Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around -15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.

Institute of Physical Biology University of South Bohemia; Nové Hrady Czech Republic

Zobrazit více v PubMed

Smallwood M, Bowles DJ. Plants in a cold climate. Philos T R Soc B. 2002;357:831–846. PubMed PMC

Moon BY, Higashi SI, Gombos Z, Murata N. Unsaturation of the membrane-lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tomato plants. Proc Natl Acad Sci USA. 1995;92:6219–6223. PubMed PMC

Moffatt B, Ewart V, Eastman A. Cold comfort: plant antifreeze proteins. Physiol Plant. 2006;126:5–16. doi: 10.1111/j.1399-3054.2006.00618.x. DOI

Heidarvand L, Amiri RM. What happens in plant molecular responses to cold stress? Acta Physiol Plant. 2010;32:419–431. doi: 10.1007/s11738-009-0451-8. DOI

Hincha DK. Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing. Philos T R Soc B. 2002;357:909–915. PubMed PMC

Öquist G, Huner NPA. Cold hardening induced resistance to photoinhibition in winter rye is dependent upon an increase capacity for photosynthesis. Planta. 1993;189:150–156.

Strand A, Hurry V, Henkes S, Huner N, Gustafsson P, Gardeström P, Stitt M. Acclimation of Arabidopsis leaves developing at low temperature: Increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin cycle and in the sucrose-biosynthesis pathway. Plant Physiol. 1999;119:1387–1397. PubMed PMC

Stitt M, Hurry V. A plant for all seasons: alteration in photosynthetic carbon metabolism during cold acclimation in Arabidopsis. Curr Opin Plant Biol. 2002;5:199–206. PubMed

Mitchell - Olds T, Schmitt J. Genetic mechanisms and evolutionary significance of natural variation in Arabidopsis. Nature. 2006;441:947–952. doi: 10.1038/nature04878. PubMed DOI

Hannah MA, Wiese D, Freund S, Fiehn O, Heyer AG, Hincha DK. Natural genetic variation of freezing tolerance in Arabidopsis. Plant Physiol. 2006;142:98–112. PubMed PMC

Zhen Y, Ungerer MC. Relaxed selection on the CBF/DREB1 regulatory genes and reduced freezing tolerance in the southern range of Arabidopsis thaliana. Mol Biol Evol. 2008;25:2547–2555. PubMed

Bodner M, Beck E. Effect of supercooling and freezing on photosynthesis in freezing tolerant leaves of Afroalpine gaint rosette plants. Oecologia. 1987;72:366–371. PubMed

Reyes-Díaz M, Ulloa N, Zúniga-Feest A, Gutiérrez A, Gidekel M, Alberdi M, Corcuera LJ, Bravo LA. Arabidopsis thaliana avoids freezing by supercooling. J Exp Bot. 2006;57:3687–3696. PubMed

Novillo F, Alonso JM, Ecker JR, Salinas J. CBF2/DREB1C is a negative regulator of CBF1/DREB1B and BF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis. Proc Natl Acad Sci USA. 2004;101:3985–3990. PubMed PMC

Steponkus PL, Lynch DV, Uemura M. The influence of cold-acclimation on the lipid-composition and cryobehavior of the plasma-membrane of isolated rye protoplasts. Philos T R Soc B. 1990;326:571–583.

Rohde P, Hincha DK, Heyer AG. Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance. Plant J. 2004;38:790–799. PubMed

Murray MB, Cape JN, Fowler D. Quantification of frost damage in plant-tissues by rates of electrolyte leakage. New Phytol. 1989;113:307–311. PubMed

Rumich-Bayer S, Krause GH. Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L. Photosynth Res. 1986;8:161–174. PubMed

Hincha DK, Sieg F, Bakaltcheva I, Köth H, Schmitt JM. Freeze-thaw damage to thylakoid membranes: specific protection by sugars and proteins. In: Steponkus PL, editor. Advances in low-temperature biology. London: JAI Press; 1996. pp. 141–183.

Agati G, Cerovic ZG, Moya I. The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: The role of the photosystem I contribution to the 735 nm fluorescence band. Photochem Photobiol. 2000;72:75–84. PubMed

Artus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin CT, Thomashow MF. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc Natl Acad Sci USA. 1996;93:13404–13409. PubMed PMC

Ehlert B, Hincha DK. Chlorophyll fluorescence imaging accurately quantifies freezing damage and cold acclimation responses in Arabidopsis leaves. Plant Methods. 2008;4:12. doi: 10.1186/1746-4811-4-12. PubMed DOI PMC

Rizza F, Pagani D, Stanca AM, Cattivelli L. Use of chlorophyll fluorescence to evaluate the cold acclimation and freezing tolerance of winter and spring oats. Plant Breeding. 2001;120:389–396.

Peguero-Pina JJ, Morales F, Gil-Pelegrin E. Frost damage in Pinus sylvestris L. stems assessed by chlorophyll fluorescence in cortical bark chlorenchyma. Ann For Sci. 2008;65(8):813P1–813P6.

Baker NR, Rosenqvist E. Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. J Exp Bot. 2004;55:1607–1621. PubMed

Neuner G, Buchner O. Assessment of foliar frost damage: a comparison of in vivo chlorophyll fluorescence with other viability tests. J Appl Bot. 1999;73:50–54.

Rapacz M. Chlorophyll a fluorescence transient during freezing and recovery in winter wheat. Photosynthetica. 2007;45:409–418.

Rapacz M, Wołniczka A. A selection tool for freezing tolerance in common wheat using the fast chlorophyll a fluorescence transcients. Plant Breeding. 2009;128:227–234.

Pospíšil P, Skotnica J, Nauš J. Low and high temperature dependence of minimum F0 and maximum Fm chlorophyll fluorescence in vivo. Biochimica et Biophysica Acta. 1998;1363:95–99. PubMed

Neuner G, Pramsohler M. Freezing and high temperature thresholds of photosystem II compared to ice nucleation, frost and heat damage in evergreen subalpine plants. Physiol Plant. 2006;126:196–204.

Hacker J, Spindelbock JP, Neuner G. Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence. Plant Cell Environ. 2008;31:1725–1733. PubMed

Nedbal L, Whitmarsh J. Chlorophyll fluorescence imaging of leaves and fruits. In: Papageorgiou G, Govindjee, editors. Chlorophyll a fluorescence: A signature of photosynthesis. Dordrech: Springer; 2004. pp. 389–407.

Matouš K, Benediktyova Z, Berger S, Roitsch T, Nedbal L. Case study of combinatorial imaging: What protocol and what chlorophyll fluorescence image to use when visualizing infection of Arabidopsis thaliana by Pseudomonas syringae? Photosynth Res. 2006;90:243–253. PubMed

Pudil P, Novovicova J, Kittler J. Floating search methods in feature selection. Pattern Recogn Lett. 1994;15:1119–1125.

Fukunaga K, editor. Introduction to statistical pattern recognition. 2nd edn. San Diego, USA: Academic Press Professional; 1990.

Martinez AM, Kak AC. PCA versus LDA. IEEE Trans Pattern Anal Mach Intell. 2001;23:228–233. doi: 10.1109/34.908974. DOI

Wang XC, Paliwal KK. Feature extraction and dimensionality reduction algorithms and their applications in vowel recognition. Pattern Recogn. 2003;36:2429–2439. doi: 10.1016/S0031-3203(03)00044-X. DOI

Mishra A, Matouš K, Mishra KB, Nedbal L. Towards discrimination of plant species by machine vision: advanced statistical analysis of chlorophyll fluorescence transients. J Fluoresc. 2009;19:905–913. PubMed

Dowgert MF, Steponkus PL. Behaviour of the plasma-membrane of isolated protoplasts during a freeze thaw cycle. Plant Physiol. 1984;75:1139–1151. PubMed PMC

Steponkus PL. Role of the plasma membrane in freezing-injury and cold acclimation. Annu Rev Plant Physiol Plant Mol Biol. 1984;35:543–584.

Barth C, Krause GH. Inhibition of photosystems I and II in chilling-sensitive and chilling-tolerant plants under light and low-temperature stress. Zeitschrift für Naturforschung C, J Biosci. 1999;54:645–657. (Ger).

Maxwell K, Johnson GN. Chlorophyll fluorescence: A practical guide. J Exp Bot. 2000;51:659–668. PubMed

Demeter S, Rozsa Z, Vass I, Hideg E. Thermo-luminescence study of charge recombination in photosystem-II at low temperatures.2. Oscillatory properties of the ZV and thermo-luminescence bands in chloroplast dark-adapted for various time periods. Biochim Biophys Acta. 1985;809:379–387.

Krause GH, Somersalo S. Fluorescence as a tool in photosynthesis research: Application in studies of photoinhibition, cold acclimation and freezing stress. Philos T R Soc B. 1989;323:281–293.

Govindjee Sixty-three years since Kautsky: chlorophyll α fluorescence. Aust J Plant Physiol. 1995;22:131–160.

Collard BCY, Jahufer MZZ, Brouwer JB, Pang ECK. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts. Euphytica. 2005;142:169–196. doi: 10.1007/s10681-005-1681-5. DOI

Codrea MC, Hakala-Yatkin M, Karlund-Marttila A, Nedbal L, Aittokallio T, Nevalainen OS, Tyystjävi E. Mahalanobis distance screening of Arabidopsis mutants with chlorophyll fluorescence. Photosynth Res. 2010;105:273–283. PubMed

Iftime D, Hannah MA, Peterbauer T, Heyer AG. Stachyose in the cytosol does not influence freezing tolerance of transgenic Arabidopsis expressing stachyose synthase from adzuki bean. Plant Sci. 2011;180:24–30. doi: 10.1016/j.plantsci.2010.07.012. PubMed DOI

Nedbal L, Soukupová J, Kaftan D, Whitmarsh J, Trtílek M. Kinetic imaging of chlorophyll fluorescence using modulated light. Photosynth Res. 2000;66:3. PubMed

Melatonin-Induced Protection Against Plant Abiotic Stress: Mechanisms and Prospects

Effects of low temperature on photoinhibition and singlet oxygen production in four natural accessions of Arabidopsis

Low temperature induced modulation of photosynthetic induction in non-acclimated and cold-acclimated Arabidopsis thaliana: chlorophyll a fluorescence and gas-exchange measurements

Chlorophyll a fluorescence, under half of the adaptive growth-irradiance, for high-throughput sensing of leaf-water deficit in Arabidopsis thaliana accessions

Automated phenotyping of plant shoots using imaging methods for analysis of plant stress responses - a review

Automated integrative high-throughput phenotyping of plant shoots: a case study of the cold-tolerance of pea (Pisum sativum L.)

Chlorophyll fluorescence emission can screen cold tolerance of cold acclimated Arabidopsis thaliana accessions