Effects of light intensity on the morphology and CAM photosynthesis of Vanilla planifolia Andrews

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1 Reserch rticle Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews Efectos de l intensidd lumínic sore l morfologí y l fotosíntesis CAM de Vnill plnifoli Andrews doi: /rfn.v70n M. Cludi Díez 1 *, Flvio Moreno 1 nd Edison Gntiv 1 ABSTRACT Key words: Chlorophyll fluorescence Gs exchnge Orchids Photosynthetic pigments Titrtle cidity Tropicl crops Vnill plnifoli is neotropicl orchid, whose fruits produce the nturl vnill, fundmentl ingredient for the food nd cosmetic industry. Becuse of its importnce in the world mrket, it is cultivted in mny tropicl countries nd recently its cultivtion hs strted in Colomi. This species requires shde for its development; however, the optiml of light conditions re unknown. This work evlutes the effect of different light intensities on CAM photosynthesis, physiology, morphology, nd growth of this species. For this, vnill seedlings were sujected to four tretments of reltive illumintion (RI) (T1=8%, T2=17%, T3=31% nd T4=67%). Most CO 2 ssimiltion occurred long night in ll tretments, which confirms tht vnill is strong CAM species. Plnts grown under high lighting (67% RI) hd lmost hlf of the photosynthesis in tretments of intermedite lighting (17 nd 31%), which is consistent with the lower nocturnl cid ccumultion in tht tretment. Likewise, the photochemicl efficiency of photosystem II (Fv / Fm) showed tht in plnts of the 67% RI occurred high rdition stress. On the other hnd, vnill plnts reched greter length, lef re, nd totl iomss when grown under intermedite rdition (17 nd 31% RI). These results suggest tht high rdition lters the functioning of vnill plnts, inhiiting photosynthesis nd growth, nd tht highly shded environments not significntly ffected the CAM photosynthesis of vnill; however, in the long-term this species showed higher photosynthesis nd growth under intermedite levels of rdition. RESUMEN Plrs clves: Fluorescenci de l clorofil Intercmio de gses Orquídes Pigmentos fotosintéticos Acidez titulle Cultivos tropicles Vnill plnifoli es un orquíde neotropicl, cuyos frutos producen l vinill nturl, un ingrediente fundmentl pr l industri limentici y cosmétic. Deido su importnci en el mercdo interncionl, se cultiv en vrios píses tropicles y recientemente h comenzdo su cultivo en Colomi. Est especie requiere somr pr su desrrollo; sin emrgo, se desconocen sus rngos óptimos de condiciones de iluminción. En este trjo evlumos el efecto de diferentes intensiddes lumínics sore l fotosíntesis CAM, l fisiologí, l morfologí y el crecimiento de est especie. Pr esto, plnts jóvenes de vinill se sometieron cutro trtmientos de iluminción reltiv (IR) (T1=8%, T2=17%, T3=31% y T4=67%). L myor proporción de similción de CO 2 ocurre durnte l noche en todos los trtmientos, lo cul confirmó que l vinill es un especie CAM fuerte. Ls plnts que crecieron jo lt iluminción (67% IR), tuvieron csi l mitd de l fotosíntesis de ls plnts en los trtmientos de iluminción intermedi (17 nd 31%), lo cul coincide con l j cumulción nocturn de ácidos en este trtmiento. Así mismo, l eficienci fotoquímic del fotosistem II (Fv / Fm) mostró que en ls plnts del 67% IR ocurrió estrés por lt rdición. De otro ldo, ls plnts de vinill lcnzron myor longitud, áre folir, y ioms totl cundo crecieron jo rdición intermedi (17 y 31% IR). Estos resultdos sugieren que l lt rdición lter el funcionmiento de ls plnts de vinill, inhiiendo l fotosíntesis y el crecimiento, y que los mientes ltmente somredos no fectn significtivmente l fotosíntesis CAM de l vinill; sin emrgo, en el lrgo plzo, est especie muestr myor fotosíntesis y crecimiento jo condiciones intermedis de rdición. 1 Fcultd de Ciencis Agrris. Universidd Ncionl de Colomi. AA Medellín, Colomi. * Corresponding uthor <mcdiez@unl.edu.co> Received: My 6, 2016; Accepted: Octoer 18, 2016 Rev.Fc.Nc.Agron. 70(1): ISSN / e-issn

2 8024 Diez MC, Moreno F, Gntiv E Vnill (Vnill plnifoli Andrews) is hemiepiphyte orchid tht fces sesonl wter stress throughout its life cycle under nturl conditions, which explin its CAM photosynthetic pthwy (Goh nd Kluge, 1989); likewise, this species fces different light environments: initilly it grows in the shdy interior of neotropicl forests, nd then, lening on the tree, reches the cnopy where flowers nd fruits t more light (Fouché nd Jouve, 1999). Since vnill plnts re exposed to sptil nd temporl (dily nd sesonl) vritions in the mount of rdition received (Fouché nd Jouve, 1999), it is expected tht this species hd high cclimtion cpcity to different light environments, such s other epiphytes nd hemiepiphytes of tropicl forests (Zotz nd Winter, 1994; Zotz nd Andrde, 2001; Hslm et l., 2003). Becuse of its importnce in the world mrket of spices, vnill is cultivted in mny tropicl countries (Bory et l., 2008). The success of this crop depends, mong other fctors, of the proper mngement of light ecuse it is the environmentl fctor with the gretest influence on plnt photosynthesis nd therefore on growth, survivl, nd ultimtely, the ility of cclimtion to different hitts (Puthur, 2005). Hence, the importnce of estlishing the cclimtion cpcity of vnill plnts to different light environments nd determining the most fvorle for their growth. The role of rdition on photosynthesis of CAM plnts is relted to the processes tht occur during the four phses of this metolism (Osmond, 1978), since the rdition cn modulte their mgnitude nd durtion (Cushmn, 2001). The intensity of photosyntheticlly ctive rdition (PAR) during the dy (Phse III), determines the rte of moiliztion of orgnic cids from the vcuole (Brrow nd Cockurn, 1982). On the other hnd, the mount of rdition during the dy lso influences the extent of CO 2 sorption t night (Phse I), ffecting the undnce of crohydrtes generted through the Clvin cycle nd glucogenesis, which re required for the provision of PEP t night (Noel nd Hrtsock, 1983). Thus, the rdition during the dy ffects the extent of cid ccumultion during the following night, nd therefore, the mplitude of dily fluctutions of cids in CAM plnts depends on rdition intensity (Lüttge, 2004). Sometimes low lighting cn e limiting for CAM photosynthesis, such s in epiphytes of cloud forests during the riny seson (Pierce et l., 2002). Excess of rdition nd over-energiztion of the CAM photosynthetic pprtus cn lso e limiting; systems of energy dissiption exhiited y C3 plnts, such s photorespirtion nd the presence of crotenes, hve lso een reported in CAM plnts (Lüttge, 2004). In regions where vnill is cultivted, locted minly in the sutropicl zone, different vlues of lighting hve recommended for the proper development of plnts: 30-80% of reltive illumintion (RI) in Mexico (Hernndez- Hernndez, 2011), 50-60% in Cost Ric (Vrel, 2011), 60% in Reunion, Comoros nd Mdgscr Islnds (Khne et l., 2008), 30-50% in Indi (Anilkumr., 1994; Srm et l, 2011; Zuin et l, 2011), 50-60% in Austrli (Exley, 2011), nd 30-50% in Indonesi nd Islnds in the south Pcific (Binchessi, 2004). The lower vlues of these rnges re generlly considered pproprite for vegettive growth nd the higher ones to stimulte flowering (Fouché nd Jouve, 1999; Puthur, 2005). However, these recommendtions re rrely sed on pulished reserch or results tht support higher growth or development of plnts in these environments. According to former reports, our hypothesis is tht RI close to 30% is the most fvorle for photosynthesis nd growth of vnill plnts in the initil phse of estlishment. To test it, the ojective of this study ws to evlute the ehvior of V. plnifoli seedlings under different lighting levels in terms of: i) vegettive growth nd morphologicl chrcteristics; ii) chrcteristics of CAM photosynthesis; iii) the response to extreme light environments, expressed s cclimtion to low light conditions s well s the susceptiility nd defense mechnisms to photo inhiition y high lighting. MATERIALS AND METHODS Experimentl site The tril ws conducted in n re of lluvil terrces of flt topogrphy in the inter-nden vlley of the Cuc river (6 o N nd W), ltitude of 540 m, on lnds of the Agriculturl Center Cotové (Universidd Ncionl de Colomi). Dt from the wether sttion locted on the sme site re: nnul men temperture of 27 C, verge nnul precipittion of 1058 mm (imodl distriution, with dry sesons from Decemer to Mrch nd from June to August) nd men reltive humidity of 75% (dt of the Cotové Sttion, IDEAM). Rev.Fc.Nc.Agron. 70(1):

3 Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews The site corresponds to the life zone tropicl dry forest (sensu Holdridge, 2000). Vnill plnts Vnill cuttings used in this reserch were out 80 cm long, tken from the picl portion of the rnches of helthy nd dult plnts of V. plnifoli from crop estlished in Sn Pedro de Urá (Antioqui, Colomi). Cuttings were hrdened in the shde for two weeks, nd then plnted in plstic continers (6 L cpcity); wooden stnd ws previously instlled in ech continer to serve s tutor for susequent plnt growth. Lighting tretments Shde houses of four different RI conditions were uilt: 8%, 17%, 31%, nd 67%. Mximum vlues of photosynthetic ctive rdition (PAR) in the shde houses occurred etween 13 nd 14 h, nd were 142, 369, 577, nd 1285 µmol m -2 s -1 for the tretments of 8, 17, 31, nd 67% RI, respectively. These vlues were otined with quntum sensors (LI-190, LI-COR, Lincoln, Nersk, USA) connected to dt logger (LI-1000, LI-COR, Lincoln, Nersk, USA) instlled for three dys in one shde house y lighting tretment, with mesurements every hour in cycles of 24 h, t the end of the first dry seson of the yer. The totl dily rdition t full sun light verged 2.4, 12.8, 28.7, nd 43.8 mol m -2 d -1 in the tretments of 8%, 17%, 31%, nd 67% RI, respectively. Shde houses were lrge enough (width: 4.5 m, length 6 m, nd height 2.4 m) to ensure ertion nd spcing etween plnts nd prevent the occurrence of diseses. Vnill plnts were grown in the shde houses for six months efore strting the mesurement of vriles to ensure cclimtion to the corresponding light environment; then the monitoring of iometric prmeters ws done for twelve months. Plnts received permnent irrigtion to void differences y drought. Averge tempertures in the shde houses vried etween 25.3 o C in the tretment of 8% RI nd 26.2 in the tretment of 67%. Vriles evluted CO 2 exchnge. Dt of CO 2 exchnge were tken in fully expnded nd helthy young leves, locted in the fourth internode from the terminl ud with portle gs exchnge system (LI 6400 XT, LI-COR Biosciences, Lincoln, NE, USA). The CO 2 concentrtion ws kept constnt t vlue of 400 µmol mol -1 nd the lock temperture t 26 C. Automtic records were tken every two hours during cycles of 24 hours to three plnts per tretment. For ech RI tretment, one curve ws estimted s the verge of the three curves performed. The net sorption/relese of CO 2 ws quntified for the dy, night nd the whole dy, y integrting the res under the 24 h curves of CO 2 exchnge (Griffiths, 1989). Night ccumultion of H +. To determine the nocturnl cid ccumultion, the chnge of titrtle cidity of lef tissues etween dusk nd dwn of the next dy ws evluted. To this, smples of 4.5 cm 2 re were tken with metl punch in young fully expnded leves of five plnts per tretment. Immeditely fter collected, smples were frozen in liquid nitrogen, rought to the lortory nd stored in n opque continer t -80 C until processing, which consisted in immersing of smples in 70 ml of ethnol (20%) nd oiling for 20 min. Titrtle cidity of the solution ws mesured s the volume of NOH (5 mm NOH) necessry to rech ph of 7.0 (Silver et l., 2005) using digitl urette (Titrette, Brnd, Wertleim, Germny) nd ph meter (Hnn 211, Woonsocket, RI, USA). With the vlues of titrtion, the H + μequivlents were clculted (expressed in terms of lef re). The chnge of titrtle cidity of lef tissue ( H + ) ws clculted s the difference etween the μequivlents of H + t dwn nd the μequivlents of H + t dusk the dy efore. Biometric prmeters of plnts. After 18 months of growth of vnill plnts in ech lighting tretment, lef re ws mesured with portle meter (LI-3000C, LI-COR Biosciences, Lincoln, NE, USA) nd the totl stem length with metric tpe of ll plnts. Besides, three plnts per repetition were hrvested in ech tretment to ssess iomss of leves, stems, nd roots; these smples were oven dried (70 C to constnt weight) nd weighed. Then, totl iomss nd specific lef re (lef re per grm of iomss) were clculted. Chlorophyll fluorescence. The mximum quntum efficiency of photosystem II (PSII), clled quntum yield potentil, ws mesured with portle fluorometer (OS30p, Optisciences, Hudson, NH, USA). This vrile expresses the reltionship etween mximl Rev.Fc.Nc.Agron. 70(1):

4 8026 Diez MC, Moreno F, Gntiv E fluorescence nd vrile fluorescence (Fv / Fm); Fv is the difference etween mximl fluorescence (Fm) nd the sl fluorescence (Fo). Mesurements were performed in fully expnded nd helthy young leves, locted t position three to seven from the terminl ud in three plnts per repetition in ech RI tretment; efore smpling, leves were drk-dpted for 30 min. Dt were tken every two hours during 24 h. Pigment nlysis. Lef discs of 1.5 cm 2 were tken in three plnts per repetition per RI tretment. After collected, discs were frozen in liquid nitrogen, trnsported to the l, nd stored t -80 C until processing. Pigment extrction ws performed with 80% cetone (Lichtenthler nd Wellurn, 1983; Poorter et l., 2011). Smples were centrifuged (5 C for 5 min) nd the superntnt ws tken for reding in Visile - UV spectrophotometer t 470, 647 nd 663 nm (Evolution 600 UV-Vis, Thermo Scientific, Wlthm, MA, USA). The following equtions developed y Lichtenthler (1987) were used to otin the concentrtion of chlorophylls (Chlo) nd totl crotenes (Tot cr) (mg/g): Chlo = (12.25 * A 663nm ) + (2.79 * A 647nm ) Chlo = (21.5 * A 647nm ) + (5.1* A 663nm ) Totl Chlo = (chlo + chlo ) ( 100 A 470nm ) ( chlo) ( chlo ) Totl cr = 198 Experimentl design nd sttisticl nlysis. Dt were nlyzed for completely rndomized design, with four RI tretments (67%, 31%, 17% nd 8%) nd four repetitions per tretment. Ech repetition consisted of shde house where six vnill plnts were instlled (24 plnts per tretment) for totl of 120 plnts for the whole tril. For the sttisticl nlysis, initilly the ssumptions of normlity nd homoscedsticity of ech vrile were evluted with the tests of Shpiro-Wilk, Kolmogorov-Smirnov, nd frequency histogrms. Then, n nlysis of vrince (ANOVA) for ech vrile ws performed. Finlly, post hoc tests to determine significnt differences mong mens were used. In order to evlute the effect RI tretments on chlorophyll fluorescence throughout the dy, split plot model over time ws (1) (2) (3) (4) used; vrinces of fluorescence were homogenized y ln (x) trnsformtion (Steel nd Torrie, 1980). RESULTS AND DISCUSSION CO 2 exchnge. Most CO 2 ssimiltion occurred t night in ll tretments. At dwn, round 6:00 h, with the sunlight of the erly hours of the morning, CO 2 ssimiltion egn to decline drsticlly until 10:00 h. From this time, with high solr rdition, CO 2 ssimiltion ws negtive; fter 16:00 h the uptke egn to increse, ut usully did not rech positive vlues until 18:00 h when it ws lredy drk, so cron ssimiltion did not occur t the end of the fternoon (Figure 1). The verge vlues of mximum instntneous ssimiltion (± SE) were 1.17 ± 0.10, 1.71 ± 0.19, 1.65 ± 0.42 nd 0.98 ± 0.09 µmol m -2 s -1 for plnts grown under 8, 17, 31 nd 67%, RI respectively, nd occurred lte in the evening etween 0 nd 6 h. At night, during the Phse I of CAM metolism (Osmond, 1978), plnts sored the lrgest proportion of CO 2 per unit of lef re, equivlent on verge to 81.2% of the totl sorption for the 24 h period (Tl 1), which confirms tht vnill is strong CAM plnt (Silver et l., 2005). Nocturnl cid ccumultion. No significnt differences occurred mong tretments in cidity vlues t the end of the fternoon (18:00 h), ut they did erly in the morning (6:00 h) (P=0.0005). Vlues of nocturnl cid ccumultion ( H + ) were significntly higher in the tretments of 8, 17 nd 31% RI s compred with the tretment of 67% (P=0.0007) (Figure 2). Biometric vriles: Men lin length ws significntly higher (P=0.0017) in the intermedite RI tretments (17 nd 31%) compred to low nd high ones (8 nd 67%): cm vs cm, respectively (Figure 3), which represents n increse of 45.8% in plnts grown under intermedite light; however, internodes ecme longer with the decrese in RI (P=0.0002); vlues were 9.7 nd 7.9 cm in plnts tht grew t RI of 8 nd 67%, respectively. Men vlues of lef re were lso significntly higher (P=0.0003) in plnts under intermedite RI tretments (17 nd 31%) compred to low nd high ones (8 nd 67%): nd 1510 cm 2, respectively (Figure 3), which represents n increse of 147.4% in plnts grown under conditions of intermedite light. Rev.Fc.Nc.Agron. 70(1):

5 Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews A B Photosynthesis (μmol CO 2 m -2 seg -1 ) PAR (μmol CO 2 m -2 seg -1 ) C Photosynthesis (μmol CO 2 m -2 seg -1 ) PAR (μmol CO 2 m -2 seg -1 ) Photosynthesis (μmol CO 2 m -2 seg -1 ) PAR (μmol CO 2 m -2 seg -1 ) D -1.0 Photosynthesis (μmol CO 2 m -2 seg -1 ) PAR (μmol CO 2 m -2 seg -1 ) Figure 1. Dily course of photosynthesis of vnill plnts growing in different light environments (n=3, SE in rs): A. 8% RI, B. 17% RI, C. 31% RI, nd D. 67% RI (left). Rdition vlues during photosynthesis dt recording (right). Blck horizontl lines in grphs on the left represent night hours. Rev.Fc.Nc.Agron. 70(1):

6 8028 Diez MC, Moreno F, Gntiv E Tle 1. Dily photosynthesis in vnill plnts growing in different light environments. Totl photosynthesis (mmol m 2 )* Night Dy 24 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.6 Reltive illumintion (%) Proportion of nigth photosynthesis (%) * Men vlues ± SE (n=3). Different letters etween light tretments in ech column indicte significnt differences (P=0.05, Duncn test). 10 μequivlents H + cm dusk dwn H + Figure 2. Titrtle cidity t dusk (18:00 h), t dwn (6:00 h), nd nocturnl cid ccumultion (ΔH + ) in vnill plnts growing in different light environments: 8% RI, 17% RI, 31% RI nd 67% RI (n=5 per light tretment). Specific lef re ws significntly greter in the most shded tretment (8% RI) (P=0.0018), with men vlue of cm 2 g -1, which represents n increse of 51, 8% with respect to the most illuminted tretment (67% RI= 68.7 cm 2 g -1 ). Similrly, root iomss ws significntly higher in intermedite light tretments (17% nd 31% RI) compred with tretments of high nd low lighting (P=0.0002), with men vlues of g vs g, respectively. Finlly, lso the totl iomss ws significntly higher (P=0.0013) in the intermedite RI tretments (17 nd 31%) compred with low nd high illumintion (8 nd 67% respectively): on verge 149, 2 vs g (176.8% increse). Chlorophyll fluorescence. Significnt differences in vlues of chlorophyll fluorescence mong illumintion tretments (P=0.0000), s well s mong hours long dy (P=0.0000) nd for the interction tretment x hour (P=0.0032). The vlues were significntly higher in the RI of 8%, followed y 17%, 31% nd 67% (Figure 4). Lef content of photosynthetic pigments. The concentrtions of chlorophyll, chlorophyll, nd totl chlorophyll were significntly higher in plnts estlished in the most shded tretment (8% RI) (P= in ll cses), followed y the intermedite (17 nd 31% RI), nd high illumintion tretment (67% RI), which hd the lowest vlues (Tle 2). The concentrtion of crotenoids showed n inverse trend: it ws significntly higher in plnts developed in high illumintion (P=0.0001), nd decresed in more shded tretments. The rtio chlorophyll / hd no significnt differences etween lighting tretments (P=0.6266), while the rtio totl chlorophyll / crotenoids ws significntly higher in plnts estlished in low light (P=0.0001). Rev.Fc.Nc.Agron. 70(1):

7 Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews Lln length (cm) Internodes length (cm) 9 8 c 500 8% 17% 31% 67% 7 8% 17% 31% 67% Folir re (cm 2 ) Specific folir re (cm 2 g -1 ) c c 500 8% 17% 31% 67% 60 8% 7% 31% 67% Totl iomss (g) % 17% 31% 67% Root iomss (g) % 17% 31% 67% Figure 3. Biometric vriles evluted in vnill plnts growing in different light environments: 8% RI, 17% RI, 31% RI nd 67% RI. Different letters on the rs indicte significnt differences (Duncn, 95%, n = 5). The results clerly show tht the light environment of growing ffects the CAM photosynthesis nd vegettive growth of vnill, with optimum illumintion rnge t intermedite to low levels ( mol m -2 d -1 of PAR, which the study re correspond to RI etween 17 nd 31%). Most vlues reported s suitle for vnill growth in other regions where this species is cultivted, rnge etween 30 nd 60% RI (Anilkumr, 1994; Binchessi, 2004; Khne et l., 2008; Exley, 2011; Hernández- Hernández, 2011; Srm et l., 2011; Vrel, 2011; Zuin et l., 2011); however, comprisons sed on RI proly re ised without knowing the PAR vlues t these sites. Results suggest tht vnill hs etter cclimtion cpcity to low light environments (though with suoptiml growth) thn to high lighting conditions. The highest sorption vlues occurred in the intermedite to low light tretments. These results gree with those of nocturnl cid ccumultion ( H + ), which were higher in the tretments of 8, 17 nd 31% RI; i.e., Rev.Fc.Nc.Agron. 70(1):

8 8030 Diez MC, Moreno F, Gntiv E plnts grown in environments RI intermedite to low (which sored more CO 2 ) trnsformed tht CO 2 into orgnic cids long night (Dodd et l., 2002) nd thus showed greter ccumultion of cids in the vcuoles. During the dy, the highest CO 2 uptke occurred in the tretments of 17 nd 31% RI, which specificlly occurred erly in the morning (Phse II of CAM metolism), ecuse during the rest of the dy (Phses III nd IV) only respirtion occurred % 17% Chlorophyll fluorescence (Fv/Fm) c d 31% 67% 0.4 Figure 4. Dily course of chlorophyll fluorescence of vnill plnts growing in different light environments: 8% RI, 17% RI, 31% RI nd 67% RI. Different letters esides ech line indicte significnt differences (Duncn, 95%, n=3 per tretment. SE in rs). As result of this lnce of cron sorption during the dy nd night, the lrgest net mount of CO 2 sored long the 24 h period occurred under the intermedite -low light tretments, with much lower vlues in the high lighting tretment. Consequently, the highest vlues of growth were otined in plnts developed in the intermedite tretments RI (17 nd 31%), with dily PAR of 8.6 nd 12.8 mol m -2 d -1, expressed s lin length, lef re, root iomss, nd totl iomss (Figure 3). In these tretments, the mximum vlues of instntneous rdition were 369 nd 577 µmol m 2 s -1, respectively, nd occurred etween the 13:00 nd 14:00 h. Accordingly, the environments which fvor the photosynthesis nd growth of vnill re pprently those with PAR levels t noon etween these two vlues, while environments of greter rdition (for exmple, the ones in the 67% or RI: 1201, 4 µmol m 2 s -1 t noon) seem to limit the photosynthesis nd development of this species. These results re consistent with vlues reported in Indi s fvorle for the vegettive development of vnill, of µmol m 2 s -1 (Puthur, 2005), lthough it would e necessry to know the totl dily PAR for this region, in order to evlute whether the dt re comprle. Plnts growing under extreme lighting conditions (high nd low), hd the lowest vlues of growth, lthough showed some mechnisms of cclimtion. On the one hnd, plnts tht grew in 8% RI showed severl dpttions to e more efficient under low light conditions, such s longer internodes nd higher specific lef re, since in low-rdition, n elongted morphology nd lrger lef ldes increse light uptke (Wlters, 2005). However, the totl lef re per plnt decresed proly s consequence of the lower resources ville under tht condition; perhps for tht reson, plnts under this light condition showed the lower growth rte over long time period (Shipley, 2002), despite no significnt differences of totl dily photosynthesis per unit lef re were found with intermedite RI tretments (Tle 1). At the other extreme, the low CO 2 ssimiltion nd growth in plnts developed under high light (67% RI) could e explined y the signs of chronic photo inhiition detected, which dmges the rection centers of PSII nd cuses photo destruction of photosynthetic pigments (Powles, 1984). The PSII efficiency (Fv / Fm) in this study declined from 0.81 ± in the tretment of 8%, indictive of sence of photo inhiition (Powles, 1984; Werner et l., Rev.Fc.Nc.Agron. 70(1):

9 Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews 2001), to 0.61 ± in the 67% RI tretment, indictive of presence of photo inhiition. Plnts from shdow environments re especilly susceptile to chronic photo inhiition when re exposed to high light intensities. Under such conditions, plnts show low potentil quntum yield of photosynthesis s result of fewer rection centers in the PSII, resulting in lower Fv / Fm throughout the dy (Murchie et l., 2009). A further indiction of irreversile dmge is tht leves of plnts grown in high light (67%) hd white spots y chlorosis, proly s result of higher chlorophyll degrdtion, ecuse leves could not effectively chnnel the high energy received in photochemicl rections, which leds to chlorophyll whitening (Anderson, 1986). The concentrtion of chlorophylls (, nd totl) ws higher in lef tissues of plnts growing in lower lighting (8% RI). Under these conditions, the synthesis of greter mount of chlorophyll is strtegy of cclimtion to increse the efficiency of photon cpture nd counterct the lower rdition reching the lef (Anderson, 1986). Perhps this high chlorophyll content ws effective to void very low vlues of totl dily photosynthesis per unit lef re, despite the limittions imposed y low rdition; this response is further evidence in fvor of the shde-tolernt chrcter of vnill. It hs een reported tht plnts dpted to low rdition hve low vlues of the chlorophyll / chlorophyll rtio (pproximtely 2) nd tht plnts dpted to high rdition hve high vlues (out 2.8 nd more) (Anderson, 1986). In this study no significnt differences in this rtio were found mong vnill plnts growing under different RI, whose verge vlue ws 2.2 (Tle 2). Low vlues of this rtio re correlted with high degree of stcking of thylkoid memrnes in the chloroplst, which increses the re of the cross section of the grn nd produces incresed risk of photo inhiition (Anderson nd Aro, 1994). This inility of vnill plnts growing under high light intensity to increse chlorophyll / chlorophyll rtio, suggests tht there were not significnt reorgniztion of thylkoid memrnes, which would llow lower stcking nd thus void photo destruction of photosynthetic pprtus in plnts exposed to high rdition (67% RI), which is further evidence of the shde- tolernt chrcter of this species. Tle 2. Lef content of photosynthetic pigments (µg cm -2 ) in vnill plnts growing in different light environments. RI Chlorophyll Chlorophyll Totl Chlorophyll Crotenoids Chlorophyll / Chlorophyll / Crotenoids 8% 23.6 ± ± ± ± ± ± % 14.2 ± ± ± ± ± ± % 17.0 ± ± ± ± ± ± % 8.5 ± 0.7 c 3.9 ± 0.4 c 12.4 ± 1.1 c 6.3 ± ± ± 0.2 c Men vlues ± SE per tretment re shown in the tle ody (n=5). Different letters etween light tretments in ech column indicte significnt differences (P=0.05, Duncn test). Crotenoids protect chlorophyll pigments ginst high rdition under conditions of light stress (Ymmoto nd Bssi, 1996) nd therefore, the rtio chlorophyll / crotenes is n indictor of the protection ginst the high rdition, since crotenes contriute to dissipte the excess of rdition (Czzonelli et l., 2011). Low vlues, indictive of greter photo oxidtive dmge, were found in plnts exposed to 67% RI (Tle 2), wheres in plnts developed under 17 nd 31% RI, were intermedite, nd higher vlues in plnts under 8% RI. Vnill plnts showed ility to cpture nd use light y incresing the concentrtion of pigments when grown t low rdition; the decrese of chlorophyll / crotenes rtio under high rdition suggests their inility to minimize the dmge cused y high rdition, which is further evidence of their inility to cclimte to high rdition environments. Since the proportion of crotenoids vs. chlorophyll decresed with incresing rdition intensity (Tle 2), vnill plnts grown in high light did not hve enough mechnisms to protect ginst the photo-destructive Rev.Fc.Nc.Agron. 70(1):

10 8032 Diez MC, Moreno F, Gntiv E dmge from high rdition. Such filure ws evidenced in the low chlorophyll content, low CO 2 ssimiltion, less chlorophyll fluorescence, nd low productivity of plnts exposed to rdition ove 800 μmol m -2 seg -1. These results confirm tht shde plnts, do not hve well developed mechnisms to counterct the effects of photo inhiition. Therefore, shde plnts such s vnill re very sensitive to high rdition; this cuses inctivtion of rection centers nd inhiition of electron trnsport, since the ntenns of their photosynthetic pprtus re unle of chnneling the light energy into the photochemicl rection centers. The excess of energy cn induce the production of rective oxygen species nd free rdicls (Powles, 1984) which rek the DNA, destroy the function of proteins, nd re responsile for peroxidtion of lipids, thus cusing dmge to the plnt metolism nd decresing the rte of photosynthesis nd growth. CONCLUSIONS Most CO 2 ssimiltion occurred long night in ll tretments, which confirms tht vnill is strong CAM plnt. Results suggest tht high rdition lters the functioning of vnill plnts, inhiiting photosynthesis nd growth, nd tht highly shded environments not significntly ffected the CAM photosynthesis of vnill; however, in the long-term this species showed higher photosynthesis nd iomss growth under intermedite levels of rdition (17-31% RI). ACKNOWLEDGEMENTS This work ws done s prt of the project Ecophysiology vnill cultivtion (Vnill plnifoli Andr.), funded y the Administrtive Deprtment of Science, Technology nd Innovtion of Colomi - COLCIENCIAS- nd the Universidd Ncionl de Colomi (contrct ). REFERENCES Anderson JM nd Aro E Grn stcking nd protection of photosystem II in thylkoid memrnes of higher plnt leves under sustined high irrdince: n hypothesis. Photosynthesis Reserch 41 (2): doi: /BF Anderson JM Photoregultion of the composition, function, nd structure of thylkoid memrnes. Annul Review of Plnt Physiology 37 (1): doi: /nnurev. pp Anilkumr AS Vnill cultivtion: A profitle gri-sed enterprise. Kerl Clling 1: Brrow SR nd Cockurn W Effects of light quntity nd qulity on the decroxyltion of mlic cid in crssulcen cid metolism photosynthesis. Plnt physiology 69 (3): doi: / pp Binchessi P Vnill: griculture nd curing techniques. A photogrphic hndook for vnill frmers. Venui Vnill Co. Snto, Vnutu. 62 p. Bory S, Grisoni M Duvl MF nd Besse P Biodiversity nd preservtion of vnill: present stte of knowledge. Genetic Resources nd Crop Evolution 55 (4): doi: / s Cushmn JC Crssulcen cid metolism. A plstic photosynthetic dpttion to rid environments. Plnt Physiology 127(4): doi: / pp Dodd AN, Borlnd AM, Hslm RP, Griffiths H nd Mxwell K Crssulcen cid metolism: plstic, fntstic. Journl of Experimentl Botny 53 (369): doi: / jexot/ Exley R Vnill Production in Austrli. pp In: Hvkin-Frenkel, D nd Belnger F (ed.). Hndook of vnill science nd technology. Wiley-Blckwell, West Sussex, UK. 339 p. Fouché JG nd Jouve L Vnill plnifoli: history, otny nd culture in Reunion Islnd. Agronomie 19 (8): Griffiths H Cron dioxide concentrting mechnisms nd the evolution of CAM in vsculr epiphytes. pp In: Lüttge, U. (ed.). Vsculr plnts s epiphytes. Springer Berlin- Heidelerg, GE. 270 p. Hslm R, Borlnd A, Mxwell K nd Griffiths H Physiologicl responses of the CAM epiphyte Tillndsi usneoides L. (Bromelicee) to vritions in light nd wter supply. Journl of Plnt Physiology 160 (6): doi: / Hernández-Hernández J Mexicn Vnill Production. pp In: Hvkin-Frenkel D. nd Belnger F (ed.). Hndook of Vnill Science nd Technology. Wiley-Blckwell, West Sussex, UK. 339 p. Holdridge LR Ecologí sd en Zons de Vid. Instituto Intermericno de Cooperción pr l Agricultur - IICA, Sn José, Cost Ric. 216 p. Khne R, Besse P, Grisoni M, Le Bellec F nd Odoux E Bouron vnill: nturl flvour with future. Chronic Horticulture 48 (2): Lichtenthler H nd Wellurn AR Determintion of totl crotenoids nd chlorophyll nd of lef extrcts in different solvents. Biochemicl Society Trnsctions 11(5): doi: /st Lichtenthler HK Chlorophylls nd crotenoids: pigments of photosynthetic iomemrnes. Methods in Enzymology 148: Lüttge U Ecophysiology of crssulcen cid metolism (CAM). Annls of Botny 93(6): doi: /o/mch087 Murchie EH, Pinto M nd Horton P Agriculture nd the new chllenges for photosynthesis reserch. New Phytologist 181(3): doi: /j x Noel PS nd Hrtsock TL Reltionships etween photosyntheticlly ctive rdition, nocturnl cid ccumultion, nd CO 2 uptke for Crssulcen Acid Metolism plnt Opunti ficusindic. Plnt physiology 71(1): doi: / pp Rev.Fc.Nc.Agron. 70(1):

11 Effects of light intensity on the morphology nd CAM photosynthesis of Vnill plnifoli Andrews Osmond CB Crssulcen Acid Metolism: curiosity in context. Annul Review of Plnt Physiology 29(1): doi: /nnurev.pp Pierce S, Winter K nd Griffiths H The role of CAM in high rinfll cloud forests: n in situ comprison of photosynthetic pthwys in Bromelicee. Plnt, Cell nd Environment 25(9): doi: /j x Poorter H nd de Jong-Vn Berkel Y Chlorophyll extrction nd determintion. In: Prometheus Wiki contriutors, pulish.csiro.u/prometheuswiki/tiki-pgehistory.php?pge=chlorophyll extrction nd determintion&preview=11; ccessed: Novemer Powles SB Photoinhiition of photosynthesis induced y visile light. Annul Review of Plnt Physiology 35 (1): doi: /nnurev.pp Puthur J Influence of light intensity on growth nd crop productivity of Vnill plnifoli Andr. Generl nd Applied Plnt Physiology 31(3-4): Srm YR, Thoms J, Ssikumr B nd Vrdrs S Vnill production in Indi. In: Odoux E nd Grisoni M (ed.). Vnill. Medicinl nd romtic plnts-industril profiles. CRC Press, Tylor nd Frncis Group, Boc Rtón, FL, USA. 420 p. Shipley B Trde-offs etween net ssimiltion rte nd specific lef re in determining reltive growth rte: reltionship with dily irrdince. Functionl Ecology 16(5): doi: /j x Silver, K, Sntigo LS nd Winter K Distriution of Crssulcen Acid Metolism in orchids of Pnm: evidence of selection for wek nd strong modes. Functionl Plnt Biology 32(5): doi: /FP04179 Steel RGD nd Torrie JH Principles nd procedures of sttistics: iometricl pproch. Mc Grow-Hill, New York, USA. 633 p. Wlters RG Towrds n understnding of photosynthetic cclimtion. Journl of Experimentl Botny 56(411): doi: /jx/eri060 Vrel E Vnill Production in Cost Ric. pp In: Hvkin-Frenkel D nd Belnger F (ed.). Hndook of Vnill Science nd Technology. Wiley-Blckwell, West Sussex, UK. 339 p. Ymmoto HY nd Bssi R Crotenoids: locliztion nd function. pp In: Ort D nd Yocum CF (ed.). Oxygenic Photosynthesis: The Light Rections. Springer, Netherlnds. doi: / _30 Zuin R, Tome M nd Liew ECY Vnill production in Indonesi. pp In: Odoux E. y Grisoni M (ed.). Vnill. Medicinl nd Aromtic Plnts-Industril Profiles. CRC Press, Tylor nd Frncis Group, Boc Rtón, FL, USA. 420 p. Zotz G nd Winter K Annul cron lnce nd nitrogen use efficiency in tropicl C 3 nd CAM epiphytes. New Phytologist 126 (3): doi: /j t04245.x Zotz G nd Andrde JL L ecologí y l fisiologí de ls epífits y ls hemiepífits. pp In: Kttn, G.H. y M.R. Gurigut (ed.). Ecologí y conservción de osques neotropicles. Liro Universitrio Regionl, Sn José, Cost Ric. 691 p. Rev.Fc.Nc.Agron. 70(1):

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