Influence of Blue Light on the Leaf Morphoanatomy

8/6/2019 Influence of Blue Light on the Leaf Morphoanatomy

http://slidepdf.com/reader/full/influence-of-blue-light-on-the-leaf-morphoanatomy 1/7



Räisänen et al., 2008!. In vitro plant culturing allows assess-ment, in controlled conditions, of the influence of light onplant development and secondary metabolite production~Rao & Ravishankar, 2002; Fila et al., 2006; Kurilcik et al.,2008; Poudel et al., 2008!.

We addressed the effects of supplementary blue light

on leaf morphology and phenolic inclusions in K. pinnatausing optical microscopy and a microchemical test in invitro culturing of plants.

MATE RI AL S AND METHODS

Plant Material

The plants used in this work were monoclonal. The matrixplant was obtained from the internal garden of Núcleo dePesquisas de Produtos Naturais, Universidade Federal doRio de Janeiro, where several specimens are grown undernatural conditions. A voucher specimen was deposited onthe Jardim Botânico do Rio de Janeiro under numberRB292.697.

Establishment of In Vitro Culture

Leaf borders of the matrix plant were cut into sections of 1 cm2 and surface-sterilized under aseptic conditions ~com-mercial detergent in aqueous solution for 15 min, 5 min insodium hypochlorite solution, washed in 70% ethanol solu-tion, and between each step, explants were rinsed withdistilled water!. Sterilized explants were transferred to auto-

claved glass flasks containing MS medium ~Murashige &Skoog, 1962! without growth regulators and supplementedwith 0.6 mM myo-inositol, 2.43 mM pyridoxine, 4.1 mMnicotinic acid, 1.48 mM thiamine, and 30 g L1 sucrose. Themedium was jellified with agar ~8 g L1! at pH 5.7 adjustedbefore autoclaving. In vitro plants were cultured under12 mmol m2 s1 ~1.6 W m2! of photosynthetic activeradiation ~PAR!. Light was supplied by white fluorescentlamps ~Sylvania 20 W—F20T12!. The spectral curves of thelamps can be viewed in Figure 1. There was no significantdifference in the photosynthetic light quantity /quality mea-sured outside and inside the flasks. Ultraviolet wavelengths

were mainly blocked by glass. The growth room tempera-ture and photoperiod were 24 6 28C and 16 h, respectively.Subsequent subcultures were done using nodal segments.

In Vitro Culture under Different Light Qualities

In vitro plants after the fourth subculture were used to study the effect of blue light on the leaf internal morphology. Twolight treatments were used: white lamps ~W plants! andwhite lamps plus blue lamps ~WB plants!. Fluorescent lampswere used for both treatments ~Sylvania 20 W day light,Sylvania 20 W blue light – F20T12! ~Fig. 1!. Eight flaskswith five plantlets were used per treatment. The growth

room temperature was 24 6 28C and photoperiod was 16 h.The PAR levels of both treatments ~white lamps and white

lamps plus blue lamps! were similar ~12 mmol m2 s1!.The fluence of both treatments was 1.6 W m2. After fourmonths, five flasks per treatment ~25 plants! were randomly taken for plant growth analysis, and the remaining threeflasks ~15 plants! were used for analysis of leaf morphology.With four months of growth, plants reached their maxi-mum growth without signs of senescence and/or nutri-tional limitation of the medium.

Morphological Parameters

Plant development was evaluated according to the following

parameters: leaf fresh weight, leaf dry weight, length of thelongest branch, and number of nodes of the longest branch.For dry weight determination, plants were dehydrated at608C for 24 h.

Anatomical analyses were performed on fully devel-oped leaves from both treatments. They were fixed in 37%formaldehyde, glacial acetic acid, and 70% ethanol solution~Johansen, 1940! and kept in 70% ethanol solution. Wholeleaves were embedded with a Leica Historesin EmbeddingKit. Sections 10 mm thick were obtained using a rotary microtome ~American Optical! and stained with toluidineblue ~O’Brien et al., 1965!. Sections were taken from themiddle third of the leaf blade and the petiole. Toluidine

Figure 1. Spectral power distribution of the ~A! white and ~B!blue fluorescent lamps.

2 Marcos Vinicius Leal-Costa et al.



blue was used to identify phenolic idioblastic cells ~Rama-lingan & Ravindranath, 1970!. The following anatomicalparameters were measured: leaf thickness, epidermal thick-ness, mesophyll thickness, and number of mesophyll celllayers.

Statistical Analysis

Statistical analysis was conducted with GraphPad Instat 3.0for Windows. Data were analyzed with Student’s t -test

~ p 0.05!.

R ESULTS

Petioles are plain-convex ~flat upper surface and roundedlower surface! in cross section under the two light treat-ments. Epidermal cells are rectangular and elliptic withconvex external periclinal walls ~parallel to the leaf surface!~Fig. 2!. Stomata are rarely found. The petiole is filled withchlorophyll and ground parenchyma. In the center, embed-

ded in the ground parenchyma, one collateral vascularbundle is present ~Fig. 2A,C!. Near to the adaxial side aretwo accessory collateral vascular bundles ~Fig. 2B,D!.

Plants grown under white light ~W plants! and plantssupplemented with blue light ~WB plants! have amphisto-matic leaves with similar epidermis. In frontal view epider-mal cells have sinuous anticlinal walls on both sides of theleaf ~Fig. 3!. The epidermis is simple with rectangular andeliptic cells in cross section ~Fig. 4!. W plants have thinnerleaves than WB plants ~Table 1!. The mesophyll is homo-geneous and densely packed in both W and WB plants,although cells are visibly larger in WB plants ~Fig. 4B!. Thenumber of mesophyll cell layers did not differ significantly

between treatments ~Table 1!. WB plants have larger chloro-plasts with conspicuous starch grains when compared to Wplants. There is a single collateral vascular bundle in themidrib ~Fig. 5! in W and WB plants.

W plants have longer branches and a larger number of nodes ~Table 2; Fig. 6A!. However, leaves are smaller in Wplants than in WB plants and are rolled up ~Fig. 6B!. Leaf fresh weight and leaf dry weight were similar for W plantsand WB plants ~Table 2!.

Toluidine blue indicated phenolic indioblastic cells as-sociated with the vascular cells in parenchyma tissue inpetioles and leaf blades of WB plants only ~Fig. 7B,D!.

DISCUSSION

Reports of blue light effects on plant development arewidespread in the scientific literature ~Spalding & Folta,2005!. Blue light effect on leaf thickness is species depen-dent. The thickness of pepper leaves increases more underblue light in combination with red light than under redlight alone ~Schuerger et al., 1997!; whereas addition of bluelight decreases leaf thickness in peach ~Rapparini et al.,

Figure 2. Transverse sections of K. pinnata petiole. ~A, B! Control

plants; ~C,D! plants cultured under supplementary blue light. avb,

accessory vascular bundle; e, epidermis; p, parenchyma tissue; vb,

main vascular bundle. Scale bars: A,C100 mm; B,D 50 mm.

Figure 3. Drawings of K. pinnata epidermis. ~A! Adaxial side; ~B!abaxial side. WB plant. Scale bar, 50 mm.

Table 1. Anatomical Parameters of Kalanchoe pinnata ~La-

marck! Persoon Cultured under White Light ~W Plants! and White

Light plus Blue Light ~WB Plants!.

W Plants WB Plants

Epidermal thickness

~adaxial side! ~ mm!

23.91 6 1.40a 31.71 6 1.62a

Epidermal thickness

~abaxial side! ~ mm!

17.35 6 1.64 17.68 6 2.30

Mesophyll thickness ~ mm! 154.32 6 7.44a 233.95 6 9.78a

Leaf thickness ~ mm! 196.00 6 8.47a 273.90 6 9.95a

Number of mesophyll

cell layers

5.45 6 0.15 5.80 6 0.15

aIndicates statistical difference. T test, p , 0.5, n15.

Blue Light and In Vitro K. pinnata Morphoanatomy 3



1999!. Thick leaves, homogeneous and densely packed meso-phyll, and large vacuoles are characteristic of succulentleaves and are often observed in crassulacean acid metabo-lism ~CAM! species ~Nelson & Sage, 2008!. These traits areof great importance in preventing CO2 leakage during theday when malate is decarboxylated and CO2 is refixed viathe Calvin-Benson cycle. Reduced CO2 conductance in CAM

Figure 4. Transverse sections of K. pinnata leaf blade. ~A! Control

plant; ~B! plant cultured under supplementary blue light. e, Epider-

mis; m, mesophyll. Scale bars: A,B 50 mm.

Figure 5. Transverse sections of K. pinnata leaf blade in the mid-

rib. ~A! Control plant; ~B! plant cultured under supplementary

blue light. e, Epidermis; m, mesophyll; vb, vascular bundle. Scale

bars: A,B100 mm.

Table 2. Morphological Parameters of Kalanchoe pinnata ~La-

marck! Persoon Cultured under White Light ~W Plants! and White

Light plus Blue Light ~WB Plants!.

W Plants WB Plants

Leaf fresh weight ~mg! 104.82 6 10.51 82.20 6 14

Leaf dry weight ~mg! 10.04 6 0.71 9.84 6 1.73

Length of the higher branch

~cm!

2.10 6 0.27a 0.71 6 0.10a

Number of nodes 6.30 6 1.87a 3.88 6 1.33a

aIndicates statistical difference. T test, p , 0.5, n 25.




plants also increases the efficiency to reuse of CO2 fromrespiration. Limited efflux of internally generated CO2 isparticularly important to CAM function during stressfulsituations, enhancing photosynthetic efficiency ~Nelson et al.,2005!. Thicker WB leaves could represent an advantageunder water stress, favoring the process of transplantationto ex vitro culturing.

Large chloroplasts with large starch grains are charac-teristic of plants under high light ~Meier & Lichtenthaler,1981; Oguchi et al., 2003!. Light also influences chloroplast

position and development ~Tholen et al., 2008!. Removingblue wavelength from spectral radiation determines someshade-type features, such as the position of chloroplasts,perpendicular to the main direction of light ~Tholen et al.,2008!. Blue light alone and blue light associated to red lightincreased starch biosynthesis in Doritaenopsis plants in com-parison to white light ~Shin et al., 2008!. WB plants alsoexhibited chloroplasts with more starch content.

Blue light inhibits dry weight accumulation on spinach,radish, and lettuce ~Yorio et al., 2001!. Tomato plants under

Figure 6. Plants cultured under ~A! white light and ~B! white light plus blue light. Observe the rolled leaf margins in

panel A. Need to show these at the same magnification for comparison.

Figure 7. ~Color online! Detail of transections of K. pinnata showing a vascular bundle in the ~A,B! petiole and ~C,D!midrib. ~A,C! Control plant; ~B, D! plant cultured under supplementary blue light. id, Phenolic idioblasts; ph, phloem;

x, xylem. Scale bars: A–D 50 mm.




blue light exhibit smaller shoots with reduction in thenumber of nodes, internode length, and fresh weight reduc-tion ~Glowacka, 2004!. Pines and narcissi are shorter underblue light than plants under white light ~Sarala et al., 2007;Wozny & Jerzy, 2007!. Blue light also reduces shoot heightin in vitro grapes without reduction in the number of nodes

~Poudel et al., 2008!. In vitro Chrysanthemum plants alsoexhibit reduced dry and fresh weight and smaller internodeslinked to blue light exposure ~Kurilcik et al., 2008!. In thiswork, WB plants were smaller and had fewer nodes than Wplants. Nevertheless, their biomass accumulation, as a mea-sure of dry weight, was equivalent. This is probably a resultof larger leaves.

Blue light often improves synthesis of phenolic com-pounds, including flavonoids, in plants. Vigna sinensis and

Phaseolus vulgaris have increased phenolic content pro-moted by blue light exposure ~El-Khawas & Khatab, 2007!.Anthocyanin accumulation is induced by blue light in Arab-dopisis thaliana ~Cominelli et al., 2008!. Phenolic idioblastsare observed in K. daigremontiana, Sedum dendroideum~Balsamo & Uribe, 1988; Duarte & Zaneti, 2002!. Densely filled cells, probably of phenolic content, are viewed in K.daigremontiana and K. pinnata ~Kondo et al., 1998!. Thesereports and the greenish reaction observed in WB plantslead us to believe that the inclusions in the idioblasts of WBplants are phenolic compounds.

CONCLUSIONS

Supplementary blue light under the intensity tested here didnot affect plant growth. However, significant morphologicaleffects were observed under supplementary blue light. Al-though W plants were larger than WB plants, they hadsmaller and thinner leaves. Shorter plants under supplemen-tary blue light are a desirable character that permits acontrolled culture in reduced spaces, including green-house culture. Additionally, accumulation of phenolic com-pounds, visible in the idioblasts using toluidine blue, wasonly found in WB plants. Overall, in vitro culture of K.

pinnata under supplementary blue light was improved,especially as a source of phenolic compounds. Further tests

on a larger scale are desirable if commercial in vitroculturing is intended for the production of phytotherapeu-tic medicines.

ACKNOWLEDGMENTS

This work was supported by “Fundação de Amparo àPesquisa do Estado do Rio de Janeiro-FAPERJ” and “Co-ordenação de Aperfeiçoamento de Pessoal de NívelSuperior-CAPES.”

R EFERENCES

Balsamo, R.A. & Uribe, E.G. ~1988!. Leaf anatomy and ultrastruc-

ture of the Crassulacean-acid-metabolism plant Kalanchoe dai-

gremontiana. Planta 173, 183–189.

Caldwell, M.M., Robberecht, R. & Flint, S.D. ~1983!. Internal

filters: Prospects for UV-acclimation in higher plants. Physiolo- gia Plantarum 58, 445–450.

Cominelli, E., Gusmaroli, G., Allegra, D., Galbiati, M., Wade,

H.K., Jenkins, G.I. & Tonelli, C. ~2008!. Expression analysis of

anthocyanin regulatory genes in response to different Light

qualities in Arabidopsis thaliana. J Plant Physiol 165, 886–894.

da-Silva, S.A.G., Costa, S.S. & Rossi-Bergmann, B. ~1999!. The

anti-leishmanial effect of Kalanchoe is mediated by nitric oxide

intermediates. Parasitology 118, 575–582.

Duarte, M.R. & Zaneti, C.C. ~2002!. Morfoanatomia de folhas

de bálsamo: Sedum dendroideum Moc. et Sessé ex DC, Crassu-

laceae. Revista Lecta 20, 153–160.

El-Khawas, S. & Khatab, H. ~2007!. Comparative studies on the

effects of differents light qualities on Vigna sinensis L. andPhaseolus vulgaris L. seedlings. Res J Agricul Biol Sci 3, 790–798.

Fila, G., Badeck, F-W., Meyer, S., Cerovic, Z. & Ghashghaie, J.

~2006!. Relationships between leaf conductance to CO2 diffu-

sion and photosynthesis in micropropagated grapevine plants,

before and after ex vitro acclimatization. J Exp Botany 57,

2687–2695.

Glowacka, B. ~2004!. The effect of blue light on the height and

habit of the tomato ~Licopersicum esculentum Mill.! transplant.

Folia Horticulturae 16, 3–10.

Jaakola, L., Määttä-Riihinen, K., Kärenlampi, S. & Hohtola,

A. ~2004!. Activation of flavonoid biosynthesis by solar radiation

in bilberry ~Vaccinium myrtillus L.! leaves. Planta 218, 721–728.

Jansen, M.A.K., Hectors, K., O’Brien, N.M., Guisez, Y. & Pot-

ters, G. ~2008!. Plant stress and human health: Do humanconsumers benefit from UV-B acclimated crops? Plant Sci 175,

449–458.

Johansen, D.A. ~1940!. Plant Microtechnique. New York: McGraw-

Hill Book Co. Inc.

Kondo, A., Nose, A. & Ueno, O. ~1998!. Leaf inner structure and

immunogold localization of some key enzymes involved in

carbon metabolism in CAM plants. J Exp Botany 49, 1953–1961.

Kurilcik, A., Miklusyte-Canova, R., Dapkunienue, S., Zilin-

skaite, S., Kurilcik, G., Tamulaits, G., Duchovskis, P. &

Zukauskas, A. ~2008!. In vitro culture of Chrysanthemum

plantlets using light-emitting diodes. Cent Eur J Biol 3, 161–167.

Lans, C.A. ~2006!. Ethnomedicines used in Trinidad and Tobago

for urinary problems and diabetes mellitus. J Ethnobiol Eth-nomed 2, 45 ~online only !.

Larcher, W. ~2000!. Ecofisiologia Vegetal . São Carlos, Brazil: RiMa

Artes e Textos.

Maffei, M., Canova, D., Bertea, C.M. & Scannerini, S. ~1999!.

UV-A effects on photomorphogenesis and essential-oil compo-

sition in Mentha piperita. J Photoch Photobio B 52, 105–110.

Medeiros, M.F.T., Fonseca, V.S. & Andreata, R.H.P. ~2004!.

Plantas medicinais e seus usos pelos sitiantes da Reserva Rio

das Pedras, Mangaratiba, RJ, Brasil. Acta Bot Bras 18, 391–399.

Meier, D. & Lichtenthaler, H.K. ~1981!. Ultrastructural devel-

opment of chloroplasts in radish seedlings grown at high- and

low-light conditions and in the presence of the herbicide ben-

tazon. Protoplasma 107, 195–207.




Meng, X., Xing, T. & Wang, X. ~2004!. The role of light in the

regulation of anthocianin accumulation in Gerbera hybrida.

Plant Growth Regul 44, 243–250.

Murashige, T. & Skoog, F. ~1962!. A revised medium for rapid

growth and bioassays of tobacco tissue cultures. Physiol Plant 15, 473–479.

Muzitano, M.F., Cruz, E.A., Almeida, A.P., da-Silva, S.A.G.,

Kaiser, C.R., Guette, C., Rossi-Bergmann, B. & Costa, S.S.~2006a!. Quercetrin: An antileishmanial flavonoid glycoside

from Kalanchoe pinnata. Planta Medica 72, 81–83.

Muzitano, M.F., Tinoco, L.W., Guette, C., Kaiser, C.R., Rossi-

Bergmann, B. & Costa, S.S. ~2006b!. The anti-leishmanial

activity assessment of unusual flavonoids from Kalanchoe pin-

nata. Phytochemistry 67, 2071–2077.

Namdeo, A.G. ~2007!. Plant cell elicitation for production of

secondary metabolites: A review. Pharmacognosy Rev 1, 69–79.

Nelson, E.A. & Sage, R.F. ~2008!. Functional constraints of CAM

leaf anatomy: Tight cell packing is associated with increased

CAM function across a gradient of CAM expression. J Exp

Botany 59, 1841–1850.

Nelson, E.A., Sage, T.L. & Sage, R.F. ~2005!. Functional leaf anatomy of plants with crassulacean acid metabolism. Funct

Plant Biol 32, 409–419.

O’Brien, T.P., Feder, N. & McCully, M.E. ~1965!. Polychromatic

staining of plant cell walls by toluidine blue O. Protoplasma 59,

368–373.

Oguchi, R., Hikosaka, K. & Hirose, T. ~2003!. Does the photo-

synthetic light-acclimation need change in leaf anatomy? Plant

Cell Environ 26, 505–512.

Poudel, R.P., Kataoka, I. & Mochioka, R. ~2008!. Effect of red-

and blue-light-emitting diodes on growth and morphogenesis

of grapes. Plant Cell Tiss Org 92, 147–153.

Räisänen, T., Ryyppö, A. & Kellomäki, S. ~2008!. Effecs of

elevated CO2 and temperature on monoterpene emission of

Scots pine ~Pinus sylvestris L.!. Atmosph Env 42, 4160–4171.Ramalingan, K. & Ravindranath, M.H. ~1970!. Histochemical

significance of green metachromasia to toluidine blue. Histoche-

mie 24, 322–327.

Rao, S.R. & Ravishankar, G.A. ~2002!. Plant cell cultures: Chem-

ical factories of secondary metabolites. Biotechnol Adv 20,

101–153.

Rapparini, F., Rotondi, A. & Baraldi, R. ~1999!. Blue light

regulation of the growth of Prunus persica plants in a long term

experiment: Morphological and histological observation. Trees

14, 169–176.

Saleh, A.A.H. ~2007!. Influence of UVAB radiation and heavy met-

als on growth, some metabolic activities and antioxidant system

in pea ~Pisum sativum! plant. Am J Plant Physiol 2, 139–154.

Sarala, M., Taulavuori, K., Taulavuori, E., Karhu, J. & Laine,

K. ~2007!. Elongation of Scots pine seedlings under blue light

depletion is independent of etiolation. Environ Exp Bot 60,340–343.

Schuerger, A.C., Brown, C.S. & Stryjewski, E.C. ~1997!. Ana-

tomical features of pepper plants ~Capsicum annum L.! grown

under red light-emitting diodes supplemented with blue or

far-red light. Ann Bot-London 79, 273–282.

Shin, K.S., Murthy, H.N., Heo, J.W., Hahn, E.J. & Paek, K.Y.

~2008!. The effect of light quality on the growth and develop-

ment of in vitro cultured Doritaenopsis plants. Acta Physiol

Plant 30, 339–343.

Shirley, B.W. ~1996!. Flavonoid biosynthesis: “New” functions for

an “old” pathway. Trends Plant Sci 1, 377–382.

Spalding, E.P. & Folta, K.M. ~2005!. Illuminating topics in plant

photobiology. Plant Cell Environ 28, 39–53.Taiz, L. & Zeiger, E. ~2009!. Fisiologia Vegetal . Porto Alegre, Brazil:

Artmed.

Taufner, C.F., Ferraço, E.B. & Ribeiro, L.F. ~2006!. Uso de

plantas medicinais como alternativa fitoterápica nas unidades

de saúde pública de Santa Teresa e Marilândia, ES. Natureza 4,

30–39 ~Online!.

Tholen, D., Boom, C., Noguchi, K., Ueda, S., Katase, T. &

Terashima, I. ~2008!. The chloroplast avoidance response de-

creases internal conductance to CO2 diffusion in Arabidopsis

thaliana leaves. Plant Cell Environ 31, 1688–1700.

Wade, H.K., Bibikova, T.N., Valentine, W.J. & Jenkins, G.I.

~2001!. Interactions within a network of phytochrome, crypto-

chrome and UV-B phototransduction pathways regulate chal-

cone synthase gene expression in Arabidopsis leaf tissue. Plant J 25, 675–685.

Wozny, A. & Jerzy, M. ~2007!. Effect of light wavelength on growth

and flowering of narcissi forced under short-day and low quan-

tum irradiance conditions. J Hortic Sci Biotech 82, 924–928.

Yorio, N.C., Goins, G.D., Kagie, H.R., Wheeler, R.M. & Sager,

J.C. ~2001!. Improving spinach, radish and lettuce growth un-

der red light-enitting diodes ~LEDs! with blue light supplemen-

tation. Hortscience 36, 380–383.


Influence of Blue Light on the Leaf Morphoanatomy

Documents

Transcript of Influence of Blue Light on the Leaf Morphoanatomy