Control of rate and physiological role of isoprene emission from plants

Abstract

Isoprene is a volatile compound produced in large amounts by some, but not all, plants by the enzyme isoprene synthase. Plants emit vast quantities of isoprene, with a net global output of 600 Tg per year, and typical emission rates from individual plants around 2% of net carbon assimilation. There is significant debate about whether global climate change resulting from increasing CO₂ in the atmosphere will increase or decrease global isoprene emission in the future. We show evidence supporting predictions of increased isoprene emission in the future but the effects could vary depending on the environment under consideration. For many years isoprene was believed to have immediate, physical effects on plants such as changing membrane properties or quenching reactive oxygen species. While observations sometimes supported these hypotheses, the effects were not always observed and the reasons for the variability were not apparent. While there may be some physical effects, recent studies show that isoprene has significant effects on gene expression, the proteome, and the metabolome of both emitting and non-emitting species. Consistent results are seen across species and specific treatment protocols. This review summarizes recent findings on the role and control of isoprene emission from plants.

Introduction

Isoprene (2-methyl-1,3-butadiene) is one of a myriad of volatile compounds that are produced by plants (Sanadze 1957; Rasmussen 1970). Volatiles have many roles in plant function but isoprene may be especially related to abiotic stress tolerance of plants. Globally, 70% of biogenic volatile organic carbon emissions (not counting methane) are accounted for by just one molecule, isoprene (Sindelarova et al. 2014). Isoprene has little to no odor at physiological concentrations compared to pleasant-smelling volatiles like pine scent or unpleasant-smelling volatiles like dimethyl disulfide. As a result, its emission from plants is not widely recognized despite its overwhelming proportion of biogenic volatile organic carbon emissions. Plant-derived isoprene enters the atmosphere in vast quantities [500 to 600 Tg per year (Guenther et al. 2006; Sindelarova et al. 2014)], which makes it an important player in atmospheric chemistry, contributing to ozone and secondary aerosol production in the troposphere and increasing the lifetime of methane (Poisson et al. 2000; Claeys et al. 2004; Zhang et al. 2007; Pike & Young 2009; Young et al. 2009). Research on isoprene emission from plants has been accelerating over the last few decades. Up until 1960 a total of 27 papers were published that can be retrieved by a search of Web of Science with the topic of isoprene and plants. In each succeeding decade the number of papers has doubled; from the beginning of 2010 to April 2019 over 1200 papers have been published on isoprene and plants.

Isoprene is produced in large amounts by some but not all plants by the enzyme isoprene synthase (Sharkey et al. 2008). Like monoterpenes, the hemiterpene isoprene is produced by the chloroplastic methylerythritol 4-phosphate (MEP) pathway (Figure 1). Isoprene synthase, which is necessary for significant isoprene emission rates, converts dimethylallyl diphosphate (DMADP) produced by the MEP pathway, to isoprene. Isoprene emission represents a loss of both carbon (typically 2% of photosynthesis) and energy (20 ATP and 14 NADPH molecules per molecule of isoprene) (Sharkey & Yeh 2001).

Figure 1: The MEP pathway in plants.

Enzymes: PEPC = phosphoenolpyruvate carboxylase, DXS = 1-deoxy-d-xylulose-5-phosphate synthase, DXR = 1-deoxy-d-xylulose-5-phosphate reductoisomerase, CMS/MCT = 4-diphosphocytidyl-2-C-methylerythritol synthase/2-C-methyl-d-erythritol-4-phosphate cytidylyltransferase, CMK = 4-(cytidine 5’-diphospho)-2-C-methyl-d-erythritol kinase, MCS = 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase, HDS = 4-hydroxy 3-methylbut-2-enyl-diphosphate synthase, HDR = 4-hydroxy-3-methylbut-2-enyl-diphosphate reductase, IDI = isopentenyl diphosphate isomerase. In isoprene emitting plants the conversion of DMADP to isoprene is catalyzed by isoprene synthase (ISPS).

Isoprene improves plant resilience to thermal, oxidative, and biotic stresses (Singsaas & Sharkey 1997; Sharkey et al. 2001; Behnke et al. 2007; Laothawornkitkul et al. 2008; Vickers et al. 2009; Velikova et al. 2011). However, the mechanism of this protection is not known (Harvey et al. 2015). It had been hypothesized that isoprene could intercalate into membranes and improve membrane stability, which could explain the thermal effects (Sasaki et al. 2007; Siwko et al. 2007; Sharkey et al. 2008; Velikova et al. 2011). Isoprene can also protect against ozone and singlet oxygen (ROS) damage (Loreto & Velikova 2001; Affek & Yakir 2002; Velikova et al. 2004) leading to the suggestion that it directly quenches ROS.

However, isoprene is highly volatile and does not dissolve into cellular components, even membranes, in great quantity (Harvey et al. 2015). Recent studies have shown that isoprene is present at very low concentration in chloroplastic membranes (0.0044 mol% isoprene under physiological conditions) and is therefore presumed to be insufficient to affect membrane dynamics or to be an efficient sink for ROS (Harvey et al. 2015). In addition, leaf discs fumigated with isoprene do not exhibit thermal protection (Logan et al. 1999; Logan & Monson 1999). Therefore, new hypotheses have been put forward to explain how isoprene protects plants from biotic and abiotic stresses. Recent studies have shown isoprene to change gene expression (Harvey & Sharkey 2016; Zuo et al. 2019), the proteome (Velikova et al. 2014; Vanzo et al. 2016), the metabolome, and metabolic fluxes (Behnke et al. 2010a; Way et al. 2013; Ghirardo et al. 2014).

This review will focus on recent advances of our knowledge on the control of isoprene emission and effects of climate change on future emission rates. Current understanding of underlying mechanisms responsible for the observed responses of isoprene to light, CO₂, and temperature will be discussed. A summary of recent findings on the physiological role and the mode of action of isoprene specifically as a signaling molecule will also be presented.

Biochemical control of isoprene emission

MEP pathway

The MEP pathway is the source of the isoprene precursor DMADP. Carbon for the MEP pathway comes primarily from photosynthesis. Some studies hypothesize that some carbon for isoprene emission can come from extra-chloroplastic sources, and the amount increases under stress (Kreuzwieser et al. 2002; Affek & Yakir 2003; Schnitzler et al. 2004; Ferrieri et al. 2005; de Souza et al. 2018; Garcia et al. 2019). This is based on the proportion of isoprene that remains unlabeled when labeled CO₂ is fed to photosynthesizing leaves. Delwiche and Sharkey (1993b) found that when plants are fed ¹³CO₂, approximately 20% of isoprene remains unlabeled, however, 20% of photosynthetic intermediates are also unlabeled. They hypothesize that all carbon for isoprene synthesis comes directly from the Calvin-Benson cycle of photosynthesis and the lack of complete labeling of isoprene reflects the lack of complete labeling of the Calvin-Benson cycle intermediates (Sharkey 2018). However, final proof of this hypothesis has not yet been published. On the other hand, when plants are fed ¹³C glucose, isoprene becomes labeled (Kreuzwieser et al. 2002) indicating that it is possible to introduce non-Calvin-Benson cycle sources of carbon for isoprene emission, although the physiological relevance is not clear. To prove that there are sources of carbon other than the Calvin-Benson cycle routinely contributing to isoprene emission will require showing that the intermediates of the Calvin-Benson have a significantly different degree of labeling than emitted isoprene. Sources of unlabeled carbon for the Calvin-Benson cycle can include starch. Leaves held in high photorespiratory conditions may begin to draw on starch reserves (Weise et al. 2006) to feed the Calvin-Benson cycle to make up for the CO₂ lost to photorespiration explaining results seen by Garcia et al. (2019). It is also known that a certain amount of ¹²CO₂ is emitted from leaves fed 99% ¹³CO₂ from unknown sources. This is called “day respiration” and presumably some of this unlabeled CO₂ gets fixed keeping the Calvin Benson cycle less than fully labeled. This day respiration is not associated with photorespiration and lowering oxygen to reduce photorespiration did not change the degree of labeling of isoprene (Delwiche & Sharkey 1993a). It remains possible that in all cases the Calvin-Benson cycle is the predominant carbon source for isoprene emission.

In addition to the close linkage between photosynthetic carbon metabolism and isoprene emission, the MEP pathway is closely linked to photosynthetic electron transport. The MEP pathway requires one CTP (with the release of CMP), one ATP, one NADPH and four ferredoxin molecules (Singsaas et al. 1993; Sharkey et al. 1996; Niinemets et al. 1999; Rasulov et al. 2009b; Behnke et al. 2013; Laffineur et al. 2013; Pallozzi et al. 2013). In leaves, the MEP pathway appears to be completely dependent on photosynthetic electron transport; if the light is abruptly turned off the production of DMADP stops so fast that post-illumination isoprene emission is used as a measure of the DMADP present in the chloroplasts at the time the light is turned off (Rasulov et al. 2009a; Rasulov et al. 2009b; Li & Sharkey 2013; Weise et al. 2013).

The MEP pathway is very strongly regulated by feedback by IDP and DMADP at DXS, competitive with thiamine diphosphate (Banerjee et al. 2013). Because of this, the metabolite concentrations can be similar despite large changes in the flux through the pathway (Ghirardo et al. 2014). Zuo et al. (2019) found a large difference in the relative amount of MEcDP in Arabidopsis versus tobacco. but the presence or absence of isoprene synthase had no effect. They concluded that when ATP was relatively more limiting, MEcDP should be low, but when reducing power (ferredoxin) is relatively low (or more oxidized) MEcDP should be high. Wright et al. (2014) found that the amount of MEcDP in leaves emitting isoprene was higher than those not emitting isoprene. They interpreted this to result from relief of feedback on DXS allowing more flux into the MEP pathway. They also found evidence for a pool of MEcDP outside of the chloroplast. MEcDP has been proposed as a retrograde signal that can affect salicylic acid signaling (Xiao et al. 2012).

Many people have reported that increasing the expression of DXS can increase the flux through the MEP pathway. Wright et al. (2014) showed that among Arabidopsis accessions 82% of the control of the flux through the MEP pathway was accounted for by variation in the amount of DXS. It is somewhat surprising that DXS amount exerts such strong control in light of the fact that it is subject to such strong feedback control. Mayrhofer et al. (2005) found that transcript level for DXS and ISPS varied coincidently through a season but that ISPS protein only accumulated late in the season and was correlated with isoprene emission.

A very strong correlation was found between ISPS and IDI (Brüggemann & Schnitzler 2002). The MEP pathway makes both DMADP and IDP and since only DMADP is a substrate for ISPS, IDP must be converted to IDP. If IDI is at all limiting, IDI could build up and inhibit DXS. By expressing IDI, the ratio of DMADP to IDP can be kept around 2 (Zhou et al. 2013) reducing the IDP inhibition of DXS.

Cross talk between the MVA and MEP pathways has been suggested so that some carbon for isoprene emission could originate in the cytosol. However, plants lacking early MEP pathway genes are unable to grow and thus any crosstalk is at such a low level that it cannot rescue plants lacking the MEP pathway (Mandel et al. 1996; Estévez et al. 2000; Phillips et al. 2008; Banerjee & Sharkey 2014). In addition, post illumination isoprene emission depletes one pool of DMADP but does not deplete all DMADP from a leaf. It is assumed [with some evidence (Weise et al. 2013)] that the dark pool of DMADP is in the cytosol and likely made by the MVA pathway. This DMADP cannot cross into the chloroplast fast enough to be seen in post illumination assays, constraining the rate at which the MVA could provide carbon for isoprene emission.

When isoprene emission is controlled by the activity of the MEP pathway DMADP concentration should be correlated with the isoprene emission rate. This has been reported (Magel et al. 2006). On the other hand, poplars with ISPS suppressed by RNAi and very little isoprene emission had very high concentration of DMADP (Behnke et al. 2010a). During development isoprene emission was most closely associated with the amount of ISPS in poplar leaves (Wiberley et al. 2005). These observations, and the high K_m for DMADP, indicate that control of the rate of isoprene emission can rest with the flux through the MEP pathway, the amount and activity of ISPS, and often, both.

Isoprene synthase (ISPS)

Isoprene synthase is a TPS-b terpene synthase with chloroplastic targeting that converts DMADP to isoprene (Silver & Fall 1995; Sharkey et al. 2013). The TPS-b isoprene synthase is unique to angiosperms, other plant groups do not have TPS-b genes. Isoprene synthase has evolved multiple times, most likely from closely related terpene synthases, especially trans β-ocimene synthases (Harley et al. 1999; Sharkey et al. 2005; Monson et al. 2013; Sharkey et al. 2013; Dani et al. 2014; Loreto & Fineschi 2015; Li et al. 2017). Trans β-ocimene synthesis and isoprene synthesis do not require rotation around the 2–3 double bond of DMADP that is required for formation of cyclic monoterpenes (Croteau & Felton 1981; Sharkey et al. 2013). The similarity in substrate shape and in reaction mechanism likely makes it easy to convert acyclic monoterpene synthases to isoprene synthase by only a few amino acid substitutions (Kampranis et al. 2007; Gray et al. 2011; Koksal et al. 2011; Gao et al. 2012; Sharkey et al. 2013; Li et al. 2017), allowing plants to maintain a balance between the energetic demands of isoprene emission and the physiological benefits under different conditions (Monson et al. 2013).

Isoprene emission has also been lost in many species (Monson et al. 2013). One example is Glycine max (soybean). Two isoprene synthase pseudogenes are present in the genome of this crop plant (Sharkey et al. 2013) while its close wild relative Glycine soja has an active isoprene synthase with a very high similarity to the coding parts of the soybean. Isoprene synthesis is found in all divisions of Plantae and all climates; it is common in fast-growing plants, particularly fast-growing hardwood trees such as Populus spp, Eucalyptus spp, and in mosses such as Campylopus introflexus (Lantz et al. 2015) and Sphagnum spp (Hanson et al. 1999), as well as a wide variety of legumes such as Peuraria montana and Mucunia pruriens. While high rates of isoprene emission are found in some monocot species such as Arundo donax and Phragmites australis, only recently was a monocot isoprene synthase cloned (Li et al. 2017). Analysis of the ISPS promoter of Ficus septica revealed the presence of cis-elements for MYC2 (jasmonic acid-JA signaling) and LHY (a circadian clock protein), which shows that isoprene synthesis can be regulated through modulation of ISPS by hormones and circadian rhythms (Parveen et al. 2019).

Control by environmental factors

The rate of isoprene emission is affected by environmental factors such as light, temperature, CO₂ concentration, and O₂ concentration. Isoprene emission changes very rapidly in response to wounding, changes in CO₂, and temperature, with short-term responses occurring within seconds (Figure 2). DMADP is produced in the chloroplast by the prokaryotic MEP pathway, also called the non-mevalonate pathway (Figure 1). While isoprene responds to many environmental factors, induced monoterpenes, which come from the same MEP pathway, are much less sensitive to environmental parameters (Feng et al. 2019).

Figure 2: The post-burning burst from Glycine soja.

At time 0 one leaflet of a trifoliolate leaf was burned for 5 seconds with a butane lighter. Assimilation and isoprene emission were tracked in an adjacent leaflet. The effect when leaves were exposed to 150 ppm ambient CO₂ (A) and 1200 ppm ambient CO₂ (B) is shown. (Unpublished data by A.T.L. and T.D.S.)

Short term

Light

Isoprene emission is light dependent (Sanadze & Kalandaze 1966; Tingey et al. 1979); the light dependence is similar to that of CO₂ fixation except that isoprene emission can continue to increase with light at higher light intensity than CO₂ fixation (Lerdau & Keller 1997). The light dependence is presumed to result from the availability of high energy intermediates such as ferredoxin, NADPH, ATP and CTP. When photosynthetic electron transport is blocked by an inhibitor, isoprene emission stops (Garcia et al. 2019). It is presumed that the MEP pathway is more dependent on reductant than ATP when compared to CO₂ fixation. The ratio of ATP per NADPH for CO₂ fixation is 1.5:1 (or more if photorespiration is considered) while for isoprene production from CO₂ it is 1.4:1 (Sharkey & Yeh 2001). Nevertheless, ATP supply also plays an important role. When isoprene emission and metabolites were measured under a variety of conditions, the relationship between isoprene emission rate and ATP had an r² of 0.8 to 0.9 while the relationship with phosphoglyceric acid, ribulose bisphosphate, and triose phosphates had r²values of 0.55 or less in both oak leaves and velvet bean (Loreto & Sharkey 1993b). Further, when methyl viologen was fed to leaves to increase the ratio of ATP to reducing power, isoprene emission at first increased (Loreto & Sharkey 1990). Photosynthetic electron transport can also result in changes in pH of the stroma and ion concentrations, both of which could potentially affect the activity of the MEP pathway or isoprene synthase (Sharkey et al. 1996; Laffineur et al. 2013; Rasulov et al. 2016).

When plants are subjected to darkness, production of DMADP stops apparently instantaneously (Weise et al. 2013). All remaining DMADP is rapidly converted to isoprene (Monson & Fall 1989a; Rasulov et al. 2009a; Li et al. 2011; Rasulov et al. 2011). About 5 min later, a second burst of isoprene emission is seen which is correlated with the amount of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) (Li & Sharkey 2013; Jud et al. 2016; Dani et al. 2017). This suggests that 4-hydroxy-3-methylbut-2-enyl-diphosphate (HMBDP) synthase (HDS) and HMBDP reductase (HDR) are initially nonfunctional after turning off the light but then become functional after a few min of darkness. These proteins usually use ferredoxin for reducing power but can also use NADPH in some cases (Wolff et al. 2003; Seemann et al. 2006; Xiao et al. 2009). After 10–30 minutes of darkness, the remaining MEcDP can be converted to HMBDP and then to DMADP/ isopentenyl diphosphate (IDP) because HDR and HDS are using reducing power from alternative sources such as NADPH from catabolism (Wolff et al. 2003; Seemann et al. 2006; Xiao et al. 2009; Li & Sharkey 2013). However, there is evidence that suggests the post-illumination burst is likely species-specific and depends on the differences in MEcDP pool sizes. For example, when Arabidopsis and tobacco engineered to emit isoprene were exposed to sudden darkness, Arabidopsis with a small pool of MEcDP did not show a post-illumination burst while tobacco with a larger pool of MEcDP did (Zuo et al. 2019).

Temperature

Isoprene emission increases with rising temperature (Sanadze & Kalandaze 1966; Sharkey et al. 1991; Rosenstiel et al. 2004; Scholefield et al. 2004; Possell & Hewitt 2011; Monson et al. 2016). Isoprene emission is also more sensitive to temperature than is photosynthesis (Table 1) and has a higher optimum – above 45°C. The effect of temperature is due to the combined effect of DMADP availability and isoprene synthase activity. The V_max of isoprene synthase has an optimum of 42–45°C, while DMADP concentration peaks at 30–35°C (Monson et al. 1992; Lehning et al. 1999; Rasulov et al. 2010; Morfopoulos et al. 2013). The Michaelis-Menten constant (K_M) of isoprene synthase for DMADP is sensitive to temperature between 30 and 40°C even though the effect is less than the turnover rate (k_cat) (Table 2). This indicates that not only does enzymatic rate change with temperature, but binding of DMADP also changes, which may explain the high temperature optimum of isoprene emission rate. In comparison, monoterpenes are often stored in oil glands (often but not always within trichomes) or resin ducts, which means that their emission is sensitive to the effect of temperature on the vapor pressure of the monoterpene and on physical disruption of the enclosing structure. Stored monoterpenes do not become labeled when ¹³CO₂ is fed to photosynthesizing leaves (Guidolotti et al. 2019).

Table 1: Q10 values for isoprene emission and assimilation.

Values were calculated at 400 ppm CO₂ for a temperature increase from 25 to 35°C. (Reproduced from Lantz et al. 2019)

Species	Q10 Isoprene	Q10 Assimilation
Tobacco	3.0	1.0
Phragmites	2.9	0.6
Sycamore	3.9	1.0
Poplar	5.7	0.7

Table 2: Q₁₀ values for k_cat, and K_M between 30 and 40°C.

Data was collected from His-tag purified Eucalyptus globulus isoprene synthase. (Unpublished data by A.T.L. and T.D.S.)

Parameter	Q10
kcat	1.66
KM	1.38

CO₂

Isoprene emission is inhibited at very low CO₂ concentration, maximal from roughly 50 to 300 μL L⁻¹ CO₂, and usually declines at higher CO₂, with the pattern being exaggerated when measured in a low oxygen atmosphere (Monson & Fall 1989b; Loreto & Sharkey 1990; Feng et al. 2019). Two hypotheses have been generally discussed to explain the short-term inhibition of isoprene emission by CO₂. The first hypothesis was that the MEP pathway could be inhibited when photosynthesis becomes limited by the rate at which phosphate is regenerated for ATP synthesis or the triose phosphate utilization (TPU) limitation (McClain & Sharkey 2019). If true, then it would be expected that CO₂-inhibition of the rate of isoprene emission might be less or even absent at higher temperature (e.g. >30°C). This is sometimes observed (Rasulov et al. 2010; Sun et al. 2013a; Potosnak et al. 2014a). However, it is not always observed and a clear separation of the short-term CO₂ suppression of isoprene emission and TPU can be seen in at least some studies (Lantz et al. 2019). The second hypothesis is that phosphoenolpyruvate carboxylase is stimulated at high CO₂ and competes with the MEP pathway for pyruvate (Rosenstiel et al. 2003; Rosenstiel et al. 2004; Wilkinson et al. 2009). However, using malate feeding and inhibitors of PEP carboxylase, Rasulov et al. (2018) concluded that the carbon competition hypothesis was not supported. In addition, using stable isotope kinetic analyses Abadie and Tcherkez (2018) found that PEP carboxylase activity falls with increasing CO₂ and that PEP carboxylase activity is correlated with the rate of photorespiration. Thus, the two leading hypotheses for the CO₂ suppression of isoprene emission have been ruled out.

A new hypothesis was put forward by Lantz et al. (2019). Stomata respond to CO₂ similarly to isoprene emission rate. There has been progress in understanding the stomatal response to CO₂. The response requires specific carbonic anhydrases (Hu et al. 2010) and a MAP kinase (NtMPK4) (Marten et al. 2008). Stomatal responses involve calcium spikes and calcium effects have been seen in isoprene responses to wound signals. It may be that mesophyll cells sense CO₂ concentration by the same or similar mechanisms found in guard cells and that signaling pathways found in guard cells also are found in mesophyll cells and regulate the MEP pathway.

Wounding

Loreto and Sharkey (1993a) showed that isoprene can be induced or lowered by transmissible wound signals. This effect can be seen even more in Glycine soja. Glycine soja has a putative isoprene synthase (XP_028221489.1) for which the protein sequence is 84% identical and 89% similar to P. montana isoprene synthase. However, under growth conditions of 25°C day temperature, Glycine soja does not emit isoprene. But, isoprene emission could be induced from a leaflet for a brief period of time by burning an adjacent leaflet for five seconds (Figure 2) (Unpublished data by A.T.L. and T.D.S.). High CO₂ caused the total isoprene release to be less than that seen in low CO₂ (Figure 2). This was also seen in P. montana. The effect was reduced by feeding 5 mM EGTA, but not 5 mM EDTA (Loreto & Sharkey 1993a)(Unpublished data of J. Allman and T. D. Sharkey). This may indicate that calcium signaling is involved in these isoprene bursts. Calcium signaling could lead to modification of isoprene synthase or release of isoprene synthase from insoluble pools. Isoprene synthase may exist in different pools, membrane bound and soluble (Wildermuth & Fall 1996; Wildermuth & Fall 1998). This observation was confirmed by Wiberley et al. (2005). Release from one pool to another in response to changes in environmental conditions (that may alter membrane stability) could lead to the apparent change in isoprene synthase activity. Effects of CO₂ on mesophyll cells of leaves independent of its effect on photosynthetic metabolism will be an important avenue of research into understanding the molecular mechanism of CO₂ responses of isoprene emission rate.

Stomatal control

Isoprene emission does not change when the stomata close (Monson & Fall 1989b; Sharkey 1991; Fall & Monson 1992). In response to stomatal closure, the resistance to isoprene escaping the leaf increases greatly, which increases internal isoprene concentration (Fall & Monson 1992). The increased concentration is proportional to the resistance such that emission rate remains constant under conditions of stomatal closure. Pallozzi et al. (2013) found that pure blue light (which resulted in stomatal closure) decreased isoprene emission compared to white light; however, the decrease was not as large as the decrease in photosynthesis in response to blue light, and the reduction in isoprene emission is likely related to decreased photosynthesis. Blue light is not as efficient in driving photosynthesis as is white or red light (McCree 1971).

Long term

In addition to the short-term environmental controls on the rate of isoprene emission from leaves there are longer term effects, probably a result of changes in gene expression and amounts of enzymes of the MP pathway and ISPS.

Light

Light availability has a large effect on the capacity for isoprene emission from leaves. The capacity for isoprene emission is typically measured with the leaf at 30°C and 1000 μm⁻² s⁻¹ photosynthetic photon flux density. By using these standard conditions, long term effects on the ability of the leaf to make isoprene can be distinguished from the short-term effects of these same parameters on the rate of isoprene emission. Low light has a much stronger inhibitory effect on the capacity for isoprene emission than the capacity for CO₂ fixation (Hanson & Sharkey 2001). A similar effect was observed in mature trees where the gradient in isoprene emission capacity from top to bottom of the canopy was much steeper than the gradient in photosynthetic capacity (Monson et al. 1994b; Harley et al. 1996; Sharkey et al. 1996). A meta-analysis of eight field campaigns indicated that considering the total daily photon flux for the two days prior to measurement improved the prediction of isoprene emission capacity (Sharkey et al. 1999).

Temperature

The history of air temperature is an even better predictor of the capacity for isoprene emission (Sharkey et al. 1999). The length of temperature history that results in the best prediction of isoprene emission capacity varies among reports from a few hours to weeks (Goldstein et al. 1998; Fuentes et al. 1999; Pétron et al. 2001). The changes in the capacity for isoprene emission caused by differences in growth temperature are correlated with amounts of ISPS (Kuzma & Fall 1993; Lehning et al. 1999).

CO₂

In addition to the instantaneous effect of CO₂ on isoprene emission rate, there is an effect of growth CO₂ on isoprene emission capacity (Pacifico et al. 2009). The changes in capacity are generally associated with ISPS in leaves. Even less is known about how CO₂ exerts its influence on the capacity for isoprene emission. Better nitrogen nutrition results in higher isoprene emission (Harley et al. 1994; Litvak et al. 1996; Funk et al. 2006) but better CO₂ nutrition results in a lower isoprene emission capacity. We note (but do not speculate on a connection at this time) that CO₂ reduces the density of stomata on leaves (Woodward 1987) (i.e. reduces the capacity for stomatal conductance) and reduces the actual stomatal conductance just as it reduces the capacity and actual isoprene emission rate.

Drought

Isoprene emission remains high even when plants are severely water stressed (Fang et al. 1996; Fortunati et al. 2008; Brilli et al. 2013). The reduced sensitivity to water stress enhances the role isoprene could play in resilience to abiotic stress during droughts (Ryan et al. 2014; Tattini et al. 2014; Arab et al. 2016; Marino et al. 2017).

Global change

Isoprene emission is highest in hot climates with short dry seasons, and these conditions may select for a higher percentage of isoprene emitting species (Taylor et al. 2018). Species that emit isoprene tend to tolerate higher temperature than non-emitting species (Taylor et al. 2019). Long dry seasons may reduce the number of isoprene emitting plants because isoprene is a larger burden under drought stress: drought does not decrease isoprene emission, while it decreases assimilation (Fang et al. 1996; Fortunati et al. 2008; Guidolotti et al. 2011; Tani et al. 2011; Potosnak et al. 2014b; Ryan et al. 2014). Isoprene emission is significant even in cold climates, and these regions will become increasingly important to estimates of global isoprene production as global temperatures increase (Tiiva et al. 2007; Ekberg et al. 2011; Lindwall et al. 2016; Svendsen et al. 2016). Far northern and southern latitudes are expected to warm more than other latitudes as a result of climate change, leading to a disproportionate response of isoprene emission to global change (Peñuelas & Staudt 2010; Pachauri et al. 2014). Furthermore, very cold temperatures disable isoprene emission in some species, and an increase in temperature may cause previously non-emitting plants to emit (Monson et al. 1994a; Schnitzler et al. 1997; Sharkey et al. 1999; Oku et al. 2014; Mutanda et al. 2016a; Mutanda et al. 2016b).

Modeling isoprene emission rates: effect of climate change

Increased drought stress, CO₂, and temperature all have effects on isoprene emission (Vanzo et al. 2015; Hantson et al. 2017; Taylor et al. 2018; Feng et al. 2019), as does changing land use (Hantson et al. 2017). Most studies are based on the conclusion that atmospheric CO₂ concentrations will increase to 800–1000 ppm by the year 2100 and temperatures will increase by 3°C globally, although larger temperature increases are expected for northern latitudes and land areas versus air over oceans (Pachauri et al. 2014). Different studies have come to different conclusions not only because of fundamental differences in their models, but because of basing their models on different studies. Arneth et al. (2007) modeled the effect of CO₂ and showed that it would suppress the increase otherwise expected from the temperature response of isoprene emission (Wilkinson et al. 2009; Young et al. 2009). Hantson et al. (2017) proposed a similar effect but also incorporated changing land use, which led to the conclusion that global isoprene emissions will greatly decrease in the next 100 years. In addition to land use changes increasing CO₂ can change the degree of land cover (i.e. leaf area index) counteracting much of the CO₂ suppression of isoprene emission from any one leaf (Constable et al. 1999; Sun et al. 2013b).

Some groups have concluded that isoprene emission will increase under future climates (Guenther et al. 2006; Monson et al. 2007; Heald et al. 2009; Keenan et al. 2011; Keenan & Niinemets 2012; Monson et al. 2012; Han et al. 2013; Pugh et al. 2013; Hsieh et al. 2017). This is because high temperature abolishes or reduces the CO₂ effect (Rasulov et al. 2009b; Rasulov et al. 2010; Potosnak et al. 2014b; Monson et al. 2016) or is more pronounced than the CO₂ effect (Lantz et al. 2019). We believe that the reduction or elimination of the CO₂ effect above 30°C is important for predicting future isoprene emissions even though the mechanism is unknown (Lantz et al. 2019). Finding a mechanism for the CO₂ suppression of isoprene emission should improve our ability to predict the effects of global change on isoprene emission.

Calfapietra et al. (2007) showed that expression of MEP pathway genes and isoprene synthase protein levels can decrease in plants grown under high CO₂ and Scholefield et al. (2004) saw a similar effect in extractable enzyme activity. Most models take into account V_max of ISPS, but do not modify equations based on the K_m for DMADP, or substrate inhibition, even though DMADP levels can be sufficiently high to cause substrate inhibition in some ISPS enzymes.

Isoprene emission can have negative impacts on human health in urban areas because it reacts with nitrogen oxides to produce tropospheric ozone (Young et al. 2009; Fiore et al. 2011; Hellen et al. 2012; Han et al. 2013; Sato et al. 2013; Wang et al. 2013; Cheung et al. 2014) and secondary aerosols (Li et al. 2018). In rural areas the reaction products include methyltetrols, which can form secondary organic aerosols. Aerosols produce a cooling effect by increasing the albedo and may increase rainfall (Claeys et al. 2004; Fuzzi et al. 2006; Kleindienst et al. 2007; Zhang et al. 2007; Day & Pandis 2011; Sato et al. 2013; Hsieh et al. 2017; Zhu et al. 2017). Thus, complex climate interactions are possible.

Physiological roles of isoprene in plants

Isoprene has a number of effects on the physiology of plants and these have been probed with a variety of techniques, leading to widely varying hypotheses for the mechanism of isoprene. For many years it was thought that isoprene dissolved into membranes, strengthening them or quenching ROS. Using the octanol/water partitioning coefficient and Henry’s coefficient for partial pressure above a liquid medium, it was calculated that the concentration inside membranes of isoprene emitting leaves should be small and empirical measurements confirmed this (Harvey et al. 2015). As a result, other possible mechanisms of action have attracted much more attention recently. The effect of isoprene on treated and untreated plants has been tested in three ways: (1) by removing isoprene emission by knocking down isoprene synthase or with treatment with fosmidomycin (which inhibits DXS, the enzyme that catalyzes the first committed step in isoprene emission, Figure 1); (2) by transgenically modifying non-emitting plants to emit isoprene constitutively; and (3) by fumigating non-emitting plants with physiologically relevant levels of isoprene in the air around leaves. In the following section we discuss the effects of isoprene on abiotic and biotic stress tolerance and plant growth. New evidence that shows coordinated modulation of isoprene and other growth regulators in executing the above functions and the ability of isoprene to act as a signaling molecule to alter gene expression networks is presented in the following sections.

Resilience to abiotic and biotic stress

Sharkey et al. (2001) and Velikova and Loreto (2005) observed that in plants treated with fosmidomycin, thermotolerance was reduced and the effect was rescued by exogenous isoprene. In a later experiment Velikova et al. (2011) used both transgenic, isoprene-emitting Arabidopsis as well as fosmidomycin-treated Platanus orientalis leaves to confirm that thermotolerance as measured by thylakoid membrane stability was improved by the presence of isoprene. Vickers et al. (2009) used transgenic, emitting Nicotiana tabacum plants to show that isoprene also improves plant responses to oxidative stress; the reaction of isoprene with reactive oxygen species was confirmed in later studies (Jardine et al. 2012; Velikova et al. 2012). However, the appearance of the isoprene oxidation product methyl vinyl ketone, which had been taken as evidence of isoprene quenching of ROS in leaves has now been shown to come from other sources (Cappellin et al. 2019). Oxidation of unsaturated fatty acids are one source of methylvinyl ketone emissions (Kai et al. 2012). Knocking down isoprene emission using RNAi in Populus x canescens also demonstrated the effect of isoprene on thermotolerance (Behnke et al. 2007; Muller et al. 2015; Velikova et al. 2015).

In particular, isoprene emission improves tolerance of leaves exposed to rapidly changing temperatures, such as sunflecks or changing wind (Singsaas & Sharkey 1998; Harley et al. 1999; Singsaas et al. 1999; Behnke et al. 2010b; Behnke et al. 2013; Vanzo et al. 2015). Harvey et al. (2015) showed that isoprene does not accumulate in thylakoid membranes at sufficiently high concentrations to produce the membrane stabilization effects shown by Siwko et al. (2007) and Velikova et al. (2012). An alternative hypothesis that is sometimes suggested is that isoprene acts as a heat dissipater – that is, emission acts as a form of evaporative cooling, reducing leaf temperature (Pollastri et al. 2014; Sanadze 2017). However exogenous isoprene would not produce this effect, and the amount of isoprene released from the leaf relative to the amount of water transpired is so minute that it would not significantly affect cooling. At 30°C and 400 ppm CO₂ (basal conditions for isoprene emission), isoprene has a ΔH_vap of 26 kJ mol⁻¹, while water has a ΔH_vap of 43 kJ mol⁻¹ (Reid & Zwolinksi 1971). A high isoprene emission rate under these conditions is 126 nmol m⁻² s⁻¹; a typical conductance for water vapor at 30°C is 0.21 mol m⁻² s⁻¹, which would result in an evaporation rate of 9 mmol m⁻² s⁻¹ if the dew point were 25°C (Sharkey et al. 1996; Pollastri et al. 2014). This means that isoprene cools at a rate of 4.3 × 10⁻³ J m⁻² s⁻¹ compared to water’s 3.7 × 10² J m² s⁻¹, or nearly five orders of magnitude more cooling by water evaporation than isoprene volatilization.

When tobacco plants expressing ISPS were exposed to Manduca sexta larvae, the caterpillars selected against the isoprene emitting lines (Laothawornkitkul et al. 2008). The tobacco lines with the highest isoprene emission rates showed the highest resistance to feeding by the caterpillars indicating a role for isoprene in protecting plants from biotic stress (Laothawornkitkul et al. 2008).

Effects on plant growth

Isoprene enhances leaf chlorophyll and carotenoid contents. This enhancement has been observed in Arabidopsis and tobacco expressing ISPS (Zuo et al., 2019) and in Salix viminalis (basket willow) (Harris et al., 2016). RNAi-mediated suppression of ISPS reduced chlorophyll and carotenoid contents in grey poplar (Behnke et al., 2007). Because isoprene, the phytol tail of chlorophyll, and carotenoids are all derived from the chloroplastic DMADP pool, it had been thought that an increase in isoprene emission would lead to a decrease in the synthesis of chlorophyll and carotenoids. Since these compounds are more abundant in plants making isoprene, regulation of the amounts of these compounds must be more complex and could include effects of isoprene on gene expression. Isoprene-mediated promotion of cytokinin biosynthesis and signaling can also lead to chlorophyll biosynthesis and retention (Zuo et al. 2019).

Recent studies show that isoprene can have a growth effect under normal unstressed growth conditions, although the effect can be variable. For example, while hypocotyl, cotyledon, and leaf growth were enhanced in Arabidopsis expressing ISPS, a suppression of leaf and stem growth was observed in tobacco expressing ISPS (Figure 3). Faster growth rates on a leaf area basis under moderate heat stress (Loivamäki et al., 2007) and better growth under severe heat stress (Sasaki et al., 2007) was reported in Arabidopsis transformed with a grey poplar ISPS. Isoprene mediated growth benefits may be apparent in some species only under specific environmental conditions and isoprene mediated changes in gene expression can explain how isoprene leads to positive growth effects.

Figure 3. Leaf growth in Arabidopsis and tobacco expressing ISPS.

A comparison of Arabidopsis rosette size reflecting projected leaf area (A-C) and leaves after being separated from the rosettes reflecting apparent total leaf area (D-F) in 35-day old Arabidopsis non-emitting empty vector control (EV-B3) and isoprene emitting lines (expressing ISPS) (B2 and C4) is presented. For (A)-(F) all plants were grown in growth chambers under a light intensity of 200 μmol m⁻² s⁻¹. Photographs depicting the front and top views of tobacco plants taken during the 27^th day (G, H), 41^st day (I, J), and 54^th day (K) of plant growth are shown. NE: Non-emitting tobacco; IE: Isoprene-emitting tobacco. (Modified and reproduced from Zuo et al., 2019)

Mechanism through which isoprene exerts its physiological effects: isoprene signaling

Harvey and Sharkey (2016) observed that exogenous isoprene alone can alter gene expression with similar patterns as constitutive expression of isoprene synthase. Fosmidomycin and fumigation experiments typically saw effects after the plants were exposed for an hour or more, which is sufficient time for changes in gene expression to take place (Sharkey et al. 2001; Velikova & Loreto 2005; Velikova et al. 2008; Harvey & Sharkey 2016). Recently, analysis of differentially expressed genes in three model systems: Arabidopsis and tobacco engineered to emit isoprene (Zuo et al. 2019) and wild-type Arabidopsis fumigated with isoprene (Harvey and Sharkey, 2016) revealed that isoprene can alter expression of important genes required for synthesis and signaling of jasmonic acid (JA), cytokinin (CK), gibberellic acid (GA), ethylene, auxin, abscisic acid (ABA) (only during drought), and salicylic acid (SA). Isoprene increased expression of genes associated with enhanced GA and CK synthesis and signaling as well as genes related to JA and ethylene synthesis; GA and CK are growth promoters and JA and ethylene can suppress growth while increasing stress tolerance. Enhancement of CK biosynthesis by Arabidopsis expressing ISPS has been also been observed (K.G.S. Dani, S. Pollastri, M. Reichelt, T.D. Sharkey, and F. Loreto, unpublished). The upregulation of GA-mediated growth and JA-mediated defense signaling simultaneously in Arabidopsis expressing ISPS can result in plants that have enhanced growth as well as defense (Figure 4). Breaking the growth defense tradeoff has been seen in other systems (Campos et al. 2016). Because JA signaling has an inhibitory effect on growth that normally leads to a growth defense trade-off, the growth effect of isoprene can vary depending on which pathway is increased more by isoprene. In addition, isoprene altered the expression of genes that promote embryo growth, seed germination, seedling establishment, and pigment biosynthesis.

Figure 4. Proposed model for how isoprene signaling can affect GA-mediated growth regulation and JA-mediated defense responses.

Transcriptomic data revealed that isoprene can alter genes required for both GA accumulation and JA synthesis (genes shown in green font) which can promote both GA-mediated growth and JA-mediated defense simultaneously. We speculate that the observed growth enhancement in Arabidopsis engineered to emit isoprene is likely a result of upregulation of PIF. However, interactions between GA and JA pathways occur through DELLA and JAZ proteins (Campos et al. 2016). JA synthesis leads to the degradation of JAZ proteins that release the inhibition of transcription factors to enhancing defense related processes (Campos et al. 2016). Antagonistic interactions between JAZ and DELLA proteins play a part in regulating the growth-defense trade-off mediated by GA and JA (Campos et al. 2016). Therefore, one possible explanation for the observed variations in isoprene mediated growth effects in different species is the likely effect of isoprene on growth-defense trade-off. Genes belonging to other signalling pathways whose expression was altered by isoprene were omitted from this diagram for the sake of simplicity. Dotted lines and genes written in green font denote isoprene responsive gene expression revealed during the present study. Asterisks (*) denote differentially expressed genes in both Arabidopsis expressing ISPS and Arabidopsis fumigated by isoprene, but, not differentially expressed in tobacco. Upregulation and down regulation of gene expression is denoted by pointed and blunt ended arrows, respectively (DFL2 and TEM1 expression was downregulated in the presence of isoprene in Arabidopsis). Solid lines denote signalling pathways that are well established. Abbreviations: BBD - Bifunctional nuclease in basal defense response; CBF - CRT/DRE binding factor; COP1 - Constitutively photomorphogenic 1, a ubiquitin ligase; CRPK1- Cold-responsive protein kinase 1; DELLA - PIF transcription factor repressors; DFL2 - Dwarf in light 2, a GH3-related (auxin response related) protein; FT - Flowering locus T; GA - gibberellic acid; HY5 - Elongated hypocotyl 5; JA - jasmonic acid; JAZ - Jasmonate ZIM-domain repressors; JMT - Jasmonic acid carboxyl methyltransferase; LOX - Lipoxygenase; MARD1 - Mediator of ABA (abscisic acid)-regulated dormancy 1, a novel zinc-finger protein; MYC2 - a basic helix-loop-helix transcription factor and a master regulator of JA signaling; MYB59 - MYB domain protein 59; OPR3 - Oxophytodienoate-reductase 3; PHYB - Phytochrome-B; PIF - Phytochrome-interacting factors, TEM1 - Tempranillo 1, a RAV transcription factor; TZF5 -Tandem CCCH zinc finger protein 5; 14-3-3 - highly conserved acidic proteins of the 14-3-3-protein family. (Reproduced from Zuo et al., 2019)

Zuo et al. (2019) also showed that isoprene leads to changes in expression of genes that improve tolerance to heavy metals, soil acidity, high light, heat, salt, drought, and oxidative stress, herbivory and wounding in plants. While expression of genes important for phenylpropanoid biosynthesis was upregulated in Arabidopsis and tobacco expressing ISPS and Arabidopsis fumigated with isoprene (Zuo et al. 2019), a lower expression was observed in grey poplar showing RNAi-mediated suppression of ISPS under moderate heat stress with corresponding decreases in total phenolic and condensed tannin content, and increased H₂O₂ (Behnke et al. 2010a). Zuo et al. (2019) concluded that isoprene likely executes its effects on growth and stress tolerance by altering expression of genes associated with synthesis of growth regulators and their signaling, and genes associated with abiotic and biotic stress responses. Based on this recent data, these authors have proposed a novel role for isoprene as a signaling molecule.

The evolutionary advantages of selecting a volatile hydrocarbon such as isoprene as a signaling molecule can only be speculated. Some investigators speculate that the volatility of isoprene helps it to act as a long-distance signal to prime neighboring plants. However, the dilution of isoprene once it is emitted from a leaf is very significant likely limiting the range over which one plant could signal another. In addition, if such a strategy were to be evolutionarily adaptive it would require that adjacent plants be significantly similar to the emitting plant. Altruism considerations in evolutionary theory could justify one plant signaling another to improve the fitness of the second plant only if the second plant harbored many of the same genes as the signaling plant. If it did, it likely would have an isoprene synthase and so would not require a signal from a neighbor. The volatility of isoprene may also allow it to attract pollinators, or predators, or repel other herbivores or pathogens.

Mechanostimulation and wounding of Medicago truncatula led to an increase in transcription of the JA biosynthesis gene 1-aminocyclopropane-1-carboxylate oxidase 2 (ACO2) and 1-deoxy-D-xylulose 5-phosphate synthase 2 (DXS2–1) of the MEP pathway, with subsequent increases in JA biosynthesis (Tretner et al. 2008). Recently, Parveen et al. (2019) showed that application of JA to Ficus septica reduces ISPS expression, protein content, and isoprene emission while DXS is increased. Analysis of ISPS promoter regions revealed the presence of cis-elements that can be targeted by transcription factors to alter gene expression in response to JA (Parveen et al. 2019). Zuo et al. (2019) showed that expression of isoprene synthase and fumigation by isoprene leads to an enhancement of genes involved in JA biosynthesis and signaling. Overall, these data show that isoprene emission and JA biosynthesis is likely coordinately regulated in the presence of stress, e.g., herbivory or wounding. While isoprene enhances JA biosynthesis and signaling, JA can enhance DXS expression and suppress ISPS expression and isoprene biosynthesis.

Xiao et al. (2012) showed that MEcDP can act as a retrograde signaling molecule, increasing SA concentration and expression of stress response genes. In non-emitting poplar, Ghirardo et al. (2014) measured the flux through the MEP pathway; their data suggest that increasing isoprene emission can lead to an overall increase in MEcDP. Mechanistically, this is because DMADP and IDP inhibit DXS, leading to decreased flux through the MEP pathway (Banerjee et al. 2013; Banerjee & Sharkey 2014). Isoprene emission may decrease DMADP pools relieving the feedback and increasing flux through the MEP pathway.

Use of isoprene synthase for crop improvement

Isoprene emission can improve plant growth in Arabidopsis thaliana and leads to changes in genes important for stress tolerance (Behnke et al. 2010a; Harvey & Sharkey 2016; Mutanda et al. 2016b; Parveen et al. 2019; Zuo et al. 2019). As such, it may be beneficial to express isoprene synthase in crop plants in order to improve resilience. However, this has several potential drawbacks. Notably, no modern crop plant releases isoprene - in fact, Glycine max appears to have lost isoprene emission in the relatively recent past based on the similarity of gene fragments in the Glycine max genome and functional isoprene synthase in the Glycine soja genome (Sharkey 2013; Sharkey et al. 2013). This may be because the cost of isoprene emission outweighs the benefit under the relatively stable, safe conditions of a tended field (Monson et al. 2013). In N. tabacum, isoprene emission was correlated with decreased growth. Therefore, more research needs to be done to determine if this would be a net positive or net negative for crop plants under higher temperature conditions. There is an atmospheric cost as well. Increasing isoprene emissions can alter the local climate and lead to production of photochemical smog (Behnke et al. 2012; Beltman et al. 2013; Han et al. 2013; Wang et al. 2013).

Zuo et al. (2019) showed that the DMADP pools in non-emitting plants, e.g., Arabidopsis and tobacco expressing ISPS is sufficient to sustain isoprene production in various environments. However, species that are engineered to emit isoprene do not have as high emission rates as native emitters. We observed that transgenic Arabidopsis thaliana emit around 3 nmol m² s⁻¹, while N. tabacum emit up to 10 nmol m² s⁻¹ (Zuo et al. 2019). Native emitters such as Phragmites australis and Populus tremuloides emit 30–60 nmol m² s⁻¹, and under some conditions emitting species, particularly tropical or subtropical species, can emit over 100 nmol m² s⁻¹ isoprene (Sharkey et al. 1996). The Jamaican caper (Capparis cynophallophora) emitted isoprene at a rate of 269 nmol m² s⁻¹. It may be that the MEP pathways in emitting species have a higher capacity than in non-emitting plants to adapt to the high demand of isoprene synthase.

Conclusions and future directions

Isoprene emission may increase over the next century as increasing temperatures lead to higher emission rates. New mechanistic hypotheses are necessary to explain the suppression of isoprene emission by CO₂ as it is not linked to electron transport or increased PEPC activity. The interactions among temperature, CO₂, leaf area index, and land use changes (among others) makes prediction of future isoprene emission difficult. What is more, these predictions will vary both temporally and spatially. There may be more isoprene emission early in the growing season as thresholds for emission are crossed sooner and there may be regions in which warming is particularly severe, leading to the temperature response overcoming the CO₂ suppression while in other areas the temperature increase will be more moderate and the CO₂ suppression will be relatively more important. It is probably best to make regional predictions for the future of isoprene emission. A better understanding of the mechanisms of control of isoprene emission rate, especially to CO₂ changes, will be very useful in this debate.

There has been a significant paradigm shift in looking for biophysical explanations for the action of isoprene to gene expression explanations. It is not clear why some experiments supported the biophysical hypotheses; it may be that there are both biophysical and gene expression effects of isoprene. While gene expression data has revealed a strong relationship between GA and JA signaling pathways and isoprene, the role of isoprene within the intricate networks of growth- and stress-related signaling pathways remains to be resolved. If isoprene is in fact a signaling molecule, research into whether introduction of isoprene can affect plant growth and resilience in non-emitting species could lead to crop improvement.

In order to cause changes in gene expression that lead to the observed stress tolerance, the plant must be detecting isoprene. There is no known receptor for isoprene, but it may be possible to find one by using forward genetics. If a gene is necessary for isoprene fumigation to produce changes in gene expression, it may be a target of isoprene directly. Expression of the phenylalanine ammonia lyase (PAL) gene may be a potential output used to search for mutants lacking components of the isoprene receptor pathway, as it is known to respond to both fumigation and constitutive expression of isoprene synthase (Harvey & Sharkey 2016; Zuo et al. 2019). A luciferase reporter under control of the PAL promoter would serve as a useful output for a high-throughput forward genetics screen.

Significant progress in understanding isoprene emission has been made recently but in more than one case the progress has been to prove that long-held hypotheses are not supported by experiments and that new hypotheses and new experiments are needed to understand the phenomenon of isoprene emission from plants