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Environmental and Experimental Botany 93 (2013) 45– 54
Contents lists available at SciVerse ScienceDirect
Environmental and Experimental Botany
jou rn al h om epa ge: www.elsev ier .com/ locate /envexpbot
ifferent mechanisms trigger an increase in freezing tolerance inestuca pratensis exposed to flooding stress
arbara Jurczyka,∗, Tomasz Krepskia, Arkadiusz Kosmalab, Marcin Rapacza
University of Agriculture in Krakow, Faculty of Agriculture and Economics, Department of Plant Physiology, Podłuzna 3, 30-239 Cracow, PolandInstitute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, Poland
a r t i c l e i n f o
rticle history:eceived 21 December 2012eceived in revised form 3 June 2013ccepted 4 June 2013
eywords:old acclimationloodingrost tolerance
a b s t r a c t
Increased precipitation and snow melt during warmer winters may lead to low temperature flooding andice encasement formation. These conditions are stressful to plants and may affect their winter survivaland spring regrowth. The aim of this study was to assess the effects of low temperature flooding onfrost tolerance, photosynthetic performance, osmotic potential, water soluble carbohydrate content andexpression of CBF6, Cor14b and LOS2 genes in four genotypes of Festuca pratensis with distinct levels of frosttolerance. It was shown that plants cold acclimated under flooding increase their frost tolerance fasterand/or to a greater extent than in non-flooded controls. Changes in the induction kinetics of transcriptionfactors encoding genes are connected with transient growth of frost tolerance in two out of the four
ranscript levelater soluble carbohydrates
genotypes, irrespective of their frost tolerance. A significant and stable increase in frost tolerance observedin the genotype with the lowest tolerance under control conditions was related to higher carbohydrateconcentration in the flooded plants. In more frost tolerant genotypes, low temperature flooding alsoimproved their resistance to low-temperature induced photoinhibition of photosynthesis. In conclusion,low-temperature flooding of the plant roots and crowns may boost cold acclimation efficiency in F.pratensis, but this effect is genotype-dependent and varies according to the background.
. Introduction
The environmental stresses faced by plants in the winter seem toe enhanced by global warming. While low temperatures and frost
vents have probably become less frequent over most areas (IPCC,007), other detrimental phenomena, like increased winter precip-tation (Osborn and Hulme, 2002) and snow melt during warmer
Abbreviations: CA, cold acclimation; CBF/DREB, C-repeat bindingactor/dehydration-responsive element-binding protein; F0 and F0′ , fluores-ence when all PSII reaction centres are open in dark- and light-acclimated leaves,espectively; Fd, fluorescence decrease; Fm and Fm′ , fluorescence when all PSIIeaction centres are closed in dark- and light-acclimated leaves, respectively; Fs,teady state fluorescence in light exposed leaves; Fv and Fv′ , variable fluorescencen dark- and light-acclimated leaves, respectively; Fv/Fm, apparent quantum yieldf PSII; PSII, photosystem II; Fv′ /Fm′ , photosystem II antenna trapping efficiency;p8 and Fp13, Festuca pratensis genotypes with lower frost tolerance, accordingo Kosmala et al. (2009); Fp1 and Fp37, Festuca pratensis genotypes with higherrost tolerance, according to Kosmala et al. (2009); LOS2, low expression of osmot-cally responsive genes 2 (bifunctional enolase with transcriptional repressionctivity); tEL50, temperature at which 50% of the plants were killed by frost; NPQ,on-photochemical quenching of chlorophyll fluorescence; qP, photochemicaluenching of chlorophyll fluorescence; STZ/ZAT10, zinc finger protein; WSC, wateroluble carbohydrates.∗ Corresponding author. Tel.: +48 12 4253301; fax: +48 12 4253202.
E-mail addresses: [email protected] (B. Jurczyk), [email protected]. Krepski), [email protected] (A. Kosmala), [email protected] (M. Rapacz).
098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.06.003
© 2013 Elsevier B.V. All rights reserved.
winters, may lead to low temperature flooding and the formation ofice encasement. These stresses may affect plant overwintering andyield. It was shown that low temperature flooding is not as damag-ing as high temperature flooding (Beard and Martin, 1970). Growthat higher temperature is accompanied by reduced oxygen solubil-ity and higher enzyme activity, but if the exposure is protracted,accumulating anaerobic products may damage the overwinteringplants (Beard, 1964; Pomeroy and Andrews, 1979; McKersie et al.,1982).
Exposure to low temperature increases the tolerance of manyspecies to freezing stress and to ice stress, and this is termedan acclimation process. Plant cold acclimation (CA) is associatedwith various biological changes. A major change in gene expres-sion pattern (Gilmour et al., 1998) triggers a set of metabolic andphysiological reactions, including the accumulation of compatiblesolutes, such as soluble sugars (Hoffman et al., 2010), and changesin photosynthetic machinery (Rapacz et al., 2008). It was shownthat flooding of the soil surface during low temperature growthdisturbed CA process of winter wheat, but this effect was genotype-dependent (Pomeroy and Andrews, 1989).
The accumulation of water soluble carbohydrates (WSC) has
been associated with improved freezing tolerance in many grassspecies (Harrison et al., 1997; Patton et al., 2007). Water solublecarbohydrates are accumulated in the leaf and crown tissuesvacuoles (Pollock and Cairns, 1991), and act as osmolites lowering
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6 B. Jurczyk et al. / Environmental a
he crystallization temperature inside cells. Main storage carbo-ydrates in temperate forage grasses are fructans (Pontis, 1989;airns et al., 2002), and their major role is maintaining high photo-ynthesis rate in low temperature conditions by decreasing sucroseccumulation in photosynthetically active cells (Thorsteinssont al., 2002). High photosynthetic activity protects plants fromold-induced photoinhibition of photosynthesis (Huner et al.,993). Freezing tolerance is related to the tolerance against theold-induced photoinhibition of photosynthesis. This is a conse-uence of common mechanisms shared by acclimation to freezingtress and cold-induced photoinhibition of photosynthesis (Rapaczt al., 2004, 2011). Comparative proteomic research published byosmala et al. (2009) revealed that most of the differentially accu-ulated proteins in cold acclimated Festuca pratensis genotypes of
iverse frost tolerance were directly involved in photosynthesis.Festuca pratensis is the most frost-tolerant species within the
olium–Festuca species complex, and may be a possible effectiveonor of frost tolerance alleles for closely related Lolium multiflo-um (Italian ryegrass) (Kosmala et al., 2006). Studies involving F.ratensis (Kosmala et al., 2009; Jurczyk et al., 2012) have shown itsmproved frost tolerance conferred by the CA, and that is why thislant is considered an excellent model for studying CA mechanisms
n grasses.The aim of the present study was to verify the hypothesis
hat low temperature flooding modifies the CA process, leadingo changes in frost tolerance of F. pratensis. We focused on dif-erent aspects of CA: acclimation of the photosynthetic apparatuso cold, changes in WSC concentration, changes in the osmoticotential and induction kinetics of some genes. Three genes of F.ratensis were selected for molecular tests; two of them encodingranscription factors, activating the cold regulated genes expres-ion (CBF6 and LOS2), and one effector gene, encoding a protectiverotein (FpCor14b) (Lee et al., 2002; Chinnusamy et al., 2007;akayama et al., 2007). It has been reported that the induction ofpCor14b, CBF6 and LOS2 genes in F. pratensis is controlled not onlyy temperature, but also by light and the time of day when the
ow-temperature shift occurs. Specific induction of CBF6 expres-ion was detected after first few hours of cold treatment (Jurczykt al., 2012).
. Material and methods
.1. Plant materials and stress treatments
The experiments were performed on the clones of four F. praten-is (Huds.) cv. Skra genotypes, Fp1, Fp8, Fp13 and Fp37, selecteds previously described (Kosmala et al., 2009). Fp1 and Fp37 werehown to be high frost tolerant and Fp8 and Fp13 to be low frostolerant genotypes. The selection was based on: (i) plant’s abilityo regrow after freezing at -8, -11 and −14 ◦C, following CA (4/2 ◦C,0/14 h photoperiod, 200 �mol m−2 s−1 PPFD), and estimated usingarsen’s (Larsen, 1978) visual score, and (ii) plant’s tEL50 (tempera-ure causing a 50% electrolyte leakage) after different periods of CAFlint et al., 1967; Kosmala et al., 2009). The three year old plantsere grown in an open-air vegetation room and at the beginning of
he experiment they were about 17 cm high. The plants were grownn 20 cm diameter pots, and the substrate was a mixture of loamoil: sand: peat (1:1:1; v:v:v). In the autumn they were transferredo an air-conditioned greenhouse and grown at 20 ◦C in daylight,hich was (if necessary) increased to 12 h and supplemented auto-atically on cloudy days to a PAR of 200 �mol m−2 s−1 using Agro
PS lamps (Philips, Brussels, Belgium). Relative humidity in thereenhouse was maintained at about 60%. Water was supplied asequired and plants were fertilized once a week with a half-strengthoagland’s solution.erimental Botany 93 (2013) 45– 54
The experiments were performed in two independent series.Plants were transferred from the greenhouse to controlled environ-ment chambers (+15 ◦C, 12/12 h photoperiod, 200 �mol m−2 s−1
PAR provided by Agro HPS lamps, Philips). Ten clones of each geno-type were divided into two groups (flooded and control). In the caseof the flooded plants the pots were partially filled with tap water toobtain the effect of partial submergence (2 cm above soil level), astate that was maintained until the end of the experiment. After twoweeks the plants flooded at 15 ◦C and control plants were subjectedto CA (21 days at 4/2 ◦C, 10/14 h photoperiod, 200 �mol m−2 s−1
PAR provided by Agro HPS lamps, Philips). The experiment was con-cluded after three weeks. The temperature and light intensity givenabove were measured on the upper surface of the leaves using aPAR/temperature microsensor, an integral part of the FMS2 chloro-phyll fluorescence measuring system (Hansatech, Kings Lynn, UK).
2.2. Frost tolerance
Samples (about 3-cm fragments from the middle part of theyoungest, but fully developed leaf) were collected after 7, 14 and21 days of CA. Frost tolerance was determined as described ear-lier (Kosmala et al., 2009). Leaf fragments were placed into 20 cm3
plastic vials on ice (5 cm3 of frozen deionized water) to ensure icenucleation in conductivity vessels. The total volume of plastic vialswas 20 cm3. The vessels were stored in darkness for 1.5 h at −3, −6,−9, −12, −15 ◦C in a programmed freezer. The temperature waslowered at a rate of 2 ◦C h−1. Freezing temperatures were main-tained for 1.5 h and then the temperature was increased up to 0 ◦Cat a rate of 3 ◦C h−1. After thawing, damage to the leaf tissue wasestimated based on electrical conductance measurements (CC501,Elmetron, Zabrze, Poland). To achieve 100% electrolyte leakage,leaves of the control plants were frozen in liquid nitrogen. The per-centage of electrolyte leakage (%EL) was determined according toFlint et al. (1967) and tEL50 was calculated using linear regressionfitted to the central (linear) part of the relationship between thefreezing temperature (at least three points) and %EL. The resultsare the means of two experimental series (20 replicates for eachfreezing temperature/genotype/treatment).
2.3. Photosynthetic acclimation to cold
Measurements of chlorophyll fluorescence parameters wereperformed before CA and after 7, 14 and 21 days of CA in floodedand control plants, using a pulse amplitude modulation chloro-phyll fluorescence imaging system FluorCAM (PSI, Brno, CzechRepublic). Four leaves from each genotype (flooded and con-trol) were detached, attached to a piece of black paper withpaper adhesive tape, and placed for measurements inside theFluorCAM chamber. Chlorophyll fluorescence induction kineticsand quenching parameters were evaluated at 20 ◦C following anexperimental protocol comprising 20 min of dark adaptation andthe measurements of: F0 (minimal fluorescence intensity mea-sured in the dark-adapted leaves with all PSII reaction centresopened), Fm (maximum fluorescence intensity of the dark-adaptedleaves with all PSII reaction centres closed) after a light saturat-ing pulse of about 2000 �mol m−2 s−1, Fs (steady state fluorescencein the actinic radiation-exposed leaves) after 400 s of actiniclight exposure (150 �mol m−2 s−1) combined with saturating lightpulses every 25 s, Fd (fluorescence decrease), Fm′ (maximum flu-orescence intensity with all PSII reaction centres closed in thelight-adapted leaves) during the last saturating pulse and F0′ (min-imal fluorescence intensity with all PSII reaction centres opened
in the light-adapted leaves) measured with actinic light sourceswitched off after a far-red light pulse. Variable fluorescence(Fv) was calculated as Fm − F0. Photochemical quenching coeffi-cient (qP) was calculated according to Schreiber et al. (1994) as
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B. Jurczyk et al. / Environmental a
P = (Fm′ − Fs)/(Fm′ − F0′ ). Non-photochemical quenching (NPQ) wasalculated as NPQ = (Fm − Fm′ )/Fm′ (Bilger and Bjorkman, 1991).pparent quantum yield of PSII was calculated as Fv/Fm. The resultsre the means of two experiments (12 replicates in total).
.4. Osmotic potential and WSC in leaves
Osmotic potential was measured in the cell sap before CA andfter 7, 14 and 21 days of CA in both the flooded and control plants.eaf samples (about 5 cm fragments of the middle part of a fullyeveloped leaf without any signs of ageing) were put into syringesn paper discs and frozen in liquid nitrogen. After thawing the cellap was squeezed onto the paper discs with the syringe piston.he osmotic potential (�0) was measured using a dewpoint micro-oltometer (HR 33T, Vescor, Logan, UT) supplied with C-52 Samplehamber. Samples for WSC analysis (0.25 g from the middle part of
fully developed leaf without any signs of ageing) were collectedrom plants before CA and after 6, 13 and 20 days of CA from bothhe flooded and control plants. WSC leaf levels were determined byhe anthrone method (Ashwell, 1975). The results are the means ofwo experiments (8 and 10 replicates in total for osmotic potentialnd WSC, respectively).
.5. Analysis of transcript levels for CBF6, Cor14b and LOS2 genes
Samples (about 0.04 g from the middle part of the youngest, butully developed leaf) were collected from the Fp1, Fp8, Fp13 andp37 plants after 1, 2, 4 and 6 h of CA and on the 21st day of CA (1,, 4 and 6 h after dawn) from the flooded and control plants. Afterollection the samples were immediately frozen in liquid nitrogennd stored at -80 ◦C. The RNeasy Plant Mini Kit (Qiagen, Hilden,ermany) was used for RNA purification. The purified RNA sam-le was incubated with gDNA Wipeout Buffer to remove genomicNA contamination (included in the QuantiTect Reverse Transcrip-
ion Kit, Qiagen). After that, the RNA was subjected to reverseranscription reaction, using a QuantiTect Reverse Transcriptionit. The final concentration and quality of cDNA were determinedpectrophotometrically (Ultrospec 2100 Pro, supplied with ultra-icrovolume cell, Amersham Biosciences, Buckinghamshire, UK).uantitative PCR analysis was performed using 7500 real-time PCR
ystem (Applied Biosystems, Foster City, CA, USA) as described byurczyk et al. (2012). We used the Actin gene as a reference geneo normalize the amount of cDNA added to each PCR (An et al.,996). Primer sequences and probes were designed using Primerxpress software version 3.0 on the basis of appropriate F. praten-is sequences: Actin EST (the amplicon sequence was checked andonfirmed), the sequence of LOS2 gene (unpublished, provided by
r H. Rudi, Norwegian University of Life Sciences, the ampliconequence was checked and confirmed), the sequence of the CBF6ene and the Cor14b gene. Sequences of primers, probes and originf the sequences are given in Table 1. All primers and probes wereable 1equences of primers and probes.
Gene Primers and probes Sequence (5′->3′)
CBF6 forward CTTCGCAGAACGAreverse GGTCCCATCCCATspecific probe FAM-CGTTCGAGC
Cor14b forward AGACCCAGATCGAreverse GCACGGCCTGGGAspecific probe FAM-TCGGAGGAG
LOS2 forward AGATCGTAGGAGAreverse TGCAGGTCTTCTCspecific probe FAM-CCCCACAGG
Actin forward GTCGAGGGCAACAreverse CCAGTGCTGAGCGspecific probe FAM-TTCTCCTTGA
erimental Botany 93 (2013) 45– 54 47
supplied by Applied Biosystems. Data were analyzed using 7500real-time PCR Sequence Detection Software version 1.3 (AppliedBiosystems). We used the relative standard curve method (AppliedBiosystems) to estimate relative gene expression. The results basedon six biological replicates (3 from each experiment) are presentedas a fold-change in the expression of a particular gene in treatedsamples with Actin as the endogenous control (reference) gene.
2.6. Statistical calculations
The tEL50 values were estimated from a linear regression fit-ted to the central (linear) part of the sigmoid relationship betweenthe freezing temperature and the electrolyte leakage, using at leastthree temperatures together with coefficient intervals for P < 0.05(Statistica 10.0, Statsoft, Tulsa, OK).
General influence of different factors on chlorophyll fluores-cence parameters, WSC concentration and osmotic potential wastested using the GLM module of Statistica 10.0 (Statsoft, Tulsa, OK).In the case of general effects studied in time course during the pro-gressive cold acclimation different lengths of CA were analyzed asrepeated measurements.
Statistical significance of the differences between the meanswas tested with Tukey’s HSD test and homogeneity groups wereindicated for P < 0.05.
Standard error for relative gene expression was calculated usingApplied Biosystems 7500 System Sequence Detection Software ver-sion 1.3. Wilcoxon paired difference test (Statistica 10.0) based onbiological replications, was used for assessing statistical signifi-cance (P < 0.05) of normalized gene expression from the floodedand non-flooded plants (Yuan et al., 2006).
3. Results
3.1. Frost tolerance
The effect of flooding on freezing tolerance depended on geno-type and CA period, but all the observed effects were positive(Fig. 1). No influence was noticed for Fp1, the genotype with highfrost tolerance, while for the second high frost tolerant genotype,Fp37, the positive effect of flooding was perceived only on the sev-enth day of CA. For the flooded low frost tolerant Fp13 genotype,tEL50 was by 3.2 ◦C and 2.8 ◦C lower than in the control plants after14 and 21 days of CA, respectively, and in the second low frost tol-erant genotype, Fp8, after 14 days of CA flooding decreased tEL50 by1.1 ◦C.
3.2. Changes in chlorophyll fluorescence parameters
Low temperature flooding affected also the pattern of changesobserved in the photosynthetic apparatus during CA (Fig. 2).Reduced apparent quantum yield of PSII (Fv/Fm) during CA was
Source
CAATTCG DQ996012.1ATCACTGATGGAAGT-MGBTGGCTTCT AJ512944.1AGAGGCGCG-MGBTGACCTTCTTGT not published, provided by dr H. Rudi
ACTGATTGCGTTGCCA-MGBTATGCAA GO859520.1GGAAATTGTCACGGAC-MGB
48 B. Jurczyk et al. / Environmental and Experimental Botany 93 (2013) 45– 54
Fig. 1. The effect of low temperature flooding on frost tolerance of F. pratensis. Two weeks before cold acclimation 5 clones of each genotype were flooded to 2 cm abovesoil level and maintained in this state until the end of the experiment. Cold acclimation lasted for 21 days in a controlled environment (4/2 ◦C, 10/14 h photoperiod,2 −2 −1
aa
Fflp(
00 �mol m s PAR, HPS lamps Agro, Philips). tEL50 means the temperature at which 5nd the coefficient intervals for P = 0.05 were calculated on the basis of freezing tests mare higher frost-tolerant (HFT) and Fp8 and Fp13 are lower frost-tolerant (LFT) genotypes
ig. 2. Changes in the apparent quantum yield of PSII (Fv/Fm) during cold acclimation oooded to 2 cm above soil level, and maintained in this state until the end of the experimehotoperiod, 200 �mol m−2 s−1 PAR, HPS lamps Agro, Philips). Fp1 and Fp37 are higheKosmala et al., 2009). The data represent the means from 12 replicates ± standard error.
0% of the total electrolytes were released from the leaf tissues. The values of tEL50
de in two replicates at five temperatures (−3, −6, −9, −12, −15 ◦C). Fp1 and Fp37 (Kosmala et al., 2009).
f F. pratensis. Two weeks before cold acclimation 5 clones of each genotype werent. Cold acclimation lasted for 21 days in a controlled environment (4/2 ◦C, 10/14 h
r frost-tolerant (HFT) and Fp8 and Fp13 are lower frost-tolerant (LFT) genotypesHomogeneity groups for P < 0.05 (Tukey’s HSD test) were indicated with letters.
nd Exp
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B. Jurczyk et al. / Environmental a
bserved in lower frost tolerant genotypes (Fp8, Fp13), irrespec-ive of flooding (P < 0.000001 and P = 0.000023, respectively). For
ore frost tolerant Fp37 genotype a drop in Fv/Fm ratio during CAas seen in the control plants, while in the flooded plants only
transitory decrease in Fv/Fm ratio was observed. No influence of
ooding on Fv/Fm ratio was detected in high frost tolerant genotypep1 (Fig. 2).The effects of low temperature flooding were also noticed forhotochemical and non-photochemical chlorophyll fluorescence
ig. 3. Changes in the chlorophyll fluorescence quenching parameters: non-photochemicaold acclimation of F. pratensis. Two weeks before cold acclimation 5 clones of each genohe experiment. Cold acclimation lasted for 21 days in a controlled environment (4/2 ◦C, 1re higher frost-tolerant (HFT) and Fp8 and Fp13 are lower frost-tolerant (LFT) genotypesrror. Homogeneity groups for P < 0.05 (Tukey’s HSD test) were indicated with letters.
erimental Botany 93 (2013) 45– 54 49
quenching coefficients (Fig. 3). NPQ decreased during the first weekof CA, and then recovered up to or above the initial level at the endof the experiment. Flooding declined the rate of this recovery in allgenotypes, but not its efficiency, and in the case of the most frosttolerant Fp37 the highest NPQ value was observed in flooded plants
after 21 days of CA. On the other hand, flooding reduced the rise inqP observed in Fp37 at the end of the experiment. Additionally, afterseven days of CA, qP values noticed in the flooded plants were lowerthan in the control plants of Fp8, Fp13 and Fp37 genotypes.l quenching coefficient (NPQ) and photochemical quenching coefficient (qP) duringtype were flooded to 2 cm above soil level, and maintained in this state the end of0/14 h photoperiod, 200 �mol m−2 s−1 PAR, HPS lamps Agro, Philips). Fp1 and Fp37
(Kosmala et al., 2009). The data represent the means from 12 replicates ± standard
50 B. Jurczyk et al. / Environmental and Experimental Botany 93 (2013) 45– 54
Fig. 4. Changes in leaf water soluble carbohydrates and cell sap osmotic potential during cold acclimation of F. pratensis. Two weeks before cold acclimation 5 clones of eachgenotype were flooded to 2 cm above soil level, and maintained in this state until the end of the experiment. Cold acclimation lasted for 21 days in a controlled environment( ◦ −2 −1
and
g icatesH
3p
mabdtt
4/2 C, 10/14 h photoperiod, 200 �mol m s PAR, HPS lamps Agro, Philips). Fp1enotypes (Kosmala et al., 2009). The data represent the means from 8 and 10 replomogeneity groups for P < 0.05 (Tukey’s HSD test) were indicated with letters.
.3. Changes in the WSC leaf concentration and cell sap osmoticotential
Two weeks of flooding at 15 ◦C before CA lowered WSC accu-ulation in leaves by eight times (P = 0.00014) (Fig. 4). WSC
ccumulation in the flooded plants during CA was 36% higher than
efore CA (P = 0.020895). On the other hand CA under control con-itions resulted in more than double drop in WSC (P = 0.00216). Onhe 21st day of CA the WSC level was higher in the leaves of allhe flooded genotypes (Fig. 4). The highest (over 21 times) growthFp37 are more frost-tolerant (HFT) and Fp8 and Fp13 are less frost-tolerant (LFT) in the case of water soluble carbohydrates and osmotic potential ± standard error.
in WSC content during CA was observed in flooded Fp13 plants, inwhich freezing tolerance was also most increased by flooding.
The changes of the leaf osmotic potential differed from thoseobserved for soluble carbohydrates (Fig. 4). In plants cold-acclimated under control conditions the osmotic potential wasdiminished during the first and the second week of CA in Fp1 and
Fp37 genotypes, respectively, and then increased again. A slightgrowth of the osmotic potential was seen in control Fp8 plantsduring the whole CA. No changes in the osmotic potential wereobserved in control Fp13 plants. In genotypes characterized by
B. Jurczyk et al. / Environmental and Experimental Botany 93 (2013) 45– 54 51
Fig. 5. Changes in the relative expression of the CBF6 gene during cold acclimation (4 ◦C) of F. pratensis. The expression level was calculated using Actin as a reference gene. Twoweeks before cold acclimation 5 clones of each genotype were flooded to 2 cm above soil level, and maintained in this state until the end of the experiment. Cold acclimationlasted for 21 days in a controlled environment (4/2 ◦C, 10/14 h photoperiod, 200 �mol m−2 s−1 PAR, HPS lamps Agro, Philips). Fp1 and Fp37 are higher frost-tolerant (HFT)a e datd
lpwa
3
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nd Fp8 and Fp13 are lower frost-tolerant (LFT) genotypes (Kosmala et al., 2009). Thifferent for the flooded and control plants (Wilcoxon two group test, P = 0.05).
ower freezing tolerance, flooding boosted the values of osmoticotential both before (P = 0.000008) and during CA (P = 0.000012),hereas in more tolerant Fp1 and, especially Fp37 genotype, such
rise was visible only during CA (P < 0.000001).
.4. Induction kinetics of CBF6, FpCor14b and LOS2 genes
The expression of the CBF6 gene was cold induced during therst hours of CA (Fig. 5). Flooding increased the CBF6 transcriptccumulation in flooded Fp1, Fp8 and Fp37 plants (Fig. 5). Duringhe first day of CA it was visible after 2 h of CA in Fp37, after 2 and
h in Fp8 and after 4 h in Fp1. On the 21st day of CA the morn-ng induction of CBF6 was not observed with the exception of theooded Fp37. In this case the relative CBF6 expression was alsoigher when compared to non-flooded plants. Moreover, flooding
ntensified CBF6 relative expression in Fp1 6 h after dawn (Fig. 5).A decrease in the LOS2 transcript level triggered by low tem-
erature flooding was observed in Fp37 and in Fp8 plants after h of CA and in Fp1 plants after 2 h of CA (Fig. 6). Some flooding-
nduced changes in the LOS2 transcript level were also seen duringhe 21st day of CA. Increased LOS2 expression was detected inp37 genotype 1, 2 and 4 h after dawn and in Fp8 genotype 2 and
h after dawn. On the other hand, reduced LOS2 expression wasbserved in Fp8 and Fp13 plants 1 and 2 h after dawn, respectivelyFig. 6).
During the first day of CA flooding limited the expression ofpCOR14b after 4 and 6 h of CA in the Fp1 and Fp13 genotypesnd also after 6 h of CA in Fp8 and Fp37 plants (Fig. 7). During the1st day of CA, flooded plants with higher frost tolerance showed
levated FpCOR14b expression when compared to the non-floodedontrol 2 and 4 (Fp1) and 4 and 6 (Fp37) hours after dawn, whereasn less frost tolerant Fp8 and Fp13 genotypes the expression of thisene was higher in flooded plants only at the beginning of the day.a represent the means from 6 replicates ± standard error. Values with asterisks are
4. Discussion
Andrews and Pomeroy (1981) proved that low temperatureflooding reduced cold hardiness in fully submerged wheat and bar-ley plants. The authors also reported that wheat cultivars continuedtheir cold hardening during the less severe flooding treatments, butat a reduced rate. Our results indicate that low temperature flood-ing of the roots and crowns (2 cm above the soil level) may improvefrost tolerance of F. pratensis. However, it is hard to determinethe common mechanism of this phenomenon. In general, flood-ing increased cold-induced WSC accumulation in the leaves, butthe very high WSC concentration following two and three weeksof CA may be related to elevated frost tolerance observed dur-ing low temperature flooding only in Fp13 plants. These plantsshowed both the highest raise in WSC concentration when com-pared to the control, and the highest increase in freezing tolerance.Similar findings were reported by Bertrand et al. (2003), whoclaimed that higher carbohydrate reserves under oxygen deficiencyin timothy favoured its winter survival and spring regrowth. Theysuggested that this was due to carbohydrate import from the rootsto the shoot bases. Different patterns of carbohydrate accumu-lation in the leaves of flooded and control plants found in ourstudy may suggest that WSC drop in control plants is caused byWSC translocation from leaves to other plant organs. Moreover,the elevated WSC concentration in leaves of flooded F. praten-sis plants during CA was not accompanied by reduced osmoticpotential, which may suggest either a higher rate of WSC poly-merization to high molecular weight fructans (Dionne et al., 2001)or that the increase in WSC concentration was associated with
a decrease in other than sugars osmolyte(s). This may also sug-gest that soluble sugars are accumulated mainly in the apoplastand their main function is membrane protection (Valluru et al.,2008).
52 B. Jurczyk et al. / Environmental and Experimental Botany 93 (2013) 45– 54
Fig. 6. Changes in the relative expression of the LOS2 gene during cold acclimation (4 ◦C) of F. pratensis. The expression level was calculated using Actin as a reference gene. Twoweeks before cold acclimation 5 clones of each genotype were flooded to 2 cm above soil level, and maintained in this state until the end of the experiment. Cold acclimationlasted for 21 days in a controlled environment (4/2 ◦C, 10/14 h photoperiod, 200 �mol m−2 s−1 PAR, HPS lamps Agro, Philips). Fp1 and Fp37 are higher frost-tolerant (HFT)and Fp8 and Fp13 are lower frost-tolerant (LFT) genotypes (Kosmala et al., 2009). The data represent the means from 6 replicates ± standard error. Values with asterisks aredifferent for the flooded and control plants (Wilcoxon two group test, P = 0.05).
Fig. 7. Changes in the relative expression of the Cor14b gene during cold acclimation (4 ◦C) of F. pratensis. The expression level was calculated using Actin as a referencegene. Two weeks before cold acclimation 5 clones of each genotype were flooded to 2 cm above soil level, and maintained in this state until the end of the experiment.Cold acclimation lasted for 21 days in a controlled environment (4/2 ◦C, 10/14 h photoperiod, 200 �mol m−2 s−1 PAR, HPS lamps Agro, Philips). Fp1 and Fp37 are higherfrost-tolerant (HFT) and Fp8 and Fp13 are lower frost-tolerant (LFT) genotypes (Kosmala et al., 2009). The data represent the means from 6 replicates ± standard error. Valueswith asterisks are different for the flooded and control plants (Wilcoxon two group test, P = 0.05).
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Flooding decreased photoinhibitory damage in high frost tol-rant Fp1 and Fp37 plants. Freezing tolerance seems to belosely related to the tolerance to cold-induced photoinhibitionf photosynthesis, which is a consequence of the acclimationechanisms common to both stresses (Rapacz et al., 2007).
he increase in non-photochemical quenching, coupled with aecrease in photochemical quenching in flooded Fp37 plants athe end of CA, suggests a higher rate of excess light energyissipation. This strategy may result in photoinhibition avoid-nce and improved photosynthetic efficiency observed under lowemperature flooding. The non-photochemical mechanism of pho-osynthetic acclimation to cold was shown to be a major protective
echanism induced during CA of F. pratensis (Humphreys et al.,007). Our results suggest that this mechanism is activated alsonder low temperature flooding, although the activation seems toe unrelated to changes in the frost tolerance. Reduced photoin-ibitory damage observed in freezing tolerant Fp1 and Fp37 plantsfter 21 days of low temperature flooding may also be connectedith higher COR14b transcript level. This appears to confirm the
esults obtained by Rapacz et al. (2008), who claimed that the levelf COR14b expression in barley was related to the differences in theolerance of cold-induced photoinhibition of photosynthesis, butot to plant frost tolerance.
On the other hand, the reduction in apparent quantum yieldf PSII (Fv/Fm) at the end of experiment was seen in flooded Fp13lants, suggesting increased photoinhibition. This can be explainedy high WSC concentration noticed in this genotype, which couldownregulate the rate of photosynthetic carbon reaction.
A transient boost in freezing tolerance observed in Fp8 and Fp37lants after 2 and 1 week of CA under flooding, respectively, wasonsistent with the changes in the induction kinetics of CBF6. Ele-ated CBF6 transcript level was detected in flooded Fp8 and Fp37lants after the second hour of CA, under specific cold inductiononditions (Gilmour et al., 1998; Jurczyk et al., 2012), as com-ared to the control. Similar increase was not detected in thep1 genotype, in which low-temperature flooding did not changehe freezing tolerance. In Arabidopsis more than 40 target genesctivated by cold stress and acting downstream to CBF-family tran-cription factors have been identified (Seki et al., 2002; Fowler andhomashow, 2002; Maruyama et al., 2004). Therefore we specu-ate that intensified expression of CBF6 in flooded Fp8 and Fp37lants at the beginning of CA may trigger further downstream genexpression, contributing to the observed transient increase in frostolerance. We also noticed raised CBF6 expression during the 21stay of CA, in the first and second hour after dawn in Fp37 geno-ype, and in the sixth hour after dawn in Fp1 genotype. Theseesults may indicate that under low temperature flooding the F.ratensis CBFs act not only at the beginning of the CA process,ut might also regulate the expression of downstream genes aftereveral weeks of low temperature treatment. However, the CBF6nduction at the sixth hour of the day may cause different effects,ecause the expression of genes acting downstream of CBFs mayndergo circadian changes (Bieniawska et al., 2008; Jurczyk et al.,012).
The CBF-targeted genes are induced under indirect control ofOS2/enolase. CBFs activate transcriptional repressor STZ/ZAT10hich is repressed by LOS2 (Lee et al., 2002; Chinnusamy et al.,
007), so that CBFs and LOS2 act synergistically in the control ofold-regulated genes. After six hours of CA (on the first day of CA)educed LOS2 transcript level was detected in flooded against con-rol Fp8 and Fp37 plants. Elevated CBF6 transcript level observed inhe same genotypes at the beginning of the day and a concomitant
ecrease in LOS2 transcript level suggest that during the first hoursf CA under flooding CBF6 and LOS2 act together in configuring lowemperature flooding transcriptome, which may contribute to theransient improvement in frost tolerance observed for Fp8 and Fp37erimental Botany 93 (2013) 45– 54 53
genotypes. Lal et al. (1991) reported raised transcript level of theenolase gene in maize roots after 24 h of anaerobic stress. On the21st day of CA heightened LOS2 expression triggered by low tem-perature flooding was observed in Fp37 plants 1, 2 and 4 h afterdawn, and in Fp8 plants 2 and 6 h after dawn. Considering the aboveit seems that, following prolonged cold treatment, LOS2 may raisethe CBF6 transcript accumulation upregulated by low-temperatureflooding.
Summing up the study results it may be concluded thatimproved frost tolerance observed in F. pratensis exposed to flood-ing stress before and during cold acclimation may be triggeredby different mechanisms. Transient growth in frost tolerance inflooded Fp8 and Fp37 genotypes correspond to the induction kinet-ics of the CBF6 gene. A significant and stable increase in frosttolerance observed in Fp13 plants was related to enhanced watersoluble carbohydrates accumulation. Additionally, low tempera-ture flooding may strengthen the resistance to low temperatureinduced photoinhibition of photosynthesis.
We intend to continue our investigation of plant responses tolow temperature flooding. Our long term objective is to elucidatethe mechanisms of forage grasses adaptation to this condition.Further work to identify regulatory components involved in lowtemperature flooding, as well as a comprehensive transcriptomicand proteomic research seem to be a promising research fields.
Acknowledgements
This study was supported by the University of Agriculture inKrakow, grant DS3113. The authors thank Professor Neil Jones fromAberystwyth University UK, for critical reading and correction ofthe manuscript. Thank also dr H. Rudi, Norwegian University of LifeSciences, for providing the sequence of LOS2 gene of F. pratensis.
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