Arabidopsis thaliana SYNAPTOTAGMIN 1 (AtSYT1) was shown to be involved in responses to different environmental and biotic stresses. We investigated gas exchange and chlorophyll a fluorescence in Arabidopsis wild-type (WT, ecotype Col-0) and atsyt1 mutant plants irrigated for 48 h with 150 mM NaCl. We found that salt stress significantly decreases net photosynthetic assimilation, effective photochemical quantum yield of photosystem II (ΦPSII), stomatal conductance and transpiration rate in both genotypes. Salt stress has a more severe impact on atsyt1 plants with increasing effect at higher illumination. Dark respiration, photochemical quenching (qP), non-photochemical quenching and ΦPSII measured at 750 µmol m-2 s-1 photosynthetic photon flux density were significantly affected by salt in both genotypes. However, differences between mutant and WT plants were recorded only for qP and ΦPSII. Decreased photosynthetic efficiency in atsyt1 under salt stress was accompanied by reduced chlorophyll and carotenoid and increased flavonol content in atsyt1 leaves. No differences in the abundance of key proteins participating in photosynthesis (except PsaC and PsbQ) and chlorophyll biosynthesis were found regardless of genotype or salt treatment. Microscopic analysis showed that irrigating plants with salt caused a partial closure of the stomata, and this effect was more pronounced in the mutant than in WT plants. The localization pattern of AtSYT1 was also altered by salt stress.

Department of Biochemistry and Biotechnology Institute of Biotechnology Faculty of Biotechnology and Food Sciences Slovak University of Agriculture 949 76 Nitra Slovakia

Department of Biophysics Faculty of Science Palacký University 783 71 Olomouc Czech Republic

Department of Botany Institute of Biology and Ecology Faculty of Science Pavol Jozef Šafárik University 041 54 Kosice Slovakia

Department of Experimental Plant Biology Institute of Botany Plant Science and Biodiversity Center Slovak Academy of Sciences 845 23 Bratislava Slovakia

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Lamers J., van der Meer T., Testerink C. How plants sense and respond to stressful environments. Plant Physiol. 2020;182:1624–1635. doi: 10.1104/pp.19.01464. PubMed DOI PMC

Bornschein G., Schmidt H. Synaptotagmin Ca2+ sensors and their spatial coupling to presynaptic Cav channels in central cortical synapses. Front. Mol. Neurosci. 2019;11:494. doi: 10.3389/fnmol.2018.00494. PubMed DOI PMC

Herdman C., Moss T. Extended-Synaptotagmins (E-Syts); the extended story. Pharmacol. Res. 2016;107:48–56. doi: 10.1016/j.phrs.2016.01.034. PubMed DOI

Craxton M. Genomic analysis of synaptotagmin genes. Genomics. 2001;77:43–49. doi: 10.1006/geno.2001.6619. PubMed DOI

Craxton M. Synaptotagmin gene content of the sequenced genomes. BMC Genom. 2004;5:43. doi: 10.1186/1471-2164-5-43. PubMed DOI PMC

Schapire A.L., Voigt B., Jasik J., Rosado A., Lopez-Cobollo R., Menzel D., Salinas J., Mancuso S., Valpuesta V., Baluska F., et al. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability. Plant Cell. 2008;20:3374–3388. doi: 10.1105/tpc.108.063859. PubMed DOI PMC

Kawamura Y., Uemura M. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. Plant J. 2003;36:141–154. doi: 10.1046/j.1365-313X.2003.01864.x. PubMed DOI

Yamazaki T., Kawamura Y., Minami A., Uemura M. Calcium-dependent freezing tolerance in Arabidopsis involves membrane resealing via synaptotagmin SYT1. Plant Cell. 2008;20:3389–3404. doi: 10.1105/tpc.108.062679. PubMed DOI PMC

Pérez-Sancho J., Vanneste S., Lee E., McFarlane H., del Valle A.E., Valpuesta V., Friml J., Botella M.A., Rosado A. The Arabidopsis SYT1 is enriched in ER-PM contact sites and confers cellular resistance to mechanical stresses. Plant Physiol. 2015;168:132–143. doi: 10.1104/pp.15.00260. PubMed DOI PMC

Lee E., Vanneste S., Pérez-Sancho J., Benitez-Fuente F., Strelau M., Macho A.P., Botella M.A., Friml J., Rosado A. Ionic stress enhances ER–PM connectivity via phosphoinositide-associated SYT1 contact site expansion in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2019;116:1420–1429. doi: 10.1073/pnas.1818099116. PubMed DOI PMC

Yan Q., Huang Q., Chen J., Li J., Liu Z., Yang Y., Li X., Wang J. SYTA has positive effects on the heat resistance of Arabidopsis. Plant Growth Reg. 2017;81:467–476. doi: 10.1007/s10725-016-0224-5. DOI

Lewis J.D., Lazarowitz S.G. Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport. Proc. Natl. Acad. Sci. USA. 2010;107:2491–2496. doi: 10.1073/pnas.0909080107. PubMed DOI PMC

Levy A., Zheng J.Y., Lazarowitz S.G. Synaptotagmin SYTA forms ER-plasma membrane junctions that are recruited to plasmodesmata for plant virus movement. Curr. Biol. 2015;25:2018–2025. doi: 10.1016/j.cub.2015.06.015. PubMed DOI PMC

Uchiyama A., Shimada-Beltran H., Levy A., Zheng J.Y., Javia P.A., Lazarowitz S.G. The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses. Front. Plant Sci. 2014;5:584. doi: 10.3389/fpls.2014.00584. PubMed DOI PMC

Yuan C., Lazarowitz S.G., Citovsky V. The plasmodesmal localization signal of TMV MP is recognized by plant synaptotagmin SYTA. mBio. 2018;9:e01314-18. doi: 10.1128/mBio.01314-18. PubMed DOI PMC

Cabanillas D.G., Jiang J., Movahed N., Germain H., Yamaji Y., Zheng H., Laliberté J.F. Turnip mosaic virus uses the SNARE protein VTI11 in an unconventional route for replication vesicle trafficking. Plant Cell. 2018;30:2594–2615. doi: 10.1105/tpc.18.00281. PubMed DOI PMC

Kim H., Kwon H., Kim S., Kim M.K., Botella M.A., Yun H.S., Kwon C. Synaptotagmin 1 negatively controls the two distinct immune secretory pathways to powdery mildew fungi in Arabidopsis. Plant Cell Physiol. 2016;57:1133–1141. doi: 10.1093/pcp/pcw061. PubMed DOI

Siao W., Wang P., Voigt B., Hussey P.J., Baluska F. Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites. J. Exp. Bot. 2016;67:6161–6171. doi: 10.1093/jxb/erw381. PubMed DOI PMC

Ishikawa K., Tamura K., Ueda H., Ito Y., Nakano A., Hara-Nishimura I., Shimada T. Synaptotagmin-associated endoplasmic reticulum-plasma membrane contact sites are localized to immobile ER tubules. Plant Physiol. 2018;178:641–653. doi: 10.1104/pp.18.00498. PubMed DOI PMC

Lee E., Santana B.V.N., Samuels E., Benitez-Fuente F., Corsi E., Botella M.A., Perez-Sancho J., Vanneste S., Friml J., Macho A., et al. Rare earth elements induce cytoskeleton-dependent and PI4P-associated rearrangement of SYT1/SYT5 endoplasmic reticulum–plasma membrane contact site complexes in Arabidopsis. J. Exp. Bot. 2020;71:3986–3998. doi: 10.1093/jxb/eraa138. PubMed DOI PMC

Ruiz-Lopez N., Pérez-Sancho J., del Valle A.E., Haslam R.P., Vanneste S., Catalá R., Perea-Resa C., Van Damme D., García-Hernández S., Albert A., et al. Synaptotagmins maintain diacylglycerol homeostasis at endoplasmic reticulum-plasma membrane contact sites during abiotic stress. Plant Cell. 2021;33:2431–2453. doi: 10.1093/plcell/koab122. PubMed DOI PMC

Van Zelm E., Zhang Y., Testerink C. Salt tolerance mechanisms of plants. Annu. Rev. Plant Biol. 2020;71:403–433. doi: 10.1146/annurev-arplant-050718-100005. PubMed DOI

Yang Z., Li J.L., Liu L.N., Xie Q., Sui N. Photosynthetic regulation under salt stress and salt-tolerance mechanism of sweet sorghum. Front. Plant. Sci. 2020;10:1722. doi: 10.3389/fpls.2019.01722. PubMed DOI PMC

Pan T., Liu M., Kreslavski V.D., Zharmukhamedov S.K., Nie C., Yu M., Kuznetsov V.V., Allakhverdiev S.I., Shabala S. Non-stomatal limitation of photosynthesis by soil salinity. Crit. Rev. Environ. Sci. Technol. 2021;51:791–825. doi: 10.1080/10643389.2020.1735231. DOI

Awlia M., Nigro A., Fajkus J., Schmoeckel S.M., Negrão S., Santelia D., Trtílek M., Julkowska M.M., Panzarová K. High-throughput non-destructive phenotyping of traits that contribute to salinity tolerance in Arabidopsis thaliana. Front. Plant. Sci. 2016;7:1414. doi: 10.3389/fpls.2016.01414. PubMed DOI PMC

Chaves M.M., Flexas J., Pinheiro C. Photosynthesis under drought and salt stress: Regulation mechanisms from whole plant to cell. Ann. Bot. 2009;103:551–560. doi: 10.1093/aob/mcn125. PubMed DOI PMC

Nawaz K., Hussain K., Majeed A., Khan F., Afghan S., Ali K. Fatality of salt stress to plants: Morphological, physiological and biochemical aspects. Afr. J. Biotechnol. 2010;9:5475–5480.

Ashraf M.H.P.J.C., Harris P.J. Photosynthesis under stressful environments: An overview. Photosynthetica. 2013;51:163–190. doi: 10.1007/s11099-013-0021-6. DOI

Deinlein U., Stephan A.B., Horie T., Luo W., Xu G., Schroeder J.I. Plant salt-tolerance mechanisms. Trends. Plant Sci. 2014;19:371–379. doi: 10.1016/j.tplants.2014.02.001. PubMed DOI PMC

Safdar H., Amin A., Shafiq Y., Ali A., Yasin R., Shoukat A., Hussan M.U., Sarwar M.I. A review: Impact of salinity on plant growth. Nat. Sci. 2019;17:34–40.

Arif Y., Singh P., Siddiqui H., Bajguz A., Hayat S. Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiol. Biochem. 2020;156:64–77. doi: 10.1016/j.plaphy.2020.08.042. PubMed DOI

Lawson T., Vialet-Chabrand S. Speedy stomata, photosynthesis and plant water use efficiency. New Phytol. 2019;221:93–98. doi: 10.1111/nph.15330. PubMed DOI

Lawson T., Blatt M.R. Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. Plant Physiol. 2014;164:1556–1570. doi: 10.1104/pp.114.237107. PubMed DOI PMC

Vialet-Chabrand S.R.M., Matthews J.S., McAusland L., Blatt M.R., Griffiths H., Lawson T. Temporal dynamics of stomatal behavior: Modeling and implications for photosynthesis and water use. Plant Physiol. 2017;174:603–613. doi: 10.1104/pp.17.00125. PubMed DOI PMC

Ball M.C., Anderson J.M. Sensitivity of photosystems II to NaCl in relation to salinity tolerance. Comparative studies with thylakoids of the salt tolerant mangrove, Avicennia marina, and the salt-sensitive pea, Pisum sativum. Funct. Plant. Biol. 1986;13:689–698. doi: 10.1071/PP9860689. DOI

Takahashi S., Murata N. How do environmental stresses accelerate photoinhibition? Trends Plant Sci. 2008;13:178–182. doi: 10.1016/j.tplants.2008.01.005. PubMed DOI

Pompelli M.F., Ferreira P.P.B., Chaves A.R.M., Figueiredo R.C.B.Q., Martinse A.O., Jarma-Orozco A., Bhatt A., Batista-Silva W., Endres L., Araújo W.L. Physiological, metabolic, and stomatal adjustments in response to salt stress in Jatropha curcas. Plant Physiol. Biochem. 2021;168:116–127. doi: 10.1016/j.plaphy.2021.09.039. PubMed DOI

Ouerghi Z., Cornic G., Roudani M., Ayadi A., Brulfert J. Effect of NaCl on photosynthesis of two wheat species (Triticum durum and T. aestivum) differing in their sensitivity to salt stress. J. Plant Physiol. 2000;156:335–340. doi: 10.1016/S0176-1617(00)80071-1. DOI

Steduto P., Albrizio R., Giorio P., Sorrentino G. Gas-exchange response and stomatal and non-stomatal limitations to carbon assimilation of sunflower under salinity. Environ. Exp. Bot. 2000;44:243–255. doi: 10.1016/S0098-8472(00)00071-X. PubMed DOI

Sui N., Yang Z., Liu M., Wang B. Identification and transcriptomic profiling of genes involved in increasing sugar content during salt stress in sweet sorghum leaves. BMC Genom. 2015;16:534. doi: 10.1186/s12864-015-1760-5. PubMed DOI PMC

Hu T., Yi H., Hu L., Fu J. Stomatal and metabolic limitations to photosynthesis resulting from NaCl stress in perennial ryegrass genotypes differing in salt tolerance. J. Am. Soc. Hortic. Sci. 2013;138:350–357. doi: 10.21273/JASHS.138.5.350. DOI

Bai J., Qin Y., Liu J., Wang Y., Sa R., Zhang N., Jia R. Proteomic response of oat leaves to long-term salinity stress. Environ. Sci. Pollut. Res. Int. 2017;24:3387–3399. doi: 10.1007/s11356-016-8092-0. PubMed DOI

Najar R., Aydi S., Sassi-Aydi S., Zarai A., Abdelly C. Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula. Plant Biosyst.-Int. J. Deal. Asp. Plant Biol. 2019;153:88–97. doi: 10.1080/11263504.2018.1461701. DOI

Sarabi B., Fresneau C., Ghaderi N., Bolandnazar S., Streb P., Badeck F.W., Citerine S., Tangama M., David A., Ghashghaie J. Stomatal and non-stomatal limitations are responsible in down-regulation of photosynthesis in melon plants grown under the saline condition: Application of carbon isotope discrimination as a reliable proxy. Plant Physiol. Biochem. 2019;141:1–19. doi: 10.1016/j.plaphy.2019.05.010. PubMed DOI

Franzoni G., Cocetta G., Trivellini A., Ferrante A. Transcriptional regulation in rocket leaves as affected by salinity. Plants. 2020;9:20. doi: 10.3390/plants9010020. PubMed DOI PMC

Pavlovič A., Singerová L., Demko V., Šantrůček J., Hudák J. Root nutrient uptake enhances photosynthetic assimilation in prey-deprived carnivorous pitcher plant Nepenthes talangensis. Photosynthetica. 2010;48:227–233. doi: 10.1007/s11099-010-0028-1. DOI

Zörb C., Herbst R., Forreiter C., Schubert S. Short-term effects of salt exposure on the maize chloroplast protein pattern. Proteomics. 2009;9:4209–4220. doi: 10.1002/pmic.200800791. PubMed DOI

Razavizadeh R., Ehsanpour A.A., Ahsan N., Komatsu S. Proteome analysis of tobacco leaves under salt stress. Peptides. 2009;30:1651–1659. doi: 10.1016/j.peptides.2009.06.023. PubMed DOI

Kang G., Li G., Zheng B., Han Q., Wang C., Zhu Y., Guo T. Proteomic analysis on salicylic acid-induced salt tolerance in common wheat seedlings (Triticum aestivum L.) Biochim. Biophys. Acta. 2012;1824:1324–1333. doi: 10.1016/j.bbapap.2012.07.012. PubMed DOI

Ma H., Song L., Shu Y., Wang S., Niu J., Wang Z., Yu T., Gu W., Ma H. Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes. J. Proteom. 2012;75:1529–1546. doi: 10.1016/j.jprot.2011.11.026. PubMed DOI

De Abreu C.E.B., dos Santos Araújo G., de Oliveira Monteiro-Moreira A.C., Costa J.H., de Brito Leite H., Moreno F.B.M.B., Prisco J.T., Gomes-Filho E. Proteomic analysis of salt stress and recovery in leaves of Vigna unguiculata cultivars differing in salt tolerance. Plant Cell Rep. 2014;33:1289–1306. doi: 10.1007/s00299-014-1616-5. PubMed DOI

Wang L., Liu X., Liang M., Tan F., Liang W., Chen Y., Lin Y., Huang L., Xing J., Chen W. Proteomic analysis of salt-responsive proteins in the leaves of mangrove Kandelia candel during short-term stress. PLoS ONE. 2014;9:e83141. doi: 10.1371/journal.pone.0083141. PubMed DOI PMC

Fatehi F., Hosseinzadeh A., Alizadeh H., Brimavandi T., Struik P.C. The proteome response of salt-resistant and salt-sensitive barley genotypes to long-term salinity stress. Mol. Biol. Rep. 2012;39:6387–6397. doi: 10.1007/s11033-012-1460-z. PubMed DOI

Wang J., Meng Y., Li B., Ma X., Lai Y., Si E., Yang K.E., Xu X., Shang X., Wang H., et al. Physiological and proteomic analyses of salt stress response in the halophyte Halogeton glomeratus. Plant Cell Environ. 2015;38:655–669. doi: 10.1111/pce.12428. PubMed DOI PMC

Liu Y.L., Shen Z.J., Simon M., Li H., Ma D.N., Zhu X.Y., Zheng H.L. Comparative proteomic analysis reveals the regulatory effects of H2S on salt tolerance of mangrove plant Kandelia obovata. Int. J. Mol. Sci. 2020;21:118. doi: 10.3390/ijms21010118. PubMed DOI PMC

Hui-Hui Z., Guang-Liang S., Jie-Yu S., Xin L., Ma-Bo L., Liang M., Nan X., Guang-Yu S. Photochemistry and proteomics of mulberry (Morus alba L.) seedlings under NaCl and NaHCO3 stress. Ecotoxicol. Environ. Saf. 2019;184:109624. doi: 10.1016/j.ecoenv.2019.109624. PubMed DOI

Xu J., Lan H., Fang H., Huang X., Zhang H., Huang J. Quantitative proteomic analysis of the rice (Oryza sativa L.) salt response. PLoS ONE. 2015;10:e0120978. doi: 10.1371/journal.pone.0120978. PubMed DOI PMC

Pang Q., Chen S., Dai S., Chen Y., Wang Y., Yan X. Comparative proteomics of salt tolerance in Arabidopsis thaliana and Thellungiella halophila. J. Proteome Res. 2010;9:2584–2599. doi: 10.1021/pr100034f. PubMed DOI

Maslova T.G., Markovskaya E.F., Slemnev N.N. Functions of Carotenoids in Leaves of Higher Plants. Biol. Bull. Rev. 2021;11:476–487. doi: 10.1134/S2079086421050078. DOI

Agati G., Brunetti C., Fini A., Gori A., Guidi L., Landi M., Sebastiani F., Tattini M. Are flavonoids effective antioxidants in plants? Twenty years of our investigation. Antioxidants. 2020;9:1098. doi: 10.3390/antiox9111098. PubMed DOI PMC

Geissler N., Hussin S., Koyro H.W. Interactive effects of NaCl salinity and elevated atmospheric CO2 concentration on growth, photosynthesis, water relations and chemical composition of the potential cash crop halophyte Aster tripolium L. Environ. Exp. Bot. 2009;65:220–231. doi: 10.1016/j.envexpbot.2008.11.001. DOI

Lutts S., Kinet J.M., Bouharmont J. NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann. Bot. 1996;78:389–398. doi: 10.1006/anbo.1996.0134. DOI

Pinheiro H.A., Silva J.V., Endres L., Ferreira V.M., de Albuquerque Câmara C., Cabral F.F., dos Santos Filho B.G. Leaf gas exchange, chloroplastic pigments and dry matter accumulation in castor bean (Ricinus communis L.) seedlings subjected to salt stress conditions. Ind. Crops Prod. 2008;27:385–392. doi: 10.1016/j.indcrop.2007.10.003. DOI

Li G., Wan S., Zhou J., Yang Z., Qin P. Leaf chlorophyll fluorescence, hyperspectral reflectance, pigments content, malondialdehyde and proline accumulation responses of castor bean (Ricinus communis L.) seedlings to salt stress levels. Ind. Crops Prod. 2010;31:13–19. doi: 10.1016/j.indcrop.2009.07.015. DOI

Yang J.Y., Zheng W., Tian Y., Wu Y., Zhou D.W. Effects of various mixed salt-alkaline stresses on growth, photosynthesis, and photosynthetic pigment concentrations of Medicago ruthenica seedlings. Photosynthetica. 2011;49:275–284. doi: 10.1007/s11099-011-0037-8. DOI

Gadallah M.A.A. Effects of proline and glycinebetaine on Vicia faba responses to salt stress. Biol. Plant. 1999;42:249–257. doi: 10.1023/A:1002164719609. DOI

Gautam S., Singh P.K. Salicylic acid-induced salinity tolerance in corn grown under NaCl stress. Acta Physiol. Plant. 2009;31:1185–1190. doi: 10.1007/s11738-009-0338-8. DOI

Karnik R., Waghmare S., Zhang B., Larson E., Lefoulon C., Gonzalez W., Blatt M.R. Commandeering channel voltage sensors for secretion, cell turgor, and volume control. Trends Plant Sci. 2017;22:81–95. doi: 10.1016/j.tplants.2016.10.006. PubMed DOI PMC

Südhof T.C. Neurotransmitter release: The last millisecond in the life of a synaptic vesicle. Neuron. 2013;80:675–690. doi: 10.1016/j.neuron.2013.10.022. PubMed DOI PMC

Jezek M., Blatt M.R. The membrane transport system of the guard cell and its integration for stomatal dynamics. Plant Physiol. 2017;174:487–519. doi: 10.1104/pp.16.01949. PubMed DOI PMC

Eisenach C., Chen Z.H., Grefen C., Blatt M.R. The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K+ channel activity with vegetative growth. Plant J. 2012;69:241–251. doi: 10.1111/j.1365-313X.2011.04786.x. PubMed DOI

Boyes D.C., Zayed A.M., Ascenzi R., McCaskill A.J., Hoffman N.E., Davis K.R., Görlach J. Growth stage–based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell. 2001;13:1499–1510. doi: 10.1105/TPC.010011. PubMed DOI PMC

Lešková A., Kusá Z., Labajová M., Krausko M., Jásik J. The Photoconvertible Fluorescent Protein Dendra2 Tag as a Tool to Investigate Intracellular Protein Dynamics. Methods Mol. Biol. 2019;2019:201–2014. PubMed

Lešková A., Labajová M., Krausko M., Zahradníková A., Baluška F., Mičieta K., Turňa J., Jásik J. Endosidin 2 accelerates PIN2 endocytosis and disturbs intracellular trafficking of PIN2, PIN3, and PIN4 but not of SYT1. PLoS ONE. 2020;15:e0237448. doi: 10.1371/journal.pone.0237448. PubMed DOI PMC

Maxwell K., Johnson G.N. Chlorophyll fluorescence—A practical guide. J. Exp. Bot. 2000;51:659–668. doi: 10.1093/jexbot/51.345.659. PubMed DOI

Martínez-García J.F., Monte E., Quail P.H. A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant. J. 1999;20:251–257. doi: 10.1046/j.1365-313x.1999.00579.x. PubMed DOI

Lichtenthaler H.K. Chlorophylls and carotenoids:Pigments of photosynthetic biomembranes. Meth. Enzymol. 1987;148:350–382.

Pękal A., Pyrzynska K. Evaluation of aluminium complexation reaction for flavonoid content assay. Food Anal. Meth. 2014;7:1776–1782. doi: 10.1007/s12161-014-9814-x. DOI