Research Article The Epiphytic Fern Elaphoglossum luridum...

Research ArticleThe Epiphytic Fern Elaphoglossum luridum (Feacutee) Christ(Dryopteridaceae) from Central and South AmericaMorphological and Physiological Responses to Water Stress

Bruno Degaspari Minardi Ana Paula Lorenzen VoytenaMarisa Santos and Aacuteurea Maria Randi

Department of Botany Federal University of Santa Catarina CP 476 88049-900 Florianopolis SC Brazil

Correspondence should be addressed to Aurea Maria Randi amrandiccbufscbr

Received 22 July 2014 Revised 3 September 2014 Accepted 17 September 2014 Published 20 October 2014

Academic Editor Aryadeep Roychoudhury

Copyright copy 2014 Bruno Degaspari Minardi et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Elaphoglossum luridum (Fee) Christ (Dryopteridaceae) is an epiphytic fern of the Atlantic Forest (Brazil) Anatomical andphysiological studies were conducted to understand how this plant responds to water stress The E luridum frond is coriaceusand succulent presenting trichomes relatively thick cuticle and sinuous cell walls in both abaxial and adaxial epidermis Threetreatments were analyzed control water deficit and abscisic acid (ABA) Physiological studies were conducted through analysisof relative water content (RWC) photosynthetic pigments chlorophyll a fluorescence and malate content No changes in RWCwere observed among treatments however significant decreases in chlorophyll a content and photosynthetic parameters includingoptimal irradiance (119868opt) andmaximumelectron transport rate (ETRmax) were determined by rapid light curves (RLC) No evidenceof crassulacean acid metabolism (CAM) pathway was observed in E luridum in response to either water deficit or exogenousapplication of ABA On the other hand malate content decreased in the E luridum frond after ABA treatment seeming todownregulate malate metabolism at night possibly through tricarboxylic acid (TCA) cycle regulation

1 Introduction

Even in humid rainforests epiphytic plants without directcontact with the soil are exposed to recurrent droughtand xerophytic features have been found and studied frommany distinct taxonomic groups [1] Epiphytes compriseapproximately 10 of the vascular flora being found almostexclusively in tropical forests which provide up to 25 ofspecies [2 3] According to Madison [4] epiphytic plants donot have direct connections with the ground In an ecologicalcontext epiphytism is an interaction between plants in whichthe epiphytic plant is dependent on the substrate suppliedby the host plant (phorophyte) while obtaining nutrientsdirectly from atmospheric moisture without emitting haus-torium structures [5] Epiphytism favors the capture of lightirradiation limiting at the same time the availability of waterfor plants [6 7]

Study of the ecophysiology of epiphytes has receivedconsiderable attention especially for the adaptations thatenable their survival on phorophytes with limited waterand no root contact with the soil These studies are mainlyconcentrated in the Bromeliaceae [8] although the numberof epiphytic bromeliads is fewer than half the number ofepiphytic ferns [7] Epiphytism is pronounced among fernsin that about 29 of fern species regularly occur in treecrowns [7] While about a third of all ferns are classified asepiphytes only a small proportion of these can be classifiedas xerophytic and separating these from mesic species isparticularly difficult [7 9]

Among the wide variety of epiphytic ferns of the Brazilianforests Elaphoglossum luridum (Fee) Christ (Dryopteri-daceae) is particularly notable [10] It occurs preferentiallyin humid forests on large trees Among pantropical gen-era Elaphoglossum is one of the largest containing about

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 817892 9 pageshttpdxdoiorg1011552014817892

2 The Scientific World Journal

600 species mostly in the Neotropics and some 85 ofthese species are epiphytes [7 11] The name of the genusderives from the Greek elaphos (deer) + glossa (tongue)so named for the shape of its fronds According to Macieland Pietrobom [12] E luridum is distinguished from otherspecies of the genus by its leathery bladed epidermis andribs with black scales on both surfaces especially in thebasal region This species occurs in Costa Rica Panamathe Lesser and Greater Antilles Trinidad Guiana FrenchGuiana Suriname Colombia Ecuador Peru and Bolivia InBrazil it is found in Amazonas Para Minas Gerais Rio deJaneiro Sao Paulo Parana Santa Catarina and Rio Grandedo Sul [12]

Drought tolerance is gained through adaptations in wateruptake water loss water storage and in many ferns desicca-tion tolerance which can be divided into two major groupsthe poikilohydrous (desiccation-tolerant) and the homoiohy-drous (relatively desiccation-intolerant) species [7 9] Xero-phytes are homoiohydrous and can be drought endurers anddrought avoiders [7 9]They can show productive ephemeralfoliage suitable only for wet season activity or they are activeyear round by virtue of desiccation-resistant leaves or greenstems with considerable water storage capacity [7] HoweverFarrant et al [13] comment that desiccation tolerance in fernshas been poorly studied

ABA H2O2 and SA can be used to induce plant defense

responses against drought stress [14 15] The ABA levelincreases as a result of drought stress and plays an importantrole in regulating the responses of plants to drought stress[16] The CAM pathway is one of the most important adapta-tions of some epiphytic ferns towater deficit In the facultativehalophyte Mesembryanthemum crystallinum L (Aizoaceae)ABA and water stress induce the facultative CAM pathway[17]

This work aimed to provide more insight into theresponses of the epiphytic fern E luridum to water stressTo accomplish this anatomical and physiological studieswere conducted Specifically three treatments were analyzedcontrol water deficit and ABA Physiological studies wereconducted through analysis of RWC photosynthetic pig-ments RLC photosynthetic parameters and malate contentafter exposure to water stress and ABA treatment in a CAMcontext

2 Material and Methods

21 Plant Collection and Acclimation Plants of E luridumwere collected from the natural habitat in the EnvironmentalConservation Unit ldquoDesterrordquo (UCAD) located in north-western Florianopolis Santa Catarina Brazil (27∘3110158405010158401015840S 0848∘3010158404410158401015840W 03) an area of 495 hectares comprising about1 of the total area of the island The unit is managedby the Federal University of Santa Catarina (UFSC) Plantswere transported to the greenhouse of the Department ofBotanywhere theywere acclimated for sixmonthsThe plantswere tied and placed in brackets on the walls During thetest period the temperature of the greenhouse varied from20 to 30∘C during the day and around 15∘C at night The

relative humidity was monitored and ranged between 45 and83 The photosynthetic photon flux density (PPFD) insidethe greenhouse alternated between 43 and 85 120583molmminus2 sminus1Irradiance was measured at the middle region of the frondswith LI-190 sensor (LI-COR Instruments USA) connected tothe LI-250 light meter (LI-COR Instruments USA) whichwas placed close to plants The plants were watered dailywith distilled water to maintain high humidity Once a weekplants were irrigated with Hoaglandrsquos solution (20) (vv)[18] After the acclimation period the plants were dividedinto three groups each subjected to different treatments(1) control plants irrigated daily for two weeks (2) plantssubjected to drought stress for a period of one week (withoutirrigation) and (3) plants irrigated 5 times a week with asolution of 10120583MABA for a period of fifteen days Treatmentswere made in triplicate

22 Anatomical Analysis of Fronds For in vivo structuralanalysis of the fronds we performed longitudinal and para-dermic cross-sections both done freehand with a razor bladeusing polystyrene as support material These sections wereplaced on a slide with water and covered with a coverslipThe stomata count was performed in triplicate in the medianregion of the abaxial surface with the aid of an opticalmicroscope Leica DM2500 (Leica Wetzlar Germany) andimages were captured with an attached digital camera (LeicaDFC295 Leica Wetzlar Germany)

Structural analysis of the fronds was also performed onsamples fixed in 25 glutaraldehyde in sodium phosphatebuffer 01M pH 72 washed in the same buffer dehydrated inan ethanol series and preserved in ethanol 70∘ GL [19] Somesamples were infiltrated in a mixture of polyethylene glycol(PEG) 1500 and 70 ethanol (1 1) for 24 hours in an oven at60∘C They were incubated again in an oven for 24 hours inpure PEG 1500 Following this incubation the samples wereembedded in PEG 1500 Other samples were infiltrated inhydroxyethyl methacrylate (Jungrsquos Historesin Leica WetzlarGermany) The samples preserved in ethanol 70∘ GL weredehydrated in an ethanol series up to 96∘ GL and processedaccording to the manufacturerrsquos instructions The samplesembedded in PEG and Historesin were sectioned by rotarymicrotome (Leica RM 2125 RT Leica Wetzlar Germany)and stained with safranin [20] or Sudan IV for lipids [21]Twenty measurements of cuticle thickness were performedThe material was examined under an optical microscope(Leica DM2500 Leica Wetzlar Germany) and images werecaptured with an attached digital camera (Leica DFC295Leica Wetzlar Germany)

For scanning electronmicroscopy analysis frond sampleswere subjected to total dehydration in ethanol series andkept in ethyl ether for 48 h at 20∘C Afterwards the samplecontainers were opened and kept in laminar flow to favorthe complete evaporation of the etherThe samples were thenplaced on aluminum brackets with the aid of double-sidedcarbon tape Frond samples were metalized with 30 nm ofgold in a Bal-Tec CED 030 metalizer (Bal-Tec AG Balz-ers Liechtenstein) A scanning electron microscope (SEM)(JEOL JSM-6390LV JEOL Tokyo Japan) was used to analyzethe samples

The Scientific World Journal 3

All anatomical analyses were performed with medianregion of control plants fronds disregarding ribs

23 Determination of RWC and Malate Contents After eachtreatment 10 g fresh mass (FM) of frond discs for eachreplicate (119899 = 3) was removed and placed in flasks containingdistilled water for 180min under a PPFD of 20120583molmminus2 sminus1at 25 plusmn 2∘C to obtain the turgid mass (TM) Frond segmentswere then dried in an oven at 60∘C for 24 h and subsequentlyweighed to obtain the dry mass (DM) The RWC valueswere calculated in fully expanded mature fronds and wereexpressed by the equationRWC = [(FMminusDM)(TMminusDM)]lowast100 [22]

Malate contents were evaluated according to Mollering[23] in order to verify differences in malate content betweennight and day (mM) (ΔMalate = (malate) night minus (malate)day) Three samples (10 g each) were taken from fresh frondmaterial at 600 h and at 1800 h from each treatment Theywere immediately immersed in liquid nitrogen (minus192∘C) withthe aim of conserving and stopping all enzymatic reactionsuntil analysis For the extraction samples were homogenizedin 10mL of distilled water at 98∘C kept in water bath at the98∘C for 10min and subsequently centrifuged at 3500 g for10min The precipitate was discarded and the extracts werekept at room temperature Aliquots of 100 120583L were used toquantify the malate through the K-LMALL enzymatic kit(Megazyme International Ireland Limited Ireland) Analyseswere performed in a spectrophotometer (Biospectro SP-220EQUIPAR Ltda Curitiba Brazil) at 340 nm and in triplicateMalate content was expressed in 120583mol gminus1 DM

24 Analysis of Photosynthetic Pigment Contents and Pho-tosynthetic Parameters The photosynthetic pigments wereanalyzed according to Lichtenthaler [24] using samples of10 g of fresh material (119899 = 3) For extraction frondsamples were taken from liquid nitrogen and immediatelyhomogenized in 10mL of aqueous acetone (80 vv) for5min using a mortar The homogenate was centrifuged for10min at 3500 g and the volume was adjusted to 10mLThe absorbances at 470 nm 646 nm and 663 nm were ana-lyzed with a UV-Vis spectrophotometer (SP-220 BiospectroEQUIPAR Ltda Curitiba Brazil)

The emission of chlorophyll a fluorescence of E luridumfrond was evaluated using a pulse amplitude-modulatedfluorometer (Diving-PAM Underwater Fluorometer WalzEffeltrich Germany) equipped with an optical fiber 55mmin diameter and a blue diode (470 nm) as the light sourceDuring the testing period the recorded temperature was 25plusmn2∘CThe analyses were carried out between 1000 and 1200 hFor each of the three treatments including control waterdeficit and application of 10 120583MABA plants were previouslyacclimated to the dark for 25min before the applicationof the first pulse of saturating light The experiments wereperformed in triplicate (119899 = 3) Transient chlorophylla fluorescence was not measured Ten rapid curves wereobtained for each replicate (thirty photosynthesis curvesfor each treatment) The minimum fluorescence (119865

119900) was

obtained by exposing samples to a measured modulated

light (ML) (01 120583mol photons mminus2 sminus1) and the maximumfluorescence (119865

119898) in the dark-adapted samples was obtained

by applying a saturating light (SL) pulse (9000120583molmminus2 sminus1)Using the RLC option the light curves were obtainedby applying a series of eight pulses of saturating light(SL) each followed by exposure to crescent actinic light(AL) (2ndash2250120583molmminus2 sminus1 PPFD) The parameters of pulse-amplitude modulation (PAM) that is electron transportrate (ETR) were calculated using the WinControl software[25] During the measurement of RLC each SL after anAL period produced a light-adapted maximum fluorescence(1198651015840119898) and light acclimated steady-state fluorescence (1198651015840) The

ETR between photosystems II and I (PSII and PSI) wasestimated using the following equation ETR=ΦPSIItimesPPFDtimes 05 times 084 where ΦPSII is the effective photochemicalquenching yield of PSII obtained by (1198651015840

119898minus 1198651015840)1198651015840

119898 PPFD is

the photosynthetic photon flux density during exposure toactinic light 05 is related to the incident photon betweenPSIIand PSI and 084 is the fraction of incident quanta absorbedby the frond [26ndash30]

All analyses were performed with median region offronds disregarding ribs

25 Statistical Analysis Data were analyzed by Excel andBioEstat software Data were expressed as mean plusmn standarddeviation The analysis of variance (multifactor ANOVA)was followed by the mean comparison test (Tukey 5) fordata with normal distribution and homoscedasticity [31]To quantitatively compare RLCs using parametric statisticssome descriptive parameters were used namely maximumelectron transport rate (ETRmax) and 119868opt (optimal irradi-ance) ETR data were plotted as rapid light curves (RLC =119875 versus 119868 where 119875 is the photosynthesis in ETR and 119868is the irradiation pulses applied to draft the curves) Bothparameters were plotted in a waiting-in-line equation (119910 =119909 sdot 119890minus119909) using an Excel macro (Microsoft Office Excel 2010)

[32] The empirical model for 119875 (ETR) versus 119868 (irradiance)was first used by Gloag et al [33] who applied a suitablemathematical model for photosynthesis (119875 = 119860 sdot 119896

119908sdot 119868 sdot

119890minus119896119908sdot119868) This equation calculated the ETRmax The ETRmax

occurs at an irradiance value of 1119896119908 the 119868opt The adjusted

values for constants (119860) and (119896119908) were determined using the

equation described by Gloag et al [33]

3 Results

31 Anatomical Analysis of Frond The epidermal cells ofthe fronds of E luridum have various shapes tending toelongation in the longitudinal direction of the frond andthe anticlinal cell walls are sinuous (Figure 1(a))The stomataare of the polocytic type in which the guard cells havea subsidiary cell in a horseshoe shape (Figures 1(b) and1(c)) Multicellular trichomes like scales with long branches(Figure 1(d)) occur on both sides of the frond but in highernumbers on the abaxial surface The frond blade has anaverage thickness of 054mm (Figure 2(a)) The mesophyll(Figure 2(a)) consists of parenchyma tending to palisade andspongy parenchyma presenting cells with large vacuoles


(a) (b)

(c) (d)

50120583m50120583m

10120583m 100120583m

Figure 1 Details of SEM and LM images of Elaphoglossum luridum epidermis (a) SEM image of adaxial epidermis cells (b) LM image ofpolocytic stomata and epidermal abaxial cells with sinuous anticlinal walls (c) SEM image of the polocytic stomata on the abaxial surface(d) SEM image of branched trichome on the abaxial epidermis

the spongy mesophyll fills the remaining part presentingrounded chlorophyllous cells and large intercellular spacesranging from ten to eleven layers of cells presenting a largenumber of pits (Figure 2(b)) The cuticle is relatively thick(21 plusmn 027 120583m) showing a light positive Sudan IV reactionto cutin in the peripheral layer (Figure 2(c)) The frond isof the hypostomatic type with stomata restricted to theabaxial epidermis randomly oriented showing a density of 33stomata per mm2 (Figure 2(d))Themeristele (Figure 2(e)) isbounded externally by pericycle with one or two cell layerscontaining a concentric vascular structure of the anficrivaltype The xylem positioned internally to phloem has a V-shape

32 Determination of RWC and Malate Contents No statis-tically significant difference was observed in RWC in the Eluridum frond between control plants and plants under waterdeficit or ABA treatments However an increase of 328RWC was seen in plants irrigated with ABA compared toplants subjected to water deficit (Table 1) No fluctuationswere observed in malate contents between night and day incontrol plants and plants subjected to water stress Howevera decrease in total malate contents was observed at 600 hin plants treated with ABA for 15 days compared to controlplants and plants subjected to water stress (Figure 3)

33 Analysis of Photosynthetic Pigment Contents and Photo-synthetic Parameters In the E luridum frond results of this

Table 1 Changes in the RWC in fronds of Elaphoglossum luridumunder different treatments control water deficit (seven days) andABA-treated fronds (fifteen days) (10 120583M)The data are presented asmean plusmn SD Lowercase letters indicate the treatments differentiatedby ANOVA followed by Tukeyrsquos test 119875 gt 005 (119899 = 3)

Treatment RWC ()Control 8937 plusmn 060ab

Water deficit 8722 plusmn 136b

ABA 9050 plusmn 056a

study show that the contents of total chlorophyll as well aschlorophyll a and b were significantly lower under waterstress treatment for seven days and under ABA treatment forfifteen days compared to the control (Figure 4) The analysisof chlorophyll a fluorescence in E luridum (Table 2) showedsignificant differences among the photosynthetic parametersanalyzed 119868opt ETRmax and the alpha 120572 (photosyntheticefficiency) under water stress for seven days and ABAirrigation for fifteen days compared to control plants Theseresults can also be observed in rapid light curves (Figure 5)where the values of 119868opt ETRmax and 120572 declined during stresstreatments Water stress caused a 268 reduction in 119868optand after ABA application a reduction of 169 ETRmax wasalso observed under water stress (505) and ABA treatment(325) For 120572 the reduction was 361 for water stress and204 for ABA treatment


(a)

(b)

ab

adpp

sp

100120583m

p

50120583m

(c)

adct

ep

20120583m

(d)st

50120583m (e)en

50120583m

Figure 2 Cross-sections of Elaphoglossum luridum frond in LM (a) Mesophyll consists of parenchyma tending to palisade (pp) spongyparenchyma (sp) and uniseriate epidermis on both sides of the frond (b) Details of the abaxial tissues showing pits in the spongy mesophyllcell walls (c) Details of the cuticle showing a light positive Sudan IV reaction (d) Details of stomata on the abaxial surface (e) Details of thevascular tissue Ab abaxial epidermis ad adaxial epidermis ct cuticle en endodermis ep epidermis p pit pp palisade parenchyma spspongy parenchyma and st stomata

4 Discussion

E luridum presents coriaceus and succulent fronds showinga parenchyma tending to the palisade and chlorophyllousspongy parenchyma which presents large vacuoles to storewater The relatively thick cuticle in the outer periclinal wallsof epidermal cells is an aspect that should contribute to themaintenance of water balance In general cuticle thicknessvaried from 01 to 10120583m considering leaves fruits andother primary organs of higher plants [34 35] According toHietz and Briones [8] these features are commonly found inepiphytic ferns and are part of a list of attributes responsiblefor maintaining water during recurring periods of droughtSinuous anticlinal cell walls were observed in the epidermis ofE luridum frond Krauss [36] commented that the tendencytoward sinuosity represents mechanical adaptations to avoidcollapse during expansion and contraction of the leaf bythe entrance and exit of water E luridum shows branchedtrichomes in the epidermis of the foliar blade Scales withsimilar anatomical structure were also found in Pleopeltis

mexicana (Fee) fronds and Elaphoglossum petiolatumBonapwhich probably increase the capacity of fronds to absorbwater without increasing cuticle water loss [8] The scaleshave evolved independently in ferns and bromeliads but theyare treated as an important structure for water absorption inepiphytes [8]

Plants of E luridum were able to maintain RWC around90 after water stress This species is homoiohydrous Tauszet al [37] observed similar results for epiphytic fernsincluding Elaphoglossum glaucumTMoore and E petiolatumBonap which are able to maintain high contents of RWCunder short-term stress Hietz and Briones [8] demonstratedthat the leathery fronds of E glaucum showed a lower rateof cuticle transpiration among the studied species In Eluridum frond the exogenous application of ABA for a periodof fifteen days slightly increased RWC in contrast to plantssubjected to drought stress In fact ABA does play a rolein the control of water balance in ferns When fronds ofPolypodium virginianum L a desiccation-tolerant fern wereincubated in ABA for 24 h prior to silica-drying the amount


Table 2 Rapid light curve (RLC) parameters plotted as ETR (119875) versus irradiance (119868) in sporophytes ofElaphoglossum luridum under differenttreatments The data are presented as mean plusmn SD Lowercase letters indicate the treatments differentiated by ANOVA (between columns)followed by Tukeyrsquos test 119875 gt 005 (119899 samplecurves = 330) 119860 scaling constant for the height of the light curve ETRmax maximum electrontransport rate 119868opt optimal irradiance and119870

119908 scaling constant for the 119909-axis of the light curve

Control Water deficit ABA stress119868opt (120583mol photon mminus2 sminus1) 1208 plusmn 810a 8840 plusmn 670b 1004 plusmn 330b

ETRmax (120583mol electron mminus2 sminus1) 760 plusmn 90a 370 plusmn 80b 510 plusmn 20b

Alpha (120572) 0172 plusmn 0008a 0110 plusmn 001c 0137 plusmn 0001b

(119860) 1960 plusmn 350 1020 plusmn 240 1390 plusmn 60(119870119908) 00008 plusmn 00001 00011 plusmn 00001 00001 plusmn 000004

Correlation coefficient 119903 0978 0956 0966119899 samplecurves 330 330 330

a a

b

aa

a

0

1

2

3

4

Control Water deficit ABA

Mal

ate (

120583m

ol gminus

1D

M)

06001800

Figure 3 Daily fluctuation (600hndash1800 h) in malate content offronds of Elaphoglossum luridum under different treatments Thedata are presented as mean plusmn SD Lowercase letters indicate thegroups differentiated by ANOVA followed by Tukeyrsquos test 119875 gt 005(119899 = 3)

a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000

Chl a Chl b Car Total chl

ControlWater deficitABA

Pigm

ents

(120583g g

minus1

DM

)

Figure 4 Chlorophyll (Chl a Chl b and total Chl) and carotenoid(Car) contents in fronds of Elaphoglossum luridum under differenttreatments The data are presented as mean plusmn SD Lowercase lettersindicate the groups differentiated by ANOVA followed by Tukeyrsquostest 119875 gt 005 (119899 = 3)

0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)

Irradiance (120583mol mminus2 sminus1 PAR)

Figure 5 Rapid light curve (RLC) plotted as ETR (119875) versusirradiance (119868) in fronds of Elaphoglossum luridum under differenttreatments The data are presented as mean plusmn SD Lowercase lettersindicate the groups differentiated by ANOVA followed by Tukeyrsquostest 119875 gt 005 (119899 = 3)

of water lost was reduced resulting in survival of the frondsupon subsequent rehydration [38] Ruszala et al [39] foundthat stomatal responses of the lycophyte Selaginella uncinatato ABA and CO

2are directly comparable to those of the

flowering plant Arabidopsis thalianaAfter water stress and ABA treatment malate content

found in E luridum seems to be many times lower thanthe content found in plants having the CAM pathwaysuggesting the absence of CAM pathway in E luridumfrond based on daynight variation In M crystallinum L(Aizoaceae) malate contents were higher than 75120583molsdotgminus1FM after ABA treatment [17] In pineapple Δmalate reachedalmost 500120583molsdotgminus1 DM after 25 days of water stress [40]On the other hand malate content decreased in fronds ofE luridum after ABA treatment seeming to downregulatemalate metabolism The ABA mode of action is linked


to diurnal stomatal movements the elevated level of ABAbiosynthesis in the dark phase of the day is responsible forstomatal closure In the evening ABAbiosynthesis outweighsABA catabolism in the guard cells leading to stomatalclosure Under drought stress conditions ABA could reach aconcentration high enough to cause ion efflux and inhibitionof sugar uptake by the guard cells ABA can also stimulate glu-coneogenic conversion of malate into starch thus reducingstomata apertures [41] Based on these lines of evidence wesuggest that ABA could reduce malate contents in fronds ofE luridum not only in guard cells but also in the mesophyllcells especially at the night

In this paper we observed a small decrease in chlorophyllcontents but not in carotenoid contents after water stressand ABA treatment in E luridum frond Tausz et al [37]working with photosynthetic pigments in different speciesof epiphytic ferns in a Mexican rainforest noted a responsesimilar to that obtained in this work that is that totalchlorophyll content decreased under water stress inducedin Polypodium plebeium Schltdl and Cham E petiolatumPhlebodium areolatum and Asplenium cuspidatum Lam Thereduction in chlorophyll content can be attributed to the highdegradation rate of these pigments which is higher than thebiosynthesis under drought stress conditions [42] On theother hand chlorophyll was retained inMohria caffrorum (L)Dev (Anemiaceae) during drying in both DT and DS formsof the plant [13]

Drought stress is potentially harmful by enhancing theproduction of reactive oxygen species (ROS) [43] Droughtstress is known to inhibit photosynthetic activity in tissuesDownregulation of PSII activity results in an imbalancebetween the generation and utilization of electrons [44] Inthis study water deficit and application of ABA decreasedETRmax and 119868opt and seemed to negatively regulate the rateof electron transport in fronds of E luridum Similarly inMcaffrorum the quantumefficiency of photosystem II (FVFM)of DT leaves declined once the water level dropped below70 RWC but it recovered to predesiccation levels uponrehydration suggesting that little damage had occurred inthe photosynthetic apparatus in those tissues or in thealternative that rapid repair had occurred [13] Accordingto Maxwell and Johnson [45] the drop of RLC under stressindicates photoinhibition of PSIITherefore our data suggestthat both water stress and ABA moderately inhibited PSIIactivity in frond

Our results show that E luridum frond is coriaceus andsucculent presenting trichomes relatively thick cuticle andsinuous cell walls in both abaxial and adaxial epidermisSince it is homoiohydrous it is able to maintain RWC undermild water stress and after application of ABA but at thesame time these factors reduced chlorophyll contents andETR This species did not show the ability to regulate the C

3

metabolism required for CAM under water stress and ABAbut it did show morphological adaptations strong enoughto withstand periods of drought E luridum responded toABA maintaining higher levels of RWC and lower contentsof nocturnal malate

Abbreviations

119860 Scaling constant for the height of the light curveABA Abscisic acidAL Actinic lightCAM Crassulacean acid metabolismDM Dry massDS Desiccation-sensitiveDT Desiccation-tolerantETR Electron transport rateETRmax Maximum electron transport rateFM Fresh mass119865119900 Minimum fluorescence119865119898 Maximum fluorescence119865V Variable fluorescence1198651015840

119898 Light adapted maximum fluorescence1198651015840 Light acclimated steady-state fluorescence

FLM Fluorescence microscopy119865V119865119898 Maximum photochemical efficiency of PSII119868 Irradiance119868opt Optimal irradiance119870119908 Scaling constant for the 119909-axis of the light curve

LM Light microscopyML Modulated lightPAM Pulse-amplitude modulation119875 PhotosynthesisPAR Photosynthetically active radiationPEG Polyethylene glycolPPFD Photosynthetic photon flux densityPS PhotosystemRCL Rapid light curveRWC Relative water contentSA Salicylic acidSEM Scanning electron microscopySL Saturating light pulsesΦPSII Actual photochemical efficiency of PSIITCA Tricarboxylic acid cycleTM Turgid mass

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The first authors would like to acknowledge the scholar-ship provided by CAPES as well as the Council for theImprovement of Universities Staff PNADB 2009 Networkin Epiphytic of Atlantic Forest systematic ecology andconservation Aurea Maria Randi and Marisa Santos wouldlike to acknowledge CNPq (National Council for Scientificand Technological Development) for the research grants

References

[1] U Luttge Vascular Plants as Epiphytes Evolution and Ecophysi-ology Springer Heilderberg Germany 1989


[2] J W Kress ldquoA symposium the biology of tropical epiphytesrdquoSelbyana vol 9 pp 1ndash22 1986

[3] J Nieder J Prosperı and G Michaloud ldquoEpiphytes and theircontribution to canopy diversityrdquo Plant Ecology vol 153 no 1-2 pp 51ndash63 2001

[4] M Madison ldquoVascular epiphytes their systematic occurrenceand salient featuresrdquo Selbyana vol 2 pp 1ndash13 1977

[5] B C Bennet ldquoPatchiness diversity and abundance relation-ships of vascular epiphytesrdquo Selbyana vol 9 pp 70ndash75 1986

[6] D H Benzing ldquoThe vegetative basis of vascular epiphytismrdquoSelbyana vol 9 pp 23ndash43 1986

[7] D H BenzingVascular Epiphytes Cambridge University PressCambridge UK 1990

[8] P Hietz and O Briones ldquoCorrelation between water relationsand within-canopy distribution of epiphytic ferns in a Mexicancloud forestrdquo Oecologia vol 114 no 3 pp 305ndash316 1998

[9] P Hietz ldquoFern adaptations to xeric environmentsrdquo in FernEcology K Mehltreter L Walker and J M Sharpe Eds pp140ndash170 Cambridge University Press New York NY USA2010

[10] A R Smith K M Pryer E Schuettpelz P Korall H Schneiderand P G Wolf ldquoA classification for extant fernsrdquo Taxon vol 55no 3 pp 705ndash731 2006

[11] RM Tryon andA F Tryon Ferns andAllied Plants with SpecialReference to Tropical America Springer New York NY USA1982

[12] S Maciel andM R Pietrobom ldquo Dryopteridaceae and Lomari-opsidaceae (Polypodiopsida) from Embraparsquos Eastern Amazo-nia field station Moju Para Brazilrdquo Rodriguesia vol 61 pp405ndash414 2010

[13] J M Farrant A Lehner K Cooper and S Wiswedel ldquoDes-iccation tolerance in the vegetative tissues of the fern Mohriacaffrorum is seasonally regulatedrdquoThe Plant Journal vol 57 pp65ndash79 2009

[14] J Dat S Vandenabeele E Vranova M Van Montagu DInze and F Van Breusegem ldquoDual action of the active oxygenspecies during plant stress responsesrdquo Cellular and MolecularLife Sciences vol 57 no 5 pp 779ndash795 2000

[15] H-Q Feng H-Y Li and K Sun ldquoEnhanced expression ofalternative oxidase genes is involved in the tolerance of rice(Oryza sativa L) seedlings to drought stressrdquo Zeitschrift furNaturforschung vol 64 no 9-10 pp 704ndash710 2009

[16] K Shinozaki and K Yamaguchi-Shinozaki ldquoGene expressionand signal transduction in water-stress responserdquo Plant Physi-ology vol 115 no 2 pp 327ndash334 1997

[17] C Chu Z Dai M S B Ku and G E Edwards ldquoInductionof crassulacean acid metabolism in the facultative halophyteMesembryanthemum crystallinum by abscisic acidrdquo Plant Phys-iology vol 93 no 3 pp 1253ndash1260 1990

[18] D R Hoagland and D I Arnon ldquoThe water culture method forgrowing plants without soilrdquoCalifornia Agricultural Experimen-tal Station vol 347 pp 1ndash39 1938

[19] S E Ruzin Plant Microtechnique and Microscopy OxfordUniversity Press New York NY USA 1999

[20] J E Kraus and M Arduin Manual Basico de Metodos emMorfologia Vegetal Editora Universidade Rural Rio de JaneiroBrazil 1997

[21] D Gerlach Botanische Mikrotechnik Eine Einfuhrung GeorgThieme Verlag Stuttgart Germany 1984

[22] H D Barrs and P E Weatherley ldquoA re-examination of therelative turgidity technique for estimating water deficits inleavesrdquoAustralian Journal of Biological Sciences vol 15 pp 413ndash428 1962

[23] H Mollering ldquoL (-) malaterdquo inMethods of Enzymatic AnalysisHU Bergmeyer Ed vol 7 pp 39ndash47 VHCVerlagsgesellschaftWeinheim Germany 1985

[24] H K Lichtenthaler ldquoChlorophylls and carotenoids pigmentsof photosynthetic biomembranesrdquoMethods in Enzymology vol148 pp 350ndash382 1987

[25] B Genty J M Briantais and N R Baker ldquoThe relationshipbetween the quantumyield of photosynthetic electron transportand quenching of chlorophyll fluorescencerdquo Biochimica etBiophysica Acta vol 990 pp 87ndash92 1989

[26] O Bjorkman andBDemmig ldquoPhoton yield ofO2evolution and

chlorophyll fluorescence characteristics at 77 K among vascularplants of diverse originsrdquo Planta vol 170 no 4 pp 489ndash5041987

[27] A J White and C Critchley ldquoRapid light curves a newfluorescence method to assess the state of the photosyntheticapparatusrdquo Photosynthesis Research vol 59 no 1 pp 63ndash721999

[28] J W Runcie and M J Durako ldquoAmong-shoot variability andleaf-specific absorptance characteristics affect diel estimates ofin situ electron transport ofPosidonia australisrdquoAquatic Botanyvol 80 no 3 pp 209ndash220 2004

[29] U Schreiber ldquoPulse-amplitude (PAM) fluorometry and satu-ration pulse methodrdquo in Chlorophyll Fluorescence A Signatureof Photosynthesis G C Papageorgiou and Govindjee EdsAdvances in Photosynthesis and Respiration Series pp 279ndash319 Kluwer Academic Publishers Dordrecht The Netherlands2004

[30] P J Ralph and R Gademann ldquoRapid light curves a powerfultool to assess photosynthetic activityrdquo Aquatic Botany vol 82no 3 pp 222ndash237 2005

[31] J H Zar Biostatistical Analysis Prentice Hall Upper SaddleRiver NJ USA 1996

[32] R J Ritchie ldquoFitting light saturation curves measured usingmodulated fluorometryrdquo Photosynthesis Research vol 96 no 3pp 201ndash215 2008

[33] R S Gloag R J Ritchie M Chen A W D Larkumand R G Quinnell ldquoChromatic photoacclimation photosyn-thetic electron transport and oxygen evolution in the Chloro-phyll 119889-containing oxyphotobacterium Acaryochloris marinardquoBiochimica et Biophysica Acta Bioenergetics vol 1767 no 2 pp127ndash135 2007

[34] P J Holloway ldquoStructure and histochemistry of plant cuticularmembranesrdquo in The plant cuticle D F Cutler K L Alvin andC E Price Eds pp 1ndash32 Academic Press London UK 1982

[35] M Riederer and L Schreiber ldquoProtecting against water lossanalysis of the barrier properties of plant cuticlesrdquo Journal ofExperimental Botany vol 52 no 363 pp 2023ndash2032 2001

[36] B H Krauss ldquoAnatomy of the vegetative organs of the pineap-ple Ananas comosus (L) Merr IImdashthe leafrdquo Botanical Gazettevol 110 pp 333ndash404 1949

[37] M Tausz P Hietz and O Briones ldquoThe significance ofcarotenoids and tocopherols in photoprotection of seven epi-phytic fern species of aMexican cloud forestrdquoAustralian Journalof Plant Physiology vol 28 no 8 pp 775ndash783 2001

[38] T L Reynolds and J D Bewley ldquoAbscisic acid enhances theability of the desiccation-tolerant fern Polypodium virginianum


to withstand dryingrdquo Journal of Experimental Botany vol 44no 269 pp 1771ndash1779 1993

[39] E M Ruszala D J Beerling P J Franks et al ldquoLand plantsacquired active stomatal control early in their evolutionaryhistoryrdquo Current Biology vol 21 no 12 pp 1030ndash1035 2011

[40] L Freschi M A Rodrigues M A S Tine and H MercierldquoCorrelation between citric acid andnitratemetabolisms duringCAM cycle in the atmospheric bromeliad Tillandsia pohlianardquoJournal of Plant Physiology vol 167 no 18 pp 1577ndash1583 2010

[41] A Daszkowska-Golec and I Szarejko ldquoOpen or close the gatemdashstomata action under the control of phytohormones in droughtstress conditionsrdquo Frontiers in Plant Science vol 4 pp 1ndash162013

[42] J Yang J Zhang Z Wang Q Zhu and L Liu ldquoWheatWater deficit-induced senescence and its relationship to theremobilization of pre-stored carbon in wheat during grainfillingrdquo Agronomy Journal vol 93 no 1 pp 196ndash206 2001

[43] J Sang M Jiang F Lin S Xu A Zhang and M Tan ldquoNitricoxide reduces hydrogen peroxide accumulation involved inwater stress-induced subcellular anti-oxidant defense in maizeplantsrdquo Journal of Integrative Plant Biology vol 50 no 2 pp231ndash243 2008

[44] D Peltzer E Dreyer and A Polle ldquoDifferential temperaturedependencies of antioxidative enzymes in two contrastingspecies Fagus sylvatica andColeus blumeirdquoPlant Physiology andBiochemistry vol 40 no 2 pp 141ndash150 2002

[45] K Maxwell and G N Johnson ldquoChlorophyll fluorescencemdashapractical guiderdquo Journal of Experimental Botany vol 51 no 345pp 659ndash668 2000

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


600 species mostly in the Neotropics and some 85 ofthese species are epiphytes [7 11] The name of the genusderives from the Greek elaphos (deer) + glossa (tongue)so named for the shape of its fronds According to Macieland Pietrobom [12] E luridum is distinguished from otherspecies of the genus by its leathery bladed epidermis andribs with black scales on both surfaces especially in thebasal region This species occurs in Costa Rica Panamathe Lesser and Greater Antilles Trinidad Guiana FrenchGuiana Suriname Colombia Ecuador Peru and Bolivia InBrazil it is found in Amazonas Para Minas Gerais Rio deJaneiro Sao Paulo Parana Santa Catarina and Rio Grandedo Sul [12]

Drought tolerance is gained through adaptations in wateruptake water loss water storage and in many ferns desicca-tion tolerance which can be divided into two major groupsthe poikilohydrous (desiccation-tolerant) and the homoiohy-drous (relatively desiccation-intolerant) species [7 9] Xero-phytes are homoiohydrous and can be drought endurers anddrought avoiders [7 9]They can show productive ephemeralfoliage suitable only for wet season activity or they are activeyear round by virtue of desiccation-resistant leaves or greenstems with considerable water storage capacity [7] HoweverFarrant et al [13] comment that desiccation tolerance in fernshas been poorly studied

ABA H2O2 and SA can be used to induce plant defense

responses against drought stress [14 15] The ABA levelincreases as a result of drought stress and plays an importantrole in regulating the responses of plants to drought stress[16] The CAM pathway is one of the most important adapta-tions of some epiphytic ferns towater deficit In the facultativehalophyte Mesembryanthemum crystallinum L (Aizoaceae)ABA and water stress induce the facultative CAM pathway[17]

This work aimed to provide more insight into theresponses of the epiphytic fern E luridum to water stressTo accomplish this anatomical and physiological studieswere conducted Specifically three treatments were analyzedcontrol water deficit and ABA Physiological studies wereconducted through analysis of RWC photosynthetic pig-ments RLC photosynthetic parameters and malate contentafter exposure to water stress and ABA treatment in a CAMcontext

2 Material and Methods

21 Plant Collection and Acclimation Plants of E luridumwere collected from the natural habitat in the EnvironmentalConservation Unit ldquoDesterrordquo (UCAD) located in north-western Florianopolis Santa Catarina Brazil (27∘3110158405010158401015840S 0848∘3010158404410158401015840W 03) an area of 495 hectares comprising about1 of the total area of the island The unit is managedby the Federal University of Santa Catarina (UFSC) Plantswere transported to the greenhouse of the Department ofBotanywhere theywere acclimated for sixmonthsThe plantswere tied and placed in brackets on the walls During thetest period the temperature of the greenhouse varied from20 to 30∘C during the day and around 15∘C at night The

relative humidity was monitored and ranged between 45 and83 The photosynthetic photon flux density (PPFD) insidethe greenhouse alternated between 43 and 85 120583molmminus2 sminus1Irradiance was measured at the middle region of the frondswith LI-190 sensor (LI-COR Instruments USA) connected tothe LI-250 light meter (LI-COR Instruments USA) whichwas placed close to plants The plants were watered dailywith distilled water to maintain high humidity Once a weekplants were irrigated with Hoaglandrsquos solution (20) (vv)[18] After the acclimation period the plants were dividedinto three groups each subjected to different treatments(1) control plants irrigated daily for two weeks (2) plantssubjected to drought stress for a period of one week (withoutirrigation) and (3) plants irrigated 5 times a week with asolution of 10120583MABA for a period of fifteen days Treatmentswere made in triplicate

22 Anatomical Analysis of Fronds For in vivo structuralanalysis of the fronds we performed longitudinal and para-dermic cross-sections both done freehand with a razor bladeusing polystyrene as support material These sections wereplaced on a slide with water and covered with a coverslipThe stomata count was performed in triplicate in the medianregion of the abaxial surface with the aid of an opticalmicroscope Leica DM2500 (Leica Wetzlar Germany) andimages were captured with an attached digital camera (LeicaDFC295 Leica Wetzlar Germany)

Structural analysis of the fronds was also performed onsamples fixed in 25 glutaraldehyde in sodium phosphatebuffer 01M pH 72 washed in the same buffer dehydrated inan ethanol series and preserved in ethanol 70∘ GL [19] Somesamples were infiltrated in a mixture of polyethylene glycol(PEG) 1500 and 70 ethanol (1 1) for 24 hours in an oven at60∘C They were incubated again in an oven for 24 hours inpure PEG 1500 Following this incubation the samples wereembedded in PEG 1500 Other samples were infiltrated inhydroxyethyl methacrylate (Jungrsquos Historesin Leica WetzlarGermany) The samples preserved in ethanol 70∘ GL weredehydrated in an ethanol series up to 96∘ GL and processedaccording to the manufacturerrsquos instructions The samplesembedded in PEG and Historesin were sectioned by rotarymicrotome (Leica RM 2125 RT Leica Wetzlar Germany)and stained with safranin [20] or Sudan IV for lipids [21]Twenty measurements of cuticle thickness were performedThe material was examined under an optical microscope(Leica DM2500 Leica Wetzlar Germany) and images werecaptured with an attached digital camera (Leica DFC295Leica Wetzlar Germany)

For scanning electronmicroscopy analysis frond sampleswere subjected to total dehydration in ethanol series andkept in ethyl ether for 48 h at 20∘C Afterwards the samplecontainers were opened and kept in laminar flow to favorthe complete evaporation of the etherThe samples were thenplaced on aluminum brackets with the aid of double-sidedcarbon tape Frond samples were metalized with 30 nm ofgold in a Bal-Tec CED 030 metalizer (Bal-Tec AG Balz-ers Liechtenstein) A scanning electron microscope (SEM)(JEOL JSM-6390LV JEOL Tokyo Japan) was used to analyzethe samples







119900) was






119898minus 1198651015840)1198651015840

119898 PPFD is





119908sdot 119868 sdot





3 Results



(a) (b)

(c) (d)

50120583m50120583m

10120583m 100120583m











(a)

(b)

ab

adpp

sp

100120583m

p

50120583m

(c)

adct

ep

20120583m

(d)st

50120583m (e)en

50120583m


4 Discussion












a a

b

aa

a

0

1

2

3

4


Mal

ate (

120583m

ol gminus

1D

M)

06001800


a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000



Pigm

ents

(120583g g

minus1

DM

)


0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)












3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







119900) was






119898minus 1198651015840)1198651015840

119898 PPFD is





119908sdot 119868 sdot





3 Results



(a) (b)

(c) (d)

50120583m50120583m

10120583m 100120583m











(a)

(b)

ab

adpp

sp

100120583m

p

50120583m

(c)

adct

ep

20120583m

(d)st

50120583m (e)en

50120583m


4 Discussion












a a

b

aa

a

0

1

2

3

4


Mal

ate (

120583m

ol gminus

1D

M)

06001800


a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000



Pigm

ents

(120583g g

minus1

DM

)


0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)












3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

50120583m50120583m

10120583m 100120583m











(a)

(b)

ab

adpp

sp

100120583m

p

50120583m

(c)

adct

ep

20120583m

(d)st

50120583m (e)en

50120583m


4 Discussion












a a

b

aa

a

0

1

2

3

4


Mal

ate (

120583m

ol gminus

1D

M)

06001800


a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000



Pigm

ents

(120583g g

minus1

DM

)


0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)












3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)

ab

adpp

sp

100120583m

p

50120583m

(c)

adct

ep

20120583m

(d)st

50120583m (e)en

50120583m


4 Discussion












a a

b

aa

a

0

1

2

3

4


Mal

ate (

120583m

ol gminus

1D

M)

06001800


a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000



Pigm

ents

(120583g g

minus1

DM

)


0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)












3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









a a

b

aa

a

0

1

2

3

4


Mal

ate (

120583m

ol gminus

1D

M)

06001800


a

aa

a

b

b a

b

b

b a

b

0

500

1000

1500

2000

2500

3000



Pigm

ents

(120583g g

minus1

DM

)


0

25

50

75

100

0 500 1000 1500 2000 2500


ETR

(120583m

ol m

minus2

sminus1)












3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3


Abbreviations







Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article The Epiphytic Fern Elaphoglossum luridum...

Documents

Transcript of Research Article The Epiphytic Fern Elaphoglossum luridum...