Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line
Luguang Wu A and Robert G. Birch A B
+ Author Affiliations
- Author Affiliations
A Botany Department, School of Biological Science, The University of Queensland, Brisbane, Qld 4072, Australia.
B Corresponding author. Email: r.birch@uq.edu.au
Functional Plant Biology 37(12) 1161-1174 https://doi.org/10.1071/FP10055
Submitted: 12 March 2010 Accepted: 6 September 2010 Published: 17 November 2010
Abstract
Transgenic sugarcane (Saccharum officinarum L. interspecific hybrids) line N3.2 engineered to express a vacuole-targeted sucrose isomerase was found to accumulate sucrose to twice the level of the background genotype Q117 in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose levels declined over successive subcultures, but the enhanced sucrose accumulation was stable. Detailed physiological characterisation revealed multiple processes altered in line N3.2 in a direction consistent with enhanced sucrose accumulation. Striking differences from the Q117 control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose-cleavage enzymes (particularly sucrose synthase breakage activity), and enhanced levels of symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate (G6P). Sucrose-non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates sucrose synthase breakage activity while downregulating SPS in plants) was significantly lower in line N3.2 during the period of fastest sucrose accumulation. For the first time, T6P is also shown to be a negative regulator of SnRK1 activity from sugarcane sink cells, hinting at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane.
Additional keywords: invertase, isomaltulose, parallel activation, Saccharum, SnRK1, sucrose isomerase, sucrose phosphate synthase, sucrose synthase, suspension culture, trehalose-6-phosphate.
References
Albertson PL, Peters KF, Grof CPL (2001) An improved method for the measurement of cell wall invertase activity in sugarcane tissue. Australian Journal of Plant Physiology 28, 323–328.Atanassova R, Leterrier M, Gaillard C, Agasse A, Sagot E, Coutos-Thevenot P, Delrot S (2003) Sugar-regulated expression of a putative hexose transport gene in grape. Plant Physiology 131, 326–334.
| Sugar-regulated expression of a putative hexose transport gene in grape.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXnvVGntQ%3D%3D&md5=9ab28e86fd68ec2f9c8ad25ff6e9e952CAS | 12529540PubMed |
Baena-González E, Rolland F, Thevelein JM, Sheen J (2007) A central integrator of transcription networks in plant stress and energy signalling. Nature 448, 938–942.
| A central integrator of transcription networks in plant stress and energy signalling.Crossref | GoogleScholarGoogle Scholar | 17671505PubMed |
Baroja-Fernandez E, Munoz FJ, Montero M, Etxeberria E, Sesma MT, et al (2009) Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield. Plant & Cell Physiology 50, 1651–1662.
| Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFaksr%2FN&md5=580dd4d89d0ef7a09589318a371fd08fCAS | 19608713PubMed |
Birch RG (2007) Metabolic engineering in sugarcane: assisting the transition to a bio-based economy. In ‘Applications of plant metabolic engineering’. (Eds RA Verpoorte, W Alfermann, TS Johnson) pp. 249–281. (Springer: Berlin)
Birch RG, Wu L (2004) Method for increasing product yield. International Patent Specification WO 2004/099403 A1.
Börnke F, Hajirezaei M, Heineke D, Melzer M, Herbers K, Sonnewald U (2002) High-level production of the non-cariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development. Planta 214, 356–364.
| High-level production of the non-cariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development.Crossref | GoogleScholarGoogle Scholar | 11855640PubMed |
Botha FC, Sawyer BJB, Birch RG (2001) Sucrose metabolism in the culm of transgenic sugarcane with reduced soluble acid invertase activity. In ‘Proceedings of the International Society of Sugarcane Technologists XXIV congress’. (Ed. DM Hogarth) pp. 588–591. (Australian Society of Sugar Cane Technologists: Mackay)
Carter JL, Garrard LA, West SH (1973) Effect of gibberellic acid on starch degrading enzymes in leaves of Digitaria decumbens. Phytochemistry 12, 251–254.
| Effect of gibberellic acid on starch degrading enzymes in leaves of Digitaria decumbens.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE3sXotVymsQ%3D%3D&md5=15e130b0eda1d82824a8b5867b642d2cCAS |
Chong BF, Bonnett GD, Glassop D, O’Shea MG, Brumbley SM (2007) Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink. Plant Biotechnology Journal 5, 240–253.
| Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXktFOqsbY%3D&md5=d57d97dd1794042aaf158e0dfc475dcbCAS | 17309679PubMed |
Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Research 5, 213–218.
| Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XjsFamsbo%3D&md5=05c2edef5d262b34f14efa90b7f5001dCAS | 8673150PubMed |
Delatte TL, Selman MHJ, Schluepmann H, Somson GW, Smeekens SCM, de Jong GJ (2009) Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry. Analytical Biochemistry 389, 12–17.
| Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXltVOktrk%3D&md5=76f176770413e520d932f55aae2908e5CAS | 19275873PubMed |
Dickson RE (1979) Analytical procedures for the sequential extraction of C-14-labeled constituents from leaves, bark and wood of cottonwood plants. Physiologia Plantarum 45, 480–488.
| Analytical procedures for the sequential extraction of C-14-labeled constituents from leaves, bark and wood of cottonwood plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1MXkslWhsbw%3D&md5=ba7f5bad3dd58b2f0f05b5c33c862aeaCAS |
Doehlert DC, Huber SC (1983) Regulation of spinich leaf sucrose phosphate synthase by glucose-6-phosphate, inorganic phosphate, and pH. Plant Physiology 73, 989–994.
| Regulation of spinich leaf sucrose phosphate synthase by glucose-6-phosphate, inorganic phosphate, and pH.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2cXotFyntA%3D%3D&md5=0b128c41e0e972469abf41d217666cbfCAS | 16663357PubMed |
Ebrahim MKH, Zingsheim O, Veith R, Abo-Kassem EEM, Komor E (1999) Sugar uptake and storage by sugarcane suspension cells at different temperatures and high sugar concentrations. Journal of Plant Physiology 154, 610–616.
Fernie AR, Roessner U, Geigenberger P (2001) The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers. Plant Physiology 125, 1967–1977.
| The sucrose analog palatinose leads to a stimulation of sucrose degradation and starch synthesis when supplied to discs of growing potato tubers.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjtFKqurw%3D&md5=779f11376d42eb97c100893d727e3d0aCAS | 11299376PubMed |
Ferreira SJ, Kossmann J, Lloyd JR, Groenewald JH (2008) The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines. Biotechnology Journal 3, 1398–1406.
| The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsV2qtrnI&md5=28830e864c72da13976aa767dddec90bCAS | 18729045PubMed |
Gnanasambandam A, Birch RG (2004) Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis. Plant Cell Reports 23, 435–447.
| Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtVagtr%2FK&md5=ee4bda92da5fcc6bf60081fd2a29cfcdCAS | 15372194PubMed |
Groenewald JH, Botha FC (2008) Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes. Transgenic Research 17, 85–92.
| Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsVGltLfE&md5=4486992b13a29985bd2172bf27c53f45CAS | 17431807PubMed |
Halford NG, Hey SJ (2009) Snf1-related protein kinases (SnRKs) act within an intricate network that links metabolic and stress signalling in plants. The Biochemical Journal 419, 247–259.
| Snf1-related protein kinases (SnRKs) act within an intricate network that links metabolic and stress signalling in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXjs1ynt78%3D&md5=06579b74d771383979a8449a5c87dfe5CAS | 19309312PubMed |
Hansom SP, Bower RS, Zhang L, Potier BA, Elliott AR, et al. (1999) Regulation of transgene expression in sugarcane. In ‘Proceedings of the International Society of Sugarcane Technologists XXIII Congress’. (Ed. V Singh) pp. 278–290. (The Sugar Technologists Association of India: New Delhi)
Jacobsen KR, Fisher DG, Maretzki A, Moore PH (1992) Developmental changes in the anatomy of the sugarcane stem in relation to phloem unloading and sucrose storage. Botanica Acta 105, 70–80.
Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Current Opinion in Plant Biology 7, 235–246.
| Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXjvVams7s%3D&md5=5d6feac569699356d3708e655261062fCAS | 15134743PubMed |
Komor E (2000) The physiology of sucrose storage in sugarcane. In ‘Carbohydrate reserves in plants: synthesis and regulation’. (Eds AK Gupta, N Kaur) pp. 35–53. (Elsevier: Amsterdam)
Kotake T, Yamaguchi D, Ohzono H, Hojo S, Kaneko S, Ishida HK, Tsumuraya Y (2004) UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts. The Journal of Biological Chemistry 279, 45 728–45 736.
| UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXovVCnsLY%3D&md5=bc0d7831068c64d0e743a2ddf514a7a0CAS |
Loreti E, Alpi A, Perata P (2000) Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos. Plant Physiology 123, 939–948.
| Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXlt1SlsbY%3D&md5=9aa72dd29ec31a9ed1acd9a5600c1ad9CAS | 10889242PubMed |
Lunn JE, Hatch MD (1997) The role of sucrose-phosphate synthase in the control of photosynthate partitioning in Zea mays leaves. Australian Journal of Plant Physiology 24, 1–8.
| The role of sucrose-phosphate synthase in the control of photosynthate partitioning in Zea mays leaves.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXit12mu7w%3D&md5=41a1b5193961a356030cbe0f2fc7ee01CAS |
Lunn JE, MacRae E (2003) New complexities in the synthesis of sucrose. Current Opinion in Plant Biology 6, 208–214.
| New complexities in the synthesis of sucrose.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjs1Gitr8%3D&md5=fd8ec85a7bbcc1ecb29eb179b1136b84CAS | 12753969PubMed |
Ma H, Albert H, Paull R, Moore P (2000) Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells. Australian Journal of Plant Physiology 21, 1021–1030.
Matsuoka K, Nakamura K (1991) Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proceedings of the National Academy of Sciences of the United States of America 88, 834–838.
| Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXhtFylsr4%3D&md5=26ca9ab7398fa772706cd4178616d484CAS | 1992474PubMed |
Moore PH (1995) Temporal and spatial regulation of sucrose accumulation in the sugarcane stem. Australian Journal of Plant Physiology 22, 661–679.
| Temporal and spatial regulation of sucrose accumulation in the sugarcane stem.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXos1eru70%3D&md5=8faa193e34ccf00a65ae6ceb9e6e2cabCAS |
Morandini P (2009) Rethinking metabolic control. Plant Science 176, 441–451.
| Rethinking metabolic control.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFGktLc%3D&md5=b4b0cb6ad4f9539a97c68eb91f394e03CAS |
Paul MJ, Primavesi LF, Jhurreea D, Zhang YH (2008) Trehalose metabolism and signaling. Annual Review of Plant Biology 59, 417–441.
| Trehalose metabolism and signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXntFaqsb0%3D&md5=c4d15f67747d2fbae971b4a5d53c828cCAS | 18257709PubMed |
Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling in plants: conserved and novel mechanisms. Annual Review of Plant Biology 57, 675–709.
| Sugar sensing and signaling in plants: conserved and novel mechanisms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XosVKht7k%3D&md5=09d490db7f78dc516b719928d061af6bCAS | 16669778PubMed |
Rossouw D, Bosch S, Kossmann J, Botha FC, Groenewald JH (2007) Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation. Functional Plant Biology 34, 490–498.
| Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmtVSksrg%3D&md5=7f018ea3738696c736f275aed1b14795CAS |
Schafer WE, Rohwer JM, Botha FC (2005) Partial purification and characterisation of sucrose synthase in sugarcane. Journal of Plant Physiology 162, 11–20.
| Partial purification and characterisation of sucrose synthase in sugarcane.Crossref | GoogleScholarGoogle Scholar | 15700416PubMed |
Schluepmann H, Dijken AV, Aghdasi M, Wobbes B, Paul M, Smeekens S (2004) Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation. Plant Physiology 135, 879–890.
| Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltlKju7k%3D&md5=734cb988e777de45192f6e99a5ae3e34CAS | 15181209PubMed |
Sinha AK, Hofmann MG, Romer U, Kockenberger W, Elling L, Roitsch T (2002) Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato. Plant Physiology 128, 1480–1489.
| Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XivFyksrs%3D&md5=cf78764b547385f2fe74bbe92f71ee05CAS | 11950996PubMed |
Taylor PWJ, Ko H-L, Adkins SW, Rathus C, Birch RG (1992) Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids). Plant Cell, Tissue and Organ Culture 28, 69–78.
| Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids).Crossref | GoogleScholarGoogle Scholar |
Toroser D, Plaut Z, Huber SC (2000) Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate. Plant Physiology 123, 403–412.
| Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXjsFemsLs%3D&md5=5bfd1aa842576be7a880ab36054baba4CAS | 10806257PubMed |
Turner W, Botha FC (2002) Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane. Archives of Biochemistry and Biophysics 407, 209–216.
| Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xot1KmtLc%3D&md5=9f36f3279f01c7c6f0e5208066c4ba32CAS | 12413493PubMed |
Van Handel E (1968) Direct microdetermination of sucrose. Analytical Biochemistry 22, 280–283.
| Direct microdetermination of sucrose.Crossref | GoogleScholarGoogle Scholar | 5641848PubMed |
Vargas WA, Salerno GL (2010) The Cinderella story of sucrose hydrolysis: alkaline/neutral invertases, from cyanobacteria to unforeseen roles in plant cytosol and organelles. Plant Science 178, 1–8.
| The Cinderella story of sucrose hydrolysis: alkaline/neutral invertases, from cyanobacteria to unforeseen roles in plant cytosol and organelles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVWmu7bE&md5=0228f837a2d2cc98e2f30877713d229cCAS |
Vickers JE, Grof CPL, Bonnett GD, Jackson PA, Morgan TE (2005) Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane clones in the field. Australian Journal of Agricultural Research 56, 57–68.
| Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane clones in the field.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXoslKmsw%3D%3D&md5=0e641e316644df52221ff26fff6bc8baCAS |
Walsh KB, Sky RC, Brown SM (2005) The anatomy of the pathway of sucrose unloading within the sugarcane stalk. Functional Plant Biology 32, 367–374.
| The anatomy of the pathway of sucrose unloading within the sugarcane stalk.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjsFyksbk%3D&md5=8d8ffe5fe867a56d1fc2a082b2308fa1CAS |
Welbaum GE, Meinzer FC, Grayson RL, Thornham KT (1992) Evidence for and consequences of a barrier to solute diffusion between the apoplast and vascular bundles in sugarcane stalk tissue. Australian Journal of Plant Physiology 19, 611–623.
| Evidence for and consequences of a barrier to solute diffusion between the apoplast and vascular bundles in sugarcane stalk tissue.Crossref | GoogleScholarGoogle Scholar |
Wendler R, Veith R, Dancer J, Stitt M, Komor E (1991) Sucrose storage in cell-suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation. Planta 183, 31–39.
| Sucrose storage in cell-suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXlvV2rsA%3D%3D&md5=bd76a119bef9de29e5729c1d118afe56CAS |
Winter H, Huber SC (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Critical Reviews in Plant Sciences 19, 31–67.
| Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXhtlajtLY%3D&md5=69d689f2777f2720c8607be9a8f50b5aCAS |
Wu L, Birch RG (2005) Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene. Applied and Environmental Microbiology 71, 1581–1590.
| Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXisVOitr8%3D&md5=36f5fd0e6495ca1a081d6102e594094dCAS | 15746363PubMed |
Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotechnology Journal 5, 109–117.
| Doubled sugar content in sugarcane plants modified to produce a sucrose isomer.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXitV2jur8%3D&md5=a1fd0ea540ab67e41c7eaaccd89f8ee4CAS | 17207261PubMed |
Zhang YH, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RAC, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ (2009) Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiology 149, 1860–1871.
| Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXks1Wgu7o%3D&md5=3dab098deab1418cc7234903023c44f7CAS | 19193861PubMed |
