Draft - University of Toronto T-Space · Draft 2 16 Abstract 17 Many spring geophytes exhibit greater growth at colder than at warmer temperatures. Previous 18 studies have suggested

Draft

Thermal acclimation of leaf respiration as a way to reduce

source-sink imbalance at low temperature in Erythronium americanum, a spring ephemeral

Journal: Botany

Manuscript ID cjb-2017-0168.R1

Manuscript Type: Article

Date Submitted by the Author: 31-Oct-2017

Complete List of Authors: Dong, Yanwen; Universite Laval Faculte des sciences et de genie, Département de biologie; Université de Lorraine, INRA, UMR 1137, Écologie et Écophysiologie Forestières, Faculté des Sciences Gérant, Dominique; Université de Lorraine, INRA, UMR 1137, Écologie et Écophysiologie Forestières, Faculté des Sciences Lapointe, Line; Université Laval, Biologie

Is the invited manuscript for consideration in a Special

Issue? : N/A

Keyword: <i>Erythronium americanum</i>, source–sink relationship, sink limitation, thermal acclimation

https://mc06.manuscriptcentral.com/botany-pubs

Botany

Draft

1

Thermal acclimation of leaf respiration as a way to reduce source-sink 1

imbalance at low temperature in Erythronium americanum, a spring 2

ephemeral 3

4

Yanwen Dong1,2, Dominique Gérant2 and Line Lapointe1 5

1 Département de Biologie and Centre d’Étude de la Forêt, Université Laval, Québec, QC, Canada 6

2 Université de Lorraine, INRA, UMR 1137, Écologie et Écophysiologie Forestières, Faculté des 7

Sciences, F- 54500 Vandœuvre-lès-Nancy, France 8

9

Corresponding author: 10

Line Lapointe 11

Tel: +1 418-656-2822 12

Fax: +1 418-656-2043 13

Email: [email protected] 14

15

Page 1 of 34


Botany

Draft

2

Abstract 16

Many spring geophytes exhibit greater growth at colder than at warmer temperatures. Previous 17

studies have suggested that there is less disequilibrium between source and sink activity at low 18

temperature, which delays leaf senescence and leads to higher accumulation of biomass in the 19

perennial organ. We hypothesize that dark respiration acclimates to temperature at both leaf and 20

bulb level, mainly via the alternative pathway, as a way to reduce source-sink imbalance. 21

Erythronium americanum was grown under three temperature regimes, 8/6 °C, 12/8 °C and 22

18/14 °C (day/night). Plant respiratory rates were measured at both growth and common 23

temperature to determine whether differences were due to direct effects of temperature on 24

respiratory rates or to acclimation. Leaf dark respiration exhibited homeostasis, which together 25

with lower assimilation at low growth temperature, most likely reduced the quantity of C 26

available for translocation to the bulb. No temperature acclimation was visible at the sink level. 27

However, bulb total respiration varied through time, suggesting potential stimulation of bulb 28

respiration as sink limitation builds up. In conclusion, acclimation of respiration at the leaf level 29

could partly explain the better equilibrium between source and sink activity in low-temperature 30

grown plants, whereas bulb respiration responds to source-sink imbalance. 31

32

Keywords: source–sink relationship, thermal acclimation, sink limitation, Erythronium 33

americanum 34

35

Page 2 of 34


Botany

Draft

3

Introduction 36

Temperature affects protein synthesis and enzyme activity, which in turn influence the rates of 37

metabolic reactions, such as photosynthesis and respiration (Raison 1980; Atkin et al. 2005b). 38

However, some plants adjust their metabolic rates to partly compensate for the negative impact of 39

changing conditions in an attempt to maintain their growth rate over a broader range of 40

temperatures. This process is called acclimation (Levitt 1972; Berry and Bjorkman 1980). In 41

many instances, the acclimation process can be extended to achieve homeostasis, i.e., where rates 42

of metabolic processes are identical in plants that are grown at contrasting temperatures when 43

measured at their respective growth temperatures (Atkin et al. 2000b). Homeostasis has been 44

demonstrated in many global warming studies (Atkin et al. 2000a; Atkin and Tjoelker 2003), 45

where differences in growth temperatures are not too large, thereby allowing for complete 46

adjustment of the different metabolic rates. Indeed, it has been reported that both leaf total dark 47

respiration (leaf RT) and net assimilation (A) can acclimate to the extent that the leaf RT / A 48

quotient remains fairly stable once the leaves have adjusted to the new growth condition (Dewar 49

et al. 1999; Loveys et al. 2003). 50

Both leaf RT and photosynthetic rates respond in a substrate-dependent manner. While 51

photosynthesis is feedback-inhibited by accumulation of carbohydrates within the leaf (Foyer et 52

al. 1990; Goldschmidt and Huber 1992; Strand et al. 1997), RT may be stimulated by an increase 53

in substrate availability (Atkin and Tjoelker 2003). One of the factors that can stimulate leaf 54

carbohydrate accumulation is a reduced rate of translocation between leaves and sink organs 55

(Krapp and Stitt 1995; Ainsworth and Bush 2011). Reduction in C translocation rates has been 56

demonstrated in winter wheat (Triticum aestivum L.) and sunflower plants (Helianthus annuus L.) 57

that developed at lower temperatures compared to rates that were measured in warm-grown plants 58

(Paul et al. 1990; Leonardos et al. 2003). Yet, not all species exhibit such reductions during cold 59

acclimation. Oilseed rape (Brassica napus L.) plants that were grown at 13 °C exhibited greater C 60

translocation rates than those that were grown at 30 °C (Paul et al. 1990) while spring crocus 61

Page 3 of 34


Botany

Draft

4

(Crocus vernus [L.] Hill) that was grown at 12 °C and 18 °C exhibited similar rates of 62

translocation from leaves to corms, early in the season (Badri et al. 2007). Despite potential 63

metabolic adjustment during the acclimation process, carbohydrate accumulation could still take 64

place under low temperature, which could increase the leaf RT / A quotient (Atkin et al. 2005a; 65

Campbell et al. 2007). 66

Another condition where carbohydrates could accumulate within the leaves is under conditions 67

of sink-limited growth (Basu et al. 1999; Hoch et al. 2002). Under such conditions, stimulating 68

leaf RT could reduce source-sink imbalances, which are known to induce early leaf senescence 69

through feedback inhibition (Gandin et al. 2009). Such increases in respiration could be due, in 70

part, to increased electron flow to the alternative respiratory pathway (Vanlerberghe and 71

McIntosh 1992; Gonzàlez-Meler et al. 1999; Florez-Sarasa et al. 2007). One of the potential roles 72

for the alternative respiratory pathway is the consumption of excess carbohydrates that are not 73

used for energy production, growth and maintenance processes in tissues, namely the “energy 74

overflow” hypothesis proposed by Lambers (1982). Indeed, several studies have reported results 75

that were consistent with the hypothesis that the alternative pathway acts to burn excess 76

carbohydrate under many stress conditions, namely drought and light stress (Giraud et al. 2008), 77

low N availability (Noguchi and Terashima 2006), and macronutrient stress (Sieger et al. 2005). 78

Gandin et al. (2009) have previously shown that the capacity of the alternative pathway (Ralt) was 79

strongly stimulated in the bulb of spring ephemerals under sink-limited conditions that were 80

caused by plant exposure to elevated CO2 concentrations. Although the non-energy conserving 81

nature of the alternative pathway would be expected to negatively affect plant growth, its positive 82

effects in the maintenance of metabolic and signalling homeostasis might more than offset its 83

negative effects (Vanlerberghe 2013). 84

In spring ephemerals, the growth of the perennial organ (bulb or corm, according to the 85

species) was shown to be higher at low temperature compared to that recorded at warmer 86

temperatures (Lapointe and Lerat 2006; Badri et al. 2007; Lundmark et al. 2009; Gandin et al. 87

Page 4 of 34


Botany

Draft

5

2011; Bernatchez and Lapointe 2012). As the new bulb/corm accumulates carbohydrates, sink 88

limitation builds up, inducing feedback inhibition of photosynthesis and eventually leaf 89

senescence (Badri et al. 2007; Gandin et al. 2011). It has been suggested that an enhanced 90

bulb/corm growth at low temperature is due to the capacity of the plant to maintain a better 91

equilibrium between source and sink activities over time (Gandin et al. 2011). Therefore, sink 92

limitation and consequent feedback inhibition on photosynthetic activity is postponed at lower 93

temperatures resulting in a longer leaf life duration and a longer period of bulb/corm growth. 94

However, a number of questions remain pending. Could this equilibrium be linked not only to 95

reduced assimilation, but also to increased respiration at low growth temperature? Could Ralt be 96

involved in the modulation of leaf or bulb RT as a response to temperature? Could respiration be 97

modulated both at the leaf and at the bulb level to more efficiently balance the amount of C that is 98

translocated from leaf to sink and the amount of C that is used at the sink level? Finally, could 99

leaf or bulb RT be modulated through time as sink limitation builds up? 100

The main objective of the present study was thus to determine if both RT and Ralt are 101

stimulated at low growth temperature in spring ephemerals to better adjust the C that is required 102

for growth with the C available from assimilation. If so, does it occur in the leaf, in the bulb or in 103

both organs? We assessed the relative contribution of the capacity of the cytochrome (Rcyt) and 104

alternative (Ralt) pathways as a function of growth temperature throughout the season in both leaf 105

and bulb of the spring ephemeral, yellow trout lily (Erythronium americanum Ker-Gawl.). Plants 106

were grown under three temperature regimes, i.e., 8/6 °C, 12/8 °C and 18/14 °C (day/night). In 107

forests nearby Quebec City, the 12/8 °C temperature regime represents the mean day and night 108

temperatures during the early growth period of this species (first two weeks of May), while the 109

highest growth temperature represents mean day and night temperatures at the end of its growing 110

period (first two weeks of June). The 12/8 °C temperature regime seems not to be the optimal 111

growth temperature since a better bulb growth was previously observed at 8/6 °C than at 12/8 °C 112

by Gandin et al. (2011). Respiratory rates were measured at both growth temperatures and at a 113

Page 5 of 34


Botany

Draft

6

common temperature (12 °C) to discriminate the effect of the measured temperature from the 114

acclimation process to growth temperature. Soluble sugar concentrations were also recorded in 115

both leaf and bulb to determine if respiratory rates are modulated as a function of substrate 116

availability. 117

118

Material and methods 119

Plant material and experimental design 120

Bulbs of E. americanum were collected from a maple forest near Saint-Augustin-de-Desmaures 121

(QC, Canada; 46°48’N, 71°23’W) in September 2012. About 650 bulbs of similar diameter (6-8 122

mm) were selected and planted individually in 10-cm diameter plastic pots containing Turface 123

(calcined clay granules, Applied Industrial Materials Corp., Buffalo Grove, IL, USA) as substrate. 124

Pots were then placed in a cold room (4-5 °C) for five months of cold stratification. Substrate 125

moisture content was checked weekly and pots were watered when the top 5 cm of Turface had 126

dried. At the end of February, all pots were moved to a growth chamber that was set at 8/6 °C 127

(day/night) in darkness for about one week of acclimation. Thereafter, about 150 pots were 128

randomly transferred to each of three growth chambers (PGW36, Conviron Inc., Winnipeg, MB, 129

Canada) under the following light conditions: photoperiod of 14 h and a photosynthetic photon 130

flux density (PPFD) of 300 µmol. m-2. s-1. The temperature regimes that were used in the 131

experiment were based upon a study by Gandin et al. (2009); three regimes were tested: 8/6 °C 132

(day/night), 12/8 °C, and 18/14 °C, with relative humidities (RH) of 50 %, 65 %, and 75 %, 133

respectively. RH was modulated as a function of temperature to maintain a constant vapour 134

pressure deficit (VPD) among growth chambers. Plants were watered regularly and fertilised 135

weekly with 10 % Hoagland’s solution for optimal plant growth (Lapointe and Lerat 2006). 136

137

Plant phenological stages 138

Page 6 of 34


Botany

Draft

7

Plant phenology was recorded throughout the growing season (Fig. 1) and was organised 139

according to well-known phenological periods: (i) leaf expansion, hereafter referred to as period I 140

(leaf sprouting and unfolding; old bulb acting as a source); (ii) green leaf period, which is divided 141

into period II (leaf expansion completed; continued shrinkage of the old bulb continues; the new 142

bulb is visible at the end of this period) and III (most new bulb growth occurs during this period); 143

(iii) leaf senescence period, which is divided into period IV (beginning of leaf senescence up to 144

mid-senescence; leaf changes from exhibiting a yellow tip to about half-yellow; bulb biomass is 145

no longer increasing) and V (mid- to complete leaf senescence; bulb enters into dormancy). In the 146

present study, the different variables were measured at specific sampling stages (identified as T1 147

to T5) during the season, which covered the different phenological stages of both leaf and bulb 148

(Fig. 1). 149

150

Leaf assimilation measurements 151

Net assimilation rates (A) were measured using a portable photosynthesis system (Li-Cor 6400, 152

Li-Cor Inc. Lincoln, NE, USA). Light was set at 300 µmol. m-2. s-1, which was similar to light 153

levels under growth conditions, and airflow was set at 200 µmol. s-1. Temperature and relative 154

humidity conditions were similar to those recorded in the growth chambers. Measurements were 155

performed on five plants (i.e., five leaves) in each growth chamber. All measurements took place 156

on leaves that had been exposed to at least 2 h of daytime lighting in the growth chambers. 157

158

Leaf and bulb respiration measurements 159

One hundred mg of fresh leaf discs (6 mm in diameter) and bulb pieces (4×4×1 mm) were 160

sampled from five plants per growth chamber per sampling stage, rinsed and vacuum infiltrated in 161

a syringe with reaction medium. The medium contained 100 mM mannitol, 10 mM HEPES (4-(2-162

hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM MES (2-(N-morpholino)ethanesulfonic 163

acid, pH 6.6) and 0.2 mM CaCl2, according to the method described by Jolivet et al. (1990). For 164

Page 7 of 34


Botany

Draft

8

the samples that were harvested at 50 % of leaf senescence (T5), a 50:50 mixture of green and 165

yellow leaf sections were used. The fragments were transferred to a dissolved O2 electrode 166

incubation chamber (Rank Brothers Ltd., Cambridge, UK). O2 uptake was measured with this 167

Clark-type polarographic electrode in 4 mL of air-saturated reaction medium under the day 168

growth temperatures (i.e., 8 °C, 12 °C and 18 °C, respectively), and at a common temperature of 169

12 °C. Aqueous KCN (1 mM) and salicylhydroxamic acid (SHAM, 10 mM), which was 170

dissolved in methoxy-ethanol, were used as inhibitors of the cytochrome pathway and of the 171

alternative pathway, respectively. The O2 electrode chamber was covered with aluminium foil 172

during leaf measurement, to ensure that leaf dark respiratory rates were being measured. 173

For both organs, total respiratory rate (RT) was measured at first without any inhibitors. The 174

capacity of the alternative pathway (Ralt) was determined using the equation: Ralt = RT+KCN – Rres, 175

where RT+KCN is the respiratory rate that was measured after the addition of the inhibitor KCN, 176

and Rres (residual respiratory rate) was measured by adding the second inhibitor SHAM. The 177

capacity of the cytochrome pathway (Rcyt) was determined by the equation: Rcyt = RT+SHAM – Rres, 178

where RT+SHAM is the respiratory rate that was measured when the inhibitor SHAM was added 179

first. 180

All leaf gas exchange data (assimilation and respiration measurements) were converted to the 181

same units (µmol O2. min-1. gDW-1). Given that the number of replicates in the assimilation 182

measurements (n = 5) and the respiration measurements (n = 2 to 4) were unequal, and done on 183

different samples, we used a permutation approach to calculate between 10 and 20 different 184

quotients of leaf dark respiration to net assimilation (leaf RT / A) per growth temperature and 185

phenological stage. All these estimated quotients were used as replicates in the statistical analysis. 186

We considered that there were only 5 replicates when calculating the standard error of the mean 187

in order to avoid under-estimating it. 188

189

Non-structural carbohydrate concentrations 190

Page 8 of 34


Botany

Draft

9

For each treatment and at each sampling stage, five plants were harvested 3 hours after the 191

beginning of the light period. Leaves and bulbs were separated and flash-frozen in liquid 192

nitrogen. Plant material was then freeze-dried, weighed and ground into fine powder in a ball mill 193

(Qiagen Inc., Toronto, Canada). Soluble sugars were analysed in both leaf and bulb samples, 194

whereas starch was only analysed in bulb samples, given that leaves in this species do not contain 195

significant quantities of starch (Gandin et al. 2009). Fifty mg of freeze-dried ground material was 196

macerated for 20 minutes in a methanol/chloroform/water solution (MCW 12:5:3, v/v) at 65 °C. 197

After homogenisation using a Polytron (Kinematica, Lucerne, Switzerland), the mixture was 198

centrifuged at 3 500 rpm for 10 minutes at 4 °C. The supernatant was then harvested and stored 199

temporarily on ice. In order to complete the sugars extraction from the remaining pellet, 200

homogenisation and centrifugation were repeated, and the second supernatant was added to the 201

first one. Total soluble sugars were analysed by reaction of 100 µL of the supernatant with 1.5 202

mL of freshly prepared anthrone solution (Hansen and Møller 1975) in warm water (60 °C) for 20 203

minutes. After cooling, the absorbance was determined at 620 nm with a spectrophotometer 204

(Beckman DU640, Beckman Coulter, USA). Glucose was used as standard. 205

The pellet containing starch was gelatinised in boiling water for 90 minutes, then hydrolysed 206

at 55 °C for 60 minutes in the presence of amyloglucosidase (Sigma-Aldrich, St. Louis, MO). 207

After cooling and centrifugation (3 500 rpm for 5 minutes), starch concentration was determined 208

colorimetrically at 415 nm on a glucose-equivalent basis using p-hydroxybenzoic acid hydrazide 209

(Blakeney and Mutton 1980). 210

211

Statistical analysis 212

Two-way ANOVAs were carried out to assess both the effect of growth temperature and 213

phenological stage on non-structural carbohydrate concentrations in the leaf and bulb, leaf and 214

bulb respiration measurements (RT, Rcyt and Ralt), leaf net assimilation (A) and the quotient of RT / 215

A in the leaf. These analyses were followed by Tukey HSD tests for multiple comparisons when 216

Page 9 of 34


Botany

Draft

10

main effect or the interaction was statistically significant. Statistical analysis was performed using 217

SAS version 9.4 (SAS Institute Inc., Cary, NC) and graphs were generated with Prism 6.0 218

(Graphpad Software Inc., La Jolla, CA). 219

220

Results 221

Plant phenology 222

Growth duration of E. americanum decreased as growth temperature increased, lasting 73 days, 223

56 days and 43 days for plants grown under the 8/6 °C, 12/8 °C and 18/14 °C regimes, 224

respectively (Fig. 1). Similar responses were exhibited for the individual phenological stages 225

(periods I to V). Leaf expansion (period I) lasted 9 days, 7 days and 5 days for plants grown at 8/6 226

°C, 12/8 °C and 18/14 °C, respectively. New bulb growth started within the core of the old bulb at 227

T2 (period III of the green-leaf period), which occurred at days 19, 15 and 11 for plants grown at 228

8/6 °C, 12/8 °C and 18/14 °C, respectively (Fig. 1). Initiation of leaf senescence (T4), which also 229

corresponds to the termination of new bulb growth, was recorded at days 47, 34 and 24 for plants 230

grown at 8/6 °C, 12/8 °C and 18/14 °C, respectively. Leaf senescence (periods IV + V) also lasted 231

longer under the coolest growth temperature regime (Fig. 1). 232

233

Leaf dark respiratory rates 234

Leaf RT was fairly constant throughout the growing season, except in senescing leaves (T5) where 235

it decreased (Fig. 2A and Table 1). Rcyt was highest at the beginning of the growth season when 236

the leaf was expanding, and gradually decreased with time until senescence (Fig. 2B and Table 1). 237

Ralt remained fairly constant over most of the season (Fig. 2C and Table 1), except during leaf 238

senescence where it declined. Plants grown at the three different growth temperatures exhibited 239

similar RT and Ralt when measured at their respective growth temperatures (Figs. 2A, 2C and 240

Table 1), whereas Rcyt was generally higher in plants grown at the highest growth temperature 241

(Fig. 2B and Table 1). 242

Page 10 of 34


Botany

Draft

11

When respiratory rates were measured at a common temperature (12 °C), RT was stimulated in 243

cold grown plants, but not through an increase in the capacity of either the cytochrome or the 244

alternative respiratory pathway (Figs. 2D, 2E and Table 1). An enhanced effect of low growth 245

temperature on Ralt was observed only at T2 (Fig. 2F). 246

247

Leaf A and RT / A quotient 248

Net assimilation (A) increased quickly from leaf unfolding (T1) to reach maximum rates at T3, 249

after which A decreased continuously until T5 (Fig. 3A and Table 1). Leaf A was significantly 250

higher in plants grown at warmer temperatures than in those grown at the cooler treatment (Fig. 251

3A and Table 1). The leaf RT / A quotient was high early in the season in plants regardless of their 252

growth temperature regime, then decreased to reach a minimum at either T3 (8/6 °C and 18/14 °C) 253

or T5 (12/8 °C). The RT / A quotient was significantly higher in plants grown at 8/6°C compared 254

to the other treatments. This was observed throughout the growing season, except at stage T4 255

where the quotients were similar among growth temperatures (Fig. 3B and Table 1). For plants 256

that were grown at 8/6 °C, leaf RT represented as much as 37 % of A early in the season, and 257

reached as low as 9 % at T3. The decrease of the leaf RT / A quotient throughout the season was 258

more moderate at the two warmer growth temperatures, from 16 % to 7 % at 12/8 °C and from 259

17 % to 6 % at 18/14 °C. 260

261

Bulb respiratory rates 262

Bulb respiratory rates are presented on a starch-free dry-mass basis (µmol O2. min-1. g starch-free 263

DW-1). This allowed us to gain a clearer picture of the changes in respiratory rates as a function of 264

metabolite concentrations in the soluble fraction of the cells, given that starch concentrations can 265

reach very high values towards the end of the season (Gandin et al. 2011). 266

As growth temperature × stage interactions were significant for each bulb respiration variable 267

measured at the growth temperature, we cannot describe general patterns that apply to all three 268

Page 11 of 34


Botany

Draft

12

growth temperature regimes (Table 1). At the coolest growth temperature, RT did not differ 269

through time; whereas Rcyt was lower early and late in the season (T1 and T5), Ralt was lower only 270

at T2 (Figs. 4A, 4B, 4C and Table 1). In plants that were grown at 12/8 °C, RT was lowest at T2 271

and increased from T2 to T4 where it reached a maximum. In contrast, Rcyt did not differ through 272

time, whereas Ralt was lowest at T3 and T5 (Figs. 4A, 4B, 4C and Table 1). In plants that were 273

grown under the warmest temperature regime, RT was also lowest at T2 and increased thereafter 274

to reach its maximum at T4. Both Rcyt and Ralt exhibited a similar pattern as RT that is, increasing 275

with time from T2 to T4 (Figs. 4A, 4B, 4C and Table 1). In general, all three respiratory variables 276

were higher in plants grown at the two warmer temperatures, although there were some inversions 277

at specific phenological stages. The most obvious one was at T2, where both RT and Rcyt were 278

higher in plants that were grown at the coolest temperature. 279

When measured at a common temperature (12 °C), growth temperature × stage interactions 280

were also significant for all variables (Table 1). Differences among plants grown under different 281

temperature regimes were less frequent than when respiratory rates were measured at their 282

respective growth temperatures. RT and Rcyt only differed early and late in the season (T1 and 283

T5), where plants that were grown at the warmest temperature exhibited the highest values (Figs. 284

4D, 4E and Table 1). Ralt was enhanced under the 12/8 °C regime, except at T5, where rates were 285

highest in plants grown at the coolest temperature (Figs. 4F and Table 1). 286

287

Soluble sugar concentrations 288

Concentrations of soluble sugars (SS) increased gradually in leaves during the growing season at 289

all three growth temperatures (Fig. 5A and Table 1), and sink limitation appeared to build up 290

earlier at warmer temperatures. Indeed, maximum SS concentrations were recorded a few days 291

prior to leaf senescence (T3) for plants that were grown at 18/14 °C, at the beginning of leaf 292

senescence (T4) for those that were grown at 12/8 °C, and only at mid-senescence (T5) for those 293

that were grown at 8/6 °C. These changes through time represented a two-fold increase for the 8/6 294

Page 12 of 34


Botany

Draft

13

°C and 12/8 °C grown plants, and a 1.5-fold increase for the 18/14 °C grown plants. For most of 295

the season, leaves of plants that were grown at the lower temperature contained higher SS 296

compared to those grown at higher temperatures. This was particularly obvious during the period 297

of leaf senescence (T4 and T5), where SS concentrations increased as growth temperature 298

decreased. 299

SS concentrations in the bulb are presented as a function of starch-free dry mass (mg. g starch-300

free DW-1), as was the case for bulb respiratory rates. At all three growth temperatures, SS 301

concentrations remained fairly constant while the old bulb was transferring C towards the leaf (T1 302

and T2); SS then increased to reach a maximum at T3, i.e., when the new bulb was growing and 303

accumulating starch (Fig. 5B and Table 1). However, these changes were not significant in plants 304

grown at the lowest temperature, given that their SS concentrations were also higher, early in the 305

season, than in plants grown at warmer temperatures. During leaf senescence, when the bulb was 306

no longer increasing in size, bulb SS concentrations declined about three-fold for plants that were 307

grown at 8/6 °C and 12/8 °C, and about two-fold for those that were grown at 18/14 °C. 308

Therefore, bulb SS concentrations were higher in plants that were grown at higher temperatures at 309

the onset (T4) and during leaf senescence (T5). Growth temperature modulated SS concentrations 310

differently in the leaf and bulb: maximum concentrations were not reached at the same 311

phenological stage, except for the plants grown at the warmest temperature regime. Towards the 312

end of the season, plants grown at warmer temperatures accumulated more SS in their bulbs, but 313

less in their leaves compared to plants grown at cooler temperatures. 314

315

Discussion 316

Thermal acclimation of respiration 317

Leaf RT of E. americanum exhibited homeostasis, which suggests that acclimation occurred in the 318

leaf in response to growth temperature. Similar results of leaf RT acclimation to changes in 319

growth temperature have been reported in arctic herb Saxifraga cernua (McNulty and Cummins 320

Page 13 of 34


Botany

Draft

14

1987), arctic-alpine Ranunculus glacialis (Arnone and Körner 1997), and many other species 321

(Loveys et al. 2003). In contrast, in Vigna radiata leaves and Glycine max cotyledons, plants 322

grown at cool or warm conditions exhibited different RT when measured at their respective 323

growth temperature, whereas no differences in respiration were observed when measured at the 324

same temperature (Gonzàlez-Meler et al. 1999). Indeed, when respiration was measured at a 325

common temperature of 12 °C, plants grown at the coolest temperature had higher leaf RT than 326

plants grown at warmer temperatures (Fig. 2D) suggesting an improved respiratory capacity to 327

compensate for the slower rates that occur at low temperature. However, it appears that 328

acclimation occurred only in plants grown at 8 °C, as plants grown at either 12/8 °C or 18/14 °C 329

exhibited similar leaf RT when rates were measured at a common temperature (Fig. 2D). This 330

result is consistent with what has been reported in Saxifraga cernua (McNulty and Cummins 331

1987), where the RT in cool-grown plants was much higher than in warm-grown plants when 332

measured at an intermediate temperature. Similarly, in the leaves of five temperate ruderal species 333

(Collier and Cummins 1990) and in Vigna radiata hypocotyls (Gonzàlez-Meler et al. 1999), RT of 334

cooler-grown plants was consistently greater than that from warmer-grown plants at any given 335

measurement temperature. 336

Thermal acclimation of respiration does not seem to occur in the bulb. Firstly, bulb RT was 337

sometimes lower, sometimes higher in the 8/6 °C plants than in the two other groups of plants, 338

depending on the phenological stage (Fig. 4A) indicating that homeostasis was not reached. 339

Secondly, bulb RT was very similar among plants grown at the different temperature regimes, 340

when measured at a common temperature (Fig. 4D). The two instances where RT differed among 341

growth temperatures (T1 and T5) could not be explained by thermal acclimation since one would 342

expect thermal acclimation to reduce RT in plants grown under higher temperature regime, while 343

we recorded increased RT values in these plants. There are very few studies reporting respiratory 344

rates of either corm, bulb or tuber as a function of growth temperature; the two we are aware of 345

were done on tissue culture in vitro. RT remains relatively constant in potato callus grown at 346

Page 14 of 34


Botany

Draft

15

different temperatures (8 to 28 °C) when measured at a common temperature (28 °C), although 347

the capacity of the alternative pathway increases exponentially with growth temperature 348

(Hemrika-Wagner et al. 1983). Yamagishi (1998) also reported similar RT in bulblets of Lilium 349

japonicum Thunb. grown at either 20 or 26 °C when measured at a common temperature. 350

Therefore, we conclude that bulb respiration did not acclimate to the different temperature 351

regimes. 352

Previous work in E. americanum has shown that A decreased as growth temperature decreased 353

(Gandin et al. 2011); the same trends were recorded in the current study (Fig. 3A). Leaf RT 354

acclimation at low growth temperature, associated with low A, resulted in high leaf RT / A 355

quotients at low growth temperature, which suggests that a reduced amount of carbohydrates was 356

available for translocation to the bulb. Despite the reduced amount of C available for 357

translocation, SS remained slightly higher in leaves of plants grown at low temperature than in 358

those of plants grown at the two other temperature regimes, suggesting that not only was there 359

less C available for translocation to the sink, but translocation rates were also most likely slower. 360

Translocation rates have previously been shown to be similar at 12 °C and 18 °C in another 361

spring geophyte (Badri et al. 2007), but it is possible that lower temperatures, such as 8 °C, do 362

slow down translocation. For instance, in winter wheat leaves, a proportionally lower C-export 363

rate has been found at 5 °C than at 20 °C (Leonardos et al. 2003). In summary, temperature 364

acclimation of respiration occurred only at the leaf level and only in the low-temperature-grown 365

plants. This thermal acclimation of respiration combined with reduced assimilation rates at low 366

temperature, further decreased the amount of C available for translocation to the sink. By 367

reducing the amount of C available for translocation, plants grown at low temperature would 368

establish a better equilibrium between source and sink activity, prolonging leaf life duration until 369

feedback inhibition of photosynthesis would induce leaf senescence. 370

We initially hypothesized that Ralt would be involved in the modulation of RT at both leaf and 371

bulb level as a response to temperature; yet similar low leaf Ralt were observed in plants grown at 372

Page 15 of 34


Botany

Draft

16

the different temperature regimes, whereas the growth temperature effect on bulb Ralt was not 373

consistent. Similar leaf Ralt among growth temperatures suggested that leaf Ralt thermally 374

acclimated, but differences were not large enough to be detected when measured under a common 375

temperature. Similar results were observed in white spruce (Picea glauca) roots where Ralt was 376

not affected by growth temperatures or measurement temperatures between 4 and 18 °C, and 377

represented 23 % of the total capacity of electron transport (Weger and Guy 1991). However, a 378

stimulation of Ralt at low growth temperatures has been reported in leaves of the perennial herb 379

Saxifraga cernua (McNulty and Cummins 1987) and of several temperate ruderal species (Collier 380

and Cummins 1990). The fact that growth rate was much less affected by low temperature in E. 381

americanum (Gandin et al. 2011) than in the species cited above might partly explain why leaf 382

Ralt was not strongly stimulated at low temperatures. When sink activity is decreased, C 383

accumulates in the leaves where it could stimulate Ralt. Gandin et al. (2009) have shown that bulb 384

Ralt can be strongly stimulated when E. americanum plants were grown under elevated CO2 385

concentrations (high source activity), indicating that bulb Ralt can be modulated in response to 386

source-sink imbalance. In warmer-grown plants, where C translocation was most likely higher 387

than in cool-grown plants, we did not detect a consistent increase in bulb Ralt. We conclude that 388

phenological stage has a stronger impact on the bulb respiratory rates than growth temperature. 389

390

Respiration as a function of phenological stage 391

Numerous studies have shown that plant respiration is modulated during organ development, with 392

higher rates of respiration in young compared to mature tissues (Azcon-Bieto et al. 1983; 393

McDonnell and Farrar 1993; Atkin and Cummins 1994; Armstrong et al. 2006). The rate of 394

respiration is often linearly related to relative growth rates of the tissues, which reflects 395

modulation in the production of energy and carbon skeletons to fulfil the needs for biosynthesis 396

and cellular maintenance (Lambers et al. 1998). In E. americanum, leaf RT was relatively constant 397

throughout the period leading up to senescence (Table 1), which would indicate that most of leaf 398

Page 16 of 34


Botany

Draft

17

growth was already achieved at T1. Nevertheless, leaf Rcyt was higher at T1, then decreased to 399

reach a stable level up to the beginning of leaf senescence. Florez-Sarasa et al. (2007) previously 400

demonstrated that growth respiration of Arabidopsis thaliana rosette leaves was largely 401

dependent on the activity of the cytochrome pathway, which would suggest that growth processes 402

requiring ATP still occurred at T1 in the leaf of E. americanum. We expected bulb Rcyt to be high 403

at T2 while cells were actively dividing within the new bulb, but this was not the case, except at 404

the coolest growth temperature (Table 1). At T2, the new bulb was very small; we posit that most 405

respiration originated from the old bulb that was being emptied by then and was less 406

metabolically active. 407

Unlike leaf RT, bulb RT exhibited a general increase through time, as the new bulb increased in 408

size and accumulated starch, except at the coolest growth temperature. The current results bring 409

support to the hypothesis that bulb RT increases as sink limitation builds up through time. Under 410

high CO2, where sink limitation was stronger, the increase in RT over time was more pronounced 411

than under ambient CO2 (Gandin et al. 2009) also pointing toward a modulation of bulb RT by 412

source-sink imbalance. Steingröver (1981) reported that Ralt was higher during early growth than 413

during extensive SS and biomass accumulation in the taproot of carrot (Daucus carota L.). 414

Similarly, Ralt decreased during inulin accumulation in storage roots of Hypochaeris radicata 415

(Lambers and Van de Dijk 1979). These authors concluded that « This suggests that sugars are 416

oxidized 'wastefully’ only if sugar supply from the shoots is in excess of the amount than can be 417

utilized for energy production, for structural growth or for storage ». In plants where most of the 418

growth of the perennial organ takes place later in the season, long after the perennial organ has 419

been developed, sink limitation is more likely to occur early in the season (Steingröver 1981). On 420

the other hand, in spring ephemerals where growth and storage occur concomitantly, sink 421

limitation is most likely to build up through time as growth slows down. However, regardless of 422

the timing of sink limitation, RT of the perennial organ appears to be strongly modulated by 423

source-sink balance. The improved balance between source and sink activity in cool-grown plants 424

Page 17 of 34


Botany

Draft

18

of E. americanum most likely explains why bulb RT did not steadily increase with time in these 425

plants compared to those grown at higher temperature. 426

As the respiratory rates that we measured were completely dependent upon endogenous 427

substrate levels, bulb RT could also vary with carbohydrate availability (Atkin and Tjoelker 2003), 428

which originated either from C stored in the old bulb or C translocated from the leaf. Changes in 429

bulb RT and bulb SS concentrations were not always related to each other, given that bulb SS 430

concentrations started decreasing between T3 and T4, while bulb RT continued to increase up to 431

T4 (Figs. 4A and 5B). At the lowest growth temperature, modulation of bulb SS concentrations 432

and bulb RT over time did not match one another. Similarly, the reduction in RT in taproot during 433

carbohydrate storage was not accompanied by a reduction in reducing sugar concentrations in 434

Hypochaeris radicata (Lambers and Van de Dijk 1979), nor in carrot (Steingröver 1981). Other 435

researchers have also reported conflicting results between carbohydrate levels within a tissue and 436

either RT (Atkin et al. 2000b) or Ralt (Wang et al. 2011). Taking into account the relative growth 437

rate of the storage organ, and thus its sink strength appears to better explain the modulation of its 438

respiratory rates over time than soluble sugar availability. 439

In conclusion, we recorded some acclimation of respiration at the leaf level, but only for RT, 440

not for either the capacity of the cytochrome or the alternative pathway of respiration. Low 441

temperature acclimation of leaf RT might partly contribute to the improved growth of the bulb 442

recorded at low growth temperature in spring ephemerals. Low temperature grown plants 443

modulated their leaf RT to reach leaf homeostasis; they also exhibited reduced A which lead to 444

higher leaf RT / A and most likely to reduced quantity of C translocated to the bulb at any given 445

time. This reduced amount of C translocated to the bulb would help maintain source and sink 446

activity in balance for a longer period of time at cooler temperatures, thus extending the duration 447

of the growth period. No temperature acclimation of either RT or Ralt was visible at the sink level, 448

and bulb RT did not vary in concert with SS concentrations. However, bulb RT did vary through 449

time at the two warmer temperatures, suggesting that bulb RT was being stimulated as sink 450

Page 18 of 34


Botany

Draft

19

limitation builds up. At the lowest temperature, bulb RT remained constant throughout the season, 451

in accordance with the better equilibrium between source and sink activity at that temperature. 452

453

Acknowledgements 454

This research was financially supported by a Discovery grant from the Natural Sciences and 455

Engineering Research Council of Canada (NSERC) to LL. The authors gratefully acknowledge 456

Mr. François Larochelle for his expert technical assistance in growth chamber experiments, Mr. 457

Gaétan Daigle for advice on statistical analyses, and Dr. William F.J. Parsons for English 458

language revision. The authors also sincerely thank all colleagues in the laboratory who 459

participated in plant harvesting: Moustafa A. Khalf, Julie Bussières, Pierre-Paul Dion, Jade 460

Boulanger Pelletier, Karolane Pitre and Dominique Manny. 461

462

References 463

Ainsworth, E.A., and Bush, D.R. 2011. Carbohydrate export from the leaf: a highly regulated 464

process and target to enhance photosynthesis and productivity. Plant Physiol. 155(1): 64-69. 465

doi: 10.1104/pp.110.167684. 466

Armstrong, A.F., Logan, D.C., and Atkin, O.K. 2006. On the developmental dependence of leaf 467

respiration: responses to short-and long-term changes in growth temperature. Am. J. Bot. 468

93(11): 1633-1639. doi: 10.3732/ajb.93.11.1633. 469

Arnone III, J.A., and Körner, C. 1997. Temperature adaptation and acclimation potential of leaf 470

dark respiration in two species of Ranunculus from warm and cold habitats. Arct. Alp. Res. 471

29(1): 122-125. doi : 10.2307/1551842. 472

Atkin, O.K., and Cummins, W.R. 1994. The effect of nitrogen source on growth, nitrogen 473

economy and respiration of two high arctic plant species differing in relative growth rate. 474

Funct. Ecol. 8(3): 389-399. doi: 10.2307/2389833. 475

Page 19 of 34


Botany

Draft

20

Atkin, O.K., and Tjoelker, M.G. 2003. Thermal acclimation and the dynamic response of plant 476

respiration to temperature. Trends Plant Sci. 8(7): 343-351. doi: 10.1016/S1360-477

1385(03)00136-5. 478

Atkin, O.K., Edwards, E.J., and Loveys, B.R. 2000a. Response of root respiration to changes in 479

temperature and its relevance to global warming. New Phytol. 147(1): 141-154. doi: 480

10.1046/j.1469-8137.2000.00683.x. 481

Atkin, O.K., Holly, C., and Ball, M. 2000b. Acclimation of snow gum (Eucalyptus pauciflora) 482

leaf respiration to seasonal and diurnal variations in temperature: the importance of changes in 483

the capacity and temperature sensitivity of respiration. Plant Cell Environ. 23(1): 15-26. doi: 484

10.1046/j.1365-3040.2000.00511.x. 485

Atkin, O.K., Bruhn, D., Hurry, V.M., and Tjoelker, M.G. 2005a. The hot and the cold: 486

unravelling the variable response of plant respiration to temperature. Funct. Plant Biol. 32(2): 487

87-105. doi: 10.1071/FP03176. 488

Atkin, O.K., Bruhn, D., and Tjoelker, M.G. 2005b. Response of plant respiration to changes in 489

temperature: mechanisms and consequences of variations in Q10 values and acclimation. In 490

Plant Respiration. Edited by H. Lambers and M. Ribas-Carbo. Springer. pp. 95-135. 491

Azcon-Bieto, J., Lambers, H., and Day, D.A. 1983. Respiratory properties of developing bean and 492

pea leaves. Funct. Plant Biol. 10(3): 237-245. doi: 10.1071/PP9830237. 493

Badri, M.A., Minchin, P.E.H., and Lapointe, L. 2007. Effects of temperature on the growth of 494

spring ephemerals: Crocus vernus. Physiol. Plant. 130(1): 67-76. doi: 10.1111/j.1399-495

3054.2007.00882.x. 496

Basu, P., Sharma, A., Garg, I., and Sukumaran, N. 1999. Tuber sink modifies photosynthetic 497

response in potato under water stress. Environ. Exp. Bot. 42(1): 25-39. doi: 10.1016/S0098-498

8472(99)00017-9. 499

Page 20 of 34


Botany

Draft

21

Bernatchez, A., and Lapointe, L. 2012. Cooler temperatures favour growth of wild leek (Allium 500

tricoccum), a deciduous forest spring ephemeral. Botany, 90(11): 1125-1132. doi: 501

10.1139/B2012-089. 502

Berry, J., and Bjorkman, O. 1980. Photosynthetic response and adaptation to temperature in 503

higher plants. Annu. Rev. Plant Physiol. 31(1): 491-543. doi: 504

10.1146/annurev.pp.31.060180.002423. 505

Blakeney, A.B., and Mutton, L.L. 1980. A simple colorimetric method for the determination of 506

sugars in fruit and vegetables. J. Sci. Food Agric. 31(9): 889-897. doi: 507

10.1002/jsfa.2740310905. 508

Campbell, C., Atkinson, L., Zaragoza-Castells, J., Lundmark, M., Atkin, O., and Hurry, V. 2007. 509

Acclimation of photosynthesis and respiration is asynchronous in response to changes in 510

temperature regardless of plant functional group. New Phytol. 176(2): 375-389. doi: 511

10.1111/j.1469-8137.2007.02183.x. 512

Collier, D.E., and Cummins, W.R. 1990. The effects of low growth and measurement temperature 513

on the respiratory properties of five temperate species. Ann. Bot. 65(5): 533-538. Available 514

from https://doi.org/10.1093/oxfordjournals.aob.a087966 [accessed 8 September 2017] 515

Dewar, R.C., Medlyn, B.E., and Mcmurtrie, R. 1999. Acclimation of the 516

respiration/photosynthesis ratio to temperature: insights from a model. Glob. Change Biol. 517

5(5): 615-622. doi: 10.1046/j.1365-2486.1999.00253.x. 518

Florez-Sarasa, I.D., Bouma, T.J., Medrano, H., Azcon-Bieto, J., and Ribas-Carbo, M. 2007. 519

Contribution of the cytochrome and alternative pathways to growth respiration and 520

maintenance respiration in Arabidopsis thaliana. Physiol. Plant. 129(1): 143-151. doi: 521

10.1111/j.1399-3054.2006.00796.x. 522

Foyer, C., Furbank, R., Harbinson, J., and Horton, P. 1990. The mechanisms contributing to 523

photosynthetic control of electron transport by carbon assimilation in leaves. Photosyn. Res. 524

25(2): 83-100. doi: 10.1007/BF00035457. 525

Page 21 of 34


Botany

Draft

22

Gandin, A., Lapointe, L., and Dizengremel, P. 2009. The alternative respiratory pathway allows 526

sink to cope with changes in carbon availability in the sink-limited plant Erythronium 527

americanum. J. Exp. Bot. 60(15): 4235-4248. doi: 10.1093/Jxb/Erp255. 528

Gandin, A., Gutjahr, S., Dizengremel, P., and Lapointe, L. 2011. Source-sink imbalance increases 529

with growth temperature in the spring geophyte Erythronium americanum. J. Exp. Bot. 530

62(10): 3467-3479. doi: 10.1093/Jxb/Err020. 531

Giraud, E., Ho, L.H., Clifton, R., Carroll, A., Estavillo, G., Tan, Y.F., Howell, K.A., Ivanova, A., 532

Pogson, B.J., and Millar, A.H. 2008. The absence of ALTERNATIVE OXIDASE1a in 533

Arabidopsis results in acute sensitivity to combined light and drought stress. Plant Physiol. 534

147(2): 595-610. doi: 10.1104/pp.107.115121. 535

Goldschmidt, E.E., and Huber, S.C. 1992. Regulation of photosynthesis by end-product 536

accumulation in leaves of plants storing starch, sucrose, and hexose sugars. Plant Physiol. 537

99(4): 1443-1448. doi: 10.1104/pp.99.4.1443. 538

Gonzàlez-Meler, M.A., Ribas-Carbo, M., Giles, L., and Siedow, J.N. 1999. The effect of growth 539

and measurement temperature on the activity of the alternative respiratory pathway. Plant 540

Physiol. 120(3): 765-772. doi: 10.1104/pp.120.3.765. 541

Hansen, J., and Møller, I. 1975. Percolation of starch and soluble carbohydrates from plant tissue 542

for quantitative determination with anthrone. Anal. Biochem. 68(1): 87-94. doi: 10.1016/0003-543

2697(75)90682-X. 544

Hemrika‐Wagner, A.M., Verschoor, E.J., and Plas, L.H. 1983. Alternative pathway respiration in 545

vivo of potato tuber callus grown at various temperatures. Physiol. Plant. 59(3): 369-374. doi : 546

10.1111/j.1399-3054.1983.tb04217.x. 547

Hoch, G., Popp, M., and Körner, C. 2002. Altitudinal increase of mobile carbon pools in Pinus 548

cembra suggests sink limitation of growth at the Swiss treeline. Oikos, 98(3): 361-374. doi: 549

10.2307/3547177. 550

Page 22 of 34


Botany

Draft

23

Jolivet, Y., Pireaux, J.C., and Dizengremel, P. 1990. Changes in properties of barley leaf 551

mitochondria isolated from sodium chloride treated plants. Plant Physiol. 94(2): 641-646. doi: 552

10.1104/pp.94.2.641. 553

Krapp, A., and Stitt, M. 1995. An evaluation of direct and indirect mechanisms for the “sink-554

regulation” of photosynthesis in spinach: changes in gas exchange, carbohydrates, metabolites, 555

enzyme activities and steady-state transcript levels after cold-girdling source leaves. Planta, 556

195(3): 313-323. Available from https://link.springer.com/article/10.1007/BF00202587 557

[accessed 8 September 2017]. 558

Lambers, H. 1982. Cyanide-resistant respiration: a non-phosphorylating electron-transport 559

pathway acting as an energy overflow. Physiol. Plant. 55(4): 478-485. doi: 10.1111/J.1399-560

3054.1982.Tb04530.X. 561

Lambers, H., Chapin III, F.S., and Pons, T.L. 1998. Plant physiological ecology. J. Agron. Crop 562

Sci. 184(2): 143–144. doi: 10.1046/j.1439-037x.2000.00378-1.x. 563

Lambers, H., and Van der Dijk, S.J. 1979. Cyanide‐resistant root respiration and tap root 564

formation in two subspecies of Hypochaeris radicata. Physiol. Plant. 45(2): 235-239. doi : 565

10.1111/j.1399-3054.1979.tb01693.x. 566

Lapointe, L., and Lerat, S. 2006. Annual growth of the spring ephemeral Erythronium 567

americanum as a function of temperature and mycorrhizal status. Can. J. Bot. 84(1): 39-48. 568

doi: 10.1139/B05-140. 569

Leonardos, E.D., Savitch, L.V., Huner, N., Öquist, G., and Grodzinski, B. 2003. Daily 570

photosynthetic and C‐export patterns in winter wheat leaves during cold stress and 571

acclimation. Physiol. Plant. 117(4): 521-531. doi: 10.1034/j.1399-3054.2003.00057.x. 572

Levitt, J. 1972. Responses of plants to environmental stresses. Academic Press, New York. 573

Loveys, B.R., Atkinson, L.J., Sherlock, D.J., Roberts, R.L., Fitter, A.H., and Atkin, O.K. 2003. 574

Thermal acclimation of leaf and root respiration: an investigation comparing inherently fast‐575

Page 23 of 34


Botany

Draft

24

and slow‐growing plant species. Glob. Change Biol. 9(6): 895-910. doi: 10.1046/j.1365-576

2486.2003.00611.x. 577

Lundmark, M., Hurry, V., and Lapointe, L. 2009. Low temperature maximizes growth of Crocus 578

vernus (L.) Hill via changes in carbon partitioning and corm development. J. Exp. Bot. 60(7): 579

2203-2213. doi: 10.1093/jxb/erp103. 580

McDonnell, E., and Farrar, J.F. 1993. Respiratory characteristics of isolated barley mitochondria 581

and intact barley roots. J. Exp. Bot. 44(9): 1485-1490. doi: 10.1093/jxb/44.9.1485. 582

McNulty, A.K., and Cummins, W.R. 1987. The relationship between respiration and temperature 583

in leaves of the arctic plant Saxifraga cernua. Plant Cell Environ. 10(4): 319-325. doi: 584

10.1111/j.1365-3040.1987.tb01612.x. 585

Noguchi, K., and Terashima, I. 2006. Responses of spinach leaf mitochondria to low N 586

availability. Plant Cell Environ. 29(4): 710-719. doi: 10.1111/j.1365-3040.2005.01457.x. 587

Paul, M.J., Lawlor, D.W., and Driscoll, S.P. 1990. The effect of temperature on photosynthesis 588

and carbon fluxes in sunflower and rape. J. Exp. Bot. 41(226): 547-555. doi: 589

10.1093/jxb/41.5.547. 590

Raison, J.K. 1980. Effect of low temperature on respiration. In The Biochemistry of plants: a 591

comprehensive treatise (USA). Edited by D.D. Davies. Academic Press, New York. pp. 613-592

626. 593

Sieger, S.M., Kristensen, B.K., Robson, C.A., Amirsadeghi, S., Eng, E.W.Y., Abdel-Mesih, A., 594

Møller, I.M., and Vanlerberghe, G.C. 2005. The role of alternative oxidase in modulating 595

carbon use efficiency and growth during macronutrient stress in tobacco cells. J. Exp. Bot. 596

56(416): 1499-1515. doi: 10.1093/jxb/eri146. 597

Steingröver, E. 1981. The relationship between cyanide-resistant root respiration and the storage 598

of sugars in the taproot in Daucus carota L. J. Exp. Bot. 32(130):911-919. doi: 599

10.1093/jxb/32.5.911. 600

Page 24 of 34


Botany

Draft

25

Strand, Å., Hurry, V., Gustafsson, P., and Gardeström, P. 1997. Development of Arabidopsis 601

thaliana leaves at low temperatures releases the suppression of photosynthesis and 602

photosynthetic gene expression despite the accumulation of soluble carbohydrates. Plant J. 603

12(3): 605-614. doi: 10.1046/j.1365-313X.1997.00605.x. 604

Vanlerberghe, G.C. 2013. Alternative oxidase: a mitochondrial respiratory pathway to maintain 605

metabolic and signaling homeostasis during abiotic and biotic stress in plants. Int. J. Mol. Sci. 606

14(4): 6805-6847. doi: 10.3390/ijms14046805. 607

Vanlerberghe, G.C., and McIntosh, L. 1992. Lower growth temperature increases alternative 608

pathway capacity and alternative oxidase protein in tobacco. Plant Physiol. 100(1): 115-119. 609

doi: 10.1104/pp.100.1.115. 610

Wang, J., Rajakulendran, N., Amirsadeghi, S., and Vanlerberghe, G.C. 2011. Impact of 611

mitochondrial alternative oxidase expression on the response of Nicotiana tabacum to cold 612

temperature. Physiol. Plant. 142(4): 339-351. doi: 10.1111/j.1399-3054.2011.01471.x. 613

Weger, H.G., and Guy, R.D.1991. Cytochrome and alternative pathway respiration in white 614

spruce (Picea glauca) roots. Effects of growth and measurement temperature. Physiol. Plant. 615

83(4): 675-681. doi: 10.1111/j.1399-3054.1991.tb02486.x. 616

Yamagishi, M. 1998. Effects of culture temperature on the enlargement, sugar uptake, starch 617

accumulation, and respiration of in vitro bulblets of Lilium japonicum Thunb. Sci. Hortic. 618

73(4): 239-247. doi: 10.1016/S0304-4238(97)00159-3. 619

620

Page 25 of 34


Botany

Draft

26

Table 1. Summary of two-way ANOVA on the effects of stage (T1 to T5) and growth temperature (8/6 °C, 12/8 °C and 18/14 °C)

on leaf and bulb respiratory rates (RT, Rcyt and Ralt), leaf net assimilation (A), leaf dark respiration to assimilation ratio (leaf RT /

A), and leaf and bulb soluble sugar (SS) concentrations. Bulb respiratory rates and bulb SS concentrations are presented on a

starch-free bulb dry-mass basis. F-values are presented, followed by significance level (*, **, and *** denote a significant

difference at P < 0.05, < 0.01, and < 0.001, respectively). Multiple comparisons among temperatures and among stages are also

shown. Lowercase letters denote significant differences (P < 0.05) among stages (comparisons in each row), and uppercase letters

refer to significant differences (P < 0.05) among temperatures (comparisons in each column). Absence of lowercase letters in rows

(stage comparisons) and uppercase letters in columns (temperature comparisons) indicates non-significant differences. GT, growth

temperature; MT, measurement temperature.

Variables GT MT

F-values 1 Multiple comparisons

T°C Stage

T°C ×

Stage for Stage only

2 and T°C × Stage for T°C

only 3 T1 T2 T3 T4 T5

Leaf RT 8/6°C 8°C

0.5 12.0*** 0.8

a

a

a

a

b

12/8°C 12°C 18/14°C 18°C

8/6°C 12°C 6.0** 12.2*** 0.2

a

a

a

a

b

A

12/8°C 12°C B

18/14°C 12°C B

Leaf Rcyt 8/6°C 8°C 4.3 * 31.7*** 1.5

a

b

b

b

c

B

12/8°C 12°C B

18/14°C 18°C A

8/6°C 12°C 0.4 18.7*** 1.0

b

a

bc

cd

d

12/8°C 12°C

18/14°C 12°C

Leaf Ralt 8/6°C 8°C 0.9 6.3*** 1.6

ab

ab

a

a

b

12/8°C 12°C

18/14°C 18°C

8/6°C 12°C

0.0 20.5*** 2.9 * c B a A b ab c

12/8°C 12°C a AB a B a a b

18/14°C 12°C a A a C a a b

Leaf A 8/6°C 8°C 230.1*** 187.3*** 16.4***

e C c C a B b B d B

12/8°C 12°C c B b B a B b B c A

18/14°C 18°C d A c A a A b A e A

Leaf RT / A 8/6°C 8°C 52.5*** 57.2*** 6.9***

a A b A d A cd c A

12/8°C 12°C a B b B b A b c B

18/14°C 18°C a B bc C d B b cd B

Leaf SS 8/6°C

21.8*** 19.9*** 4.2 **

c bc A b b A a A

12/8°C

c bc AB ab a A ab B

18/14°C

b b B a a B c C

Page 26 of 34


Botany

Draft

27

Table 1 (continued)

Variables GT MT

F-values 1 Multiple comparison

T°C Stage

T°C ×

Stage for Stage only

2 and T°C × Stage

for T°C

only 3 T1

T2

T3

T4

T5

Bulb RT 8/6°C 8°C

9.1** 9.26 ** 7.4*** B A B B

12/8°C 12°C ab AB d B bc a A cd B

18/14°C 18°C ab A c B b a A b A

8/6°C 12°C

6.6 ** 9.0 ** 3.2 * B B

12/8°C 12°C ab B d bc a cd B

18/14°C 12°C a A c b b b A

Bulb Rcyt 8/6°C 8°C

1.0 1.8 4.8 ** b a A a a B ab A

12/8°C 12°C AB B B

18/14°C 18°C b c B b a A b A

8/6°C 12°C

2.5 0.8 3.7 ** b B ab a a ab B

12/8°C 12°C AB

B

18/14°C 12°C a A b b b a A

Bulb Ralt 8/6°C 8°C

2.6 3.2 * 7.0*** a b B a A a B a A

12/8°C 12°C a a A b B a A b B

18/14°C 18°C b ab AB ab A a A ab A

8/6°C 12°C

0.2 1.5 6.4*** b b B ab ab B a A

12/8°C 12°C a a A b a A b B

18/14°C 12°C AB

B AB

Bulb SS 8/6°C

4.8 * 30.5*** 6.6*** a a A a b B b B

12/8°C ab bc B a ab AB c B

18/14°C b b B a ab A b A

1 Significance level: *, ** and *** denote a significant difference at P < 0.05, 0.01 and 0.001, respectively.

2 Multiple comparisons for Stage effect were shown only when Stage effect was significant, but T °C × Stage

interaction effect was not significant.

3 Multiple comparisons for T °C effect were shown only when T °C effect was significant, but T °C × Stage

interaction effect was not significant.

Page 27 of 34


Botany

Draft

28

Figure Captions

Figure 1. Representative illustration of plant phenology of E. americanum grown at 8/6 °C, 12/8 °C and

18/14 °C. Shown from left to right: duration of leaf expansion (period I), duration of green leaf (period II

and III) and leaf senescence period (period IV and V). Arrows indicate the sampling stages T1 to T5.

Numbers indicate the duration (in days) of each stage.

Figure 2. Total dark respiratory rate (leaf RT, µmol O2. min-1. g DW-1, A and D), capacity of the

cytochrome pathway (leaf Rcyt, µmol O2. min-1. g DW-1, B and E) and capacity of the alternative pathway

(leaf Ralt, µmol O2. min-1. g DW-1, C and F) in the leaves of plants grown at 8/6 °C (white), 12/8 °C (grey)

and 18/14 °C (black) during the green leaf period and leaf senescence (T1 to T5), measured at growth

temperature (A, B and C) and a common temperature of 12 °C (D, E and F). Means ± standard error of the

mean (SEM) are presented (n = 2 to 4). * and ** denote that the respiratory rates differed for at least two

out of the three growth temperatures at P < 0.05 and < 0.01, respectively, within each stage when

Temperature × Stage interaction effect was significant. See Table 1 for results of multiple test

comparisons.

Figure 3. Leaf net assimilation (leaf A, µmol O2. min-1. g DW-1, A) and quotient of leaf total dark

respiration to leaf net assimilation (leaf RT / A quotient, B) of E. americanum grown at 8/6 °C (white),

12/8 °C (grey) and 18/14 °C (black) during the green leaf period and leaf senescence (T1 to T5). Means ±

standard error of the mean (SEM) are presented (n = 5). *** denotes that A differed for at least two out of

the three growth temperatures at P < 0.001 within each stage. See Table 1 for results of multiple test

comparisons. Different letters refer to significant differences (P < 0.05) among temperatures.

Figure 4. Total respiratory rate (bulb RT, µmol O2. min-1. g starch-free DW-1, A and D), capacity of the

cytochrome pathway (bulb Rcyt, µmol O2. min-1. g starch-free DW-1, B and E) and capacity of the

Page 28 of 34


Botany

Draft

29

alternative pathway (bulb Ralt, µmol O2. min-1. g starch-free DW-1, C and F) in the bulb of plants grown at

8/6 °C (white), 12/8 °C (grey) and 18/14 °C (black) during the green leaf period and leaf senescence (T1

to T5), measured at growth temperature (A, B and C) and a common temperature 12 °C (D, E and F).

Means ± standard error of the mean (SEM) are presented (n = 2 to 4). *, **, and *** denote that the

respiratory rates differed for at least two out of the three growth temperatures at P < 0.05, < 0.01 and <

0.001, respectively, within each stage. See Table 1 for results of multiple test comparisons.

Figure 5. Soluble sugar concentrations in the leaf (mg. g DW-1, A) and bulb (mg. g starch-free DW-1, B)

of E. americanum plants grown at 8/6 °C (white), 12/8 °C (grey) and 18/14 °C (black) during the green

leaf period and leaf senescence (T1 to T5). Data are expressed as mean ± standard error of the mean

(SEM) (n = 5). *, **, and *** denote that the respiratory rates differed for at least two out of the three

growth temperatures at P < 0.05, < 0.01, and < 0.001, respectively, within each stage. See Table 1 for

results of multiple test comparisons.

Page 29 of 34


Botany

Draft

Figure 1. Representative illustration of plant phenology of E. americanum grown at 8/6 °C, 12/8 °C and 18/14 °C. Shown from left to right: duration of leaf expansion (period I), duration of green leaf (period II and III) and leaf senescence period (period IV and V). Arrows indicate the sampling stages T1 to T5.

Numbers indicate the duration (in days) of each stage.

146x84mm (300 x 300 DPI)

Page 30 of 34


Botany

Draft

Figure 2. Total dark respiratory rate (leaf RT, µmol O2. min-1. g DW-1, A and D), capacity of the cytochrome pathway (leaf Rcyt, µmol O2. min-1. g DW-1, B and E) and capacity of the alternative pathway (leaf Ralt, µmol

O2. min-1. g DW-1, C and F) in the leaf of plants grown at 8/6 °C (white), 12/8 °C (grey) and 18/14 °C

(black) during the green leaf period and leaf senescence (T1 to T5), measured at growth temperature (A, B and C) and a common temperature 12 °C (D, E and F). Means ± standard error of the mean (SEM) are

presented (n = 2 to 4). * and ** denote that the respiratory rates differed for at least two out of the three growth temperatures at P < 0.05 and < 0.01, respectively, within each stage when Temperature × Stage

interaction effect was significant. See Table 1 for results of multiple test comparisons.

177x162mm (300 x 300 DPI)

Page 31 of 34


Botany

Draft

Figure 3. Leaf net assimilation (leaf A, µmol O2. min-1. g DW-1, A) and quotient of leaf total dark respiration to leaf net assimilation (leaf RT / A quotient, B) of E. americanum grown at 8/6 °C (white), 12/8 °C (grey)

and 18/14 °C (black) during the green leaf period and leaf senescence (T1 to T5). Means ± standard error of

the mean (SEM) are presented (n = 5). *** denotes that A differed for at least two out of the three growth temperatures at P < 0.001 within each stage. See Table 1 for results of multiple test comparisons. Different

letters refer to significant differences (P < 0.05) among temperatures.

189x202mm (300 x 300 DPI)

Page 32 of 34


Botany

Draft

Figure 4. Total respiratory rate (bulb RT, µmol O2. min-1. g starch-free DW-1, A and D), capacity of the cytochrome pathway (bulb Rcyt, µmol O2. min-1. g starch-free DW-1, B and E) and capacity of the alternative

pathway (bulb Ralt, µmol O2. min-1. g starch-free DW-1, C and F) in the bulb of plants grown at 8/6 °C

(white), 12/8 °C (grey) and 18/14 °C (black) during the green leaf period and leaf senescence (T1 to T5), measured at growth temperature (A, B and C) and a common temperature 12 °C (D, E and F). Means ± standard error of the mean (SEM) are presented (n = 2 to 4). *, **, and *** denote that the respiratory

rates differed for at least two out of the three growth temperatures at P < 0.05, < 0.01 and < 0.001, respectively, within each stage. See Table 1 for results of multiple test comparisons.

183x177mm (300 x 300 DPI)

Page 33 of 34


Botany

Draft

Figure 5. Soluble sugar concentrations in the leaf (mg. g DW-1, A) and bulb (mg. g starch-free DW-1, B) of E. americanum plants grown at 8/6 °C (white), 12/8 °C (grey) and 18/14 °C (black) during the green leaf

period and leaf senescence (T1 to T5). Data are expressed as mean ± standard error of the mean (SEM) (n

= 5). *, **, and *** denote that the respiratory rates differed for at least two out of the three growth temperatures at P < 0.05, < 0.01, and < 0.001, respectively, within each stage. See Table 1 for results of

multiple test comparisons.

192x221mm (300 x 300 DPI)

Page 34 of 34


Botany

Documents

Draft - University of Toronto T-Space · Draft 2 16 Abstract 17 Many spring geophytes exhibit greater growth at colder than at warmer temperatures. Previous 18 studies have suggested