CC BY
173
Journal of Stress Physiology & Biochemistry, Vol. 6 No. 3 2010, pp. 114-122 ISSN 1997-0838 Original Text Copyright © 2010 by Kiarostami, Mohseni, Saboora,
ORIGINAL ARTICLE
Biochemical changes of Rosmarinus officinalis under salt stress Kiarostami*, Kh., Mohseni, R., Saboora, A
Department of Biology,Faculty of Science,Alzahra University, Tehran,Iran
*Email- su_kiarostami@yahoo.com
Received June 3, 2010
The effects of salinity on some growth and physiological parameters in rosemary were investigated. 2 month-old plants were subjected to three salt treatments (50, 100, and 150 mM) by adding NaCl to the pots.
Plant growth parameters not affected by low concentrations of NaCl, but it decreased with higher concentrations. The content of photosynthetic pigments decreased at all salinity levels. The Na+ content of leaves increased, whereas the K+ content decreased with the progressive increase in NaCl concentration. Salinity increased proline and malondealdehyde contents. Stress induced by NaCl caused an accumulation of proline, total phenolic and antioxidant in rosemary plant.
key words: Antioxidant/Ionic content/Photosynthetic pigments/ Rosmarinus officinals/ Salt stress.
ORIGINAL ARTICLE
Biochemical changes of Rosmarinus officinalis under salt stress Kiarostami*, Kh., Mohseni, R., Saboora, A
Department of Biology,Faculty of Science,Alzahra University, Tehran,Iran
*Email- su_kiarostami@yahoo.com
Received June 3, 2010
The effects of salinity on some growth and physiological parameters in rosemary were investigated. 2 month-old plants were subjected to three salt treatments (50, 100, and 150 mM) by adding NaCl to the pots.
Plant growth parameters not affected by low concentrations of NaCl, but it decreased with higher concentrations. The content of photosynthetic pigments decreased at all salinity levels. The Na+ content of leaves increased, whereas the K+ content decreased with the progressive increase in NaCl concentration. Salinity increased proline and malondealdehyde contents. Stress induced by NaCl caused an accumulation of proline, total phenolic and antioxidant in rosemary plant.
key words: Antioxidant/Ionic content/Photosynthetic pigments/ Rosmarinus officinals/ Salt stress.
Rosmarinus officinalis , related to the lamiaceae family of plants. Rosemary is a native of the Mediterranean region of Europe and the Near East . Today rosemary is cultivated in nearly all countries as medicinal and ornamental plant. The herb rosemary has been used as a food spice and as a medicine since ancient times (Moss, et al 2003) Rosemary is used as an antispasmodic in real colic and dysmenorrhoea, in relieving respiratory disorders and to stimulate growth of hair. Rosemary extract relaxes smooth muscles and has choleretic ,hepatoprotective and antitumerogenic activity(Al-Sereiti et al 1999). It is also antiviral, inhibiting even
HIV protease at very low concentrations (Aruma et al 1996).
The potent antioxidant properties of rosemary extracts have been attributed to its phenolic compounds, mainly rosmarinic acid and diterpenes carnosic acid and carnosol (Sergi Munne-Bosch and Leonor Alegre 2002 ,
Troncoso et al 2005). Rosemary is grown in many parts of the world including saline area. Salinity is one of the most important factor limiting plant growth and yield. About 25% of world's total area (including 15% of Iran's lands)
spectrophotometrically by Lichtenthaler formula.
are saline. These areas contain about 33% of worlds and 50% of Iran's irrigated lands respectively (Kamkar et al 2004). In saline lands where salinity reduce agriculture area, the search for new crop cultivation is interesting in agricultural research .Salt tolerant plants can be used to produce economically important materials such as antioxidants and essential oils. Environmental condition and genetic influence plants secondry products (Aziz Eman et al 2008 , Peter, 2004). We investigated the effect of salt stress on rosemary in order to cultivation this plant at saline area and better landscape use in Iran.
MATERIALS AND METHODS Sample preparation: A pot experiment was conducted in Alzahra University ,Tehran, Iran. Rosemary plants were grown in greenhouse 25 ± 2°C under 16 h photoperiod with supplementary light and irrigated three times a week. After two months the plants were treated with 50, 75, 100 and 150 mM NaCl. Treatments initiate with 20 mM NaCl at first week. Every week the pots were treated with 25 mM NaCl . Treatments were completed during three consecutive weeks and the pots irrigated with tap water after every three days. 7 day after the last day of salt treatment the plants were harvested and analyzed.
Analysis of growth
Three plants per each replication were randomly harvested for growth measurement. Plants height and fresh weight were recorded. The fresh weight of shoots were measured immediately. The dry weight were measured by drying the plants at 80°C to give a constant weight.
Photosynthetic pigment determination Chlorophyll a, chlorophyll b and carotenoids were extracted in 80% acetone and were determined
( Lichtenthaler 1994)
Proline determination
Proline was quantified by using ninhydrin reagent and measured according to Bates et al (1973). Proline was extracted from 0.5 g of leaf in 10 ml of 3% sulfosalicylic acid. Two ml of extract was reacted with 2 ml acid- ninhydrin and 2 ml of glacial acetic acid for 75 min at 100 °C. The reaction was terminated in an ice bath . The reaction mixture was extracted with 4 ml of toluene and vortexed. The absorbance of toluene layer was spectrophotometrically determined at a wavelength of 520 nm. Concentration was determined from a standard curve and calculated on a fresh weight basis (^mol proline /g fw -1).
Cation analysis
The cations ( K+,Na+) were determined in dried ground leaves by Flame photometer (Jenway , Model PFP7) after acid digestion (Yash,p.K. 2000). K+ and Na+ contents were quantified based on a standard curve.
Determination of malondealdehyde
Malondealdehyde (MDA) levels were measured by the method of Heat and Packer (1968)
0.5 g of fresh leaf was homogenized in 2.5 mL of 0.1% (w/v) trichlroacetic acid (TCA), and centrifuged at 1000 rpm for 5 min. For every 1 ml of supernatant 4 mL of 20% TCA containing 0.5% TBA was add. The mixture were incubated for 30 min at 95°C, chilled on ice, and centrifuged at 4000 g for 10 min. the absorbance of the supernatant was measured at 532 nm using a Philips PU 8620 spectrophotometer. Unspecific absorption at 600 nm was subtracted from the 532 nm values. The concentration of MDA was calculated by using a molar extinction coefficient of 156 mM-1 cm.
Phenolic compounds
phenolics were extracted by the method of Conde with some modification( Conde et al 1995).1 g of
dried powdered sample was extracted with 20 ml methanol 80 % at 70 °C in water bath for 3 h. After filtration the solvent was evaporated to dryness using a rotary vacuum evaporator and the residue re dissolved in 10 ml of water. The aqueous solution used for quantitative determination with UV/VIS spectrophotometer.
The reaction mixture was prepared by mix in potassium ferrycyanid 83.4 mM 4- aminoantipiren 20.8 mM, NaHCO3 0.25 mM in a ration of 100:100:700 ^L.100 ^L of sample was then added . Absorbance was measured using spectrophotometer at 510 nm and plotted against a standaed curve obtained from gallic acid (Faust et al 1967).
Antioxidant assay
For DPPH assay, the method of Oktay was adopted with some modifications .4mL of a 0.004 % solution of DPPH in methanol 80% was mixed with 1ml of various concentrations of methanolic extract (0.02-0.1 mg/mL). The reaction mixture was vortex and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. The ability to scavenge DPPH radical was calculated by the following equation:
( RSC %) = (Ablank - Asample) /(A blank) x 100 where ( RSC %)= DPPH radical scavenging activity (%)
AbsMankl is the absorbance of DPPH radical + methanol (1 ml DPPH and 3 mL methanol) , Abssample is the absorbance of DPPH radical + sample extract
IC 50 Value was also calculated ( Oktay et al, 2003).
Statistical analysis
All data were analysed using SPSSS software, when analysis of variance showed significant
difference between means, Duncans multiple range test was applied to compare the means at p < 0.5.
RESULTS and DISCUSSION
Growth analysiss
The NaCl salinity reduced growth of rosemary plants and growth parameters decreased as salt stress increased. Growth inhibition is a common response to salinity ( Pardia 2005). The inhibitory effects of salt stress on growth parameters were also reported by other researchers using various plants ( Shaddad and Heikal 1982, Soussi et al 1998, Chaparzadeh et al 2004, Jenifer. and Franklin 2002 ).
Salt stress can lead to stomatal closure, which reduce CO2 assimilation in the leaves and inhibit carbon fixation and lead to reduction in photosynthesis rate and plant growth.
Photosynthetic pigments
Salinity caused a decrease in Chlorophyll and carotenoid content in rosemary plant.
Photosynthetic pigments content decreased progressively as salt stress increased. Salinity had more effect on Chl b content. The lowest pigments levels were observed in plants treated with 150 mM NaCl.
Salt stressed plants contained less carotenoid than the controls , however there was no significant difference in carotenoid content between various concentration of NaCl.
Similarly Munjal and Gosswami(1995), Weimburg (1975) Soussi et al (1998 ) , Xu ,Xinwen et al( 2008) and Jenifer and Franklin (2002) reported that salinity caused a decrease in chlorophyll and carotenoids level in other plants.
Table 1. Salt stress effects on rosemary growth parameters and biochemistry
NaCl mM 0 50 100 150
Freshweight (g) 23.66 ±0.5a 22.03± 0.25a 19.5±0.29a 13.26±.06bc
Dry weight (g) 1.7±0.21a 1.5±0.26a 0.71± 0.2b 0.33±0.4b
Plant height(cm) 23.66±2.7a 22.03±0.37a 19.58±0.74a 13.26±3.1cb
Na+ % 5.27±.13e 5.7±.12de 7.34±.17c 10.44±.31a
K+% 1.01±.05a 0.843±.06ab 0.76±0.07bc 0.31±0.1d
K+/Na+ 0.19±.01a 0.14±.01b 0.1±0.01c 0.3±0.1e
Chla (mg/g fw) 16.64±0.8a 17.83±0.3a 11.77±0.8b 7.73±1.1c
Chl b(mg/g fw) 18.11±2.3a 9.49±0.5b 7.96±2.9bc 3.57±0.53c
Chl t(mg/g fw) 36.47±0.82a 26.6±1.1b 17.96±1.38 d 10.68±1.39f
Chl b/ chl a 1.07±0.22a 1.47±0.06b 2.05±1.1a 2.04±0.33a
carotenoids(mg/g fw) 11.64±2.3 a 8.72±0.54 ab 8.85±2.91 ab 7.14±0.53 b
proline ^mol /g fw 0.12±0.01 c 0.28±0.003 a 0.51±0.003 a 0.47±0.04 a
MDA(nmol/g) 0.00025±0.01d 0.0018±0.02d 0.00062±0.04b7 0.00056±0.03 b
Phenolics(mg/g) 0.913±0.1e 1.59±0.19d 4.22±0.01a 3.47±0.03b
IC50 mg/ml 51.95±1.36d 45.98±0.62c 9.19±0.62a 10.7±0.57a
Decrease in chlorophylls level under salt stress may be due to reduction in pigment biosynthesis or enzymatic chlorophyll degradation (Xu ,Xinwen et al 2008 , Yang et al 2009)
The elevation of chl b/chl a ratio under saline condition suggested salinity had most adverse effect on Chlprophyll b content. Similar results obtained by Stoeva and Kaymakanova (2008) on Phaseolus vulgaris under sult stress.
The Chlprophyll level is an index of the photosynthesis (Xu et al 2008) and decrease in Chlprophyll level lead to reduction in growth parameters.
Slight reduction in carotenoids contents may be due to their protective role against reactive oxygen species. Salinity can lead to oxidative stress and causing significant decrease to photosynthetic systems. Carotenoids can protect photosynthetic system against reactive oxygen species generate
under salt stress (Parida and Das 2005, Parviz and Satava Waiz 2008).
Ionic content
Salinity caused decrease in K+ content and K+/Na+ ratio in treated plants however Na+ content increased at all salt levels.
Under salinity stress high NaCl uptake competes with the uptake of K+ and lead to reduction of K+/Na+ ratio and Na+ toxicity. Decrease in K+ accumulation under salt stress has been reported in other plants, including wheat, Sorghum, beat, barely and rice (Zaho and Ren 2007).
Na+ accumulation in salt stressed plants lead to low water potential, Change in essential ion uptake and ionic imbalance and reduces leaf expansion photosynthetic rate and limit growth (Zaho and Ren, 2007, Sairam et al 2002 ,Jenifer et al 2002,), Greenway and Munns 1980, Sharma 1997) The
increase of Na+ uptake in shoots of salt-treated plants did not drastically affect their growth status.
It appeared that rosemary is moderately salt tolerant (Tounekti et al 2008). This tolerance is apparently due to the ability of these plants to accumulate Na+ in their leaves and to maintain turgor and osmotic adjustment.
Proline
Proline content of salt stressed plants was higher than of the control although salt stressed plants having no significant increase in proline content .It is possible that the level of salt in the medium was not sufficient to increase proline. High accumulation of proline in leaves are important adaptive mechanism of salt tolerance. Proline acts as an osmolyte and reduces the osmotic potential, thus reducing toxic ion uptake ( Hare et al 1998)
Under salinity rosemary plant accumulated Na+ to maintain leaf turgor, Althoug they need synthesis of organic solute especially proline. Proline and Na+ accumulation is a mechanism used for maintaining turgor and reducing the adverse effect of salt stress (Bandeh - hagh et al 2008).
Proline may be act as radical scavenger and protects cells against salt induced oxidative stress (Hong et al 2000). Since total phenolic content and antioxidant activity of salt stressed plants increased, it seems that the phenolic antioxidant system of rosemary reducing the effect of salt stress and proline content did not significantly increased due to salt stress.
Malondealdehyde
MDA content in rosemary plants was poorly increased under saline condition.
MDA is regarded as a marker for evaluation of lipid peroxidation that increases with environmental stress. Under normal physiological conditions low concentrations of lipid peroxidation products are found in tissues and cells. In the presence of
oxidative stress more lipid peroxidation products are formed due to cell damage
The increase in lipid peroxidation may be due to the incapability of antioxidants to scavenger reactive oxygen species results from salt stress. The present results were agreed with the results of Ben Amor et al (2005), and Chaparzade et al 2004). A little increase in MDA level may be as a result of an effective antioxidant protection system in rosemary plant.
Phenolic compounds and antioxidant activity
Antioxidant activity and total phenolic content increased in salinized plants. Leaf phenolic content was significantly increased at 50 mM NaCl, and decreased at 150 mM NaCl. IC50 in 100 mM NaCl-treated plants was 83% lower than that observed in control plants. Phenolic compounds are commonly found in plants and have different biological effects. Plants can synthesize and accumulate phenolic compounds in response to stress (Dixon and Paiva, 1995, Mamdouh et al 2002)., Rosemary is a good source of phenolic antioxidant compounds .The antioxidant activity of phenolic compounds can play an important role in neutralizing ROS (Zheng and Wang, 2001). Reactive oxygen species contribute to various environmental stress including salinity.
We observed a correlation between the phenolic content and antioxidant activity. The close correlation between antioxidant activity and phenolic content has been demonstrated by the other workers (Liu et al 2007,Verzellon et al 2007,Naciye et al 2008).
Rosemary leaf contains phenolic acids (2-3% rosemarinic, chlorogenic, and caffeic). Rosmarinic and carnosic acid were reported as major antioxidant active phenolic compounds in rosemary plants (Almela et al., 2006). An increase in total phenolic content and antioxidant activity of rosemary under salinity can reduce oxidative stress. It can be interesting for production of antioxidant compounds in rosemary plants under salt stress. These results
confirm by others (Riadh et al 2007 , Riadeh et al
2007).
REFERENCES
Almela, L., Sanchez-Munoz, B., Fernandez-Lopez,
J.A., Rocaa, M.J. and Virginia, R. (2006) Liquid chromatograpic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material. J. Chromatogr., 7,221229.
Al-Sereiti, MR., Abu-Amer, KM and Sen, P.(1999) Pharmacology of rosemary (Rosmarinus officinalis) and its therapeutic potentials. Indian .J. Exp. Biol.,37(2), 124-30.
Aruma, OI., Spencer,JP., Rossi, R., Aeschbach ,R., Khan ,A., Mahmood ,N., Munoz, A., Murcia ,
A., Butler , J. and Halliwell, B. (1996) An evaluation of extracts of rosemary and provencal herbs. Food. Chem. Toxicol., 34(5), 449-56.
Aziz Eman ,E., Al-Amier, H. and Craker Lyle, E. (2008) Influence of Salt Stress on Growth and Essential Oil Production in Peppermint, Pennyroyal, and Apple Mint. J. Herb. Spic.
Med. Plants, 14, (1, 2) 77 - 87.
Bandeh - hagh, A., Toorchi, M., Mohammadi, A., Chaparzadeh,N.,Hosseini Salekdeh,G. and Kazemnia,H.(2008) Growth and osmotic adjustment of canola genotypes in response to salinity. J .Food. Agri. Envir., 6(2), 201-208.
Bates, L.E., Waldern, R.P. and Teare, ID.( 1973) Rapid determination of free proline for water stress studies. Plant. Soil., 39, 205-207.
Ben Amor, N., Ben Hamed, K. and Grinon , C. (2005) Physiological and antioxidant
responses of perennial halophyte Crithmum maritimum to salinity. Plant. Sci., 168 ,889899.
Chaparzadeh, N.,M.L.D., Amico, R.A., Khavari-Najad, R. and F. Navarizzo, (2004). Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiol. Biochem., 42,695-701.
Conde, E., E. Cadahia, M.C. Garcia-Vallejo and M.B. Fernandz de Simon,1995.Polyphenolic composition of wood extracts from Eucalyptus camaldulensis , E.globulus and E.rudis . Holzforschung., 49,41-47.
Dixon, R.A. and Paiva, N.L. (1995) Stress-Induced Phenylpropanoid Metabolism. Plant Cell., 7, 1085 - 1097.
Faust, S.D. and Mikulewicz, E.W.( 1967). Factors influencing the condensation of 4-aminoantipyrine with derivatives of hydroxybenzene - II. Influence of hydronium ion concentration on absorptivity. Wat. Res.,
1, 509-522.
Greenway ,H., Munns, R. (1980) Mechanism of salt tolerance in nonhalophytes.
Ann.Rev.Plant.Physiol.,31,149-190.
Hare, P.,Cress ,WA. and Van Staden ,J.( 1998) Dissecting the roles of osmolyte accumulation during stress. Plant. Cell. Environ., 21,535553.
Heath, R.L. and Packer, L.( 1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys., 125, 189-198.
Hong, Z., Lakkinel, K., Zhang, Z. and Verma, DPS. (2000) Removal of feedback inhibition of pyrroline-5-carboxylase synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol.,122,1129-1136.
Jenifer, A. and Franklin- Janus, J. (2002) Growth and elemental composition of Jak pine seedlings treated with sodium chloride and sodium
sulfate. Trees structure and function., 16(4-5),325-330.
Kamkar, B., Kafi, M. and Nassiri, M .(2004) Determination of most sensitive developmental period of wheat (Triticum aestivum) to salt stress to optimize saline water utilization. 4th International Crop Science Congress,Brisbane,Australia.
Lichtentaler, H. K. (1994). Chlorophyll and carotenoids pigments of photosynthetic biomembranes. Method in enzymology, 148: 350-382.
Liu, X., Dong, M., Chen, X., Jiang, M., Lv, X. and Yan, G. (2007) Antioxidant activity and phenolic of an endophytic Xylaria sp. From Ginkgo biloba. Food chemistry.,105,548-554.
Mamdouh, M., Mahmoud, Y., Omar, E and Zeinab,
B. (2002) Kinetin regulation of growth and secondary metabolism in waterlogging and salinity treated Vigna sinensis and Zea mays. Acta. Physiol. Plant., 24( 1), 19-27.
Moss, M., Cook, J., We, Snesk, etal. (2003) Aromas rosemary and lavender essential oils differentially affect cognition and mood in healthy adults., IntJ Neurosci.,2003,113:15-38.
Munjal, R. and Goswami, C.L. ( 1995) Response of chloroplastic pigments to NaCl and GA3 during cotton cotyledonary leaf growth and maturity. Agric. Sci. Digest., 15,146-150.
Naciye, E., Guler, A. and Erol, A (2008) Antioxidant activity of rosemary extract, blackseed essential oil , carnosic acid, rosamarinic acid and sesamol., Food chem.,. 110(1), 76-82.
Oktay , M., Gulcin, L. and Kufrevioglu , OL.(2003) Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebeen. Wissen. Technolo.,36,263-271.
Parida, A.K. and Das , A.B.(2005) Salt tolerance and salinity effect on plants : a review. Ecotoxicol. Environ.Saf., 60,324-349.
Parviz , A., Satyawati, S.(2008) Salt stress and phtobiochemical responses of plants- a review. Plant.Soil .Environ.,54:89-99.
Peter, K.V. (2004) Handbook of herbs and spice. Vol.2. Woodhead publishing Ltd and CRC press LLC.page 245.
Riadh, K., Wided, M., Ahmed, D., Hanen, F., Claude, G. and Chedly, A.( 2007) Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritime., Plant.Physi. Biochem., 45 ( 3-4), 244-249.
Sairam, R.K., Veerrabhadra, K., Srivastava, G.C. (2002) Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, Antioxidant activity and osmolyte concentration. Plant. Sci, 163,1037-1046.
Shadad, M.A. and Heikal, M.M.D. (1982) Interactive effect of gibberellic acid and salinity on kidney bean. Bull.Fac. Sci. Assiut Univ.,II(1):65-81.
Sharma , SK .(1997) Plant growth, photosynthesis and ion uptake in chickpea as influenced by salinity. Ind. J.Plant. Physiol., 2,171-173.
Soussi, M., Ocana , A. and Liuch, C. (1998) Effects of salt stress on growth , Photosynthesis and nitrogen fixation in chick- pea (Cicer arietinum L.). J. Exp.Bot., 49,1329-1337.
Sergi, M. and Leonor, A (2002).Subcellular Compartmentation of the Diterpene Carnosic Acid and Its Derivatives in the Leaves of Rosemary. Plant .Physiol., 125, 1094-1102.
Stoeva, N. and Kaymakanova, M. (2008) Effect of salt stress on the growth and photosynthesis rate of bean plants (Phaseolus vulgaris L.) .J.C.Europ.Agr.,9,384-391.
Troncoso, N., Sierra, H., Carvajal, L., Delpiano, P. and Gunther, G. (2005) Fast high performance liqiuid chromatography and ultraviolet-visible quantification of principle phenolic antioxidants in fresh rosemary. J. chromat.,1100,20-50
Tounekti, T., Vadel, AM., Bedoui, A. and Khemira,
H. (2008) NaCL stress affects growth and essential oil composition in rosemary (Rosmarinus officinalis). J .Hort. Sci. Biotech.,83(2), 267-273.
Verzeloni, E.,Tagliazucchi, D. and Conte, A. (2007) Relationship between the antioxidant properties and the phenolic and flavonoid content in traditional balsam vinegar. Food. chem., 105, 564-571.
Weimberg, R.( 1975) Effect of growth in highly salinized media on the enzymes of the
photosynthetic appartures in pea seedlings. Plant .Physiol.,6, 8-12.
Yang, F., Xiao, X.,Zhang, Sh. And Li, Ch.( 2009) Salt stress responses in populous cathagana Rehder. Plant .Sci.,176,669-677.
Yash, P.K. (2000) Handbook of reference methods for plant analysis. CRC press,pp 44-45.
Xu , X., Xu , H., Wang, Y., Wang , X., Qiu, Y. and Xu, Bo.( 2008) The effect of salt stress on the chlorophyll level of the main sand - binding plants in the shelterbelt along the Tarim Desert Highway. Chin .Sci. Bul.,53,109-111.
Zaho, GQ., Mab, B.L. and Ren, C.Z. (2007) Growth ,gas exchanges, chlorophyll fluorescence and ion content of naked oat in response to salinity. Crop. Sci. ,47,123-131.
Zheng, W. and Wang, S.Y.( 2001) Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food. Chem., 49, 5165-5170.
