elevated temperature and co stimulate late-season · in the northern hemisphere (ipcc, 2014), which...

Elevated Temperature and CO2 Stimulate Late-SeasonPhotosynthesis But Impair Cold Hardening in Pine[OPEN]

Christine Y. Chang, Emmanuelle Fréchette, Faride Unda, Shawn D. Mansfield, and Ingo Ensminger*

Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada L5L 1C6 (C.Y.C.,E.F., I.E.); Graduate Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, CanadaM5S 3G5 (C.Y.C., E.F., I.E.); Department of Wood Science, University of British Columbia, Vancouver, BritishColumbia, Canada V6T 1Z4 (F.U., S.D.M.); and Graduate Department of Ecology and Evolutionary Biology,University of Toronto, Toronto, Ontario, Canada M5S 3B2 (I.E.)

ORCID IDs: 0000-0001-6609-3527 (C.Y.C.); 0000-0002-2588-7942 (E.F.); 0000-0003-0842-8741 (F.U.); 0000-0002-0175-554X (S.D.M.);0000-0001-9014-2499 (I.E.).

Rising global temperature and CO2 levels may sustain late-season net photosynthesis of evergreen conifers but could also impairthe development of cold hardiness. Our study investigated how elevated temperature, and the combination of elevatedtemperature with elevated CO2, affected photosynthetic rates, leaf carbohydrates, freezing tolerance, and proteins involved inphotosynthesis and cold hardening in Eastern white pine (Pinus strobus). We designed an experiment where control seedlingswere acclimated to long photoperiod (day/night 14/10 h), warm temperature (22°C/15°C), and either ambient (400 mL L21) orelevated (800 mmol mol21) CO2, and then shifted seedlings to growth conditions with short photoperiod (8/16 h) and lowtemperature/ambient CO2 (LTAC), elevated temperature/ambient CO2 (ETAC), or elevated temperature/elevated CO2 (ETEC).Exposure to LTAC induced down-regulation of photosynthesis, development of sustained nonphotochemical quenching,accumulation of soluble carbohydrates, expression of a 16-kD dehydrin absent under long photoperiod, and increasedfreezing tolerance. In ETAC seedlings, photosynthesis was not down-regulated, while accumulation of soluble carbohydrates,dehydrin expression, and freezing tolerance were impaired. ETEC seedlings revealed increased photosynthesis and improvedwater use efficiency but impaired dehydrin expression and freezing tolerance similar to ETAC seedlings. Sixteen-kilodaltondehydrin expression strongly correlated with increases in freezing tolerance, suggesting its involvement in the development ofcold hardiness in P. strobus. Our findings suggest that exposure to elevated temperature and CO2 during autumn can delaydown-regulation of photosynthesis and stimulate late-season net photosynthesis in P. strobus seedlings. However, this comes atthe cost of impaired freezing tolerance. Elevated temperature and CO2 also impaired freezing tolerance. However, unless thefrequency and timing of extreme low-temperature events changes, this is unlikely to increase risk of freezing damage in P.strobus seedlings.

Land surface temperature is increasing, particularlyin the northern hemisphere (IPCC, 2014), which isdominated by boreal and temperate forests. At higherlatitudes, trees rely on temperature and photoperiodcues to detect changing seasons and to trigger cessationof growth and cold hardening during the autumn(Ensminger et al., 2015). For boreal and temperate ev-ergreen conifers, cold hardening involves changes incarbohydrate metabolism, down-regulation of photo-synthesis, accumulation of cryoprotective metabolites,

and development of freezing tolerance (Crosatti et al.,2013; Ensminger et al., 2015). These processes minimizefreezing damage and enable conifers to endure winterstresses. However, rising temperatures result in asyn-chronous phasing of temperature and photoperiodcharacterized bydelayed arrival offirst frosts (McMahonet al., 2010), which may impact the onset and develop-ment of cold hardening during autumn.

Short photoperiod induces the cessation of growth inmany tree species (Downs and Borthwick, 1956; Heide,1974; Repo et al., 2000; Böhlenius et al., 2006). As aconsequence, carbon demand in sink tissue decreasestoward the end of the growing season, and the bulk ofphotoassimilate is translocated from source tissues tostorage tissues (Hansen and Beck, 1994; Oleksyn et al.,2000). In addition, cryoprotective soluble sugars, in-cluding sucrose, raffinose, and pinitol, accumulate inleaf tissues to enhance freezing tolerance (Strimbecket al., 2008; Angelcheva et al., 2014). Thus, by winter,leaf nonstructural carbohydrates are mainly comprisedof mono- and oligosaccharides, and onlyminimal levelsof starch remain (Hansen and Beck, 1994; Strimbecket al., 2008). The concurrent decrease of photoassimilate

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ingo Ensminger ([email protected]).

C.Y.C. and I.E. designed the study; C.Y.C. and E.F. performedmeasurements and samplings; C.Y.C. analyzed pigments; F.U. ana-lyzed carbohydrates with input from S.D.M; C.Y.C. analyzed thedata; C.Y.C. and I.E. wrote the manuscript; all authors reviewedthe final manuscript.

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http://orcid.org/0000-0001-6609-3527

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http://orcid.org/0000-0003-0842-8741

http://orcid.org/0000-0002-0175-554X

http://orcid.org/0000-0001-9014-2499

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and demand for metabolites that occur during the ces-sation of growth also impacts the citric acid cycle thatmediates between photosynthesis, respiration, andprotein synthesis. The citric acid cycle generates NADHto fuel ATP synthesis via mitochondrial electrontransport, as well as amino acid precursors (Shi et al.,2015). In C3 plants, the enzyme phosphoenolpyruvatecarboxylase (PEPC) converts phosphoenolpyruvate tooxaloacetic acid in order to supplement the flow ofmetabolites to the citric acid cycle and thus controls theregulation of respiration and photosynthate partition-ing (O’Leary et al., 2011).Cessation of growth, low temperature, and presum-

ably short photoperiod decrease the metabolic sink forphotoassimilates, resulting in harmful excess light en-ergy (Öquist and Huner, 2003; Ensminger et al., 2006)and increased generation of reactive oxygen species(Adams et al., 2004). During autumn and the develop-ment of cold hardiness, conifers reconfigure the pho-tosynthetic apparatus in order to avoid formation ofexcess light and reactive oxygen species. This involves adecrease in chlorophylls and PSII reaction center coreprotein D1 (Ottander et al., 1995; Ensminger et al., 2004;Verhoeven et al., 2009), as well as aggregation of light-harvesting complex proteins (Ottander et al., 1995;Busch et al., 2007). Additionally, photoprotective ca-rotenoid pigments accumulate in leaves, especially thexanthophylls, zeaxanthin, and lutein that contribute tononphotochemical quenching (NPQ) via thermal dis-sipation of excess light energy (Busch et al., 2007;Verhoeven et al., 2009; Demmig-Adams et al., 2012).Prolonged exposure to low temperature induces sus-tained nonphotochemical quenching (NPQS), wherezeaxanthin constitutively dissipates excess light energy(Ensminger et al., 2004; Demmig-Adams et al., 2012;Fréchette et al., 2015).In conifers, freezing tolerance is initiated during early

autumn in response to decreasing photoperiod (Rostadet al., 2006; Chang et al., 2015) and continues to developthrough late autumn in response to the combination ofshort photoperiod and low temperature (Strimbeck andSchaberg, 2009; Chang et al., 2015). In addition tochanges in carbohydrate content, freezing tolerancealso involves the expression of specific dehydrins(Close, 1997; Kjellsen et al., 2013). Members of thedehydrin protein family are involved in responses toosmotic, salt, and freezing stress (Close, 1996). Dehy-drins have been associated with improved freezingtolerance in many species including spinach (Kayeet al., 1998), strawberry (Houde et al., 2004), cucumber(Yin et al., 2006), peach (Wisniewski et al., 1999), birch(Puhakainen et al., 2004), and spruce (Kjellsen et al.,2013). In angiosperms, a characteristic Lys-rich dehy-drinmotif known as the K-segment interacts with lipidsto facilitate membrane binding (Koag et al., 2003;Eriksson et al., 2011). Several in vitro studies havedemonstrated dehydrin functions including preventionof aggregation and unfolding of enzymes (using Vitisriparia; Hughes and Graether, 2011), radical scavenging(using Citrus unshiu; Hara et al., 2004), and suppression

of ice crystal formation (usingPrunus persica;Wisniewskiet al., 1999). To date, dehydrin functions have not beendemonstrated in planta.

Rising temperatures since the mid-twentieth centuryhave delayed the onset of autumn dormancy and in-creased length of the growing season in forests acrossthe northern hemisphere (Boisvenue and Running,2006; Piao et al., 2007; McMahon et al., 2010). Studieshave shown that elevated temperatures ranging from+4°C to +20°C above ambient can delay down-regulation of photosynthesis in several evergreen co-nifers. Consistent findings were apparent amongclimate-controlled chamber studies exposing Pinusstrobus seedlings to a sudden shift in temperature and/or photoperiod (Fréchette et al., 2016), as well aschamber studies exposing Picea abies seedlings to sim-ulated autumn conditions using a gradient of decreas-ing temperature and photoperiod (Stinziano et al.,2015). Similar findingswere also demonstrated in open-top chamber experiments exposing mature Pinus syl-vestris to a gradient of decreasing temperature andnatural photoperiod (Wang, 1996). Elevated tempera-ture (+4°C above ambient) also impaired cold harden-ing in Pseudotsuga menziesii seedlings (Guak et al., 1998)and mature P. sylvestris (Repo et al., 1996) exposed to adecreasing gradient of temperature and natural pho-toperiod using open-top chambers. In contrast, a recentstudy showed that smaller temperature increments(+1.5°C to +3°C) applied using infrared heaters did notdelay down-regulation of photosynthesis or impairfreezing tolerance in field-grown P. strobus seedlingsthat were acclimated to larger diurnal and seasonaltemperature variations (Chang et al., 2015). For manytree species, photoperiod determines cessation ofgrowth (Tanino et al., 2010; Petterle et al., 2013), lengthof the growing season (Bauerle et al., 2012), and de-velopment of cold hardiness (Welling et al., 1997; Liet al., 2003; Rostad et al., 2006). However, the effects ofclimate warming on tree phenology are complex andcan be unpredictable due to species- and provenance-specific differences in sensitivity to photoperiod andtemperature cues (Körner and Basler, 2010; Basler andKörner, 2012; Basler and Körner, 2014).

The effect of elevated CO2 further increases uncer-tainties in the response of trees to warmer climate.Similar to warmer temperature, elevated CO2 may alsodelay the down-regulation of photosynthesis in ever-greens and extend the length of the growing season, asdemonstrated in mature P. sylvestris (Wang, 1996). El-evated CO2 increases carbon assimilation (Curtis andWang, 1998; Ainsworth and Long, 2005) and biomassproduction (Ainsworth and Long, 2005) during thegrowing season. The effects could continue during theautumn if dormancy or growth cessation is delayed,which suggests that elevated CO2 may increase annualcarbon uptake. However, long-term exposure to ele-vated CO2 can also down-regulate photosynthesisduring the growing season (Ainsworth and Long,2005). Prior studies that have attempted to determinethe impact of a combination of elevated CO2 and/or

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Elevated Temperature and CO2 Impair Cold Hardening

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temperature on cold hardening in evergreens havelargely focused on freezing tolerance, with contrastingresults. Open-top chamber experiments showed that acombination of elevated temperature and CO2 bothdelayed and impaired freezing tolerance of P. menziesiiseedlings (Guak et al., 1998) and evergreen broadleafEucalyptus pauciflora seedlings (Loveys et al., 2006) butdid not affect freezing tolerance of mature P. sylvestris(Repo et al., 1996). A recent field experiment examiningmature trees revealed that Larix decidua, but not Pinusmugo, exhibited enhanced freezing damage followingsix years of exposure to combined soil warming andelevated CO2 (Rixen et al., 2012). In contrast, a climate-controlled study showed that exposure to elevated CO2advanced the date of bud set and improved freezingtolerance in Picea mariana seedlings (Bigras and Bertrand,2006). In a second study on similar seedlings conductedby the same authors, exposure of trees to elevated CO2also enhanced freezing tolerance but impaired the ac-cumulation of sucrose and raffinose (Bertrand andBigras, 2006). These previous experiments used exper-imental conditions where temperature and photope-riod gradually decreased. While this approach aims tomimic natural conditions, it is difficult to distinguishspecific responses to either photoperiod or tempera-ture. Because of the contrasting findings from previousstudies, we designed an experiment aiming to separatethe effects of photoperiod, temperature, and CO2 on awide range of parameters that are involved in coldhardening in conifers.

Our study aimed to determine (1) how induction anddevelopment of the cold hardening process is affectedby a shift from long to short photoperiod under warmconditions and (2) how the combination of warm airtemperature and elevated CO2 affects photoperiod-induced cold hardening processes in Eastern whitepine (P. strobus). To assess the development of coldhardening, we measured photosynthetic rates, changesin leaf carbohydrates, freezing tolerance, and proteinsinvolved in photosynthesis and cold hardening over36 d. Assuming that both low temperature and shortphotoperiod cues are required to induce cold hardeningin conifers, we hypothesized that warm temperatureand the combination of warm temperature and ele-vated CO2 would prevent seedlings growing underautumn photoperiod from down-regulating photo-synthesis. We further hypothesized that warm tem-perature and the combination of warm temperatureand elevated CO2 would impair the development offreezing tolerance, due to a lack of adequate phasing ofthe low temperature and short photoperiod signals.

RESULTS

Photosynthetic Gas Exchange

Under long photoperiod, net photosynthetic CO2assimilation (Anet), measured at growth temperature,was approximately 20% higher in seedlings grown at

elevated CO2 compared to seedlings grown at ambientCO2 (Fig. 1A, day 0). After transfer to short photoperiodtreatments, Anet decreased in the low temperature/ambient CO2 (LTAC) seedlings by 50% from day 0 today 1 and remained significantly lower than seedlingstransferred to elevated temperature/ambient CO2(ETAC) or elevated temperature/elevated CO2 (ETEC)conditions for the entire experiment (Table I). In com-parison, seedlings transferred to ETAC retained ratherconstant Anet throughout the experiment. Seedlingstransferred to ETEC performed significantly higher Anet(20%–30%) than seedlings grown under ETAC through-out the experiment, except for a brief decrease in Anet thatoccurred on days 1 and 4 of treatment.

At day 0, stomatal conductance (gs; Fig. 1B) andrespiration (Rd; Fig. 1C) did not differ between treat-ments, while intrinsic water use efficiency (IWUE; Fig.1D) was higher in seedlings grown at elevated CO2. InLTAC seedlings, IWUE decreased rapidly at day 1 andstabilized, while gs showed some fluctuations through-out the experiment. In contrast, ETAC seedlingsretained similar gs and IWUE at the beginning and endof the experiment. Seedlings transferred to ETECexhibited significantly lower gs and significantly higherIWUE, in comparison with seedlings grown underETAC (Table I).

At day 0, there was no difference in dark respiration(Rd) between seedlings grown at ambient and elevatedCO2 (Fig. 1C). However, after transfer to short photo-period conditions, Rd increased approximately 3-folduntil day 8 of the experiment in all treatments. This wasfollowed by a decrease in Rd until day 26 and a subse-quent increase in all treatments by day 36. Over thecourse of the experiment, Rd was significantly lower inLTAC seedlings than in ETAC seedlings (Table I).Nonetheless, by day 36, Rd was similar in seedlings ofall three treatments.

The maximum substrate-saturated rate of Rubiscocarboxylation (Vcmax) was also measured at a commontemperature of 25°C (Fig. 1E). At day 0, seedlingsgrown at elevated CO2 exhibited slightly lower Vcmax incomparison with seedlings grown at ambient CO2 (Fig.1E). After 36 d, seedlings grown at LTAC exhibitedsignificantly lower Vcmax, whereas Vcmax in ETAC andETEC seedlings was not significantly affected.

Chlorophyll Fluorescence

In contrast to photosynthetic gas exchange, chloro-phyll fluorescence was only responsive to low tem-peratures and was unaffected by either photoperiod orelevated CO2. On day 0, seedlings grown at elevatedCO2 levels exhibited slightly lower effective quantumyield of PSII (FPSII), and slightly higher NPQ and PSIIexcitation pressure (1 2 qP) compared with seedlingsgrown at ambient CO2. Maximum quantum yield ofPSII (Fv/Fm) andFPSII decreased in seedlings within thefirst 4 d of exposure to LTAC conditions and remainedsignificantly lower for the duration of the experiment

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when compared to seedlings exposed to ETAC or ETECtreatments (Fig. 2, A and B; Table I). In contrast, NPQ(Fig. 2C), NPQS (Fig. 2D), and 1 2 qP (Fig. 2E) signifi-cantly increased in LTAC seedlings in comparison toseedlings grown at ETAC and ETEC (Table I). Growthunder ETAC conditions did not affect Fv/Fm, FPSII,NPQ, NPQS, or 1 2 qP compared to day 0.

Photosynthetic Pigments

On day 0, needles from seedlings grown at elevatedCO2 levels contained 50% less chlorophyll (Fig. 3A) and40% higher carotenoids per chlorophyll (Fig. 3B) com-pared with seedlings grown at ambient CO2. Followingthe first day of transfer to short photoperiod, totalchlorophylls in LTAC and ETAC seedlings were de-creased by 50%, while total carotenoids increased by50% in LTAC seedlings, and ETAC seedlings wereunaffected. ETEC seedlings did not exhibit changes inchlorophylls but exhibited a transient increase in ca-rotenoids during the first 2 weeks of treatment thatsubsided by day 16. By day 36, seedlings from all threetreatments contained similar chlorophyll levels, andLTAC seedlings retained higher carotenoids than bothelevated temperature treatments.

On day 0, the ratio of chlorophyll a to chlorophyll b(Chl a/b) was slightly, but not significantly, lower inseedlings grown at elevated CO2 compared to seedlingsgrown at ambient CO2 (Fig. 3C). After the first day oftreatment, both LTAC and ETAC seedlings exhibiteddecreased Chl a/b, with a larger initial decrease in ETACseedlings. By day 16, Chl a/b in LTAC seedlings con-tinued to decline while ETAC seedlings recovered tovalues similar to day 0. In contrast, ETEC seedlings didnot experience an initial decrease in Chl a/b andmaintained relatively constant Chl a/b throughout theexperiment.

On day 0, seedlings grown at elevated CO2 exhibitedhigher leaf xanthophyll cycle pigments (Fig. 3D) andde-epoxidation state of the xanthophyll cycle (DEPS;Fig. 3E) than seedlings grown at elevated CO2. After thefirst day of transfer to LTAC treatment, xanthophyllcycle pigments and DEPS increased considerably andremained significantly higher in LTAC seedlings incomparison with ETAC and ETEC seedlings (Table I).Xanthophyll pool size did not respond to the ETACtreatment, but DEPS increased 3-fold from day 0 to day1 and remained elevated until the end of the experi-ment. In ETEC seedlings, xanthophyll cycle pigmentstransiently increased during the first 16 d, but DEPS didnot change. By day 36, ETEC and ETAC seedlings

Figure 1. Response of photosynthetic gas ex-change to LTAC, ETAC, and ETEC. A, Anet, netphotosynthetic CO2 assimilation; B, gs, sto-matal conductance; C, Rd, dark respiration; D,IWUE; E, Vcmax, maximum rate of Rubiscocarboxylation. Gray background indicateslong photoperiod controls; white backgroundindicates short photoperiod treatments. Mea-surements for Anet, gs, Rd, and IWUE weretaken at 1400 mmol quanta m22 s21 andgrowth conditions; A/Ci curves used to calcu-late Vcmax were taken at 1400 mmol quantam22 s21, 400 mmol mol21 CO2, and 25˚C.Points represent the average of n = 8 to 106 SE

biological replicates. Letters in E indicate sig-nificantly different groups determined by one-way ANOVA (P , 0.05).





exhibited similar xanthophyll cycle pigment contentand DEPS.

Nonstructural Carbohydrates

Seedlings grown at elevated CO2 contained almostdouble the leaf starch of seedlings grown at ambientCO2 on days 0 and 1. Leaf starch content continuouslydecreased in seedlings from all three short photoperiodtreatments over the course of the experiment (Fig. 4A).By day 36, all three treatments exhibited similar starchcontent.

Total and individual soluble carbohydrates (Fig. 4, B–F) increased in seedlings fromall three treatments duringthe first 4 days before stabilizing. Total leaf soluble car-bohydrate content did not differ among seedlings of allthree treatments (Fig. 4B; Table I). Seedlings grown un-der LTAC exhibited significantly higher leaf sucrose,hexoses including Glc and Fru, and raffinose contentcompared to seedlings grown at ETAC, but this wasoffset by significantly lower pinitol (Fig. 4, C–F; Table I).Notably, hexose (Fig. 4D) and raffinose (Fig. 4F) levelsincreased during the first week of treatment in LTACseedlings and remained elevated for the duration of theexperiment. In contrast, hexose and raffinose concen-trations were increased in seedlings grown under ETACand ETEC during the first week of treatment, but by day16, levels of both soluble carbohydrates returned tolevels similar to values recorded on day 0. Neither hex-ose nor raffinose content differed significantly betweenETAC and ETEC seedlings (Table I).

All seedlings exhibited similar amounts of pinitol onday 0, and pinitol levels increased following transferto all three short photoperiod treatments (Fig. 4F).Pinitol levels stabilized on day 16 in seedlings grownunder LTAC, while pinitol continued to accumulate inseedlings grown under ETAC and ETEC throughoutthe experiment. Pinitol content was significantly lowerin LTAC seedlings compared to ETAC seedlings, butdid not differ between ETAC and ETEC seedlings(Table I).

Proteins

On day 0, protein content for photosynthetic proteinsRbcL, Lhcb1, and D1 did not differ between seedlingsgrown at ambient or elevated CO2. RbcL did not reveala significant response to temperature, photoperiod, orCO2 (Fig. 5A). In contrast, Lhcb1 and D1 decreased by50% in LTAC seedlings after 36 d (Fig. 5, B and C).ETAC seedlings also exhibited significantly decreasedD1 and slightly decreased Lhcb1 concentrations, al-though this difference was statistically not significant.ETEC seedlings did not exhibit changes in RbcL, Lhcb1,or D1.

PEPCwas significantly decreased in seedlings grownat elevated CO2 in comparison with seedlings grown atambient CO2 at the beginning of the experiment (Fig.5D). When seedlings were transferred from long toshort photoperiod treatments, PEPC decreased in thetreatments with ambient CO2 and remained low inseedlings grown at elevated CO2, and there was no

Table I. The effects of LTAC, ETAC, and ETEC on photosynthetic gas exchange, chlorophyll fluorescence,photosynthetic pigments, and carbohydrates

Tukey’s HSD multiple comparisons of means indicate significant differences (Sig.) between treatmentpairs following two-way ANOVA (Table S1). (*** P , 0.001; ** P , 0.01; * P , 0.05; n.s., not significant).

Category ParameterLTAC vs. ETAC ETAC vs. ETEC LTAC vs. ETEC

P Sig. P Sig. P Sig.

Photosynthetic gas exchange Anet ,0.001 *** ,0.001 *** ,0.001 ***gs ,0.001 *** ,0.001 ** 0.813 n.s.Rd 0.002 ** 0.084 n.s. 0.128 n.s.E ,0.001 *** 0.002 ** ,0.001 ***IWUE 0.208 n.s. ,0.001 *** ,0.001 ***

Chlorophyll fluorescence Fv/Fm ,0.001 *** 0.049 * ,0.001 ***FPSII ,0.001 *** 0.314 n.s. ,0.001 ***NPQ 0.001 ** 0.673 n.s. ,0.001 ***NPQS ,0.001 *** 0.037 * ,0.001 ***1 2 qP ,0.001 *** ,0.001 *** ,0.001 ***

Photosynthetic pigments Total Chl ,0.001 *** ,0.001 *** 0.383 n.s.Car/Chl ,0.001 *** ,0.001 *** 0.336 n.s.Chl a/b 0.023 * 0.094 n.s. ,0.001 ***V+A+Z/Chl ,0.001 *** ,0.001 *** ,0.001 ***DEPS ,0.001 *** ,0.001 *** ,0.001 ***

Carbohydrates Starch 0.009 ** ,0.001 *** 0.094 n.s.Soluble sugars 0.247 n.s. 0.709 n.s. 0.419 n.s.Sucrose 0.011 * 0.025 * 0.742 n.s.Hexose ,0.001 *** 0.102 n.s. ,0.001 ***Pinitol 0.043 * 0.417 n.s. 0.210 n.s.Raffinose ,0.001 *** 0.697 n.s. ,0.001 ***


Chang et al.


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difference in PEPC between treatments at the end of theexperiment (Fig. 5D).We detected two dehydrins, 52 and 16 kD in size.

The 52-kD dehydrin was constitutively expressedand revealed only minor nonsignificant differencesamong treatments and over the course of the experi-ment (Fig. 5, E and F). In contrast, the 16-kD dehydrinwas not detected in samples collected from seedlingsgrown at long photoperiod (day 0) but was expressedin all three short photoperiod treatments on day36 (Fig. 5G). Furthermore, the 16-kD protein showedincreased expression in all three treatments startingon day 16 and then continued to increase until day36 (Fig. 5H). Expression of the 16-kD dehydrin wasmore than 2-fold higher in LTAC seedlings than inETAC or ETEC seedlings. Total leaf protein contentdid not vary significantly during the course of theexperiment or between treatments (SupplementalFig. S1).We further investigated the 16-kD dehydrin in or-

der to elucidate the amino acid sequence of this pro-tein using mass spectrometry. A tentative consensussequence for a K-segment of the dehydrin was gener-ated. The sequence shared 73% identity with the

angiosperm K-segment EKKGIMDKIKEKLPG (Close,1996) and 60% identity with the gymnosperm K-segment[Q,E]K[P,A]G[M,L]DKIK[A,Q][K,M][I,L]PG (Jarviset al., 1996; Supplemental Fig. S2).

Freezing Tolerance

Freezing tolerance in seedlings acclimated to longphotoperiod andwarm temperature ranged from27°Cto 210°C on day 0, with no significant difference be-tween seedlings grown at ambient or elevated CO2 (Fig.6; Supplemental Fig. S3A). After 36 d, freezing tolerancewas significantly increased in all treatments (Fig. 6;Supplemental Fig. S3B). Freezing tolerancewas230.4°Cin seedlings grown under LTAC, which was signifi-cantly lower than freezing tolerance of seedlings grownunder ETAC (226°C) and ETEC (223.9°C; Fig. 6;Supplemental Fig. S3B).

Correlation between Dehydrins and Freezing Tolerance

The relationship between the cold- and photoperiod-responsive 16-kD dehydrin and freezing tolerance was

Figure 2. Response of chlorophyll fluores-cence to LTAC, ETAC, and ETEC. A, Fv/Fm,maximum quantum efficiency of photosystemII; B, FPSII, effective quantum yield of photo-system II; C, NPQ; D, NPQS; E, 1 2 qP, exci-tation pressure at photosystem II. Graybackground indicates long photoperiod con-trols; white background indicates short pho-toperiod treatments. Measurements weretaken at growth conditions; light-adaptedmeasurements were taken at 1400 mmolquantam22 s21. Points represent the average ofn = 8 to 10 6 SE biological replicates.











further investigated using field-grown seedlings. Levelsof the 16-kD dehydrin increased during autumn (Fig.7C), particularly as minimum temperatures decreasedbelow freezing (Fig. 7A), while levels of the 52-kDdehydrin remained unchanged (Fig. 7B).

Using a mixed linear model approach, we show thatboth expression of the 16-kD dehydrin and freezingtolerance were best represented by photoperiod, fol-lowed by minimum air temperature (Tmin) and the in-teraction of photoperiod and Tmin (Table II). Freezingtolerance correlated strongly with levels of the 16-kDdehydrin (Fig. 8).

DISCUSSION

Under field conditions, simultaneous decreases inphotoperiod and temperature induce cold hardeningin conifer seedlings. To investigate the impact of ris-ing temperature and atmospheric CO2 levels, weaimed to assess (1) how the onset and development ofcold hardening are affected by a shift from longphotoperiod to short photoperiod when air temper-ature remains warm and (2) how the combination ofwarmer air temperature with elevated CO2 affectsthe progress of the short photoperiod-induced coldhardening process.

Warm Temperature Suppresses the Down-Regulation ofPhotosynthesis in Seedlings Growing underShort Photoperiod

Shifting P. strobus seedlings from long photoperiodand warm temperature to LTAC induced cold hard-ening and resulted in the expected down-regulation ofphotosynthesis (Fig. 1A), modifications in thylakoidmembrane protein composition (Fig. 5, B and C), andchanges in photosynthetic pigments (Fig. 3; Öquist andHuner, 2003; Ensminger et al., 2006; Busch et al., 2007;Chang et al., 2015). This reorganization of the chloro-plast further led to the development of sustained non-photochemical quenching (Fig. 2D), an importantmechanism for the safe dissipation of excess light en-ergy required by cold-hardy evergreen plants duringwinter (Ensminger et al., 2004; Busch et al., 2007;Verhoeven et al., 2009).

Chlorophylls decreased from day 0 to day 1 (Fig. 3A).These large decreases in leaf chlorophylls during shiftfrom summer to autumn/winter have been previ-ously observed in conifers (e.g. Ottander et al., 1995;Ensminger et al., 2004; Bigras and Bertrand 2006;Fréchette et al., 2016). It appears that P. strobus, like otherconifers, can quickly adjust thylakoid membrane orga-nization and the associated pigments in order to de-crease light absorption and photochemistry per leaf area.

Figure 3. Changes in photosynthetic leaf pig-ments in response to LTAC, ETAC, and ETEC. A,Tot Chl, total chlorophylls expressed per gramfresh weight; B, Tot Car, total carotenoidsexpressed per mol chlorophyll; C, Chl a/b, ratioof chlorophyll a to chlorophyll b; D, V+A+Z,total xanthophyll cycle pool, comprised ofviolaxanthin, antheraxanthin, and zeaxanthin;E, DEPS. Gray background indicates long pho-toperiod controls; white background indicatesshort photoperiod treatments. Points repre-sent the average of n = 8 to 10 6 SE biologicalreplicates.


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Short photoperiod alone was insufficient to inducethe down-regulation of photosynthesis. Under ETAC,photosynthetic carbon assimilation (Fig. 1A), Fv/Fm(Fig. 2A), and FPSII (Fig. 2B) remained unaffected, al-though some components of the light reactions weredown-regulated, as indicated by decreases in totalchlorophyll content (Fig. 3A) and minor losses ofLhcb1 (Fig. 5B) and D1 (Fig. 5C). Warm temperaturealso affected the ability of ETAC seedlings to quenchexcess energy in comparison with LTAC seedlings:ETAC seedlings did not increase photoprotectivepigments (Fig. 3, B andD), showed smaller increases inDEPS (Fig. 3E), and suppressed the development ofNPQS (Fig. 2D). Together, this indicates that a lowtemperature signal is required to down-regulate pho-tosynthesis and up-regulate photoprotective processesin P. strobus seedlings. Indeed, a climate-controlledstudy using Pinus banksiana seedlings observed thatlow temperature alone, but not short photoperiodalone, significantly down-regulated photosynthesis;however, the combination of short photoperiodand low temperature also induced a greater down-regulation of Fv/Fm than low temperature alone(Busch et al., 2007).In contrast, field-grown P. strobus seedlings did not

exhibit a delay in the down-regulation of photosyn-thesis (Chang et al., 2015). In the field experiment,

canopy-level air temperature was elevated by +1.5°Cduring the day and +3°C during the night, and mea-surements were taken at monthly intervals, whenphotoperiod decreased from about 14 h in August to9.5 h in November (Chang et al., 2015). Evidently field-grown plants, which experience large natural day-to-day temperature variations and which are exposed to amuch smaller temperature increment of +1.5°C/+3°C,exhibit a much weaker response of photosynthesis toshort photoperiod and warmer temperature thanseedlings grown at the controlled conditions of thisexperiment.

The Combination of Warm Temperature and Elevated CO2Stimulates Photosynthesis in Seedlings Growing underShort Photoperiod

In ETECwhite pine seedlings, rates of photosyntheticcarbon uptake were increased by 33% compared toseedlings grown at ETAC (Fig. 1A) and greatly im-proved water use efficiency (Fig. 1D). The increase inwater use efficiency was likely a consequence of de-creased stomatal conductance (Fig. 1B) under CO2 en-richment, a phenomenon that has been well-describedfor plants actively growing during the vegetativeseason (Ceulemans and Mousseau, 1994; Curtis andWang, 1998, Ainsworth and Long, 2005; Leakey et al.,

Figure 4. Changes in leaf nonstructural car-bohydrate content in response to LTAC, ETAC,and ETEC. A, Starch content, expressed aspercent dry weight; B, total soluble sugars,composed of the sum of C to F; C, Sucrosecontent; D, hexose content (Glc + Fru); E,pinitol content; F, raffinose content, expressedper unit dry weight. Gray background indi-cates long photoperiod controls; white back-ground indicates short photoperiod treatments.Points represent the average of n = 8 to 106 SE

biological replicates. Letters indicated on theinsets for D and F indicate significantly dif-ferent treatments determined by two-wayANOVA (P , 0.05).





2009), but not for plants exposed to simulated late-season conditions triggering the development of coldhardiness.

The stimulation of photosynthetic carbon uptakeunder elevated CO2 also caused an excess accumulationof leaf carbohydrates in P. strobus seedlings, manifest-ing in greater starch content (Fig. 4A) and lower Vcmax(Fig. 1E) observed in seedlings grown at elevated CO2

(EC) compared with seedlings grown at ambient CO2(AC) seedlings at the beginning of the experiment.Under short photoperiod, i.e. extended night, respira-tion is increased relative to carbon assimilation andmay cause a decline in the daily net carbon uptake. As aconsequence, depletion of carbohydrates may at leastpartially restore the carbon sink capacity in seedlingsduring cold hardening. Carbon depletion resulting

Figure 5. Changes in expression of leaf pro-teins in response to LTAC, ETAC, and ETEC. A,RbcL, Rubisco large subunit; B, Lhcb1, lightharvesting complex protein of photosystem II;C, D1, reaction center core protein of photo-system II; D, PEPC; E to H, Dhn, dehydrin. Theaverage optical density of the day 0 AC controlwas arbitrarily scaled to 1 for RbcL, Lhcb1, D1,PEPC, and 52-kD dehydrin (A–F); the averageoptical density of day 36 LTAC treatment wasscaled to 1 for 16-kD dehydrin (G–H). Barsrepresent the average of n = 8 to 10 6 SE bio-logical replicates. Letters, where present, indi-cate significantly different groups determinedby one-way ANOVA (P, 0.05). Representativeblots shownwere loadedwith 5mg total proteinper lane.


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from an extended night period has been previouslyshown in Arabidopsis (Arabidopsis thaliana) leavesunder warm growth conditions (e.g. Gibon et al.,2004), but not under cold or elevated CO2 conditions inconifers.In addition, EC seedlings exhibited higher excitation

pressure at PSII (Fig. 2E), lower chlorophylls (Fig. 3A),higher carotenoids (Fig. 3B), and higher DEPS (Fig. 3E)than AC seedlings. This indicated that an imbalancebetween light absorption and photochemistry waspresent in EC seedlings that required increased dissi-pation of excess energy. Consistent with this hypothe-sis, studies in conifer seedlings, e.g. Picea mariana(Bigras and Bertrand, 2006) and Pinus sylvestris (Wangand Kellomäki, 1997), reported high levels of NPQ inplants growing under long photoperiod and elevatedCO2 conditions, followed by a relaxation of NPQwith adecrease in photoperiod.

A Major Shift in Carbohydrate Metabolism Is Induced byShort Photoperiod

Seedlings from all three treatments exhibited degra-dation of leaf starch and the concurrent accumulation ofsucrose, which formed the bulk of soluble carbohy-drates in the leaf (Fig. 4, A–C). During autumn, thesechanges in leaf carbohydrate composition in field-grown conifers such as P. sylvestris reflect the seasonaltranslocation of carbohydrates from leaves to roots(Oleksyn et al., 2000). This seems to be largely triggeredby photoperiod and was observed in our experimentwithin the first 4 d following transfer to short photo-period, indicated by increases in leaf sucrose (Fig. 4C)and dark respiration (Fig. 1C). In contrast, a transientdecrease in leaf starch content was accomplished at amuch slower rate and over the first 2 weeks (Fig. 4A).Notably, short photoperiod induced increased res-

piration rates (Fig. 1C) and decreased PEPC levels (Fig.

5D), reflecting the significant metabolic changes thatoccur during autumn (Dauwe et al., 2012). The down-regulation of PEPC suggests that short photoperiodinduced a metabolic shift where mitochondrial respi-ration was not principally maintained using the endproducts of glycolysis. Instead, pyruvate derived fromthe conversion of malate by malic enzyme may alsohave been used to fuel the citric acid cycle, thusbypassing the anaplerotic pathway mediated by PEPC.Indeed, a study of Picea sitchensis during autumn coldacclimation revealed a strong down-regulation of PEPCtranscripts along with key enzymes involved in gly-colysis but increased transcript levels for malic enzyme,citric acid cycle enzymes, and concentrations of leafcitrate, malate, and succinic acid (Dauwe et al., 2012).Hence, our findings provide strong evidence that themetabolic shift that occurs during the development ofcold hardiness is largely triggered by short photoperiodand not low temperature.

An initial increase in hexose content during the first2 weeks of the experiment was observed in all threetreatments (Fig. 4D, inset), but persisted only in theLTAC treatment. In general, Glc and Fru are only ob-served in minor quantities in conifer needles (Hochet al., 2003; Strimbeck et al., 2008) because they act astransitory intermediates in Suc synthesis, glycolysis,and regeneration of triose phosphates in the Calvincycle (Granot et al., 2013). The relatively small but sig-nificant increase in Glc and Fru levels in LTAC seed-lings clearly suggests an impairment of metabolicactivity by low temperature. This further emphasizesthat only certain aspects of the cold hardening processcan be triggered by short photoperiod alone.

Short Photoperiod Alone Increases Freezing Tolerance andAccumulation of Some, But Not All, CryoprotectiveCarbohydrates

Low temperature acclimation and the developmentof freezing tolerance are associated with the accumu-lation of soluble carbohydrates such as Suc (Guy et al.,1992; Uemura and Steponkus, 2003), raffinose (Knauppet al., 2011), and pinitol (Angelcheva et al., 2014), whichare considered cryoprotectants. Our data suggest thatraffinose contributes more effectively than pinitol tocold hardening in P. strobus. Accumulation of Suc andpinitol was induced by short photoperiod in all threetreatments (Fig. 4, C and E). Surprisingly, LTAC seed-lings contained 20% to 30% less pinitol than ETAC andETEC seedlings by the end of the experiment (Fig. 4E).Raffinose synthesis was also induced following shift toshort photoperiod but only accumulated in LTACseedlings and was depleted in ETAC and ETEC seed-lings after the first 2 weeks (Fig. 4F). The higher accu-mulation of pinitol observed in the two elevatedtemperature treatments at the end of the experimentdid not confer enhanced freezing tolerance in compar-ison with the LTAC treatment; instead, ETAC andETEC seedlings exhibited significantly lower freezing

Figure 6. Shoot freezing tolerance at the beginning (day 0) and end (day36) of the experiment in response to LTAC, ETAC, and ETEC. Graybackground indicates long photoperiod controls; white backgroundindicates short photoperiod treatments. Bars represent average LT50 ofn = 10 seedlings, estimated using sigmoidal curves fit to the data(Supplemental Fig. S3); error bars represent 95% confidence intervals.Letters, where present, indicate significantly different groups deter-mined by extra sum-of-squares F test.






tolerance (Fig. 6). Seedlings from all three treatmentsexhibited an approximate 2- to 3-fold increase infreezing tolerance after 36 d of short photoperiod ex-posure, but maximum freezing tolerance also requireda low-temperature stimulus (Fig. 6). Elevated CO2 onlyminimally affected the accumulation of cryoprotectivecarbohydrates. After transfer to short photoperiod,ETEC seedlings contained slightly more Suc at the endof the experiment, but similar levels of hexoses, pinitol,and raffinose in comparison with ETAC seedlings (Fig.4, C–F).

The field-grown P. strobus seedlings began to developfreezing tolerance in response to decreasing photope-riod prior to low-temperature exposure, which in-creased considerably following frost exposure (Fig. 7).This is consistent with results from our previous study,in which field-grown P. strobus seedlings exhibitedprefrost development of freezing tolerance, followed bypostfrost accumulation of sucrose, raffinose, and pinitoland enhanced freezing tolerance (Chang et al., 2015). Astudy on P. sylvestris and P. abies also demonstrated thatseedlings exposed only to short photoperiod developedgreater freezing resistance than seedlings only exposedto low temperature (Christersson, 1978). These findingsindicate that the photoperiod signal is crucial for de-veloping cold hardiness prior to low-temperatureexposure.

Maximum Freezing Tolerance Is Achieved by aCombination of Short Photoperiod and Low Temperatureand Correlates with the Induction of a 16-kD Dehydrin

Several dehydrin proteins are inducible by low tem-perature in spruce, and their accumulation correlateswith the development of freezing tolerance (Kjellsenet al., 2013). We probed total leaf proteins using anantibody specific to the highly conserved K-segmentregion of dehydrins. We observed a constitutivelyexpressed protein band with an approximate size of52 kD that was present in all samples (Fig. 5, E and F). Asecond protein band, 16 kD in size, was absent underlong photoperiod conditions but was significantly in-duced in response to short photoperiod and low tem-perature (Fig. 5, G andH). Analysis of the 16-kD proteinband via mass spectrometry confirmed the presenceof a dehydrin and provided a tentative consensussequence for its K-segment: EKKGILGQVKEKLPG(Supplemental Fig. S2). The structure of the tentative

Figure 7. Changes in dehydrin protein expression and development offreezing tolerance during cold hardening in needles of field grown P.strobus seedlings. A, Photoperiod (Phot), mean (Tmean), and minimum

(Tmin) ambient air temperature measured at field site; dotted verticallines indicate sampling dates. B, Constitutively expressed 52-kD and C,autumn-induced 16-kD dehydrin levels. The average optical density ofday 36 LTAC, used as a reference, was arbitrarily scaled to 1. Repre-sentative blots shown were loaded on an equal protein basis. Barsrepresent the average of n = 5 6 SE plot replicates. D, Shoot freezingtolerance. Bars represent the average of n = 5 6 95% confidence in-terval. Letters, where present, indicate statistically different groups de-termined by one-way ANOVA (P , 0.05).


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P. strobus K-segment was more closely related tothe known angiosperm K-segment (73% identity,EKKGIMDKIKEKLPG; Close, 1996) than known vari-ants of the gymnosperm K-segment (60% identity,[Q,E]K[P,A]G[M,L]DKIK[A,Q][K,M][I,L]PG; Jarviset al., 1996). Intriguingly, this 16-kD dehydrin appearedto be associatedwith freezing tolerance, andonly appearedafter several weeks of short photoperiod treatment, witha strong induction in seedlings exposed to low temper-ature (Fig. 5H). Although present in all samples exposedto short photoperiod, its accumulation was stronglysuppressed by both warm temperature and the combi-nation of warm temperature and elevated CO2.The delayed induction of the 16-kD dehydrin sug-

gests that its synthesis requires additional factors inaddition to environmental signals, such as changes inlevels of key metabolites (e.g. Kaplan et al., 2007) oractivation of genes involved in cold hardening anddormancy (e.g. Holliday et al., 2008). Previous studieshave showed that dehydrin expression is often inducedby environmental signals (Close, 1997; Puhakainenet al., 2004; Holliday et al., 2008; Kjellsen et al., 2013),but some dehydrins are also developmentally con-trolled during seed germination (Close, 1997).We further investigated the relationship between the

accumulation of dehydrins and freezing tolerance inneedles from field-grown seedlings. We identified theconstitutively expressed 52-kD dehydrin band in latesummer, autumn, and winter (Fig. 7A). The 16-kDdehydrin was absent during late summer but wasexpressed in early autumn and strongly up-regulated inlate autumn after temperature decreased below freez-ing (Fig. 7B). In the field-grown seedlings, we observedthat both 16-kD dehydrin accumulation and freezing

tolerance were determined by photoperiod and mini-mum air temperature (Table II). Furthermore, the 16-kDdehydrin correlated strongly with freezing tolerance(Fig. 8); therefore, suppression of 16-kD dehydrin syn-thesis by warm temperature likely contributed to theimpaired freezing tolerance observed in ETAC andETEC seedlings (Fig. 6).

Autumn accumulation of dehydrins has previouslybeen reported in P. sylvestris: Korotaeva et al. (2015)reported constitutive expression of ;70-kD and 45-kDdehydrins, while a 17-kD dehydrin manifested duringspring and autumn, but not summer. Similarly, Petrovet al. (2011) reported a 15-kD dehydrin absent duringsummer, detectable during early autumn, and stronglyaccumulated by late autumn. However, our study is thefirst to our knowledge to identify a dehydrin that ap-pears to be induced by short photoperiod alone butshows maximum expression when plants are exposedto the combination of low temperature and short pho-toperiod and which further shows a strong correlationwith the development of maximum freezing tolerancein pine seedlings.

CONCLUSION

Our findings suggest that warmer temperature has agreater negative impact than elevated CO2 on the de-velopment of cold hardening upon exposure to shorterphotoperiod in P. strobus seedlings. Evidently, ele-vated temperature and the combination of elevatedtemperature and elevated CO2 can suppress the down-regulation of photosynthesis and impair the develop-ment of freezing tolerance during autumn. However,all seedlings exposed to short photoperiod, irrespectiveof temperature or CO2 level, developed sufficientfreezing tolerance to protect against typical wintertemperatures at their geographic origin. According tolong-term meteorological records, the average mini-mum daily temperature in southern Ontario over thepast 30 years was 210.1°C (Environment Canada,2016). This is well above the 225°C to 229°C range of

Table II. Best predictors of seasonal variation for LT50 and 16-kDdehydrin in P. strobus needles determined by linear mixed-effectsmodeling

P value, P value of difference between predictor model and nullmodel; Tmin, minimum daily temperature; Tmean, mean daily temper-ature. AIC scores are provided to indicate goodness-of-fit for eachpredictor model; higher DAIC scores indicate higher goodness-of-fit.

Rank Predictor AIC DAIC P value

LT50Null model 508.60

1 Photoperiod 430.88 77.72 ,0.0012 Tmin 471.78 36.83 ,0.0013 Photoperiod:Tmin 472.66 35.94 ,0.0014 Photoperiod:Tmean 488.53 20.08 ,0.0015 Tmean 495.09 13.51 ,0.001Full model Photoperiod 3 Tmin 241.03 267.57 ,0.001

Dehydrin,16 kDNull model 120.35

1 Photoperiod 62.09 58.25 ,0.0012 Tmin 83.90 36.44 ,0.0013 Photoperiod:Tmin 86.19 34.16 ,0.0014 Photoperiod:Tmean 100.27 20.08 ,0.0015 Tmean 104.75 15.60 ,0.001Full model Photoperiod 3 Tmin 47.92 72.42 ,0.001

Figure 8. Correlation between relative leaf protein content of 16-kDdehydrin (Dhn) and freezing tolerance (LT50). Each point indicates plotaverage of three individuals. R2 value indicates goodness-of-fit for anexponential relationship. P value indicates whether log-transformedslope differs significantly from zero.





freezing tolerance exhibited by seedlings from alltreatments at the end of our experiment. Extreme lowtemperature events can result in minimum tempera-tures of 235°C, such as the extreme minimum tem-perature recorded on January 16, 1994 (EnvironmentCanada, 2016), but we have shown that field-grownseedlings developed freezing tolerance to 260°C inNovember. Based on our findings, future increases intemperature and CO2 may extend the growing seasonand stimulate late-season net carbon uptake. However,unless the frequency and timing of extreme low tem-perature events changes, warming climate is unlikely toincrease risk of freezing damage in P. strobus seedlingsin Ontario.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Two replicate experiments were conducted in 2012 and 2013 using 3-year-old(3+0) Eastern white pine (Pinus strobus) seedlings from a local seed orchard(Ontario seed zone 37; Somerville Seedlings). In April, seedlings were plantedin 3-L pots filled with a mixture of sand and sphagnum peat moss (1:2 v/v) andfertilized with 28:10:10 (N:P:K) mineral fertilizer (Miracle-Gro; Scotts). Pottedseedlings were then kept outside in an experimental garden at the University ofToronto Mississauga campus and transferred to growth chambers in early July(Biochambers).

Seedlings were acclimated for 6 weeks to long photoperiod at either ambientCO2 (22°C day/15°C night, 14 h daylength, 400 mmol mol21 CO2) or elevatedCO2 (22°C day/15°C night, 14 h daylength, 800 mmol mol21 CO2). Followingacclimation to long photoperiod treatment, seedlings grown at ambient CO2

were shifted to a short photoperiod treatment of either LTAC (12°C day/5°Cnight, 8 h daylength, 400 mmol mol21 CO2) or ETAC (22°C day/15°C night, 8 hdaylength, 400 mmol mol21 CO2), while seedlings grown at elevated CO2 wereshifted to a short photoperiod treatment with ETEC (22°C day/15°C night, 8 hdaylength, 800 mmol mol21 CO2). Diurnal temperature variations in southernOntario, where the seedlings originated, often exceed 10°C (EnvironmentCanada, 2016). Therefore, we chose treatment ranges that differed by 10°C andwere physiologically relevant. However, they were higher than the smalltemperature ranges (+1.5°C to +3°C) that we have used previously in long-termmonitoring field experiments (Chang et al., 2015). The 12°C/5°C temperaturerange for the LTAC treatment represents typical chilling temperatures experi-enced during midautumn (Fig. 7A), while the 22°C/15°C range for the ETACtreatment represents conditions in which only the short photoperiod, but notlow temperature, signal is perceived. Photosynthetic active radiation at the topof the canopy was maintained at 1400 mmol quanta m22 s21 during the day andat 400mmol quanta m22 s21 during the first and last half-hour of each day. Lightwas provided using metal halide and high pressure sodium lamps. Humiditywas set to 60% relative humidity.

Measurements and samples were taken after 6 weeks of long photoperiodacclimation (day 0) prior to transferring seedlings to short photoperiod condi-tions and repeated on days 1, 4, 8, 16, 26, and 36 following transfer. The durationof the experiment andmeasuring pointswere selected to assess early-,mid-, andlate-stage responses to a change in photoperiod and temperature, given constantexposure to 400 or 800 mmol mol21 CO2. We did not perform control mea-surements with unchanged photoperiod and temperature, since no change inphotosynthesis, pigments, and proteins occur when photoperiod and temper-ature remain constant (Busch et al., 2007). All measurements were taken atgrowth temperature. Throughout the experiment, seedlings were watered ev-ery 2 to 3 d. Seedlings were rotated within each chamber once per week.

Mature current-year needle samples were obtained from field-grownseedlings during August 17, September 26, October 26, and November 16 of2013 for the analysis of leaf dehydrins and freezing tolerance. Three-year-old(3+0) seedlingsobtained fromthe sameseedorchardused in thegrowthchamberexperiment were established in five replicate field plots during May 2012 at theKoffler Scientific Reserve of the University of Toronto, located in southernOntario (44°0509N, 79°4839W). Plots were excavated to 30 cm deep, filled with amixture composed of one-third peat, one-third sand, and one-third local soil,

and tilled prior to planting. Ambient canopy temperature was recorded usinginfrared sensors (model IRT-P5, Apogee Instruments) and a CR1000 datalogger(Campbell Scientific). Five-day running averages for mean ambient tempera-ture and minimum ambient temperature were calculated using GraphPadPrism v6.04 (GraphPad Software).

Photosynthetic Gas Exchange

Gas exchange was measured at growth conditions on current-yearneedles attached to the seedlings after $2 h of exposure to growth lightusing a GFS-3000 (Walz). The measuring cuvette was set to the followinggrowth conditions: 400 or 800 mmol mol21 CO2, 12°C or 22°C, and 60%relative humidity. Dark respiration (Rd) was measured after 40 min of darkadaptation. Net photosynthetic carbon assimilation (Anet), stomatal con-ductance (gs), and evapotranspiration (E) were subsequently measured atgrowth light intensity (1400 mmol quanta m22 s21) once steady-state as-similation was achieved. IWUE was calculated as A/gs, according to Silvaand Horwath (2013).

A/Ci curves were measured on days 0 and 36. Assimilation was assessedafter 2 to 3 min of exposure to CO2 levels of 400, 300, 250, 200, 150, 100, 50; 400,550, 650, 800, and 1000 mmol mol21 CO2, based on a protocol by Long andBernacchi (2003).Measurementswere taken at 25°C and 1400mmol quantam22 s21.The initial slope of the A/Ci curve, which represents the Rubisco-limited rate ofcarboxylation (Wc), was used to calculate the maximum substrate-saturatedrate of Rubisco carboxylation (Vcmax) at 25°C using the following equation:

Wc ¼ VcmaxðCiÞCi þ Kcð1þ O

KoÞ

where Ci represents the intracellular partial pressure of CO2, O represents thepartial pressure of oxygen at 25°C, and Kc and Ko represent the Michaelis-Menten constants of Rubisco for the competing carboxylation and oxygena-tion reactions, respectively (Farquhar et al., 1980; Sage, 1990).

Chlorophyll Fluorescence

Chlorophyll fluorescence was measured at growth conditions on attachedcurrent-year needles using a Dual-PAM-100 (Walz) after $2 h of exposure togrowth light. Dark-adapted minimum PSII fluorescence (Fo) and maximumPSII fluorescence (Fm) were obtained after 40 min of dark adaptation in the leafclip, followed by assessment of light-adapted minimum PSII fluorescence (Fo9),light-adapted maximum PSII fluorescence (Fm9), and transient fluorescence (Ft)after exposure to 1400 mmol quanta m22 s21 of actinic light for 3 to 5 min.Maximum quantum yield of PSII was calculated as Fv/Fm = (Fm 2 Fo)/Fm, andeffective quantum yield of PSII was calculated as DF/Fm9 = (Fm9 2 Ft)/Fm9(Genty et al., 1989). The excitation pressure at PSII was calculated as 1 2 qP =12 (Fm92 Ft)/(Fm92 Fo9) (Maxwell and Johnson, 2000). NPQwas calculated asNPQ = (Fmrec/Fm9) 2 1 (Bilger and Björkman, 1990) with fully recoveredFm (Fmrec) estimated as Fo 3 5, according to Chang et al. (2015). Sustained non-photochemical quenching was calculated as NPQS = (Fmrec/Fm)2 1 (Ensmingeret al., 2004; Maxwell and Johnson, 2000; Porcar-Castell, 2011; Changet al., 2015).

Photosynthetic Pigments

Needle samples, collected from chamber-grown seedlings after $2 h of ex-posure to growth light, were flash-frozen in liquid nitrogen and stored at 280°C.Samples were homogenized to a fine powder using a mortar and pestle. Pho-tosynthetic pigments were extracted from 50 to 60 mg homogenized frozenneedle tissue at 4°C in 700 ml of 98% methanol buffered with 2% 0.5 M ammo-nium acetate for 2 h in the dark. The extract was centrifuged at 4°C at14,000 rpm for 5 min and the supernatant collected. The pellet then was washedtwice with 700 mL of methanol at 4°C followed by centrifugation, and the su-pernatant was again collected. Finally, the total supernatant was filtered using0.2 mM pore polytetrafluoroethylene syringe filters (Thermo Scientific).

Photosynthetic pigments were separated using a reverse-phase C30 column(5mm, 2503 4.6mm; YMCCo.) and analyzedwith an Infinity 1260 series HPLCsystem equipped with a UV-diode array detector (Agilent Technologies), asdescribed in Fréchette et al. (2016) and Junker and Esnminger (2016). Pigmentswere eluted using a gradient of methanol and water buffered with 0.2% am-monium acetate and tert-butyl methyl ether at a flow rate of 1 mL min21 at acolumn temperature of 25°C. Pigments were calibrated using standards from


Chang et al.



Sigma Aldrich and DHI Lab products. Peak detection and pigment quantifi-cation were performed using ChemStation software (Agilent Technologies).

Total chlorophyllswere expressed as the sumof chlorophylls a and b inmmol g21

fresh weight. The ratio of chlorophyll a to chlorophyll b was expressed asmol mol21. All carotenoid concentrations were normalized to chlorophyllcontent and expressed as mmol mol21. Total carotenoids were expressed as thesum of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z), neoxanthin, lutein,a-carotene, and b-carotene. Total xanthophyll cycle pigments were calculatedas the sum of V, A, and Z. The DEPS was calculated as (0.5A + Z)/(V + A + Z),according to Thayer and Björkman (1990).

Nonstructural Carbohydrates

Needle samples, collected shortly after midday from chamber-grownseedlings, were flash-frozen in liquid nitrogen and stored at 280°C. Leaf-soluble carbohydrates were extracted according to Park et al. (2009). Twohundred fifty micrograms galactitol internal standard was added to 30 to 40 mghomogenized, lyophilized needle tissue. The mixture was incubated in 4 mL ofextraction buffer (methanol:chloroform:water, 12:5:3 v/v) overnight at 4°C. Themixture was centrifuged at 6000 rpm for 10 min and the supernatant collected.The pellet was washed twice in extraction buffer followed by centrifugation.Five milliliters of water was added to the total supernatant, mixed, andcentrifuged at 4000 rpm for 4 min. Finally, the upper aqueous phasewas collected.

Two milliliters of the soluble carbohydrate extract were dried using avacuum centrifuge and resuspended in 1 mL of nanopure water. The resus-pended extract was filtered through a 0.45 mM pore nylon syringe filter(Chromatographic Specialties) and analyzed using an ICS-5000 anion-exchange HPIC (Dionex) equipped with an electrochemical pulse ampero-metric detector. Glucose, sucrose, fructose, and pinitol were eluted using aHi-Plex Ca column (Agilent Technologies) with water at a flow rate of0.270 mL min21 with a column temperature of 70°C. Raffinose was elutedusing a Carbo-Pac PA1 column (Dionex)with 150mMNaOH (isocratic) at aflowrate of 1 mL min21. For all soluble carbohydrates, postcolumn detection wasperformed using NaOH at a rate of 100 mM min21.

Leaf starch was determined using the residual tissue pellet from thesoluble sugar extraction according to Park et al. (2009). The pellet was driedovernight at 55°C. Twenty to thirty micrograms of pellet was resuspendedin 5 ml of 4% H2SO4 and autoclaved for 5 min at 121°C. Once cooled, theextract was spun at 500 rpm for 5 min and the supernatant was collected,filtered using a 0.45 mm nylon filter, and analyzed using a DX-600 anion-exchange IC/HPLC (Dionex) equipped with a Carbo-Pac PA1 column andelectrochemical pulse amperometric detector. The extract was elutedwithwaterat a flow rate of 1 ml min21 with a column temperature of 30°C. Postcolumndetection was performed usingNaOH at a rate of 100mMmin21. Peak detectionand quantification of all nonstructural carbohydrates were performed usingPeakNet software (Dionex).

Leaf starch content was expressed as percent dry weight. Soluble sugarswere expressed as mg g21 dry weight. Total soluble sugars were calculated asthe sum of glucose, fructose, sucrose, raffinose, and pinitol.

Protein Extraction, SDS-PAGE, and Immunoblotting

Proteins were extracted according to Busch et al. (2007) from 50 to 60 mghomogenized frozen needle tissue obtained from chamber-grown and field-grown seedlings. The extraction buffer consisted of 60 mM Tris-HCl, pH 6.8,4% (w/v) SDS, 15% (w/v) sucrose, 20 mM DTT, and Complete EDTA-freeproteinase inhibitor cocktail (Roche Diagnostics). Protein concentration wasassessed using the RC/DC protein assay kit (Bio-Rad Laboratories). Five mi-crograms protein per lane was loaded on 10% NuPage Novex Bis-Tris gels(Invitrogen) and separated at 120 V for 90 min at room temperature using theXCell Mini gel system. Following separation, proteins were transferred to aPVDF membrane (0.2 mm pore size; Bio-Rad) using the XCell II Blot Module(Invitrogen) for 1 h at 30 V on ice. The membrane was blocked using 5% nonfatmilk in Tris-buffered saline for 45 min at room temperature, then probed withantibodies against PsbA (1:5000), Lhcb1 (1:5000), RbcL (1:5000), PEPC (1:1000),or dehydrin (1:1000). Goat antirabbit IgG conjugated with horseradish perox-idase (1:75,000) was used as a secondary antibody for chemiluminescent proteindetection using the Amersham ECL Prime kit (GE Healthcare, Buck-inghamshire) and a ChemiDoc MP (Bio-Rad) for visualization. All antibodieswere obtained from Agrisera. Optical band density was quantified using ImageLab software (Bio-Rad).

Mass Spectrometry

Forty micrograms of total protein from a sample containing high levels ofthe 16-kD dehydrin, as previously determined bywestern blot, was loaded on a12% Bolt Bis-Tris gel (Invitrogen) and separated at 100 V for 2 h 45 min at roomtemperature using the BoltMini Gel Tank (Invitrogen). Presence of dehydrins inthe 16-kDbandwas confirmed on a replicate gel usingwestern blot, as describedabove. Following separation, the gel was stained using Coomassie Blue for 1 h,then destained overnight. The 16-kD band was subsequently excised and im-mersed in 1%acetic acid prior to analysis. Trypsin digest of the sample, followedby liquid chromatography-tandemmass spectrometry analysis, was performedat the SPARC BioCentre mass spectrometry facility of the Hospital for SickChildren (Toronto, ON, Canada). Peptide sequences were predicted by proteinBLAST against nr and Uniprot databases, using PEAKS Studio v7.5 (Bio-informatics Solutions; Ma et al., 2003). Peptides containing fragments of thedehydrin K-segment were aligned using MUSCLE (www.ebi.ac.uk/Tools/msa/muscle/; Edgar, 2004) andmanually reviewed. The aligned peptides werethen used to generate a consensus K-segment peptide using Jalview v2.9.0b2(www.jalview.org/; Waterhouse et al., 2009; Supplemental Fig. S2). Similarityamong the consensus K-segment, angiosperm K-segment, and gymnospermK-segment was calculated using protein BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM = blastp; Altschul et al., 1990).

Freezing Tolerance

Chlorophyll fluorescence was used to assess freezing tolerance using amodified protocol after Sutinen et al. (1992). Sampleswere taken from chamber-grown seedlings after 36 d of LTAC, ETAC, or ETEC treatment, and from field-grown seedlings on August 17, September 26, October 26, and November 16 of2013. Sections of current-year shoots from five seedlings per treatmentwere cut,dark-adapted for 40 min, and Fv/Fm measured. Shoots were then individuallywrapped in moist paper towel and aluminum foil and sealed in plastic bags.

Shootswere exposed toa rangeof freezing temperaturesat 5°C intervals from0°C to 240°C using a Thermotron SM-16-8200 environmental test chamber(Thermotron Industries). The initial decrease from 0°C to 21°C was achievedover 1 h, followed by a maximum cooling rate of 5°C h21 to reach target tem-perature. Each target temperature was held for 8 to 12 h. At the end of eachinterval, shoot sections were transferred to a 4°C refrigerator for 24 h, followedby transfer to RT, where the shoots were allowed to recover for 24 h. Followingthe 24 h recovery period, shoots were unwrapped and exposed to 1 h lightexposure at 800 mmol quanta m22 s21 in order to stimulate PSII, then darkadapted for 40 min (Burr et al., 2001). Fv/Fm was then recorded. Evaluation ofthe temperature at which 50% of seedlings were damaged by freezing (LT50)was assessed as the midpoint of a sigmoidal curve fit to the data, using themethod described previously (Chang et al., 2015). Fitted sigmoidal modelcurves are presented in Supplemental Fig. S3.

Statistical Analysis

The effects of treatment and time on photosynthesis, pigment content,nonstructural carbohydrates and freezing tolerance of chamber-grown seedlingswere assessed by two-way ANOVA, using the l-mer function of the lme4 package(Bates et al., 2014) in R v3.1.1 (www.r-project.org/). The ANOVA model usedtreatment and day as categorical fixed factors, and individual and replicate asrandom factors. Tukey’s HSD post-hoc tests were used to contrast betweentreatments at each time point, andwere performed using the glht function of themultcomp package (Hothorn et al., 2008) in R v3.1.1. The effects of treatment onprotein content were assessed by one-way ANOVA, using GraphPad Prismv6.04. LT50 values for the chamber-grown seedlings were compared amongtreatments with an extra sum-of-squares F test (P , 0.05), using GraphPadPrism v6.04.

Linear mixed models were used to estimate the best predictor for seasonalchanges in 16-kD levels and freezing tolerance assessed infield-grown seedlingsusing the l-mer function of the lme4 package (Bates et al., 2014) in R v3.1.1. Plotwas included as a random factor in all models. Models were scored using theAkaike information criterion (AIC) to evaluate goodness-of-fit. DAIC was cal-culated by subtracting the AIC score of each predictor from that of the nullmodel. Significance of predictors was calculated by comparing the null modelwith the predictor using ANOVA.

In order to evaluate the strength of the relationship between levels of the16-kD dehydrin and freezing tolerance, as well as to correlate dehydrin levelsand freezing tolerance to photoperiod and air temperature, R2 values were




http://www.ebi.ac.uk/Tools/msa/muscle/

http://www.ebi.ac.uk/Tools/msa/muscle/

http://www.jalview.org/



http://www.r-project.org/


obtained from nonlinear regression of the data using the exponential growthequation Y = Y0

kX, where k is the rate constant. Nonlinear regressions wereperformed using GraphPad Prism v6.04.

Supplemental Data

The following supplemental materials are available:

Supplemental Figure S1. Changes in total leaf protein content.

Supplemental Figure S2. Peptides used to generate consensus K-segmentsequence.

Supplemental Figure S3. Modeled shoot freezing tolerance.

Supplemental Table S1. The effects of treatment and time on photosyn-thetic gas exchange, chlorophyll fluorescence, photosynthetic pigments,and carbohydrates.

ACKNOWLEDGMENTS

The authors are grateful to Laura V. Junker for guidance with photosyn-thetic pigment analysis and to Tarek Bin Yameen andAlexandra Zubilewich forassistance with freezing tests.

ReceivedMay 16, 2016; accepted August 31, 2016; published September 2, 2016.

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