The thermal tolerance of photosynthetic tissues: a global systematic review and agenda for future research

1 Research Review 1 The thermal tolerance of photosynthetic tissues: a global systematic

2 review and agenda for future research 3

4 Sonya R. Geange1,2,‡,*, Pieter A. Arnold1,‡,*, Alexandra A. Catling1,3, Onoriode Coast1,4,

5 Alicia M. Cook5, Kelli M. Gowland1, Andrea Leigh5, Rocco F. Notarnicola1,

6 Bradley C. Posch1, Susanna E. Venn6, Lingling Zhu1, Adrienne B. Nicotra1

7

8 1 Research School of Biology, The Australian National University, Canberra, ACT, Australia

9 2 Department of Biological Sciences, University of Bergen, Thormøhlensgt, Bergen, Norway

10 3 School of Biological Sciences, The University of Queensland, Brisbane, QLD, Australia

11 4 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent

12 ME4 4TB, United Kingdom 13 5 School of Life Sciences, University of Technology Sydney, Broadway, NSW, Australia

14 6 School of Life and Environmental Sciences, Deakin University, Melbourne, VIC, Australia

15 ‡ Sonya R. Geange and Pieter A. Arnold should be considered joint first author

16 * Corresponding authors:

17 Sonya R. Geange (phone: +447432057249, email: sonya.geange@uib.no)

18 Pieter A. Arnold (phone: +61261252543, email: pieter.arnold@anu.edu.au)

19

20 Word counts:

21 Summary: 200 22 Main text: 8121 23 No. of figures: 5 (all in colour)

24 No. of tables: 2 25 Supporting information: Figs S1-S8, Notes S1

26 2 Summary 27 Understanding plant thermal tolerance is fundamental to predicting impacts of extreme temperature

28 events that are increasing in frequency and intensity across the globe. Extremes, not averages, drive

29 species evolution, determine survival, and increased crop performance. To better prioritise

30 agricultural and natural system research, it is crucial to evaluate how researchers are assessing the

31 capacity of plants to tolerate extreme events. We conducted a systematic review to determine how

32 plant thermal tolerance research is distributed across wild and domesticated plants, growth forms

33 and biomes, and identify crucial knowledge gaps. Our review shows that most thermal tolerance

34 research examines cold tolerance of cultivated species; ~5% of articles consider both heat and cold

35 tolerance. Plants of extreme environments are understudied, and techniques widely applied in

36 cultivated systems are largely unused in natural systems. Lastly, we find that lack of standardised

37 methods and metrics compromises the potential for mechanistic insight. Our review provides an

38 entry point for those new to the methods used in plant thermal tolerance research and bridges often

39 disparate ecological and agricultural perspectives for the more experienced. We present a

40 considered agenda of thermal tolerance research priorities to stimulate efficient, reliable, and

41 repeatable research across the spectrum of plant thermal tolerance.

42

43 Keywords: agriculture, climate change, extreme, temperature, thermal breadth, thermotolerance,

44 warming.

45 46 3 Introduction 47 As the Earth’s climate changes, our dependence on healthy vegetation systems is coming into

48 sharp focus. Temperature is arguably the most important determinant of plant species adaptation

49 and distribution across the planet (Nievola et al., 2017). Researchers seek to understand plant

50 species responses to temperature to breed crops for a growing population, gain fundamental insight

51 into physiological, ecological, and evolutionary processes, and predict responses of wild species to

52 the changing climate. There has been an ever-increasing number of publications over the last

53 century in various specialist fields of plant thermal tolerance research, but the work is scattered

54 across different fields and geographic regions. Thus, as a research community we cannot easily and

55 objectively prioritise research effort or effectively summarise what the thousands of published

56 studies tell us about plant thermal tolerance.

57 Many biological processes are fundamentally dependent on temperature: including growth,

58 reproduction and, in plants, photosynthesis. Classic studies have established that thermal limits are

59 key to establishing the distribution limits of land plants, constraining the survival of plant tissue

60 between -60°C and +60°C, where species growing in the most extreme biomes exhibit a range of

61 adaptations to function and persist (Osmond et al., 1987). Importantly, it is extreme low and high

62 temperatures that can impair physiological functions, growth, and determine survival by profoundly

63 changing the structure and fluidity of cell membranes, altering enzyme function, and destroying

64 proteins (Osmond et al., 1987; Sung et al., 2003; Hatfield & Prueger, 2015). Extreme temperature

65 events that are increasing in frequency and severity (IPCC, 2018) can affect organisms profoundly

66 and are a major driving force for selection, adaptation, and species persistence (Gutschick &

67 BassiriRad, 2003; Buckley & Huey, 2016; Lancaster & Humphreys, 2020).

68 Studies have shown that plant cold tolerance varies depending on factors such as elevation,

69 ontogeny (Marcante et al., 2012; Sierra-Almeida & Cavieres, 2012), microsites (i.e. sheltered vs

70 exposed) (Bannister et al., 2005; Briceño et al., 2014; Venn & Green, 2018), and water availability

71 (Sierra-Almeida et al., 2009; Venn et al., 2013). For example, alpine plants can withstand very low

72 temperatures and tolerate extracellular ice formation and the resulting dehydration (Sakai &

73 Larcher, 1987; Larcher, 2003). Higher heat tolerance is found at lower absolute latitudes and is

74 positively correlated with mean annual temperature (Lancaster & Humphreys, 2020). For a given

75 latitude, desert species have higher tolerance to heat relative to coastal congeneric species in situ,

76 but these differences can diminish under common garden conditions (Knight & Ackerly, 2002;

77 2003). Recent studies of Australian desert species have found that within a single desert biome,

78 species vary widely in their physiological response to high temperature (with critical temperatures

79 4 ranging from 48-54°C). Further, critical damage thresholds are driven less by macro-scale climate

80 or latitude, than by microhabitat variation, especially soil moisture variation (Curtis et al., 2016).

81 Crops are susceptible to temperature extremes and exposure to sub- and supra-optimal

82 temperatures can cause significant yield losses. The degree of susceptibility to temperature stress

83 varies with species, duration, intensity, and developmental stage. Extreme heat after seedling

84 establishment can scorch leaves, impair biochemical processes, and accelerate premature

85 senescence. Cold or heat stress coinciding with reproductive development in major cereal crops (the

86 most temperature-sensitive stage; Yoshida et al., 1981) negatively affects reproductive processes

87 and structures, which consequently reduces yield quantity and quality (Jagadish et al., 2007; Coast

88 et al., 2016). If, and to what extent, crops acclimate to thermal stress is still being tested. However,

89 research is increasingly showing that crop varieties can acclimate their physiology to both low

90 (Yamori et al., 2010) and high temperatures (Li et al., 1991; Wang et al., 2011) to varying extents,

91 similar to that observed in wild species.

92 Our rapidly changing climate means that extreme events are having major impacts on wild

93 and agricultural systems worldwide (Gitz et al., 2016; Harris et al., 2018); plant thermal tolerance

94 research must be well directed, or risk floundering at such a critical time. At one extreme – high

95 temperature – the frequency, intensity, and a-seasonality of heatwaves are breaking records

96 annually (Hewitson et al., 2014; Harris et al., 2018). Although some species exhibit a high capacity

97 to withstand higher temperatures and heatwaves than are currently experienced (Drake et al., 2018;

98 Aspinwall et al., 2019), heatwaves are predicted to exceed the thermal tolerance limits of many

99 species across a wide latitudinal range (O'Sullivan et al., 2017). Shortened growing seasons, yield

100 reductions, and crop losses have been occurring and are predicted to worsen (>40% by 2100 in

101 some regions), primarily due to increasing heat stress (Jha et al., 2014). Similarly, at the other

102 extreme – low temperature – the frequency of cold snaps is increasing in some regions, both

103 directly (e.g. through disruption of the polar vortex driving cold cells towards temperate regions;

104 Kretschmer et al., 2018) and indirectly (e.g. where warmer averages reduce snow cover and

105 increase exposure to frost; Woldendorp et al., 2008). If frosts occur during warmer conditions or if

106 there is a substantial late-season frost event, such as the 2007 spring freeze in the USA, then this

107 temperature backlash can cause substantial frost damage and widespread devastation to crops and

108 natural species alike (Jönsson et al., 2004; Gu et al., 2008). Understanding cold tolerance limits

109 may elucidate which species may be released from temperature limitation in future, for instance the

110 expansion of subtropical and tropical plants into temperate zones due to reduced frequency or

111 severity of cold snaps (Cavanaugh et al., 2014).

112 5 Thermal tolerance in practice reflects a range of interacting elements. In many regions, plants

113 may experience both hot and cold extremes, with events in each direction causing a shift in overall

114 resource allocation from growth and reproduction to protection from physiological stress (Lortie et

115 al., 2004; Mitra & Bhatia, 2008). For example, heating events are common in alpine environments,

116 where small stature plants track soil rather than air temperatures and thus heat to potentially

117 damaging levels (Squeo et al., 1991). The few studies examining heat tolerance for alpine species

118 indicate that it can be surprisingly high (~48-50°C), with species living in warmer microhabitats

119 having higher heat tolerance than species living in sheltered habitats (Buchner & Neuner, 2003;

120 Larcher et al., 2010).

121 Focusing on responses of a given species to only one of these extremes is therefore unlikely to

122 provide a comprehensive understanding of thermal tolerance or to increase our predictive power in

123 the face of climate change. Moreover, the potential for an extreme temperature event to become

124 critically stressful to a plant may depend on a range of accompanying circumstances, such as water

125 status, light conditions, or ambient temperatures prior to or following the event. Plants in cold

126 climates may shift their thermal tolerance or alter their phenology in response to average warming

127 conditions, but this may be at the cost of frost hardiness (Jönsson et al., 2004). In addition, what

128 constitutes an ‘extreme’ event for a given species or biome may be relatively benign in a different

129 context. Thus, it is essential to consider abiotic factors and the dynamics of plant thermal tolerance.

130 Here, we present the results and synthesis of a large-scale systematic review focused on the

131 tolerance of photosynthetic tissues of land plants to extreme heat and/or cold stress for both

132 cultivated and wild species across life forms, biomes, and the world. We explore the many

133 techniques that are used to measure thermal tolerance, the metrics derived from them, and the

134 widely diverging experimental conditions under which thermal tolerance is assessed. We note that

135 the concept of what constitutes ‘thermal tolerance’ is debatable. Some studies focus on reduced

136 productivity under simulated future climates, others assess repairable damage after moderate

137 chilling or heat stress, and others focus on the onset of irreparable damage following extreme

138 freezing or heatwave events. For the purposes of this review we define thermal tolerance as the

139 temperature (high or low) beyond which the plant exhibits substantial or lasting damage; we note

140 that this temperature is often estimated from (and assumed to be correlated with) the temperature at

141 which the plant invokes protective mechanisms.

142 Our objective was to review the geographic and temporal distribution of research efforts,

143 assess methodological approaches, and highlight the commonalities, ambiguities, and deficiencies

144 in global plant thermal tolerance research. Our review provides a timely synthesis of research to

145 date and bridges often disparate ecological and agricultural perspectives. We also present

146 6 recommendations and an agenda to highlight thermal tolerance research priorities and provide a go- 147 to reference to inform efficient and reliable research across the spectrum of plant thermal tolerance.

148 Our approach to the systematic review 149

A systematic review relies on synthesis of a comprehensive and repeatable literature search

150 (Lowry et al., 2013; Lortie, 2014; Gurevitch et al., 2018). We employed the Preferred Reporting

151 Items in Systematic Reviews and Meta-Analyses (PRISMA) framework (Moher et al., 2009) to

152 compile a database of articles that measured plant thermal tolerance (Fig. S1). Briefly, our literature

153 search (December 2017) of the Institute for Scientific Information (ISI) Web of Knowledge used an

154 extensive list of search terms (Supporting Information Notes S1) and yielded more than 21,000

155 articles. We first screened the titles and then the abstracts and at each step excluded articles that did

156 not include investigations into tolerance of leaves or leaf-buds of angiosperms and gymnosperms

157 exposed to potentially damaging high or low temperature events as distinct from growth conditions.

158 Each article was evaluated based on 15 criteria (Notes S1) relating to each thermal tolerance

159 assay technique being reported, important elements of experimental design, focal species, and

160 characteristics thereof. Experimental conditions for assessing thermal tolerance diverge widely and

161 methods for imposing experimental thermal stress can include mild to severe temperatures that are

162 either applied gradually (ramped), suddenly (shocked), as a sustained growth temperature, or as a

163 combination of any of these three. There is good biological justification for considering different

164 rates of exposure to change. Thus, our survey focused on characterising specific design elements of

165 the studies we included. We documented the conditions with which thermal stress was imposed to

166 determine how consistent and comparable they were.

167 Many articles reported multiple techniques to evaluate thermal tolerance. Henceforth we

168 refer to scientific publications as ‘articles’ and uses of individual techniques within an article as

169 ‘studies’. After quality checks, the dataset contained data from 1,691 unique articles comprising

170 3,743 studies of thermal tolerance assays (Fig. S1). The dataset is publicly available through the

171 figshare repository (10.6084/m9.figshare.13083662).

172 A brief history and description of plant thermal tolerance techniques

173 A broad array of techniques is used to assay thermal stress. Thermal tolerance research on

174 both cultivated and wild species became more common in the 1990s, but the rate of increase was

175 more dramatic in cultivated species, which has culminated in four-fold more thermal tolerance

176 articles on cultivated (n = 1,358) than wild species (n = 339). The technologies used to measure

177 thermal tolerance have evolved through time (Fig. 1a,b). Early studies assessed thermal tolerance

178 7 simply by quantifying visual damage. Moving forward, researchers of cultivated species were

179 consistently earlier adopters of emerging techniques, such as (epi)genetics and ‘omics (e.g.

180 metabolomics, proteomics, genomics), often 10-20 years in advance of use in wild species research

181 (Fig. 1, Notes S1). Overall, the most widely used techniques for assaying plant thermal tolerance in

182 the past 20 years have been chlorophyll fluorescence (487 studies), electrolyte leakage (468

183 studies), and a broad array of other biochemical assays (446 studies in total). In recent years, studies

184 using (epi)genetics and ‘omics, biochemical assays, and reactive oxygen species (ROS) and

185 antioxidant techniques have been rapidly increasing. These specific techniques are expanded upon

186 below and Notes S1 summarises these and the remaining thermal tolerance techniques and includes

187 relevant indicators and references.

188 Fluorescence techniques measure changes in fluorescence re-emitted from chlorophyll in the

189 photosystems in response to high or low (potentially stressful) temperature. A variety of measures

190 have been applied in this context, including minimum fluorescence (F0); maximum fluorescence

191 (FM); photosynthetic quantum efficiency (φPSII); maximum photosynthetic quantum efficiency

192 (FV/FM); non-photochemical quenching (NPQ); and chlorophyll a fluorescence transients (Maxwell

193 & Johnson, 2000). Exemplary articles have used these methods to define thermal metrics such as

194 LT50 (also T50), the temperature at which FV/FM declines to 50% of the maximum FV/FM of

195 unstressed photosystems (Curtis et al., 2014) or Tcrit, the inflection point between slow and fast rise

196 phases of the temperature-dependent increase in F0; (Knight & Ackerly, 2002). Others have

197 measured Rfd: chlorophyll fluorescence decrease ratio or vitality index, calculated on the decline of

198 FM to the fluorescence steady-state level (FS) (Perera-Castro et al., 2018). Their popularity has

199 increased in recent years as fluorescence techniques can be high throughput, but there has been little

200 explicit comparison of how the various measures differ in their interpretation.

201 Measures of electrolyte leakage are another widely applied technique; these assess change in

202 ion concentrations in response to thermal damage using electrical conductivity. These methods are

203 highly conducive to determination of thermal metrics such as critical temperatures at which 50% (or

204 other standard) change in tissue ionic conductance (gTi) or electrical conductivity (EC) is reached.

205 From these, researchers have calculated LT50, which is well correlated with frost damage (Kreyling

206 et al., 2015), and other damage indices (Id) (Whitlow et al., 1992). Tolerance metrics derived from

207 electrolyte leakage are strongly related to the climate of origin of both native and non-native species

208 (Kreyling et al., 2015) and species that are cold-sensitive release electrolytes more rapidly than

209 cold-resistant species (Patterson et al., 1976). Electrolyte leakage measures the site of physiological

210 injury at extreme temperatures and can be high-throughput, but it is potentially less sensitive than

211 chlorophyll fluorescence or gas exchange, and is limited to laboratory assays (Xu et al., 2014).

212 8 There is a wide array of biochemical measures employed in thermal tolerance research

213 including heat shock proteins (HSPs) and studies of ROS. Heat shock proteins and factors are

214 produced rapidly in response to abiotic stresses to alleviate cellular damage (Wang et al., 2004).

215 HSPs function as molecular chaperones, assist in protein folding, maintain signal transduction, and

216 prevent protein aggregation (Chen et al., 2018). Their relative abundance can be detected using

217 western blotting or slot/dot blotting. In general, more tolerant individuals or species will induce a

218 larger abundance of HSPs, or changes in gene expression associated with their production (Feder &

219 Hofmann, 1999); however, this pattern is not universal or clear-cut (Barua & Heckathorn, 2004).

220 An array of techniques including chromatography, quantitative real-time PCR, and in

221 vitro chaperone-like activity assays are used to assess heat shock responses (Chen et al., 2018).

222 Although their name suggests a specificity for heat stress, HSPs can be upregulated in response to a

223 wide range of other stresses that induce protein unfolding including cold, drought, salinity, and

224 oxidative stress (Feder & Hofmann, 1999; Barua & Heckathorn, 2004; Wang et al., 2004).

225 However, patterns of protein synthesis during cold acclimation can differ substantially to those

226 expressed during heat shock responses (Guy, 1999). Therefore, while HSP determination may aid

227 mechanistic understanding of the stress response for a given species, we are far from using such

228 techniques widely, especially for wild species.

229 ROS and antioxidants play important roles in maintaining the redox state in plant cells. ROS

230 are natural by-products of metabolic processes that can affect gene expression and contribute to

231 plant growth, signalling, development, cell cycles, programmed cell death, abiotic stress responses,

232 pathogen defence, and adaptation (Gill & Tuteja, 2010; Mittler et al., 2011). Like HSPs, ROS

233 concentrations can increase rapidly in response to diverse stimuli, including temperature extremes.

234 Increased ROS concentration following thermal stress leads to unfavourable modification of lipids,

235 proteins, and nucleic acids, resulting in cell damage and metabolic dysfunction. These impairments

236 inhibit growth, reduce fertility, and promote premature senescence. Plants produce antioxidants to

237 scavenge or detoxify ROS or their precursors and prevent free radical formation to mitigate cellular

238 damage caused by uncontrolled ROS accumulation. However, under extreme temperature stress,

239 antioxidant production can lag ROS production, making ROS a major factor in crop yield loss. A

240 wide variety of ROS and antioxidants can be assayed with various methods to assess concentration

241 or expression patterns with thermal stress (Gill & Tuteja, 2010; Mittler et al., 2011).

242 More recently, epigenetics, genomics, and other ‘omics (e.g. transcriptomics, metabolomics,

243 phenomics) have been applied in thermal tolerance research. These approaches have revealed

244 regulatory mechanisms, new gene variants and their expression and function, and have been

245 instrumental in adaptive plant breeding for resistance to abiotic stressors (Jha et al., 2014; 2017;

246 9 Shah et al., 2018). For example, identifying molecular mechanisms underlying heat stress responses

247 in silico has led to the refinement of transgenic techniques to engineer the overexpression of HSPs

248 and genes related to ROS activity and membrane stability to confer increased heat tolerance in

249 various crop species (Grover et al., 2013). However, assessing the success of these efforts is

250 confounded by various research groups applying non-standardised methods, and limited field-scale

251 phenomic capabilities (Grover et al., 2013).

252 Often what determines the adoption of an approach to assessing thermal tolerance is a

253 combination of context of the research question, conventional wisdom, and local practice. However,

254 when bodies of work are produced in isolation, in a limited number of research laboratories, or

255 focused on one biome or study organism, the potential for siloing and lack of comparability among

256 research programs arises. Thus, our review considers when and where these various techniques

257 have been applied.

258 What comprises the plant thermal tolerance literature?

259 Geographic spread 260 An examination of the geography of thermal tolerance research based on both the country of

261 affiliation of the first author and the location where the experiments were conducted (when

262 available), shows that plant thermal tolerance is researched all over the world but, unsurprisingly,

263 the distribution of this research is not uniform. The volume of articles by authors based in the USA,

264 China, and Europe, vastly outweighs contributions by other individual countries (Fig. 2; see Figs

265 S2-S5 for more detailed global and regional distributions). The patchy network of research likely

266 reflects institutional bias and availability of research funding, where most articles, even for

267 ecological research in the tropics, for example, are led by authors from developed countries (Stocks

268 et al., 2008). Many of the thermal tolerance articles on cultivated species pre-date the more recent

269 focus on climate change and trace back to developing domesticated species suited to a range of

270 growing environments.

271 Overall, articles published on wild species represent a narrower portion of global distribution

272 than do those on cultivated species (Fig. 2a,b). Wild species are understudied in many of the more

273 thermally extreme regions on Earth (e.g. north-west Asia, Middle East, Africa, South and Central

274 America, and India, Fig. 2a,c,e,g). These gaps in global coverage, particularly for heat tolerance

275 (Fig. 2g,h), mean that thermal tolerance is understudied in exactly those developing countries where

276 there is rising demand for increased crop yield and where some of the greatest climate change- 277 induced yield losses are predicted to occur (Parry et al., 2004; Tester & Langridge, 2010).

278 10 Comparative thermal tolerance studies 279 Delving deeper shows that our understanding of thermal tolerance is informed by an eclectic

280 spread of research across growth forms, and that there is relatively little broad-scale comparative

281 work. We have a far greater understanding of the thermal tolerance of species that we have bred and

282 depend on for food, timber, and fibre (n = 1,358), than those that comprise the rest of Earth’s

283 terrestrial biosphere that perform essential ecosystem services (n = 339; Fig. 3). Within the

284 literature, and for both cultivated and wild species, a greater proportion of articles investigate cold

285 (59%) than heat tolerance (35%) and there are strikingly few articles that examine both heat and

286 cold tolerance together (5%, Table 1).

287 In terms of taxonomic selection, research on cultivated species tended to focus on a single

288 species (42%) or on differences among intraspecific varieties (41%), but less often across multiple

289 species (17%; Fig. 4a). In contrast, studies on wild species were split evenly between focusing on

290 single or multiple species (44%) but investigated intraspecific diversity far less often (12%;

291 Fig. 4b). The representation of different life forms also varied between cultivated and wild systems.

292 Studies on cultivated species contained a greater proportion of graminoids (e.g. Poaceae),

293 forbs/herbs (e.g. vegetable species) and vines (e.g. viticulture), with fewer shrubs or trees (Fig. 4c).

294 In contrast, studies on wild species were more evenly spread with relatively more focus on woody

295 species (Fig. 4d).

296 The recent work of Lancaster and Humphreys (2020) demonstrates the potential for meta- 297 analytic comparison of thermal tolerance, and there remains ample opportunity to build on the

298 relatively few studies that apply a standard method of assessing thermal tolerance and take an

299 explicitly broad comparative approach. In particular, extension of excellent comparative works such

300 as O'Sullivan et al. (2017), Zhu et al. (2018), Sentinella et al. (2020), and Lancaster and Humphreys

301 (2020) into extreme biomes, across a wider range of growth forms, and considering other

302 experimental nuances is still warranted. Such efforts will lead to a better understanding of general

303 rules in thermal tolerance and have potential to explore the underlying mechanistic differences in

304 the various measures of tolerance.

305

306 Cold vs heat tolerance research 307 Studies on cultivated species covered both cold and heat tolerance across the different types

308 of cultivation, but with more studies on cold tolerance overall (Table 1, Fig. 3a). Cold tolerance was

309 more often assessed within viticulture, plantation forestry, horticultural and vegetable crops,

310 Arabidopsis, and multiple or other types of cultivation (e.g. tobacco, plants for oil). In contrast, heat

311 11 tolerance made up more than half of the studies within cereals, fibre crops, and pasture and turf

312 grasses. Cereals and fibre crops had the lowest proportion of articles that considered both heat and

313 cold tolerance simultaneously.

314 For wild species, the proportion of studies focusing on heat, cold, and both heat and cold

315 tolerance varied across biomes, but cold tolerance research made up the majority for all biomes

316 except for arid ones (Table 1; Fig. 3b). Plant responses to both cold and hot extremes may be linked

317 at localised scales via processes such as early snowmelt (Körner, 2003) or microhabitat variability

318 (Suggitt et al., 2018), or across a species’ distribution by large scale changes in global circulation

319 patterns influence extreme events (Kretschmer et al., 2018). In tropical/subtropical biomes, the

320 proportion of studies on cold and heat tolerance was more equal and these had the highest number

321 of articles that examined both heat and cold tolerance. Studies in temperate biomes made up 34% of

322 the wild dataset and these were dominated by cold tolerance studies. Articles on boreal forests were

323 focused entirely on cold tolerance, as were most articles on arctic/alpine/subalpine biomes.

324 Remarkably, heat tolerance was assessed far less often than cold tolerance in wild species; the

325 greatest proportion of heat tolerance research was conducted in the warmer biomes: arid/semi- 326 arid/savannah and tropical/subtropical, but even here, cold tolerance research was as or more

327 prevalent. Given consistent predictions of increasing frequency and intensity of heatwaves across

328 the world together with average warming (Perkins-Kirkpatrick & Gibson, 2017; Harris et al., 2018;

329 IPCC, 2018), the relatively low coverage of studies on plant heat tolerance is concerning.

330 Considerations when designing thermal tolerance experiments

331 Application of techniques 332 Our assessment of the history of thermal tolerance research indicates that there were not

333 gaping holes in coverage by cultivation type, biome, or life form in the application of techniques for

334 evaluating thermal tolerance. However, there is clearly opportunity for expanding the application of

335 many techniques into new areas and non-model systems. For example, it is perhaps not surprising

336 that HSPs have not been examined in species from the world’s coldest biomes.

337 Plant thermal tolerance arises from complex phenomena involving perception of thermal

338 stress, transmission of the information (cascade signalling), genomic regulatory processes, and then

339 physiological and biochemical changes (Urano et al., 2010; Hasanuzzaman et al., 2013). By

340 integrating approaches across scales we can shed light on the molecular mechanisms and cellular

341 pathways that lead to physiological changes and confer tolerance (comprehensively reviewed by

342 Nievola et al., 2017). Applying multidisciplinary and holistic approaches to diverse species will

343 reveal new gene variants, products, and traits for crop-breeders to target for engineering or breeding

344 12 programs to obtain new stress-tolerant varieties (Fragkostefanakis et al., 2015; Jha et al., 2017;

345 Shah et al., 2018). Our review found a range of techniques under the umbrella of biochemistry

346 (including ROS, HSPs, and other biochemistry) and ‘omics (metabolomics, transcriptomics) that

347 are commonplace in cultivated studies but rare in wild studies. We see great potential to gain better

348 mechanistic understanding in wild species by applying more of these biochemical techniques and

349 aiming to scale to the whole phenotype (e.g. Aspinwall et al., 2019).

350 The emergence of high-throughput techniques for proteomics and metabolomics (Zivy et al.,

351 2015) along with phenomics (Furbank et al., 2019) allows thermal tolerance to be assessed in both

352 controlled environments and field studies for cultivated and wild species alike. This presents the

353 opportunity to scale from mechanism to emergent phenotype (Deshmukh et al., 2014; Campbell et

354 al., 2018). Greater crosstalk among researchers studying thermal tolerance on cultivated and wild

355 species and application of these approaches to high-throughput scales would be mutually beneficial.

356 Ours is an era of evidence synthesis and meta-analyses (Gurevitch et al., 2018), in which new

357 analytical tools are released frequently. The rise of open trait databases such as TRY (Kattge et al.,

358 2020) and GlobTherm (Bennett et al., 2018) underpins efforts to consolidate knowledge and extend

359 the application and utility of individual studies to a global context. Databases hold great promise to

360 generate comparative analyses; for example, contrasting thermal metrics across species or biomes,

361 or assessing different measurement techniques for given species (e.g. Lancaster & Humphreys,

362 2020). We caution that there remain many considerations and caveats to consider in such syntheses;

363 for example, the differences in measurement conditions and the specific methods of application of

364 thermal stress, techniques to measure tolerance, and other aspects of experimental design. Armed

365 with new insights and databases, researchers can contribute improvements to the accuracy and

366 dynamic capabilities of model predictions and decision-making tools for regional-scale suitability,

367 growth, and yield of crop species as extreme events become more frequent and intense (Caubel et

368 al., 2015; Zampieri et al., 2019).

369

370 Experimental design considerations 371 It is abundantly clear that experimental designs and techniques vary widely among studies,

372 and most notably between wild and cultivated systems (Figs 5a, S6). We found that it was common

373 for research on cultivated species to compare relative performance of many varieties under a set of

374 controlled conditions, but rare to provide an explicit explanation for temperature treatment choices

375 (see Zub et al., 2012 for an exemplary exception). On the other hand, these studies also generally

376 conducted several complementary assays to achieve broader mechanistic insights. In contrast,

377 13 studies on wild species focused on identifying tolerance limits under natural conditions more than

378 understanding tolerance mechanisms; however, they generally provided explanations for their

379 chosen rates of temperature change and treatment temperatures (e.g. Sierra-Almeida & Cavieres,

380 2012). Our review demonstrated three areas that warrant careful consideration and explanation

381 when designing thermal tolerance research: how temperature stress is applied, the importance of

382 recognising thermal legacy, and accounting for interactions with other factors. These are presented

383 in detail below and summarised in Table 2A.

384 Application of temperature stress 385 Field, common-garden, glasshouse, and growth chambers each present different limitations,

386 and the specific context of growth conditions can greatly influence plant responses (Passioura,

387 2006; Poorter et al., 2016). Overall, we found that most articles (94%) imposed stress in an

388 experimentally controlled manner, such as with a temperature-controlled growth chamber or water

389 bath, as opposed to focusing on natural extreme events such as frosts or heatwaves (6%). In some

390 experimentally controlled studies, thermal stress was imposed as a controlled ramp and in others as

391 a sudden shock (Fig. 5b), each of which can induce different response mechanisms and pathways.

392 In contrast to shocks, ramping temperature allows time for hardening processes to provide some

393 thermal protection before reaching critically damaging temperatures. The application of ramp vs

394 shock approaches differed between studies of cultivated and wild species. Research on cultivated

395 species applied thermal stress as shocks more often than on wild species (Fig. S7). Within wild

396 species, most studies on cold tolerance ramped stress, whereas those researching heat tolerance

397 applied a shock more often than ramping (Fig. S7). Biochemical assays and (epi)genetics and

398 ’omics were most often conducted on plant tissue that was exposed to a temperature shock, whereas

399 studies using electrolyte leakage, assays of visual damage, and thermometry were more often

400 conducted on plant tissue that was exposed to a temperature ramp (Fig. 5b).

401 Cultivated species were assayed most often for periods of hours (1,322 studies) or longer

402 (days = 785 studies and weeks = 431 studies), whereas for wild species, shorter timeframes were

403 generally used: hours or less (415 studies). The exception was for HSPs, where stresses lasting

404 <24h were common for both cultivated and wild species. Research on wild species that did apply

405 stress over longer periods of days (89 studies) and weeks (72 studies) tended to focus on water

406 potential, ROS/antioxidants, other biochemical factors, and gas exchange (Fig. 5a). In wild species,

407 short stress intervals of 60 minutes or less were often used in association with gas exchange or

408 chlorophyll fluorescence assays (Fig. 5a). A greater proportion of studies on cultivated species

409 failed to clearly specify the maximum stress duration compared to those on wild species (Fig. 5a).

410 In some cases, these differences reflect that the type of assay dictates the stress duration and cannot

411 14 be consistent, but nonetheless such variation among studies hampers our ability to identify common

412 responses.

413 In nature, the rate and frequency of exposure to extreme temperatures varies between cold and

414 hot extremes. Leaf temperature can vary rapidly and repeatedly on a hot, calm day (Vogel, 2009),

415 such that the frequency, duration, and magnitude of the heat stress are likely to affect the impact of

416 and response to the stress. In contrast, exposure to extreme low temperatures tends to be more

417 gradual and sustained over hours or even days (Sierra-Almeida & Cavieres, 2012). Thus, there is

418 biological justification for using different rates to apply thermal stress when studying heat vs cold

419 tolerance. However, we found that in many cases, studies elected to deliver their heat or cold

420 treatments as a shock (e.g. moving a plant directly from a benign to a high or low temperature- 421 controlled growth room) without providing the rationale behind that approach. The insect thermal

422 tolerance literature is actively debating how moving to a dynamic delivery of extreme temperature

423 (i.e. ramping temperature at biologically-relevant speeds, as opposed to a quick shock) would

424 increase the relevance and impact of their research (Rezende et al., 2014), and plant researchers

425 could stand to benefit from considering a similar approach.

426 One limitation to adopting techniques used in animal thermal tolerance is the growth form of

427 plants, which determine how we measure them. In the animal literature, it is standard to measure

428 critical temperatures on small arthropods on which whole-organism tolerance can be assessed (e.g.

429 Slatyer et al., 2013; Hoffmann & Sgrò, 2018; MacLean et al., 2019). Fundamentally, whole- 430 organism measures on plants are more challenging due to their modularity, below-ground biomass,

431 and growth form variation that contribute to a complex array of alternative mechanisms to escape or

432 cope with thermal stress (Huey et al., 2002). Modular organs such as leaves are therefore targeted

433 for most thermal tolerance measurements in plants. However, this only determines limits to

434 photosynthetic performance or organ survival, rather than higher-level or probabilistic

435 measurements of whole-organism performance and survival that are more common in the animal

436 thermal tolerance literature (Rezende & Bozinovic, 2019). Seedlings will be essential to exploring

437 whether tolerance of leaves can be reasonable approximations for thermal tolerance measurements

438 for whole plants or how these approaches could be developed.

439 Adopting more realistic regimes and justifying these with data from relevant natural

440 settings, as well as providing better descriptions of the temperature ranges around set points would

441 enable a more nuanced investigation of the differences between acute vs chronic stress responses,

442 and between facultative protective responses vs signs of irreparable damage (Lai & He, 2016;

443 Trapero-Mozos et al., 2018). At present, the definition and use of ‘stress’ and ‘stressful events’ is

444 somewhat ad hoc and impedes our ability to compare results or derive generalisations (Jansen &

445 15 Potters, 2017). Differentiating damaging conditions from those that are suboptimal or induce

446 protective mechanisms is essential contextual information; researchers need to attempt to explain

447 how and why selected treatments and assays were conducted. By placing treatments in context with

448 historical, realised, or projected climatic conditions, researchers provide an opportunity for others to

449 assess the extremity of the treatments imposed relative to the biology of that species. For example,

450 what may be an extremely high temperature for vegetative growth in broccoli (Brassica oleracea

451 var. italica Plenck) is sub-optimal for maize (Zea mays L.), and sensitivity to thermal stress will also

452 vary across life-stages and with environmental history (Hatfield & Prueger, 2015).

453 Understanding thermal legacy 454 Although warmer origin species often exhibit higher heat tolerances than cooler origin species

455 under common conditions (Zhu et al., 2018; Lancaster & Humphreys, 2020), it is important to note

456 that the acclimation state of plants or tissue can substantially affect thermal tolerance and

457 understanding the potential to acclimate will be important for predicting impacts of our changing

458 climate. For example, geographic trends in thermal tolerance appear to be much stronger in

459 acclimated (hardened) plants (Lancaster & Humphreys, 2020). While we did not directly assess

460 acclimation, the term acclimation certainly frequents the literature we reviewed (Fig. S8). Thermal

461 tolerance can shift in response to changes in both continuous growth temperature and exposure to

462 extreme temperature events (Downton et al., 1984; Hamilton et al., 2008; Drake et al., 2018) and

463 changes can occur across the scale of minutes (e.g. heat shock) to months (e.g. seasonal change)

464 (Havaux, 1993; Bannister et al., 2005). Acclimation of thermal tolerance can be influenced by

465 temperature alone (Strimbeck et al., 2008), as well as other environmental conditions such as

466 photoperiod (Bannister et al., 2005) and water availability (Lu & Zhang, 1998). Thus, in addition to

467 considering interactive effects on thermal tolerance, it is crucial for studies on thermal tolerance to

468 be explicit about the thermal legacy of their study organisms.

469 Variability in background thermal regimes may have significant effects on plant responses to

470 extreme conditions (Gutschick & BassiriRad, 2003; Bita & Gerats, 2013). Furthermore, plant

471 thermal tolerance research seldom reports variability of ambient environmental factors in controlled

472 growth environments (including temperature, light, and humidity) or differences between air and

473 leaf temperatures, which can differ among species by up to 10°C in hot conditions (Wise et al.,

474 2004; Vogel, 2009). Comparisons among studies that differ in experimental designs, biomes, and

475 species may be complicated by ambiguity at best and, more concerningly by legacy, if prior thermal

476 exposure is not reported explicitly and terms to describe changes in thermal tolerance are not

477 defined carefully.

478 16 Interactions with other environmental factors

479 Average temperatures are increasing alongside more intense and frequent extreme events,

480 often with a backdrop of resource limitation. These factors will likely exacerbate the effect of

481 thermal stress with potentially long-lasting or irreversible community-level effects (Harris et al.,

482 2018). Variation in other abiotic factors may include ordinary elements such as seasonal variation in

483 temperature, light, or water availability. In many situations thermal stress from high temperatures

484 will occur with or following onset of water limitation. Nonetheless, most studies in the literature

485 focused on thermal tolerance in the absence of additional experimental variables (57%). Among the

486 studies that included additional environmental factors, the most common was the effect of a

487 controlled growth temperature prior to applying thermal stress (13%), e.g. to determine whether

488 hardening alters the effect of extreme events. Given that heat stress events often co-occur with

489 belowground resource limitations, it is concerning that an extremely small percentage of studies

490 considered how availability of water (6%) or soil nutrients (2%) affected thermal responses.

491 Likewise, we found few studies that considered the effects of light (3%), CO2 (1%), or other non- 492 climate factors (8%) on thermal stress responses. Indeed, such two- and three-way treatment

493 interactions were investigated by just 10% of all studies. Given that our changing climate will bring

494 shifts in both thermal and precipitation regimes and that drought and thermal acclimation have been

495 shown to interact (Sierra-Almeida et al., 2009; Hoover et al., 2014), it seems pertinent to consider

496 their combined impact on tissue damage, yield loss, or mortality. For studies of thermal tolerance to

497 have real-world meaning, a greater understanding of how other factors limit responses to

498 temperature is crucial.

499

500 Towards development of standard approaches and comparable thermal metrics

501 The more we can apply a set of standardised approaches across species, crop types or biomes,

502 and different thermal regimes, the greater our potential to identify general patterns in the

503 physiology, ecology, and evolution of thermal tolerance. Of course, the reality is that methods are

504 regularly fine-tuned and refined for specific study organisms and contexts. Plant thermal tolerance

505 research is most informative if the underlying premises regarding experimental conditions are well

506 justified and experimental procedures are explained unambiguously.

507 Thermal tolerance metrics are a valuable tool to support comparative research to identify

508 general patterns across species or biomes. For example, Tcrit and T50 of FV/FM, often generated via

509 measuring chlorophyll fluorescence, have been measured for hundreds of species (Notes S1; e.g.

510 Knight & Ackerly, 2002; Zhu et al., 2018; Lancaster & Humphreys, 2020). However, we found that

511 17 only 23% of studies across both cultivated (49%) and wild (17%) species either reported a metric or

512 provided information from which such a metric might be obtainable. Thus, where possible, we

513 advocate adoption of techniques that generate a thermal tolerance metric that can be used for global

514 comparative analyses.

515 The many different and nuanced approaches to researching plant thermal tolerance have

516 propagated various metrics and terms. For example, plant thermal tolerance metrics frequently do

517 not specify whether they reflect a heat or cold response (e.g. Tcrit could refer to either hot or cold

518 critical temperature). Further, measures of the same name, but derived from different thermal

519 tolerance assays will vary in their functional significance depending on the underlying physiological

520 processes that are being quantified. While measures and metrics from different tolerance assays

521 (e.g. LT50 from FV/FM and LT50 from visual damage) yield interesting intra-assay comparisons, they

522 do not always provide equivalent information, correlate well with each other, or represent

523 biologically sensible comparisons (e.g. Neuner & Pramsohler, 2006; Curtis et al., 2016). Ideally,

524 streamlining metrics and terms would allow for greater comparability across experimental

525 approaches and techniques, as is currently more commonplace in animal ecophysiology (Rezende et

526 al., 2014; Rezende & Bozinovic, 2019; Sunday et al., 2019). Exploring how different assays

527 correlate is a further vital step toward standardising approaches to evaluate thermal tolerance but

528 also for understanding the mechanistic links among patterns of response in different measures.

529 We advocate a multidisciplinary approach to assessing plant thermal tolerance. For example,

530 measure the thermal tolerance of photosynthesis directly using a method that produces a tolerance

531 metric, such as chlorophyll fluorescence or electrolyte leakage. Biochemical responses to thermal

532 extremes, particularly ROS and HSP, could then be measured to probe underlying mechanisms. To

533 better understand the impact of thermal tolerance, a holistic view to growth and seed production is

534 always useful, though we appreciate often logistically intractable. However, we note that until there

535 are more studies that investigate the thermal tolerance responses of plants to extreme events using

536 multiple approaches, we cannot infer which method generates the most reliable information or

537 metric for predictive models.

538

539 An agenda for future thermal tolerance research

540 The primary objective of this synthesis was to determine the state of knowledge in the field of

541 plant thermal tolerance research and to identify commonalities, ambiguities, and deficiencies in the

542 global literature of plant thermal tolerance measurement. By mapping topics by article titles and

543 author keywords, we can visualise the general siloing with respect to thermal tolerance assays,

544 18 species selection, and geography (Fig. S8). After decades of research, there are still remarkable

545 holes in our knowledge base, punctuated by large divides among specific sub-fields of thermal

546 tolerance research. Our systematic review found little equivalency among techniques and study

547 designs, let alone thermal metrics, indicating that cross-species comparisons remain far from

548 straightforward. Addressing these issues will be crucial as trait databases become key sources for

549 understanding plant responses to increased temperature means and extremes as the climate changes.

550 Our review has demonstrated the need to explicitly revisit not only how we study thermal

551 tolerance, but also what our priorities are while studying it. The ‘how’ has been covered above.

552 Below, we outline four broad areas that we see as priorities for empirical thermal tolerance

553 research, for which our recommendations are summarised in Table 2B. This agenda seeks to

554 provoke discussion and improve efficiency, repeatability, and comparative power in our research to

555 catalyse fundamental advances and applied outcomes.

556 1. The comparative ecology of thermal tolerance in the ecological and evolutionary

557 strategy spaces 558 Plant ecologists have made great advances in understanding how traits are related to

559 distribution of species across the globe (O'Sullivan et al., 2017; Lancaster & Humphreys, 2020;

560 Sentinella et al., 2020), but we have less understanding of how thermal ecology links to other

561 elements of plant strategy space (Vasseur et al., 2018). If we are to assess which ecosystems are

562 most at risk under climate change accurately, a greater understanding of how thermal tolerance of

563 species scales to the community level is essential. Multi-species comparative projects were under- 564 represented within our dataset and these were not comparisons of within or between community

565 variation in most cases. In the stand-out exemplary studies, there remains relatively low

566 representation of non-woody growth forms. Undoubtedly, factors such as competition, facilitation,

567 differential resource utilisation, and population demographics all modify the thermal response

568 profiles of individual species and have flow-on effects to the functioning of communities and

569 ecosystems. For example, the variation in thermal tolerance of species, growth forms, or functional

570 types has the potential to change relative survival and dominance within communities, thereby

571 leading to shifts in the distribution of species and communities (Ackerly, 2003). Such changes may

572 then alter ecosystem function at small catchment and large landscape scales. Thus, improved

573 understanding of how such variation affects community thermal tolerance in natural systems is

574 warranted.

575 2. Understanding the geography and drivers of thermal tolerance breadth

576 Published research on wild plants in alpine biomes around the world has primarily focused on

577 19 cold tolerance (e.g. Bannister, 2007; Briceño et al., 2014) while in desert plants, research on heat

578 tolerance dominates (e.g. Knight & Ackerly, 2002; Curtis et al., 2014; 2016). Yet mountain plants

579 can reach extreme high temperatures in summer (Larcher et al., 2010) and desert plants are exposed

580 to extreme cold (Lazarus et al., 2019). Little is known about thermal tolerance breadth, including

581 whether specialising for one extreme is antagonistic to the other. While responses to heat and cold

582 shock may differ or have different kinetics, some share signalling and metabolic pathways (Kaplan

583 et al., 2004) and so fundamental insight about the mechanistic determinants of thermal tolerance

584 could be revealed by comparing heat and cold tolerance. Further, thermal tolerance breadth may

585 vary with climatic affiliation; for example, being broader in widespread species or species from

586 variable or more extreme climates (Sheth & Angert, 2014).

587 Biodiversity models often assume that realised distributions reflect species’ fundamental

588 climatic tolerances, however, by underestimating thermal tolerances these models may

589 underestimate the breadth of a species’ niche (Bush et al., 2018). Thus, we propose that the thermal

590 tolerance breadth could be a better indicator of species’ fundamental climatic tolerance, and thus

591 adaptive capacity: important considerations to better predict species distributions or extinction risk

592 under climate change. Thermal tolerance breadth could also be indicative of a crop’s suitability for

593 particular agro-ecological zones and potentially a desirable trait to target in crop breeding in

594 growing regions that have both cold and hot extremes (Varshney et al., 2011). Cultivars or species

595 with narrow thermal tolerance breadth may be particularly vulnerable to changing climatic

596 conditions, especially if that narrow tolerance is associated with low genetic diversity and narrow

597 range sizes (Slatyer et al., 2013). Conversely, cultivars selected for their tolerance to temperature

598 extremes or natural species that have evolved with frequent extremes in temperature may have high

599 thermal tolerance breadth and be buffered against crop failure and extinction (Buckley & Huey,

600 2016). Thus, thermal tolerance breadth has potential to yield insight with relevance to both wild and

601 cultivated species. Such hypotheses have been tested in animals, but rarely in plants (Sheth &

602 Angert, 2014).

603 3. Influences of other factors on thermal tolerance and the potential for shared

604 mechanistic and evolutionary underpinnings 605

Few studies examine how thermal tolerance interacts with other abiotic factors that could

606 enhance or reduce susceptibility to thermal extremes. Although research that has focused on thermal

607 tolerances has yielded important information we cannot infer from these studies how plants would

608 respond to combinations of temperature and one or more other stresses (Mittler, 2006; Suzuki et al.,

609 2014). In agricultural fields and natural habitats, plants are often exposed to multiple simultaneous

610 environmental stresses. For example, heat stress frequently occurs in combination with drought.

611 20 Interactions between water limitation and thermal response are ripe for investigation (Jagadish et

612 al., 2011; Fahad et al., 2017), given that both temperature and precipitation regimes are changing

613 across much of the globe. There is growing evidence that plant thermal tolerances are underpinned

614 by molecular and metabolic processes that are both distinct to temperature stress (Rizhsky et al.,

615 2004) and common to other stresses (e.g. tricarboxylic acid-cycle intermediates increase in response

616 to temperature and drought stress; Kaplan et al., 2004). For combinations of thermal tolerance with

617 tolerance to one or more other stresses, plants require unique metabolic and signalling responses

618 (Zandalinas et al., 2018). There remains much to be learnt about the drivers of these unique

619 processes. Addressing this gap is essential for improving model parameterisation for the prediction

620 of plant responses to climate change, identification of key traits for climate-resilient crop breeding

621 programs, and the development of better adaptation strategies for managed agricultural settings and

622 natural habitats.

623 4. Understanding the sensing of and response to thermal stress along the continuum

624 from protective mechanisms to acquired damage 625

There is a complex continuum between temperatures that induce protective mechanisms and

626 those that cause irreparable damage and impact survival (Nievola et al., 2017). The relative impact

627 of a single large vs repeated small exposures outside optimal temperatures remains poorly

628 understood, and the mechanisms underlying priming or memory responses and recovery from

629 thermal stress are complex and still an active area of investigation (Bruce et al., 2007; Lämke &

630 Bäurle, 2017; Hüve et al., 2019). The extent of and mechanisms underlying the plasticity of thermal

631 tolerance are thus another area needing attention and improved analysis (Arnold et al., 2019).

632 Timeframes over which thermal tolerance acclimates in response to realistic temperature

633 fluctuations on diurnal and seasonal bases are yet to be explored in depth. Such studies will provide

634 more comprehensive insight into capacity for stress priming, recovery, and memory (Crisp et al.,

635 2016; Hilker & Schmülling, 2019). Thermal tolerance is highly responsive to changes in climate,

636 growing environment, and interactive abiotic factors and stressors, but not all observed responses

637 will be equally important. On macroscales, general trends in plant thermal tolerance can be

638 observed at a coarse resolution across a range of techniques (Lancaster & Humphreys, 2020), and

639 there is evidence that thermal tolerance plasticity is consistent across different growing

640 environments (Zhu et al., 2018). Much like determining that extreme events have greater impact on

641 selection pressure and population persistence than average warming (Buckley & Huey, 2016), it

642 will be critical to determine the relative importance of the sensitivity and variability of thermal

643 tolerance responses in dynamic environments.

644 21 Conclusions 645 A comprehensive understanding of the thermal tolerance of land plants is crucial. Our rapidly

646 changing climate demands that we pay increased attention to the importance of thermal tolerance

647 for agricultural production and efficiency, ecosystem services, and persistence of wild species. Our

648 systematic review documents geographic and temporal distributions of research efforts and

649 methodological approaches in plant thermal tolerance to date. It shows that there are substantial

650 gaps in our knowledge, and we argue that these are hindering new insights into plant thermal

651 tolerance. The lack of standardised research methods, limited transdisciplinary communication,

652 ambiguous use of terminology and metrics, and unrepresentative global coverage are

653 methodological issues that can be addressed. Conceptual advances will arise from a focus on

654 understanding how thermal tolerance varies in ecological and evolutionary strategy space, studying

655 the importance of thermal breadth, and delimiting mechanisms that underlie acclimation potential

656 and thus the ability to induce protection vs accumulate damage. Finally, we crucially need more

657 insight into how thermal tolerance interacts with and its relative importance in comparison to other

658 abiotic factors such as drought. To these ends, we have identified key design elements for effective

659 thermal tolerance research and outlined an agenda to instigate both fundamental advances and

660 applied outcomes.

661 22 Acknowledgements 662 We would like to thank Verónica Briceño, Jack Egerton, and Rachel Slatyer for insightful

663 discussions during the early phases of this research. We also thank Owen Atkin, Belinda Medlyn,

664 David Ackerly, and two other anonymous reviewers for feedback on earlier drafts of the

665 manuscript.

666

667 Author contributions 668 SRG, PAA, and ABN led the systematic review, data curation and analyses, and led manuscript

669 writing with substantial input on drafts from all authors. All authors contributed significantly to the

670 immense effort that was screening and evaluating articles in the systematic review.

671

672 ORCIDs 673 Sonya R. Geange: 0000-0001-5344-7234

674 Pieter A. Arnold: 0000-0002-6158-7752 675 Alexandra A. Catling: 0000-0002-7537-183X

676 Onoriode Coast: 0000-0002-5013-4715 677 Alicia M. Cook: 0000-0003-3594-3220

678 Kelli M. Gowland: 0000-0001-6066-3103 679 Andrea Leigh: 0000-0003-3568-2606

680 Rocco F. Notarnicola: 0000-0001-9860-6497 681 Bradley C. Posch: 0000-0003-0924-6608

682 Susanna E. Venn: 0000-0002-7433-0120 683 Lingling Zhu: 0000-0003-0489-0680

684 Adrienne B. Nicotra: 0000-0001-6578-369X

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1025 36 Figure Legends 1026 Fig. 1. (a) Thermal tolerance techniques are presented in order of appearance within the

1027 literature for cultivated (left) and wild systems (right). (b) The uptake of techniques since the

1028 1960s; a given article may use multiple techniques (studies) represented exceeds the total

1029 articles identified in the systematic review. Numbers to the right of each plotted line refer to the

1030 numbered techniques described in (c). (c) Definitions for each of the 10 techniques within the

1031 scope of this review. Techniques displayed with an adjacent circle indicate the capacity for a

1032 thermal metric to be generated. Additional information on the techniques and references are

1033 provided in Supplementary Notes S1.

1034 Fig. 2. Global distribution of plant thermal tolerance research. The choropleth map is coloured

1035 by the number of articles in the country of the first author’s affiliation. Total articles on (a)

1036 cultivated and (b) wild species; cold tolerance studies on (c) cultivated and (d) wild species;

1037 studies on cold and heat tolerance together (termed both) on (e) cultivated and (f) wild species;

1038 heat tolerance studies on (g) cultivated and (h) wild species. The number of studies varies

1039 considerably, hence each panel has a different scale for the colour gradient scale bars. The

1040 colour gradients are log-transformed. Regional maps of articles from USA, China, Europe, and

1041 wild studies by experiment location instead of author location are presented in Figs S2–S5.

1042 Fig. 3. The number of studies of thermal tolerance measures on (a) cultivated species across

1043 types of cultivation and (b) wild species across different biomes that focus on either cold

1044 tolerance, heat tolerance, or both heat and cold tolerance. Inset figures highlight the relative

1045 uptake of heat, cold, or both heat and cold tolerance approaches through time for articles on (c)

1046 cultivated and (d) wild species.

1047 Fig. 4. The proportion (and numbers) of intraspecific, single species or multiple species studies

1048 on (a) cultivated and (b) wild species. The variation in life form of the focal study organisms

1049 (forb/herb, graminoid, shrub, tree, vine, or multiple forms (for studies on multiple species)) for

1050 studies on (c) cultivated and (d) wild species.

1051 Fig. 5. (a) The maximum duration of the thermal stress imposed and (b) the type of stress (ramp,

1052 shock, not specified), expressed proportionally within each assay technique for cultivated and

1053 wild systems. Maximum durations listed in order from the longest duration on the left to shortest

1054 on the right: months, weeks, days, hours, minutes, or unspecified. For (b) we defined ramp as a

1055 rate of change in temperature less than 1oC per minute and shock as a rate of change exceeding

1056 1oC per minute. Numbers of studies are shown to the right of the proportion bars.

1057 37 Tables 1058 Table 1. Summary of the number of studies (and percentage of articles in parentheses) for

1059 thermal tolerance research on cultivated species of each type of cultivation and for wild species

1060 of each biome category investigating cold, heat, or both heat and cold tolerance.

1061 Cultivated – type of cultivation Cold Heat Heat and cold

Total Arabidopsis 201 (61.5) 106 (30.8) 21 (7.7)

328 Cereals 339 (49.6) 388 (47.3) 22 (3.4) 749 Fibre

36 (39.4) 43 (54.5) 2 (6.1) 81 Horticulture and vegetables

523 (60.4) 334 (32.4) 61 (7.1) 918 Legumes 117 (51.3)

117 (38.3) 24 (10.4) 258 Pasture and turf grasses

71 (46.1) 111 (48.3) 9 (5.6) 191 Plantation forestry

71 (66.2) 44 (25.0) 14 (8.8) 129 Viticulture 45 (63.8)

38 (27.7) 5 (8.5) 88 Other crops 146 (64.3) 70 (29.7)

16 (4.0) 232 Multiple 33 (61.3) 19 (29.0) 7 (9.7)

59 Not specified 0 (0.0) 3 (100.0) 0 (0.0) 3 Cultivated – subtotal

1,582 (56.7) 1,273 (37.7) 181 (5.6) 3,036 Wild – biome

Alpine/Arctic tundra/Subalpine 79 (74.5) 29 (21.3)

4 (4.2) 112 Arid/Semi-arid/Savannah 27 (45.0) 20 (55.0)

0 (0.0) 47 Boreal forest 45 (100.0) 0 (0.0) 0 (0.0)

45 Mediterranean 29 (52.0) 17 (40.0) 5 (8.0) 51

Temperate 179 (76.2) 54 (21.3) 5 (2.5) 238 Tropical/Subtropical

32 (61.8) 26 (23.5) 16 (14.7) 74 Multiple 65 (64.7)

39 (29.4) 9 (7.9) 113 Not specified 12 (42.9) 15 (57.1)

0 (0.0) 27 Wild – subtotal 468 (69.7) 200 (25.9)

39 (4.4) 707 All species total 2,050 (59.3) 1,473 (35.3)

220 (5.4) 3,743 Note that multiple individual uses of thermal tolerance techniques (studies) can occur in a

1062 single article; therefore, we reported both the number of studies along with percentages of

1063 articles in parentheses for each subcategory (row).

1064

1065 38 Table 2. Key considerations and recommendations for future research.

1066 A. Methodological and design considerations:

1. Application of techniques: Greater crosstalk among researchers studying thermal tolerance of cultivated and wild species would be mutually beneficial to compare and apply different techniques and develop high-throughput approaches.

2. Experimental design considerations: Careful consideration when designing thermal tolerance research, particularly on how temperature stress is applied, thermal legacy effects, and interactions with other environmental factors.

3. Development of standard approaches and comparable metrics: Test comparability of methods and metrics and use multidisciplinary approaches to generate stronger insights into both mechanisms and patterns of thermal tolerance.

B. Research priority agenda:

1. The comparative ecology of thermal tolerance in the ecological and evolutionary strategy spaces: Trait-based approaches in plant ecology should be linked to thermal tolerance to scale-up to higher-level ecosystem processes. Broad-scale comparative studies across a wider range of growth forms, biomes, and that can account for methodological differences will generate greater understanding of biogeographic patterns of tolerance.

2. Understanding the geography and drivers of thermal tolerance breadth: Prioritise measuring thermal tolerance breadth, both heat and cold tolerance, particularly in wild species in thermally extreme regions or regions where snowmelt dynamics are changing, and crop species in regions where climate vulnerability is high.

3. Influences of other factors on thermal tolerance and the potential for shared mechanistic and evolutionary underpinnings: Multi-factorial experiments are key to identifying molecular and metabolic responses and for determining which are distinct to temperature stress or common to other sources of stress.

4. Understanding the sensing of and response to thermal stress along the continuum from protective mechanisms to acquired damage: Conduct detailed investigations into the time-sensitive aspects of recovery and damage dynamics, the role of plasticity, and effects of various thermal stresses, including means, extremes, variability, and microhabitats, on plant thermal tolerance.

1067 39 Supporting Information 1068 Fig. S1 PRISMA (Preferred Reporting Items in Systematic Reviews and Meta-Analyses)

1069 diagram illustrating the number of articles identified through database searching, title and

1070 abstract screening, and full-text searching.

1071 Fig. S2 Choropleth map of the distribution of plant thermal tolerance research within the

1072 People’s Republic of China.

1073 Fig. S3 Choropleth map of the distribution of plant thermal tolerance research within the United

1074 States of America.

1075 Fig. S4 Choropleth map of the distribution of plant thermal tolerance research within Europe.

1076 Fig. S5 Choropleth map of the global distribution of plant thermal tolerance research on wild

1077 plants coloured by where the experiment was conducted, rather than the country of origin of the

1078 first author’s affiliation.

1079 Fig. S6 Number of times a thermal tolerance technique was used within types of cultivation

1080 within cultivated systems and biomes within wild systems.

1081 Fig. S7 Proportion (and number) of studies for cultivated and wild systems that employed a

1082 ramp or shock approach to initiating thermal stress, when considering cold tolerance, heat

1083 tolerance or both cold and heat tolerance.

1084 Fig. S8 Topic mapping of thermal tolerance articles using title and author keywords.

1085 Notes S1. Systematic review methods, options and justifications for reviewer screening of

1086 articles, and extended version of Fig. 1 glossary of common tools and techniques for measuring

1087 thermal tolerance in land plants.

1088 1

New Phytologist Supporting Information Article title: The thermal tolerance of photosynthetic tissues: a global systematic review and roadmap for future research

Authors: Sonya R. Geange, Pieter A. Arnold, Alexandra A. Catling, Onoriode Coast, Alicia

M. Cook, Kelli M. Gowland, Andrea Leigh, Rocco F. Notarnicola, Bradley C. Posch,

Susanna E. Venn, Lingling Zhu, Adrienne B. Nicotra

Article acceptance date: 14 September 2020

The following Supporting Information is available for this article:

Fig. S1 PRISMA (Preferred Reporting Items in Systematic Reviews and Meta-Analyses) diagram illustrating the number of articles identified through database searching, title and abstract screening, and full-text searching.

Fig. S2 Choropleth map of the distribution of plant thermal tolerance research within the

People’s Republic of China.

Fig. S3 Choropleth map of the distribution of plant thermal tolerance research within the

United States of America.

Fig. S4 Choropleth map of the distribution of plant thermal tolerance research within Europe.

Fig. S5 Choropleth map of the global distribution of plant thermal tolerance research on wild plants coloured by where the experiment was conducted, rather than the country of origin of the first author’s affiliation.

Fig. S6 Number of times a thermal tolerance technique was used within types of cultivation within cultivated systems and biomes within wild systems.

Fig. S7 Proportion (and number) of studies for cultivated and wild systems that employed a ramp or shock approach to initiating thermal stress, when considering cold tolerance, heat tolerance or both cold and heat tolerance.

Fig. S8 Topic mapping of thermal tolerance articles using title and author keywords.

Notes S1 Systematic review methods, options and justifications for reviewer screening of articles, and extended version of Fig. 1 glossary of common tools and techniques for measuring thermal tolerance in land plants.

2

Fig. S1

Fig. S1. PRISMA (Preferred Reporting Items in Systematic Reviews and Meta-Analyses) diagram illustrating the number of articles identified through database searching, title and abstract screening, and full-text searching. Indicated at the bottom of the diagram are the number of articles identified as belonging to either cultivated or wild systems.

3

Fig. S2

4

Fig. S2. Distribution of plant thermal tolerance research within the People’s Republic of

China. The choropleth map is coloured by the number of articles in the province of the first author’s affiliation. Total number of articles on (a) cultivated species and on (b) wild species; focussing on cold tolerance in (c) cultivated species and on (d) wild species; cold and heat tolerance together (termed both) on (e) cultivated species and on (f) wild species; and focussing on heat tolerance in (g) cultivated species and on (h) wild species. Note that each panel has a different scale for the colour gradient scale bars, and that the gradients are log- transformed for easier differentiation between the colours among provenances because the number of articles varies significantly. China had 334 articles on cultivated species, but only

36 on wild species. Most articles on cultivated species within China were in the central and eastern provinces, whereas the few wild articles included north-western and eastern provinces. Province abbreviations: AH = Anhui, BJ = Beijing, CQ = Chongqing, FJ = Fujian,

GS = Gansu, GD = Guangdong, GX = Guangxi, GZ = Guizhou, HI = Hainan, HE = Hebei,

HL = Heilongjiang, HA = Henan, HK = Hong Kong, HB = Hubei, HN = Hunan, NM = Inner

Mongolia, JS = Jiangsu, JX = Jiangxi, JL = Jilin, LN = Liaoning, NX = Ningxia, QH =

Qinghai, SN = Shaanxi, SX = Shanxi, SD = Shandong, SH = Shanghai, SX = Sichuan, TW =

Taiwan, TJ = Tianjin City, XZ = Tibet, XJ = Xinjiang, YN = Yunnan, ZJ = Zhejiang.

5

Fig. S3

6

Fig. S3. Distribution of plant thermal tolerance research within the United States of America (USA). The choropleth map is coloured by the number of articles in the state of the first author’s affiliation. Total number of articles on (a) cultivated species and on (b) wild species; focussing on cold tolerance in (c) cultivated species and on (d) wild species; cold and heat tolerance together (termed both) on (e) cultivated species and on (f) wild species; and focussing on heat tolerance in (g) cultivated species and on (h) wild species. Note that each panel has a different scale for the colour gradient scale bars, and that the gradients are log- transformed for easier differentiation between the colours among countries because the number of articles varies significantly. The USA had 241 articles on cultivated species, but only 80 articles on wild species. Research on cultivated species was conducted throughout the

USA, but only in four states (Hawai’i, Massachusetts, Wyoming, and West Virginia) were cold and heat tolerance investigated together. State abbreviations: AL = Alabama, AK =

Alaska, AR = Arkansas, AZ = Arizona, CA = California, CO = Colorado, CT = Connecticut,

DE = Delaware, DC = District of Columbia, FL = Florida, GA = Georgia, HI = Hawai’i, ID =

Idaho, IL = Illinois, IN = Indiana, IA = Iowa, KS = Kansas, KY = Kentucky, LA = Louisiana,

ME = Maine, MD = Maryland, MA = Massachusetts, MI = Michigan, MN = Minnesota, MS

= Mississippi, MO = Missouri, MT = Montana, NE = Nebraska, NV = Nevada, NH = New

Hampshire, NJ = New Jersey, NM = New Mexico, NY = New York, NC = North Carolina,

ND = North Dakota, OH = Ohio, OK = Oklahoma, OR = Oregon, PA = Pennsylvania, RI =

Rhode Island, SC = South Carolina, SD = South Dakota, TN = Tennessee, TX = Texas, UT =

Utah, VT = Vermont, VA = Virginia, WA = Washington, WV = West Virginia, WI =

Wisconsin, WY = Wyoming.

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Fig. S4

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Fig. S4. Distribution of plant thermal tolerance research within Europe. The choropleth map is coloured by the number of articles in the country of the first author’s affiliation. Total number of articles on (a) cultivated species and on (b) wild species; focussing on cold tolerance in (c) cultivated species and on (d) wild species; cold and heat tolerance together (termed both) on (e) cultivated species and on (f) wild species; and focussing on heat tolerance in (g) cultivated species and on (h) wild species. Note that each panel has a different scale for the colour gradient scale bars, and that the gradients are log-transformed for easier differentiation between the colours among countries because the number of articles varies significantly. Europe had reasonable coverage for wild species, with 133 articles, in addition to 433 on cultivated species. Notably, Eastern European countries were well represented in articles of cultivated species, but less so for wild species. Country abbreviations are provided for a subset of countries for orientation purposes: AT = Austria,

BY = Belarus, BG = Bulgaria, CZ = Czech Republic, EE = Estonia, FI = Finland, FR =

France, DE = Germany, GR = Greece, HU = Hungary, IT = Italy, LV = Latvia, LT =

Lithuania, NO = Norway, PL = Poland, PT = Portugal, RO = Romania, RU = Russia, RS =

Serbia, ES = Spain, SE = Sweden, TR = Turkey, UA = Ukraine, UK = United Kingdom.

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Fig. S5

Fig. S5. Global distribution of plant thermal tolerance research on wild plants. In contrast to

Figs S2-S4, the choropleth map here is coloured by the number of articles in the country where the experiment was conducted, rather than the country of origin of the first author’s affiliation, to better represent the distribution of the experimental locations of articles on wild species. For wild species (a) the total number articles, then those focusing on (b) cold tolerance, (c) cold and heat tolerance together (termed both), and (d) heat tolerance. Note that each panel has a different scale for the colour gradient scale bars. Because the number of articles varies significantly, the gradients are log-transformed for easier differentiation of the colours among countries. Comparing study location and affiliation identifies a handful of articles from otherwise unrepresented countries, though there were relatively few discrepancies between the first author’s affiliation and the location that the wild experiments were conducted. Notably, Peru, Ecuador, Antarctica, Greenland, Iceland, Papua New Guinea, and French Guiana were locations for wild experiments on cold tolerance that were not reflected in the first author’s affiliation, and 40 articles did not state any clear location information.

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Fig. S6

Fig. S6. The number of times a thermal tolerance technique was used within (a) types of cultivation within cultivated systems and (b) biomes within wild systems. Note that the scale bars for number of records differ between cultivated and wild panels.

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Fig. S7

Fig. S7. The proportion (and number) of studies for cultivated and wild systems that employed a ramp or shock approach to initiating thermal stress, when considering cold tolerance, heat tolerance or both cold and heat tolerance. Ramp approaches are defined as

≤1°C/min temperature change and shock defined as >1°C/min temperature change.

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Fig. S8

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Fig. S8. Topic mapping of thermal tolerance articles using title and author keywords. (a) 2D ordination of title and author keywords.

Each point represents one article, and articles are coloured according to the highest weighted topic for that article. (b) Bar chart showing the number of articles within each of the five topic groups. Terms above each bar indicate the five most common terms for that group. (c) The top 25 key terms for each topic group from five topic clusters (determined by 2D ordination for similarity) are presented in decreasing frequency of occurrence. Descriptive summary of the five major groupings within the thermal tolerance literature: Topic 1 was mostly focused on chilling and was strongly associated with articles on horticulture and vegetables and viticulture research. Assays that featured prominently within this grouping were membrane damage, antioxidants, and other biochemistry. Topic 2 focused heavily on freezing (as distinct from chilling) resistance, particularly within wild species. Here, electrolyte leakage and membrane damage again featured prominently, but assays of ice nucleation and carbohydrate analysis were also common. Within this group of articles, there was also a strong seasonal element, with research encompassing winter and spring freezing activity. Topic 3 was represented by articles on the heat tolerance of cultivated systems, where there was an emphasis on biochemical and molecular techniques. Articles on heat tolerance of wild species were not distinct from this group. Topic 4 primarily encompassed gas exchange articles across both cultivated and wild systems, regardless of whether they were heat or cold focused.

Topic 5 focused on cold tolerance and gene expression, often on model organisms or cultivated species. These results collectively suggest that there is some siloing with respect to thermal tolerance assays, species selection, and geography.

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Notes S1 1. Systematic review methods We conducted a search of the Institute for Scientific Information (ISI) Web of Knowledge across all subscribed Web of Science databases using an extensive list of search terms. The following words/terms were used within the title and topics tabs: Title: (cold OR freez* OR chill* OR frost* OR “low temperature*” OR froze* OR heat* OR “high temperature*” OR “extreme temperature*” OR “thermal extreme*” OR ice OR therm* OR “cool* temp*” OR “hot temp*”

OR “rising temp*” OR temp* OR “warm temp*” OR “increas* temp*” OR cool* OR warm*

OR hot) AND Topic: (tolera* OR stress* OR respon* OR avoid* OR resistan* OR acclimat*

OR harden* OR adapt* OR injur*) AND Topic: (plant* OR shrub* OR tree* OR leaf* OR bud* OR herb* OR grass* OR graminoid* OR thallus* OR moss* OR fern* OR forb* OR leaves) AND Topic: (cold OR freez* OR chill* OR frost* OR “low temperature*” OR froze*

OR heat* OR “high temperature*” OR “extreme temperature*” OR “thermal extreme*” OR ice

OR therm* OR “cool* temp*” OR “hot temp*” OR “rising temp*” OR temp* OR “warm* temp*” OR “increas* temp*” OR cool* OR warm* OR hot). Asterisks denote Boolean operators that included all words and terms that began with the specified root. Using the “refine” function in Web of Science, we limited outputs to articles published in English and within the Web of

Science categories of Plant Sciences, Ecology, Agronomy, Horticulture, Forestry, Agriculture

Multi-disciplinary, Biodiversity Conservation, or Biology. We included articles published across all years and across all indexes. Our original literature search was conducted on 14 December

2017, using world-leading database access from The Australian National University. Despite focusing only on Web of Science outputs, which may have missed or excluded some relevant articles, it yielded 21,763 articles.

We used the metagear package (Lajeunesse, 2016) in the R environment for statistical computing v3.5.1 (R Core Team, 2018) to download full-text articles and to randomly allocate the 21,763 articles to be screened by 12 co-authors. In an initial screen, titles of these articles were assessed and tagged as “yes”, “no”, or “maybe” for inclusion to the next screening step.

This was based on whether the titles indicated investigations into tolerance of leaves or leaf-buds of angiosperms and gymnosperms exposed to potentially damaging high or low temperature events as distinct from growth conditions. The co-authors then re-assessed articles grouped as

“maybe” and a consensus “yes” or “no” grouping was achieved, yielding 6,508 articles kept at

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the title level. The procedure was repeated for abstracts, which resulted in 2,877 articles retained for evaluation as full-text articles (Fig. S1).

Article evaluation criteria The criteria were:

1) Whether the article dealt with cultivated (e.g. crop plants, horticultural plants, forestry trees, and including the model species Arabidopsis) or wild species.

2) Whether the assay investigated heat, cold, or both heat and cold effects.

3) The diversity of the species measured in the study.

4) For articles on cultivated species – the type of cultivation.

5) For wild species – the biome of origin for the studied plants.

6) The life forms of species.

7) The thermal tolerance technique(s) used.

8) Whether the thermal tolerance assay was applied to leaves, leaf-buds, or both.

9) The nature of the thermal stress applied in the experiment (manipulated or natural).

10) Whether other experimental factors (water, light, etc.) were considered.

11) Whether a thermal metric was reported for the technique(s).

12) Whether stress temperature was gradually ramped or applied as a shock during thermal assay(s).

13) The maximum duration of the thermal assay.

14) Whether the thermal assay was repeated.

15) Whether to include the article or not and, if not, the reason(s) for exclusion.

The criteria for exclusion of an article were: the full-text was not easily accessible; the article was a review, not an empirical article; the methodological details were insufficient to evaluate how the study was conducted; the plants were not stressed at a non-growth temperature (e.g. slightly elevated growth temperatures but not outside average ranges); the study did not address thermal tolerance of leaves or leaf-buds; or the response variable was not relevant for evaluating thermal tolerance of leaf tissue (e.g. growth or whole-plant survival). For clarity, and to aid co-authors in their evaluation of each criterion, expanded justification reference material was drawn up (section 2 below). In addition, a glossary of common tools and techniques for measuring thermal tolerance in land plants was produced (Fig. 1c and section 3 below).

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Once all co-authors had finalised screening, the data were aggregated and rigorously checked for duplicated articles, missing values, clerical errors, and inconsistencies. Where it was necessary to add in or change missing values, the article was re-evaluated to verify that any changes to the data were appropriate. After checking the dataset, the final version contained data from 1,691 unique articles comprising 3,743 studies of thermal tolerance assays (Fig. S1).

The global distribution and concentration of articles on plant thermal tolerance were evaluated using the country of the affiliation of the first author of the article for both cultivated and wild studies. Additionally, we recorded where the experiments were conducted and whether the sample collection location information was stated for wild studies. For the most dominant regions in our database (China, USA, and Europe), we also generated province, state and country-level maps, respectively. Spatial data for generating world, China, USA and European maps were obtained from the R packages ggmap (Kahle & Wickham, 2013), ggplot2 (Wickham,

2016), maptools (Bivand & Lewin-Koh, 2019), and usmap (Di Lorenzo, 2018).

We used topic mapping with the article title and author keywords to explore topical aggregations and/or divisions within the field of thermal tolerance research. Terms were created through constructing a document term matrix, which converted all text to lower case, removed punctuation and numbers, stemmed all words, and removed words with fewer than three letters or those contributing to less than 1% of documents. Final terms reported represent the most common ‘full’ version of a stemmed term. We mapped five topic groups, which provided a balance between providing a broad overview of the field and avoided creating artificial subfields.

Topic mapping was conducted using the R package revtools (Westgate, 2019) and ISI Web of

Knowledge bibliography files. The model was run over 20,000 iterations to optimise fit using the

Latent Dirichlet Allocation (LDA) approach. The generated ordination plot clustered together articles that the algorithm defined as belonging to a similar topic group.

2. Options and justifications for reviewer screening of articles

Each article that was identified for screening at the full-text level was evaluated by the reviewers based on the 15 criteria listed in the main text. Below is an outline for the training for multiple authors to assess articles against the 15 criteria, then the criteria and extended justifications for them, along with a list of available options that the reviewers had for each criterion.

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Training dataset Full-text articles of 30 of the 2,877 articles were chosen as a training dataset for the 12 co- authors to review and evaluate based on pre-defined screening criteria (see below). The training dataset included a representative sample of articles that spanned multiple years, thermal tolerance techniques, and publication fields. The training process identified ambiguities in the screening criteria and reduced discrepancies among the individual reviewers, such that the remaining full-text articles could be randomly allocated across the reviewers for consistent screening. To ensure that reviewer decisions were similar, 20 duplicate articles were surreptitiously included in these allocations, to reveal any inconsistencies in the review process.

If a reviewer was unsure about their decisions on a given article, they could request a secondary review by another reviewer with more specific expertise.

Criteria and justifications (1) If the article dealt with cultivated or wild species

Based on the contextual information provided in the article, was the study conducted on cultivated plants (e.g. crop plants, horticultural plants, forestry trees, Arabidopsis) or on wild plant species. Wild plants could include native or invasive species, or wild plant species that were brought into or grown in laboratory or glasshouse conditions, as long as the context for the study was assessing a wild ecosystem and not of plants for direct anthropogenic use. Checked at abstract and full-text levels.

Reviewer options: Cultivated, Wild

(2) Whether the assay investigated heat, cold, or both heat and cold effects

The type of thermal tolerance assessed in the article. Heat tolerance, cold tolerance, or both heat and cold tolerance within the same article. Mild temperature differences, such as chilling stress on cold-adapted species or mild warming treatments were considered to be non-stressful or not different to growth temperatures, and were excluded as such. If reviewer was unsure about the relevant severity of the temperature stress imposition (i.e. whether the study measured tolerance per se), they requested a secondary review. Checked at abstract and full-text levels.

Reviewer options: Cold, Heat, Both heat and cold

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(3) The diversity of the species measured in the study

The level of intra- or inter-specific variation that the study investigated. Single species studies were those that reported tolerance from only a single type of a single species. Intraspecific studies reported more than one type of a single species (e.g. different cultivars, lines, varietals of the same species). Multiple species studies reported tolerance on more than one distinct species.

Transgenic or genetically modified plants were typically reported in intraspecific studies, but consensus was not reached among reviewers regarding how these plants were classified into the three categories (e.g. whether intraspecific or multiple species); such studies might represent up to 10% of the dataset based on article abstracts that contained the term “transgenic”.

Reviewer options: Single species, Intraspecific, Multiple species

(4) For studies on cultivated taxa – the type of cultivation

The category of cultivation for the species that were measured for thermal tolerance. Arabidopsis includes all studies using Arabidopsis thaliana as a model laboratory species, noting that studies on wild Arabidopsis plants were not included under this category. Cereals included all Poaceae species. Fibre included cotton and textile crops. Forestry includes species grown specifically for forestry-use (e.g. the same species used for forestry in one study may be a wild species in a different country or study context). Horticulture and vegetables included all cultured ornamental plants, fruits, tree nuts, and vegetables. Legumes included all Fabaceae species. Pasture and turf grasses included all grasses cultivated for use in grazing pastures or for lawn use. Viticulture included all grapes. Other crop included oil crops, tobacco plants, and other medicinal crops.

Multiple was for when more than one type of cultivation was used within a single study. Any plant species that was not a managed, bred species was considered to be wild. Not specified was for when all other categorising options were exhausted.

Reviewer options: Arabidopsis, Cereals, Fibre, Horticulture and vegetables, Legumes, Pasture and turf grasses, Plantation forestry, Viticulture, Other crop, Multiple, Not specified, Wild

(5) For wild species – the biome of the studied plants

The biome from which the experimental samples of wild species originate. Any non-wild species (i.e. cultivated species, see criterion 4 above) were assigned an arbitrary biome label of cultivated. In many cases, the article specified the biome from which the samples originated or at

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least provided some geographic information or context to assist the reviewer in determining biome by searching various online resources such localised information or maps based on species and location information, and global biome maps (e.g. Olson et al., 2001; Friedl et al.,

2010; Higgins et al., 2016). The categories were very broad-scale assessments of climate conditions to identify major differences among biomes. Multiple was used when the article compared across more than one biome. Not specified was used when the biome remained unclear after searching for the species/cultivar and geographic region.

Reviewer options: Alpine/Arctic tundra/Subalpine, Arid/Semi-arid/Savannah, Boreal forest,

Mediterranean, Temperate, Tropical/Subtropical, Crop, Multiple, Not specified

(6) The life-forms of species The life-form of the plant species. Tall plants with a woody stem were classified as trees. Small- medium height woody plants were classified as shrubs. Any plant with a trailing or climbing growth habit were classified as vines. Grass-like plants were classified as graminoids. All other herbaceous plants that were angiosperms or gymnosperms were classified as forbs/herbs. If the species does not fit in any life-form category (i.e. is not an angiosperm or gymnosperm), then the article was excluded due to not measuring a relevant plant species. Articles that used the same thermal tolerance technique or conditions across multiple plant life-forms were classified as multiple.

Reviewer options: Tree, Shrub, Vine, Graminoid, Herb/forb, Multiple

(7) The thermal tolerance technique(s) used The type of scientific technique that the study used to assess the thermal tolerance of leaves or buds. If the article used more than one technique, an additional row of information was entered for each relevant technique and experimental condition (see criteria 8-14 below). Further details on each technique are provided in section 3 below. Chlorophyll fluorescence measured changes in fluorescence re-emitted from chlorophyll in the photosystems. Gas exchange included the rate of CO2 uptake or O2 evolution to evaluate the ability of a leaf to recover photosynthetic capacity/respiration rate. Electrolyte leakage/membrane stability included measures of structural damage to cell membranes and electrical conductivity. Quantified visual damage estimation (or death) included calculations of the percentage of damaged leaves on a whole-plant, or proportion

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of cell death or damaged area on an individual leaf or bud. Note that articles that recorded only whole-plant survival and did not explore mechanistic drivers at leaf or bud level were excluded.

Thermometry/spectrometry included spectral or infrared imaging (including SPAD measurements of chlorophyll content), reflectance, and measurements of exothermic reactions such as ice nucleation. Reactive oxygen species (ROS) and antioxidants included oxygen radicals, oxidizing agents, and antioxidants that can affect gene expression or impact on plant responses to stress. Heat shock proteins/factors (HSPs/HSFs) included molecular chaperones and their transcriptional activators, respectively, which are induced to alleviate damage caused by high temperatures. (Epi)genetics and ’omics included any of the ‘omics (e.g. metabolomics, proteomics), protein expression, gene expression, genomics, and epigenetics. Articles on whole genomes were not considered because the genome itself does not respond to stress in the time of a stress event, where such an article would be excluded. Other biochemistry included any wet chemistry that did not fall into other category (e.g. ions, osmotic potential, chlorophyll content or sugars when measured by wet chemistry). Water potential included measures of the potential for water to move between areas of a plant, for example via osmosis or mechanical pressures.

Reviewer options: Chlorophyll fluorescence, Gas exchange, Electrolyte leakage and membrane stability, Quantified visual damage, Thermometry and spectrometry, Reactive oxygen species and antioxidants, Heat shock proteins and factors, (Epi)genetics and ‘omics, Other biochemistry,

Water potential

(8) If the thermal tolerance technique was used on leaves, buds, or both

The relevant plant tissue that was measured in the thermal tolerance assay. Included articles could have measured whole or parts of leaves, leaf buds, or both leaves and leaf buds. Articles that used non-specified tissues from seedlings were included under the classification of leaves and leaf buds, because seedlings have a leaf bud and will often have leaves. If the study measured any other plant part instead (e.g. seeds, pollen, flower buds, flowers) then it was excluded.

Reviewer options: Leaf, leaf buds, leaves and leaf buds

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(9) The conditions under which the experiment was conducted

The experimental nature of the temperature stress applied to the plants in the study.

Temperature-controlled experimentally imposed stress included controlled environment facilities or growth chambers where temperatures were controlled within set ranges. Not temperature- controlled experimentally imposed stress included semi-natural or variable settings with an imposed manipulation but not tight temperature control (e.g. ITEX, open-top chambers, or shade-cloth to protect from frost). Entirely naturally imposed stress was where the study took advantage of a natural event such as a heatwave or frost.

Reviewer options: Experimentally imposed stress that was temperature controlled,

Experimentally imposed stress that was not temperature controlled, Entirely naturally imposed stress

(10) If other experimental factors were considered

Aspects of the study experimental design in addition to thermal tolerance. These were growth or treatment conditions that were intentionally manipulated, and options were specified for potential factorial combinations of climate change relevant conditions. None refers to any study that measured thermal tolerance without applying any additional treatment. Climate change relevant conditions were considered to be temperature (growth temperature treatments, not stress conditions), light, CO2, water availability, or soil nutrients and options were provided for every factorial combination of these factors. Non-climate change relevant factors were considered to be any other factor not listed as climate change relevant (e.g. hormones or antioxidant applications, herbivory). Other combination included any other factorial combination of factors that did not fall into the possible combinations of climate change relevant factors (e.g. hormone × salt treatment). Genotypic differences were not specified here.

Reviewer options: None, Temperature, Water, Light, Nutrients/soil, CO2, Temperature × water,

Temperature × light, Temperature × nutrients/soil, Temperature × CO2, Water × light, Water × nutrients/soil, Water × CO2, Light × nutrients/soil, Light × CO2, Nutrients/soil × CO2,

Temperature × water × light, Temperature × water × CO2, Temperature × water × nutrients/soil, Temperature × light × CO2, Temperature × light × nutrients/soil, Water × light ×

CO2, Water × light × nutrients/soil, Water × CO2 × nutrients/soil, Light × nutrients/soil × CO2,

Non-climate change relevant, Other combination

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(11) Whether a thermal metric was reported for the technique(s)

Any relevant metric for the temperature at which a given quantifiable thermal event occurs (e.g. intracellular freezing that is reported with a nucleation temperature measurement). Thermal metric reported included articles that explicitly stated the thermal metric value in text or a table.

Thermal metric can be calculated included articles that contained information from which a thermal metric could be extracted or calculated (e.g. data presented in a Figure). No thermal metric reported included all other articles that did not measure or report a thermal metric.

Example thermal metrics are Tcrit, T20, NT, LT50 of tissues but not whole-plant survival.

Reviewer options: Thermal metric reported, Thermal metric can be calculated, No thermal metric reported

(12) If temperature was gradually ramped or changed as a step function during thermal assay(s)

Type of application of temperature stress from which thermal tolerance was determined. Shock was if the rate of change in temperature was greater than or equal to 1°C per minute. Ramp was used if the rate of change was slower than 1°C per minute. Not specified was used when the nature of the treatment and test temperatures were unclear or unspecified in the article.

Reviewer options: Shock, Ramp, Not specified

(13) The maximum duration of the thermal tolerance assay

The cumulative duration of the thermal tolerance assay. Minutes included all assays lasting from seconds to up to 59 minutes. Hours included all assays lasting from 60 minutes up to and including 24 hours. Days included all assays lasting from greater than 24 hours up to seven days.

Weeks included all assays lasting from greater than seven days up to 28 days. Months included any assays lasting more than 29 days. Not specified was used when the duration of the assay was not stated and could not otherwise be determined from the article text or figures.

Reviewer options: Minutes, Hours, Days, Weeks, Months, Not specified

(14) If the thermal assay was repeated Whether the thermal tolerance assays were repeated during the course of the experimental duration. Repeated was when the same assay was applied for plants at different developmental or

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growth stages. Not repeated was if the thermal tolerance assay was measured once only on the individual experimental plants.

Reviewer options: Repeated, Not repeated

(15) Whether to include the article or not and, if not, the reason(s) for exclusion

Reviewers chose to include the article or provide the main reason for excluding it. Include was used for all articles that contained appropriate data on plant thermal tolerance according to all criteria listed above. Article not accessible was used where the article full-text pdf could not be accessed from The Australian National University library access through the ISI Web of Science,

Google Scholar, or ResearchGate and similar academic platforms. Insufficient method details was used where the article did not provide enough detail or explicit information for a reviewer to confidently assess how the study was carried out and subsequently fill out one or more attribute columns where not specified was not otherwise an option. Not stressed at non-growth temperature was used for articles that did not apply what could be deemed a thermal stress (e.g. mild chilling treatments or growth under 1°C warming), which was sometimes context- or species-specific. The thermal stress should have lasted less time than a growing season and should have been at a temperature that the authors of the paper considered to be outside of normal growth temperatures (e.g. plants grown at 18°C in a laboratory or glasshouse then exposed to 30°C was considered stressful by the authors because even if that temperature was regularly encountered in the field, the temperature was much higher than the laboratory-grown plants have grown in). Review article was used for any article that was a literature review or commentary type of article that did not contain primary data from a scientific investigation. Not relevant plant species was used for any article that studied thermal tolerance of plants that were not angiosperms or gymnosperms (e.g. bryophytes). Not relevant plant part was used for any article that studied thermal tolerance of plant material other than leaves and/or leaf buds, or for studies that only measured thermal tolerance as whole-plant survival. Not relevant measure was used for articles that did not measure thermal tolerance (e.g. only measured change in growth parameters under increased temperatures). Other reason was used for other relevant reasons for excluding articles (e.g. conference proceedings, written in non-English language, methodology articles, or not about plants).

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Reviewer options: Include, Article not accessible, Insufficient method details, Not stressed at non-growth temperature, Review article, Not relevant plant species, Not relevant plant part, Not relevant measure, Other

3. Extended version of Fig. 1 glossary of common tools and techniques for measuring thermal tolerance in land plants

Techniques used to measure thermal tolerance in plant leaves and leaf buds. For each article in our systematic review, we assessed what type of thermal tolerance technique was used and whether the results could provide a specific temperature at which some physiological threshold is reached; we termed this a thermal tolerance metric (TTM). To qualify as a TTM, the metric would have to be based on the response of an organ assayed across multiple temperatures. Specific metrics vary but are generally critical values for thresholds, e.g. LT50 (lethal temperature at which 50% damage ensues).

Below, we describe the categories of techniques that we included in our systematic review and provide examples of the specific measurements and potential TTMs for each technique. We cite a small number of references here that we found to be good examples of application of the techniques.

Technique and summary (Earliest record of technique in our review)

Measures or indicators of the technique Thermal tolerance metric, TTM

Quantified visual damage estimation or death (1961)

A calculation of the percentage of damaged (discoloured or brown) leaves on the whole-plant or cellular level (e.g. leaf dry mass; leaf area (damaged vs healthy); proportion of cell death; etc.)

Measures: Microscopy for visual assessment of cells;

Photography of whole or section of leaves; visual score of damage to leaf buds (Zub et al., 2012)

TTM: LT50, the temperature of 50% necrosis of cells, buds, or leaves

Chlorophyll fluorescence (1979) Measured changes in fluorescence re-emitted from chlorophyll in the photosystems in response to high or low (potentially stressful) temperature

Measures: Minimum fluorescence (F0); Maximum fluorescence (FM); Photosynthetic quantum efficiency (φPSII); Maximum photosynthetic quantum efficiency (FV/FM); Photochemical quenching (qP); Non-photochemical quenching (NPQ); Chlorophyll a fluorescence transients (O-J- I-P) measures (Berry & Bjorkman, 1980; Strasserf &

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Technique and summary (Earliest record of technique in our review)

Measures or indicators of the technique Thermal tolerance metric, TTM

Srivastava, 1995; Maxwell & Johnson, 2000) TTM: LT50 or T50 threshold: temperature at which FV/FM declines to 50% of the maximum FV/FM of unstressed photosystems (Curtis et al., 2014); Tcrit: temperature at calculated inflection point between slow and fast rise phases of the temperature-dependent increase in F0) (Knight &

Ackerly, 2002); TS20 and LT50 or T50: temperature when F0 reaches 20 or 50%, respectively, of the F0 maximum (Tmax) (Knight & Ackerly, 2002); Rfd: chlorophyll fluorescence decrease ratio or vitality index, calculated on the decline of

FM to the fluorescence steady state level (FS) (Lichtenthaler et al., 1986; Perera-Castro et al., 2018)

Thermometry and spectrometry (1964) Thermometry: plant tissue temperatures can indicate functional parameters (e.g. point of ice nucleation on a leaf and its progression through the plant); can differentiate between the roles of extrinsic and intrinsic ice nucleating agents in the freezing process; and the effect of the freezing process on the plant form.

Spectrometric approaches are primarily used to assess pigment distributions, contents and to derive indices of photochemical health

Thermometry measures: analyses of the critical temperatures for ice formation in cells (e.g. high-resolution infrared thermography) (Wisniewski et al., 2008); thermal imaging can also be used to assess plant water status (water stressed plants with reduced stomatal conductance are generally warmer)

Spectrometry measures: reflectance at various wavelengths (visual to near infrared) - can be spatially resolved (imaging) or point based (e.g. pigments) (Lefsrud et al., 2005)

TTM: NT – Ice nucleation temperature Electrolyte leakage and membrane stability (1972)

Damaged cell membranes leak ions and other contents and damage can be measured using electrical conductivity

Measures: tissue ionic conductance (gTi), electrical conductivity (EC); damage index (Id) (Whitlow et al., 1992)

TTM: LT50 - Temperature at which 50% electrolyte leakage occurs

Gas exchange (1968) Examines changes in the rates of leaf CO2 uptake or O2 evolution as indicators of photosynthetic capacity and respiration. Measures frequently include rate of water loss

Measures: Net CO2 assimilation rate (Anet); dark respiration (Rdark) stomatal conductance (gs); intercellular CO2 concentration (ci); and transpiration rate (E) of intact leaves (von Caemmerer & Farquhar, 1981)

TTM: Tmax upper thermal limit of leaf respiratory CO2 release in darkness (O'Sullivan et al., 2013; 2017)

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Technique and summary (Earliest record of technique in our review)

Measures or indicators of the technique Thermal tolerance metric, TTM

Water Potential (1974) Quantifies the potential for water to move between one area of a plant to another through osmosis, gravity, mechanical pressure, or matrix effects such as capillary action

Measures: Recorded as negative potential ψ, relative to pure water reference. Measured using psychrometers, or pressure chambers. A unit of pressure, as a form of energy (ψ, psi,

MPa) (West & Gaff, 1976) TTM: πtlp – leaf turgor loss point or bulk turgor loss point, taken as the point at which leaf cells become flaccid – fails to maintain cell turgor pressure (Epi)genetics and ‘omics (1971)

Molecular mechanisms that alter gene expression and function without changes in the DNA sequence (chemical modification of DNA (methylation) and histones, incorporation of histone variants and long or small non- coding RNAs)

Gene expression: techniques to evaluate amounts and types of mRNA molecules in a cell (e.g. via transcriptomics), reflecting the function and enzymatic activities of the sample.

‘Omics more broadly: encompasses all the other ‘omics e.g. whole-genome detection of genes (genomics), proteins (proteomics), and metabolites (metabolomics)

Indicators: SMP (Single Methylation Polymorphism); DMR (Differentially Methylated Regions); GBS (Genome Bisulfite

Sequencing): techniques aimed to evaluate the methylation status of the cytocines in the whole genome (WGBS) or in a reduced representation of it (RRBS), taking advantage of a bisulfite treatment that converts non-methylated cytocines in uracils (van Gurp et al., 2016; Paun et al., 2019)

‘Omics techniques: Mass spectrometry (MS); SNP genotyping (genomics); RNAseq and gene expression, microarrays, gene chips (transcriptomics); gel electrophoreses, enzyme-linked immunosorbent assays (ELISAs), protein microarrays and chromatography (proteomics); nuclear magnetic resonance (NMR), and chromatography (metabolomics) (Gemperline et al., 2016)

Heat shock proteins and factors (HSPs and HSFs) (1991)

Rapidly induced proteins and factors in response to abiotic stresses and alleviate damage. HSPs function as molecular chaperones, assist in protein folding, maintain signal transduction and prevent protein aggregation (Chen et al., 2018)

Measures: Relative abundance detected using western blotting, slot/dot blotting (more tolerant individuals would induce a larger abundance of HSPs), gene expression (chromatography, quantitative real time PCR), in vitro chaperone-like activity assay, electron microscopy (Zhang et al., 2015; Chen et al., 2018)

Common HSPs measured: Hsp100, Hsp90, Hsp70, Hsp60, and small HSPs

Reactive Oxygen Species (ROS) and antioxidants (1981)

Indicators: Many aspects of the roles of ROS in plants are covered in a ROS special issue of Plant Physiology (2006)

27

Technique and summary (Earliest record of technique in our review)

Measures or indicators of the technique Thermal tolerance metric, TTM

ROS are oxygen radicals and non-radical oxidizing agents that can be converted into radicals. ROS are natural by-products of a plants metabolic processes and can affect gene expression and impact upon a plants growth, signalling, development, cell cycle, programmed cell death (PCD), abiotic stress responses, pathogen defence and adaptation (Gill & Tuteja, 2010; Mittler et al., 2011). ROS concentrations can increase rapidly in response to a multitude of stimuli including temperature extremes.

Antioxidants mitigate cellular damages caused by the accumulation of ROS and in (Gill & Tuteja, 2010; Mittler et al., 2011)

Common ROS: Free radicals: superoxide radicals, hydroxyl radicals (OH*), perhydroxy radicals (O2H*), alkoxy radicals; non-radicals: hydrogen peroxide (H2O2), singlet oxygen (O2i); others that may not be in plants: molecular oxygen (triplet ground state, O23Σ), superoxide anion (O2.-), ozone (O3)

Common antioxidants: Thiobarbituric acid (TBA), Malondialdehyde (MDA), Ascorbate or ascorbic acid or

Vitamin C (ASH), Glutathione (GSH), Ascorbate peroxidase (APX), Superoxide dismutase (SOD), Catalase (CAT),

Glutathione reductase (GR); as well as less common radicals such as: a-tocopherols, phenolic compounds, alkaloids and non-protein amino acids

Other Biochemistry (1968) Temperature tolerance can also be measured by the presence (or absence) and change in quantity of certain biochemicals

Measures: Other chemical compounds that can be used to infer thermal tolerance include:

• Volatile Organic Compounds (VOCs), e.g. isoprene

• Biogenic Volatile Organic Compounds (BVOCs), e.g. terpenes

• Organic acids (ascorbic acid, pyruvic acid, etc.) and amino acids (proline, asparagine, etc.)

• Sugars (sucrose, glucose, etc.) or sugar alcohols (xyilitol, myo-inositol, etc.), raffinose family oligosaccharides (RFOs)

• Plant hormones (abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA)

• Phenol and flavonoid contents • Nutrient/element content (e.g. N, Ca, Mg, K, Na)

• Chlorophyll content (unless measured using spectrometry)

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6. Water potential Quantifies the potential for water to move between one area of a plant to another through osmosis, gravity, mechanical pressure or matrix effects such as capillary action.

7. (Epi)genetics and ‘omics Broadly, ‘omics refers to the fields of molecular biology that are specifically associated with whole-genome detection of: genes (genomics), gene expression (transcriptomics), proteins (proteomics), and metabolites (metabolomics).

Epigenomics specifically refers to the molecular mechanisms that alter gene expression and function without changes in DNA sequence (e.g. through chemical modification of DNA (methylation) and histones, incorporation of histone variants and long or small non-coding RNAs).

8. Heat Shock Proteins (HSPs) Rapidly induced in response to abiotic stresses and alleviates damage. HSPs function as molecular chaperones, assist in protein folding, maintain signal transduc- tion and prevent protein aggregation.

9. Reactive Oxygen Species (ROS)/ Antioxidants ROS are oxygen radicals and non-radical oxidizing agents that can be converted into radicals. They are by-products of a plant’s metabolic processes which can impact upon a plant’s growth, signalling, develop- ment, cell cycle, programmed cell death, abiotic stress responses and pathogen defence and can increase rapidly in response to temperature stress. Antioxi- dants mitigate the cellular damage that

ROS cause.

10. Other biochemistry Techniques can be used to assess thermal tolerance by the presence or absence of certain biochemicals.

Techniques 1. Quantified visual damage Measures the percentage of damaged (discolored/brown) leaves or leaf area (e.g. proportion of cell death; leaf area).

2. Thermometry & Spectrometry Used to identify temperature-induced changes in plant tissue and can indicate functional parameters (e.g. leaf ice nucleation point and its progression through the plant and indices of photochemical health).

3. Gas exchange Determines the ability of a leaf to recover photosynthethic capacity, or change its rate of respiration, after exposure to stressful temperature through examination of the time stability of rate of CO2 uptake or O2 evolution.

4. Electrolyte leakage / Membrane stability Measures electrical conductivity to determine cell membrane damage/ leakiness in response to stress.

5. Chlorophyll fluorescence Refers to light re-emitted from chlorophyll and provides a sensitive indicator of temperature stress.

1960 2020 2010 2000 1990 1980 1970 0 100 200 300 400

500 1970 1980 1990 2000 2010 Year of Publication Number of Articles

Techniques over time 5 1 4 3 10 2 9 6 8 7 0 100 200

300 400 500 1960 1980 2000 2020 Year of Publication

Number of Articles 10 4 5 7 3 9 1 8 6 2 Techniques over time

Wild Quantified visual damage Chlorophyll fluorescence

Thermometry & Spectrometry Electrolyte leakage / Membrane stability

Gas exchange Water potential (Epi)genetics and ‘omics

Heat Shock Proteins (HSPs) Reactive Oxygen Species (ROS)/ Antioxidants

Cultivated Chlorophyll fluorescence Thermometry & Spectrometry

Electrolyte leakage / Membrane stability Gas exchange

Water potential (Epi)genetics and ‘omics Heat Shock Proteins (HSPs)

Reactive Oxygen Species (ROS)/ Antioxidants Quantified visual damage (a) (c) (b) (b) Wild: total

1 5 30 80 N Articles 1 10 100 300 N Articles (a) Cultivated: total (d) Wild: cold

1 5 20 45 N Articles 1 10 50 150 N Articles (c) Cultivated: cold (f) Wild: both

1 2 3 4 N Articles 1 5 10 15 N Articles (e) Cultivated: both (h) Wild: heat

1 5 15 30 N Articles 1 10 60 130 N Articles (g) Cultivated: heat

Cold Heat and Cold Heat Temperate Multiple Alpine / Arctic Tundra /

Subalpine Tropical / Subtropical Arid / Semi-arid /

Savannah Mediterranean Boreal forest Not specified

0 100 200 300 Number of Studies (b) Wild: Biome 400

800 Number of Articles 1960 1980 2000 2020 Year of Publication

0 (d) Horticulture / Vegetables Cereals Arabidopsis

Legumes Plantation forestry Viticulture Fibre Multiple

Not specified 0 250 500 750 1000 Number of Studies (a) Cultivated: Type of cultivation d)

0 400 800 1960 1980 2000 2020 Year of Publication Number of Articles (c)

Pasture grasses Other crop Forb / Herb Graminoid Multiple

Shrub Tree Vine Wild Cultivated Species Diversity Intraspecific

Multiple Species Single Species (a) (b) (c) (d) Life Form

1243 515 1278 312 87 308 7 294 112 59 65 160 1165 967

92 237 267 308 Chlorophyll fluorescence Electrolyte leakage/

Membrane stability (Epi)genetics and ‘omics Gas exchange

Heat Shock Proteins / Factors Other biochemistry Quantified visual damage

ROS and Antioxidants Thermometry / Spectrometry Water potential

Thermal Tolerance Technique Maximum Duration Not Specified

Minutes Hours Days Weeks Months (a) Ramp / Shock Not Specified

Ramp Shock 0.00 0.25 0.50 0.75 1.00 Proportion of Studies

Cultivated 0.00 0.25 0.50 0.75 1.00 Proportion of Studies

Cultivated Chlorophyll fluorescence Electrolyte leakage/

Membrane stability (Epi)genetics and ‘omics Gas exchange

Heat Shock Proteins/ Factors Other biochemistry Quantified visual damage

ROS and Antioxidants Thermometry / Spectrometry Water potential

0.00 0.25 0.50 0.75 1.00 Proportion of Studies Wild

0.00 0.25 0.50 0.75 1.00 Proportion of Studies Wild

Thermal Tolerance Technique (b) 63 190 278 265 524

69 294 398 497 458 18 69 27 150 65 12 75 21 119 151

63 190 278 265 524 69 294 398 497 458 18 69 27 150

65 12 75 21 119 151