradiance in L pumila might

improve secondary metabolites production

Conflict of Interests

The authors declare that there is no conflict of interests regar-ding the publication of this paper


Acknowledgment

The authors are grateful to the Ministry of Higher EducationMalaysia for financing this work under the Research Univer-sity Grant Scheme no 91007

References

[1] J A Jamal P J Houghton and S R Milligan ldquoTesting of Labi-sia pumila for oestrogenic activity using a recombinant yeastscreenrdquo Journal of Pharmacy and Pharmacology vol 50 no 9pp 79ndash82 1998

[2] B C Stone ldquoNotes on the genus Labisia Lindl (Myrsinaceae)rdquoMalayan Nature Journal vol 42 pp 51ndash55 1988

[3] C Wiart and F K E WongMedicinal Plants of Southeast AsiaPrentice Hall Petaling Jaya Malaysia 2nd edition 2002

[4] A J Jamia and P J Houghton ldquoDetermination of iron contentfrom Labisia pumila using inductively coupled plasma tech-niquerdquo in Proceedings of the 16th National Seminar on NaturalProducts pp 118ndash120 2000

[5] M F Wan Ezumi S Siti Amrah A W M Suhaimi and S SJ Mohsin ldquoEvaluation of the female reproductive toxicity ofaqueous extract of Labisia pumila var alata in ratsrdquo IndianJournal of Pharmacology vol 39 no 1 pp 30ndash32 2007

[6] M H Ibrahim and H Z E Jaafar ldquoPhotosynthetic capacityphotochemical efficiency and chlorophyll content of threevarieties of Labisia pumila Benth exposed to open field andgreenhouse growing conditionsrdquo Acta Physiologiae Plantarumvol 33 no 6 pp 2179ndash2185 2011

[7] P J Houghton J A Jamal and S Milligan ldquoStudies on Labisiapumila herb and its commercial productsrdquo Journal of Pharmacyand Pharmacology vol 51 pp 236ndash239 1999

[8] E Karimi H Z E Jaafar and S Ahmad ldquoPhytochemical ana-lysis and antimicrobial activities of methanolic extracts of leafstem and root from different varieties of Labisia pumila benthrdquoMolecules vol 16 no 6 pp 4438ndash4450 2011

[9] E Karimi andH Z E Jaafar ldquoHPLC andGC-MSdeterminationof bioactive compounds inmicrowave obtained extracts of threevarieties of Labisia pumila Benthrdquo Molecules vol 16 no 8 pp6791ndash6805 2011

[10] E Karimi H Z E Jaafar and S Ahmad ldquoPhenolics and flav-onoids profiling and antioxidant activity of three varieties ofMalaysian indigenous medicinal herb Labisia pumila BenthrdquoJournal of Medicinal Plant Research vol 5 no 7 pp 1200ndash12062011

[11] Z Ali and I A Khan ldquoAlkyl phenols and saponins from theroots of Labisia pumila (Kacip Fatimah)rdquo Phytochemistry vol72 no 16 pp 2075ndash2080 2011

[12] L S Chua N A Latiff S Y Lee C T Lee M R Sarmidi andR A Aziz ldquoFlavonoids and phenolic acids from Labisia pumila(Kacip Fatimah)rdquo Food Chemistry vol 127 no 3 pp 1186ndash11922011

[13] P V A Fine Z J Miller I Mesones et al ldquoThe growth-defensetrade-off and habitat specialization by plants in Amazonianforestsrdquo Ecology vol 87 no 7 supplement pp S150ndashS162 2006

[14] E Karimi E Oskoueian R Hendra A Oskoueian and H ZE Jaafar ldquoPhenolic compounds characterization and biologicalactivities of citrus aurantium bloomrdquo Molecules vol 17 no 2pp 1203ndash1218 2012

[15] E Karimi E Oskoueian R Hendra and H Z E Jaafar ldquoEva-luation of Crocus sativus L stigma phenolic and flavonoid

compounds and its antioxidant activityrdquo Molecules vol 15 no9 pp 6244ndash6256 2010

[16] M H Ibrahim H Z E Jaafar A Rahmat and Z A RahmanldquoInvolvement of nitrogen on flavonoids glutathione antho-cyanin ascorbic acid and antioxidant activities of malaysianmedicinal plant Labisia pumila Blume (Kacip Fatimah)rdquo Inter-national Journal ofMolecular Sciences vol 13 no 1 pp 393ndash4082012

[17] W Barz and J Koster ldquoTurnover and degradation of secondary(natural) productsrdquo in The Biochemistry of Plants Volume 7Secondary Plant Products pp 35ndash84 Academic Press NewYork NY USA 1981

[18] P GWaterman and S Mole ldquoExtrinsic factors influencing pro-duction of secondarymetabolites in plantsrdquo in Insect-Plant Inte-ractions vol 1 pp 108ndash134 CRC Press Boca Raton Fla USA1989

[19] M H Ibrahim H Z E Jaafar A Rahmat and Z A RahmanldquoThe relationship between phenolics and flavonoids productionwith total non structural carbohydrate and photosyntheticrate in Labisia pumila Benth under high CO

2and nitrogen

fertilizationrdquoMolecules vol 16 no 1 pp 162ndash174 2011[20] M H Ibrahim and H Z E Jaafar ldquoInvolvement of carbohydr-

ate protein and phenylanine ammonia lyase in up-regulation ofsecondarymetabolites inLabisia pumilaunder variousCO

2and

N2levelsrdquoMolecules vol 16 no 5 pp 4172ndash4190 2011

[21] M H Ibrahim and H Z E Jaafar ldquoIncreased carbon dioxideconcentration improves the antioxidative properties of themalaysian herb Kacip Fatimah (Labisia pumila Blume)rdquoMolecules vol 16 no 7 pp 6068ndash6081 2011

[22] M H Ibrahim and H Z E Jaafar ldquoEnhancement of leaf gasexchange and primary metabolites under carbon dioxide enric-hment up-regulates the production of secondary metabolites inLabisia pumila seedlingsrdquoMolecules vol 16 no 5 pp 3761ndash37772011

[23] H Z E Jaafar M H Ibrahim and E Karimi ldquoPhenolics andflavonoids compounds phenylanine ammonia Lyase and antio-xidant activity responses to elevated CO

2in Labisia pumila

(Myrisinaceae)rdquoMolecules vol 17 no 6 pp 6331ndash6347 2012[24] A Ghasemzadeh H Z E Jaafar and A Rahmat ldquoElevated car-

bon dioxide increases contents of flavonoids and phenolic com-pounds and antioxidant activities in Malaysian young ginger(Zingiber officinale Roscoe) varietiesrdquoMolecules vol 15 no 11pp 7907ndash7922 2010

[25] J Penuelas andM Estiarte ldquoCan elevated CO2affect secondary

metabolism and ecosystem functionrdquo Trends in Ecology andEvolution vol 13 no 1 pp 20ndash24 1998

[26] R Ceulemans andM Mousseau ldquoTansley review No71 Effectsof elevated atmospheric CO

2onwoody plantsrdquoNewPhytologist

vol 127 no 3 pp 425ndash446 1994[27] F A Bazzaz ldquoThe response of natural ecosystems to the rising

global CO2levelsrdquo Annual Review of Ecology and Systematics

vol 21 no 1 pp 167ndash196 1990[28] F A Bazzaz and S L Miao ldquoSuccessional status seed size and

responses of tree seedlings toCO2 light and nutrientsrdquoEcology

vol 74 no 1 pp 104ndash112 1993[29] R H Johnson and D E Lincoln ldquoSagebrush carbon allocation

patterns and grasshopper nutrition the influence of CO2

enrichment and soil mineral limitationrdquo Oecologia vol 87 no1 pp 127ndash134 1991

[30] E D Fajer M D Bowers and F A Bazzaz ldquoThe effect of nutr-ients and enrichedCO

2environments on production of carbon-


based allelochemicals in Plantago a test of the carbonnutrientbalance hypothesisrdquoTheAmericanNaturalist vol 140 no 4 pp707ndash723 1992

[31] S H Schneider ldquoScenarios of global warmingrdquo in Biotic Inter-actions and Global Change pp 9ndash23 Sinauer Sunderland UK1993

[32] S Larsson A Wiren L Lundgren and T Ericsson ldquoEffects oflight and nutrient stress on leaf phenolic chemistry in Salixdasyclados and susceptibility to Galerucella lineola (Coleo-ptera)rdquo Oikos vol 47 no 2 pp 205ndash210 1986

[33] R Sfendla H Desilets S Laliberte and A Olivier ldquoThe effectsof CO

2enrichment increased light irradiance and reduced suc-

rose concentration on acclimatization of micropropagated Am-erican ginseng plantletsrdquo Journal of Herbs Spices and MedicinalPlants vol 13 no 3 pp 97ndash106 2008

[34] Y Feng M E Warner Y Zhang et al ldquoInteractive effects ofincreased pCO

2 temperature and irradiance on themarine coc-

colithophore Emiliania huxleyi (Prymnesiophyceae)rdquoEuropeanJournal of Phycology vol 43 no 1 pp 87ndash98 2008

[35] M Lambreva K Christov and T Tsonev ldquoShort-term effect ofelevated CO

2concentration and high irradiance on the antiox-

idant enzymes in bean plantsrdquo Biologia Plantarum vol 50 no4 pp 617ndash623 2006

[36] F A Bazzaz and S L Miao ldquoSuccessional status seed size andresponses of tree seedlings toCO

2 light and nutrientsrdquoEcology

vol 74 no 1 pp 104ndash112 1993[37] P S Curtis and X Wang ldquoA meta-analysis of elevated CO

2eff-

ects onwoody plantmass form and physiologyrdquoOecologia vol113 no 3 pp 299ndash313 1998

[38] M Matsuki ldquoRegulation of plant phenolic synthesis frombiochemistry to ecology and evolutionrdquo Australian Journal ofBotany vol 44 no 6 pp 613ndash634 1996

[39] R T Palo ldquoDistribution of birch (Betula SPP) willow (SalixSPP) and poplar (Populus SPP) secondary metabolites andtheir potential role as chemical defense against herbivoresrdquoJournal of Chemical Ecology vol 10 no 3 pp 499ndash520 1984

[40] J Tuomi P Niemala F S Chapin J P Bryant and S SirinldquoDefensive responses of trees in relation to their carbonnut-rient balancerdquo inMechanisms for Woody Plant Defenses AgainstInsects Search for Pattern pp 57ndash72 Springer New York NYUSA 1988

[41] J P Bryant F S Chapin III and D R Klein ldquoCarbonnutrientbalance of boreal plants in relation to vertebrate herbivoryrdquoOikos vol 40 no 3 pp 357ndash368 1983

[42] D A Herms andW JMattson ldquoThe dilemma of plants to growor defendrdquo Quarterly Review of Biology vol 67 no 3 pp 283ndash335 1992

[43] M Estiarte J Penuelas B A Kimball et al ldquoFree-air CO2enr-

ichment of wheat leaf flavonoid concentration throughout thegrowth cyclerdquo Physiologia Plantarum vol 105 no 3 pp 423ndash433 1999

[44] P Hogy and A Fangmeier ldquoAtmospheric CO2enrichment

affects potatoes 2 Tuber quality traitsrdquo European Journal ofAgronomy vol 30 no 2 pp 85ndash94 2009

[45] S B Idso B A Kimball G R Pettit III L C Garner and R ABackhaus ldquoEffects of atmospheric CO

2enrichment on the gro-

wth and development of Hymenocallis littoralis (Amaryllidac-eae) and the concentrations of several antineoplastic and antivi-ral constituents of its bulbsrdquo The American Journal of Botanyvol 87 no 6 pp 769ndash773 2000

[46] T Lu X He W Chen K Yan and T Zhao ldquoEffects of elevatedO3andor elevated CO

2on lipid peroxidation and antioxidant

systems in Ginkgo biloba leavesrdquo Bulletin of EnvironmentalContamination and Toxicology vol 83 no 1 pp 92ndash96 2009

[47] M H Ibrahim H Z E Jaafar A Rahmat and Z A RahmanldquoEffects of nitrogen fertilization on synthesis of primary andsecondary metabolites in three varieties of Kacip Fatimah(Labisia pumila Blume)rdquo International Journal of MolecularSciences vol 12 no 8 pp 5238ndash5254 2011

[48] A K Bharti and J P Khurana ldquoMolecular characterization oftransparent testa (tt) mutants of Arabidopsis thaliana (ecotypeEstland) impaired in flavonoid biosynthetic pathwayrdquo PlantScience vol 165 no 6 pp 1321ndash1332 2003

[49] S H R Davies and S J Masten ldquoSpectrophotometric methodfor ascorbic acid using dichlorophenolindophenol eliminationof the interference due to ironrdquoAnalyticaChimicaActa vol 248no 1 pp 225ndash227 1991

[50] MH Ibrahim andH Z E Jaafar ldquoPrimary secondarymetabo-lites H

2O2 malondialdehyde and photosynthetic responses of

Orthosiphon stimaneus Benth to different irradiance levelsrdquoMolecules vol 17 no 2 pp 1159ndash1176 2012

[51] M A Martinez-Tellez and M T Lafuente ldquoEffect of high tem-perature conditioning on ethylene phenylalanine ammonia-lyase peroxidase and polyphenol oxidase activities in flavedoof chilled fortune mandarin fruitrdquo Journal of Plant Physiologyvol 150 no 6 pp 674ndash678 1997

[52] M H Ibrahim H Z E Jaafar M H Harun and M R Yus-op ldquoChanges in growth and photosynthetic patterns of oil palm(Elaeis guineensis Jacq) seedlings exposed to short-term CO

2

enrichment in a closed top chamberrdquo Acta Physiologiae Plan-tarum vol 32 no 2 pp 305ndash313 2010

[53] M Joyeux A Lobstein R Anton and F Mortier ldquoComparativeantilipoperoxidant antinecrotic and scavenging properties ofterpenes and biflavones from Ginkgo and some flavonoidsrdquoPlanta Medica vol 61 no 2 pp 126ndash129 1995

[54] V Alexieva I Sergiev S Mapelli and E Karanov ldquoThe effect ofdrought and ultraviolet radiation on growth and stress markersin pea and wheatrdquo Plant Cell and Environment vol 24 no 12pp 1337ndash1344 2001

[55] H Z E Jaafar M H Ibrahim and N F M Fakri ldquoImpact ofsoil field water capacity on secondary metabolites phenylala-nine ammonia-lyase (PAL) maliondialdehyde (MDA) andphotosynthetic responses of Malaysian Kacip Fatimah (Labisiapumila Benth)rdquoMolecules vol 17 no 6 pp 7305ndash7322 2012

[56] M H Ibrahim and H Z E Jaafar ldquoImpact of elevated carbondioxide on primary secondary metabolites and antioxidantresponses of Eleais guineensis Jacq (Oil Palm) seedlingsrdquoMolecules vol 17 no 5 pp 5195ndash5211 2012

[57] E P McDonald J Agrell and R L Lindroth ldquoCO2 and lighteffects on deciduous trees growth foliar chemistry and insectperformancerdquo Oecologia vol 119 no 3 pp 389ndash399 1999

[58] D T Tissue R B Thomas and B R Strain ldquoAtmospheric CO2

enrichment increases growth and photosynthesis of Pinus ta-eda a 4 year experiment in the fieldrdquo Plant Cell and Environ-ment vol 20 no 9 pp 1123ndash1134 1997

[59] A Ghasemzadeh H Z E Jaafar A Rahmat P E M Wahaband M R A Halim ldquoEffect of different light intensities ontotal phenolics and flavonoids synthesis and anti-oxidant activ-ities in young ginger varieties (Zingiber officinaleRoscoe)rdquo Inte-rnational Journal of Molecular Sciences vol 11 no 10 pp 3885ndash3897 2010


[60] A Ghasemzadeh and H Z E Jaafar ldquoEffect of CO2enrichment

on synthesis of some primary and secondary metabolites inginger (Zingiber officinale Roscoe)rdquo International Journal ofMolecular Sciences vol 12 no 2 pp 1101ndash1114 2011

[61] J Den-Hertog I Stulen F Fonseca and P Delea ldquoModulationof carbon and nitrogen allocation in Urtica dioica and Plantagomajor by elevated CO

2 impact of accumulation of nonstruc-

tural carbohydrates and ontogenetic driftrdquo Physiologia Plan-tarum vol 98 no 1 pp 77ndash88 1996

[62] A A Amin M Rashad and H M H El-Abagy ldquoPhysiologicaleffects of indole-3-butyric-acid and salicylic acid on growthyield and chemical constituents of onion plantsrdquo Journal ofApplied Science Research vol 3 pp 1554ndash1563 2007

[63] H Poorter Y van Berkel R Baxter et al ldquoThe effect of elevatedCO2on the chemical composition and construction costs of

leaves of 27 C3 speciesrdquo Plant Cell and Environment vol 20no 4 pp 472ndash482 1997

[64] P B Kaufman L J Cseke S Warber J A Duke and H L Bri-elmanmNatural Products from Plants CRC Press Boca RatonFla USA 1999

[65] A Ghasemzadeh H Z E Jaafar and A Rahmat ldquoSynthesis ofphenolics and flavonoids in ginger (Zingiber officinale Roscoe)and their effects on photosynthesis raterdquo International Journalof Molecular Sciences vol 11 no 11 pp 4539ndash4555 2010

[66] A Ghasemzadeh H Z E Jaafar andA Rahmat ldquoIdentificationand concentration of some flavonoid components in Malaysianyoung ginger (Zingiber officinale Roscoe) varieties by a highperformance liquid chromatography methodrdquo Molecules vol15 no 9 pp 6231ndash6243 2010

[67] M Wink Introduction Biochemistry Role and Biotechnology ofSecondary Products CRC Press Boca Raton Fla USA 1999

[68] M H Ibrahim and H Z E Jaafar ldquoReduced photoinhibitionunder low irradiance enhanced Kacip Fatimah (Labisia pumilaBenth) secondary metabolites phenyl alanine lyase and antiox-idant activityrdquo International Journal ofMolecular Science vol 13no 5 pp 5290ndash5306 2012

[69] W J Mattson R Julkunen-Tiitto and D A Herms ldquoCO2enr-

ichment and carbon partitioning to phenolics do plant resp-onses accord better with the protein competition or the growth-differentiation balance modelsrdquo Oikos vol 111 no 2 pp 337ndash347 2005

[70] H Saxe D S Ellsworth and J Heath ldquoTree and forest fun-ctioning in an enriched CO

2atmosphererdquoNew Phytologist vol

139 no 3 pp 395ndash436 1998[71] R Guo G Yuan and Q Wang ldquoEffect of sucrose and mannitol

on the accumulation of health-promoting compounds and theactivity of metabolic enzymes in broccoli sproutsrdquo ScientiaHorticulturae vol 128 no 3 pp 159ndash165 2011

[72] M J Tallis Y Lin A Rogers et al ldquoThe transcriptome of Popu-lus in elevated CO

2reveals increased anthocyanin biosynthesis

during delayed autumnal senescencerdquoNew Phytologist vol 186no 2 pp 415ndash428 2010

[73] S YWang J A Bunce and J LMaas ldquoElevated carbon dioxideincreases contents of antioxidant compounds in field-grownstrawberriesrdquo Journal of Agricultural and Food Chemistry vol51 no 15 pp 4315ndash4320 2003

[74] S Y Wang and H Jiao ldquoScavenging capacity of berry crops onsuperoxide radicals hydrogen peroxide hydroxyl radicalrsquos andsinglet oxygenrdquo Journal of Agricultural and Food Chemistry vol48 no 11 pp 5677ndash5684 2000

[75] H Wang M G Nair G M Strasburg et al ldquoAntioxidant andantiinflammatory activities of anthocyanins and their aglycon

cyanidin from tart cherriesrdquo Journal of Natural Products vol62 no 2 pp 294ndash296 1999

[76] H Tamura and A Yamagami ldquoAntioxidative activity of monoa-cylated anthocyanins isolated from Muscat Bailey A graperdquoJournal of Agricultural and Food Chemistry vol 42 no 8 pp1612ndash1615 1994

[77] B Aloni and L Karni ldquoEffects of CO2enrichment on yield car-

bohydrate accumulation and changes in the activity of antiox-idative enzymes in bell pepper (Capsicum annuum L)rdquo JournalofHorticultural Science andBiotechnology vol 77 no 5 pp 534ndash540 2002

[78] M S Islam T Matsui and Y Yoshida ldquoEffect of carbon dioxideenrichment on physico-chemical and enzymatic changes intomato fruits at various stages of maturityrdquo Scientia Horticul-turae vol 65 no 2-3 pp 137ndash149 1996

[79] V Vorne K Ojanpera L de Temmerman et al ldquoEffects of ele-vated carbon dioxide and ozone on potato tuber quality in theEuropean multiple-site experiment lsquoCHIP-projectrsquordquo EuropeanJournal of Agronomy vol 17 no 4 pp 369ndash381 2002

[80] M C de Tullio and O Arrigoni ldquoHopes disillusions and morehopes from vitamin CrdquoCellular andMolecular Life Sciences vol61 no 2 pp 209ndash219 2004

[81] R D Hancock and R Viola ldquoBiosynthesis and catabolism of L-ascorbic acid in plantsrdquo Critical Reviews in Plant Sciences vol24 no 3 pp 167ndash188 2005

[82] A Matros S Amme B Kettig G H Buck-Sorlin U Sonnew-ald andHMock ldquoGrowth at elevatedCO

2concentrations leads

to modified profiles of secondary metabolites in tobacco cvSamsunNN and to increased resistance against infection withpotato virus Yrdquo Plant Cell and Environment vol 29 no 1 pp126ndash137 2006

[83] B AWustman E Oksanen D F Karnosky et al ldquoEffects of ele-vated CO

2andO

3on aspen clones varying inO

3sensitivity can

CO2ameliorate the harmful effects of O

3rdquo Environmental Poll-

ution vol 115 no 3 pp 473ndash481 2001[84] F Porteaus J Hill A S Ball et al ldquoEffect of free air carbon

dioxide enrichment (FACE) on the chemical composition andnutritive value of wheat grain and strawrdquo Animal Feed Scienceand Technology vol 149 no 3-4 pp 322ndash332 2009

[85] S Meyer Z G Cerovic Y Goulas et al ldquoRelationships betweenoptically assessed polyphenols and chlorophyll contents andleaf mass per area ratio in woody plants a signature ofthe carbon-nitrogen balance within leavesrdquo Plant Cell andEnvironment vol 29 no 7 pp 1338ndash1348 2006

[86] A Robredo U Perez-Lopez J Miranda-Apodaca M LacuestaA Mena-Petite and A Munoz-Rueda ldquoElevated CO

2reduces

the drought effect on nitrogen metabolism in barley plantsduring drought and subsequent recoveryrdquo Environmental andExperimental Botany vol 71 no 3 pp 399ndash408 2011

[87] K M Herrmann and L M Weaver ldquoThe shikimate pathwayrdquoAnnual Review of Plant Biology vol 50 pp 473ndash503 1999

[88] G S Rogers P J Milham M Gillings and J P Conroy ldquoSinkstrength may be the key to growth and nitrogen responses inN-deficient wheat at elevated CO2rdquo Australian Journal of PlantPhysiology vol 23 no 3 pp 253ndash264 1996

[89] E AAinsworth andA Rogers ldquoThe response of photosynthesisand stomatal conductance to rising [CO

2] mechanisms and

environmental interactionsrdquo Plant Cell and Environment vol30 no 3 pp 258ndash270 2007

[90] A Wingler S Purdy J A MacLean and N Pourtau ldquoThe roleof sugars in integrating environmental signals during the reg-


ulation of leaf senescencerdquoNewPhytologist vol 161 pp 781ndash7892006

[91] M H Ibrahim and H Z E Jaafar ldquoThe relationship of nitr-ogen andCN ratiowith secondarymetabolites levels and antio-xidant activities in three varieties of malaysian Kacip Fatimah(Labisia pumila Blume)rdquoMolecules vol 16 no 7 pp 5514ndash55262011

[92] C C Wong H B Li K W Cheng and F Chen ldquoA systematicsurvey of antioxidant activity of 30 Chinese medicinal plantsusing the ferric reducing antioxidant power assayrdquo Food Chem-istry vol 97 no 4 pp 705ndash711 2006

[93] J Yu X X Tang P Y Zhang J Y Tian and H J Cai ldquoEffects ofCO2enrichment onphotosynthesis lipid peroxidation and acti-

vities of antioxidative enzymes of Platymonas subcordiformissubjected to UV-B radiation stressrdquo Acta Botanica Sinica vol46 no 6 pp 682ndash690 2004

[94] L Flohr C F Fuzinatto S P Melegari and W G Matias ldquoEff-ects of exposure to soluble fraction of industrial solid waste onlipid peroxidation andDNAmethylation in erythrocytes ofOre-ochromis niloticus as assessed by quantification of MDA andm5dC ratesrdquo Ecotoxicology and Environmental Safety vol 76no 1 pp 63ndash70 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


can change the concentration of these active constituents[16 17] Changes in the accumulation pattern of secondarymetabolites due to environmental factors are however theresult of changes in both the rate of synthesis and therate of catabolism [18 19] The activity of L-phenylalanineammonia lyase (PAL EC 4315) determines the extent towhich phenylalanine is withdrawn from primarymetabolismand enters the general phenylpropanoid pathway [17] Latelyit was found that the enrichment of L pumila with highlevels of CO

2increased the secondary metabolite production

(phenolics and flavonoids) of this plant [20ndash25] Similarresults were observed in ginger (Zingiber officianale) [24]

Atmospheric CO2concentration has increased by about

a third over preindustrial levels and is continuing to increaseat around 04 per year By the year 2100 the atmosphericpartial pressure ofCO

2is projected to be 700 parts permillion

(ppm) or higher [26] Increased CO2concentration in the

air promotes growth and accelerates physiologic processesin young trees because it causes an increase in internalcarbon availability and changes partitioning of assimilatesamong plant parts (roots stems and leaves) and primaryand secondary metabolites [27] Plant responses to CO

2will

likely be influenced by other environmental factors [28]including nutrient and water availability [29 30] and climatechange [31] Irradiance also directly affects plant growth andphytochemistry [31] and is expected to mediate effects ofelevatedCO

2onplants Both irradiance andCO

2are required

resources for photosynthesis and their levels will interactivelyaffect primary and secondary plant metabolism [31] How-ever studies on interactive effects between irradiance andCO2levels on phytochemistry of herbal plants were limited

Recently researchers have reported that environmental fac-tors such as irradiance and CO

2concentration can signifi-

cantly alter secondary metabolite synthesis and productionin plants [32ndash36] The synthesis of medicinal componentsin the plant is affected by CO

2and light intensity with

changes observed in plant morphology and physiologicalcharacteristics [37 38] Both factors were known to adjust notonly plant growth and development but also the biosynthesisof primary and secondary metabolites [39]

Two theories that best describe plant phyto-chemicalresponses to elevated CO

2and different irradiance have

receivedmuch attentionThe carbonnutrient balance (CNB)theory [40 41] proposes that allocation to storage andsecondary metabolites is determined by the balance betweenthe availability of carbon and nutrients such that carbonin excess of growth demands is allocated to carbon-basedsecondary metabolites or storage compounds The growth-differentiation balance (GDB) theory [42] is premised upongeneral patterns for relative responses of net assimilationand growth to resource availability (eg water nutrientsand light) such that conditions limiting growth more thanassimilation allow resources to be available for differentiationprocesses such as secondary metabolism

There have been several studies that investigated theeffects of elevated CO

2on plant primary and secondary

metabolites [25 43ndash45] but only a few covered the responsesof secondary metabolites under increasing CO

2and irradi-

ance Although secondarymetabolites constitute a significant

sink for assimilated carbon to date the picture is unclear asto how these compounds respond to different levels of CO

2

and irradiance However the interaction of CO2enrichment

with different irradiance levels is expected to enhance themedicinal value of this plant Hence the objective of thisstudy was to examine the effects of different CO

2and irra-

diance levels on the secondary metabolites (total flavonoidsphenolics anthocyanins and ascorbic acid) soluble sugarsleaf gas exchange characteristics phenylalanine ammonialyase (PAL) activity lipid peroxidation and antioxidantactivity (DPPH) of L pumila seedlingsThe study also aims toinvestigate at which CO

2and irradiance levels the production

of secondary metabolites was enhanced The relationshipsbetween all these parameters were also investigated

2 Materials and Methods

21 Experimental Location Plant Materials and TreatmentsThe experiment was carried out in the glasshouse complexat Field 2 Faculty of Agriculture Universiti Putra Malaysia(longitude 101∘ 441015840N and latitude 2∘ 581015840 S 68m above sealevel) with a mean atmospheric pressure of 1013 kPa Three-month-old L pumila var alata seedlings were left for amonthin the nursery to acclimatize until they were ready for thetreatments CO

2enrichment treatments were initiated when

the seedlings reached four months of age and when plantswere exposed to 400 800 or 1200120583molmol CO

2 Labisia

pumila plants were grown under four levels of shade (020 40 or 60 shade) using black net The average lightintensity passing through each of the 0 20 40 and 60shade treatments was 900 625 500 and 225120583molm2srespectively The photosynthetic photon flux density wasmeasured using LICOR-1412 quantum sensors When theseedlings had reached 4 months of age they were fertilizedwith NPK Blue Special at 15 g per plant The seedlings wereplanted in a soilless medium containing coco-peat burntpaddy husk and well composted chicken manure in a 5 5 1(vv) ratio in 25 cm diameter polyethylene bags Day andnight temperatures in the glasshouse were maintained at27ndash30∘C and 18ndash21∘C respectively The relative humiditywas maintained between 50 and 60 All seedlings wereirrigated using overhead mist irrigation which was givenfour times a day or when necessary Each irrigation sessionlasted for 7min The factorial experiment was arranged ina split plot using a randomized complete block design withthe three CO

2levels (400 800 and 1200120583molmol) as the

main plots and four levels of light intensity (900 625 500 and225120583molm2s) as the subplots replicated three times Eachexperimental unit consisted of seven seedlings with a total of252 seedlings in the experiment All plants were harvested atthe end of 15 weeks of treatments

22 Growth Chamber Microclimate and CO2Enrichment

The seedlings were raised in specially constructed growthchambers receiving 12 h photoperiod and average photosyn-thetic photon flux density of 330 120583molm2s Day and nighttemperatures were recorded at 30 plusmn 10∘C and 20 plusmn 15∘C




2








3(03mL) in a test









230∘C





119898(maximum



AA


Abs control

(1)








2+


2+ 900120583molm2s (092mg


2+


2











2













400





800





1200












400





800





1200









2(1200120583molmol)







2


2was also observed





2and






2



2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2









2and low








2+ 900120583molm2s




2and low





60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2








3(03mL) in a test









230∘C





119898(maximum



AA


Abs control

(1)








2+


2+ 900120583molm2s (092mg


2+


2











2













400





800





1200












400





800





1200









2(1200120583molmol)







2


2was also observed





2and






2



2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2









2and low








2+ 900120583molm2s




2and low





60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119898(maximum



AA


Abs control

(1)








2+


2+ 900120583molm2s (092mg


2+


2











2













400





800





1200












400





800





1200









2(1200120583molmol)







2


2was also observed





2and






2



2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2









2and low








2+ 900120583molm2s




2and low





60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





400





800





1200












400





800





1200









2(1200120583molmol)







2


2was also observed





2and






2



2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2









2and low








2+ 900120583molm2s




2and low





60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2(1200120583molmol)







2


2was also observed





2and






2



2

+ 675 120583molm2s 800120583molmol CO2

+ 675 120583molm2s400 120583molmol CO

2+ 675120583molm2s 1200120583molmol CO

2









2and low








2+ 900120583molm2s




2and low





60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60

50

40

30

20

10

0

PAL

activ

ity(n

M tr

ansc

inna

mic

mg

prot

ein

hour

)

225 500 675 900


800

1200



35

40

30

25

20

15

10

5

0

Tota

l chl

orop

hyll

cont

ent

(mg

g fre

sh w

eigh

t)


400

800

1200

225 500 675 900















2levels


2(1145



2 respectively





2and


2





400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


400 800 1200

14

12

10

8

6

4

2

0


Net

pho

tosy

nthe

sis (120583

mol

m2s

)

(a)

400 800 1200


Stom

atal

cond

ucta

nce

(

mm

olm

2s

)

40

35

30

25

20

15

10

5

0

gs

(b)

400 800 1200


Max

imum

effici

ency

of p

hoto

syste

m II

(fvfm

)

100

095

090

085

080

075

070

065

060

055

050

(c)











2



30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


30

40

50

60

70

80

20

10

0

DPP

H (

inhi

bitio

n)


400

800

1200

225 500 675 900



6

8

10

12

14

16

4

2

0

MD

A (n

mol

mL)


400

800

1200

225 500 675 900






2







2




2and low


2and low irradiance

4 Conclusions










2is attributed to







Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgment


References





















2and nitrogen



2and





2in Labisia pumila
































2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















2eff-

























2
















































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































2andO


3sensitivity can







2reduces





2] mechanisms and

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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