Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants

Authors


Summary

  • Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear.
  • Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels.
  • Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31–46%) and lipid peroxidation (30–47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting.
  • Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8–24% more biomass under drought, implying that isoprene emission incurred a yield penalty.

Introduction

Drought stress, which is predicted to increase in intensity and frequency in the future, limits crop productivity and quality (Griffiths & Parry, 2002; Dai, 2011). There has been extensive research into how plants react to this with respect to drought tolerance, drought avoidance and whole-plant water use efficiency (WUE; defined as the amount of biomass produced per unit of water consumed) (Blum, 2005; Schachtman & Goodger, 2008). During drought, the phytohormone abscisic acid (ABA) accumulates, promoting stomatal closure and inhibiting stomatal opening. This reduces water loss and can maintain plant water status (Hartung et al., 2002; Chaves et al., 2003; Mishra et al., 2006). However, this may decrease yield by limiting CO2 assimilation rate, increasing leaf temperature (through restricting transpiration) and elevating photorespiration (Noctor et al., 2002; Zhou et al., 2007; Lawlor & Tezara, 2009).

Drought also reduces the content and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme in the photosynthetic carbon cycle, and reduces ribulose-1,5-bisphosphate (RuBP) regeneration (Parry et al., 2002; Flexas et al., 2006; Lawlor & Tezara, 2009). These metabolic impairments further reduce the biochemical capacity for carbon assimilation and utilization, and decrease the maximum efficiency of photosystem II (PSII) photochemistry (Fv/Fm). Together with stomatal closure, these limitations to carbon assimilation cause a disparity between the number of photons absorbed by the light-harvesting complexes and the number used for photochemistry. (Noctor et al., 2002; Lawlor & Tezara, 2009).

Photons absorbed by the chloroplasts drive photosynthetic electron flow in thylakoid membranes and produce energy, which is used during carbon assimilation. The efficiency of PSII photochemistry is optimized when photon intensities are in balance with carbon assimilation. When photon intensities exceed that required to support carbon fixation, photosynthetic efficiency decreases (Krieger-Liszkay, 2005; Miyake et al., 2005; Asada, 2006). Electron carriers become over-reduced and the excess photon energy and electrons must be dissipated. This dissipation can occur through variable fluorescence from chlorophyll a associated with PSII, through formation of triplet states of chlorophyll (which may then form reactive oxygen species (ROS)) or as heat (nonphotochemical quenching) (DemmigAdams et al., 1996; Miyake et al., 2005; Zhou et al., 2007).

Under drought (water withheld for up to 21 d) and osmotic stress (−0.7 MPa), foliar ROS content and lipid peroxidation can significantly increase compared with well-watered plants (Begcy et al., 2011; Ozfidan et al., 2011). When water deficit occurs under high light (in excess of that required to balance carbon uptake) and carbon assimilation is suppressed, increased ROS production may permanently damage the photosynthetic apparatus, ultimately reducing crop yield and quality (Miller et al., 2010; Suzuki et al., 2012). Thus, there may be considerable potential for increasing drought tolerance, and improving whole-plant WUE (water consumption relative to CO2 uptake) by enhancing plant antioxidant defences under drought (Schachtman & Goodger, 2008; Miller et al., 2010).

Isoprene (C5H8, 2-methyl-1,3-butadiene) is a volatile organic five-carbon (C5) compound emitted by many, but not all, plant species (see review by Sharkey et al., 2007 and references therein). By either stabilization of lipid membranes or direct quenching of ROS, it protects plants exposed to stresses that cause an increase in ROS, such as transitory or prolonged elevated temperatures (Singsaas et al., 1997; Velikova et al., 2005; Sasaki et al., 2007), ozone (Ryan et al., 2009; Vickers et al., 2009a), and excess light absorbance (Magel et al., 2006). While some attention has previously been given to isoprene responses to drought, there is little agreement between studies, with decreased (Fang et al., 1996; Bruggemann & Schnitzler, 2002; Brilli et al., 2007), increased (Monson et al., 2007; Beckett et al., 2012) or no effect on (Pegoraro et al., 2004, 2006) isoprene emission reported, and no consensus as to whether isoprene may protect plants under drought stress. It has also been suggested that isoprene emission may be positively correlated with foliar ABA concentration, as the two compounds share a common biosynthetic pathway in leaves (Barta & Loreto, 2006). Therefore, it has been postulated that the emission of volatile isoprenoids, such as isoprene, may be directly associated with foliar ABA content (Barta & Loreto, 2006).

Previous studies have concentrated only either on isoprene responses to drought (Pegoraro et al., 2004, 2006; Fortunati et al., 2008) or on the level of oxidative stress induced by drought without measuring isoprene emission (Reddy et al., 2004; Lawlor & Tezara, 2009; Miller et al., 2010; Suzuki et al., 2012). Only one study, using the resurrection plant Xerophyta humilis, measured biochemical and leaf-level responses, including isoprene emission responses, to drought (Beckett et al., 2012). Isoprene and other isoprenoids were stimulated under moderate drought stress with a peak in emission at 80% leaf relative water content (RWC). Thereafter, isoprene emission declined progressively until 50% RWC, after which emission ceased. Decreases in photosynthesis and the efficiency of PSII were fully reversed by rehydration. While it was concluded that isoprene and other nonvolatile isoprenoids acted to reduce membrane damage under drought, no comparison with a non-isoprene-emitting species was made.

To examine whether isoprene mediated photosynthetic and whole-plant drought responses, non-isoprene-emitting azygous and isoprene-emitting homozygous transgenic tobacco (Nicotiana tabacum) plants (Vickers et al., 2009a,b) were grown at two different levels of water availability. Foliar hydrogen peroxide (H2O2) content, evidence of lipid peroxidation (thiobarbituric acid reactive substances (TBARS)), ABA concentration, isoprene emission, gas exchange, leaf water potential (Ψleaf) and chlorophyll a fluorescence (the estimate of PSII operating efficiency Fq′/Fm′, the estimate of PSII maximum efficiency Fv′/Fm′ and the efficiency factor Fq′/Fv′) were assessed. Whole-plant transpiration and shoot biomass were measured to determine relationships between soil/plant water status and physiological performance. As isoprene emission should decrease drought-induced ROS content and lipid peroxidation, it was hypothesized that isoprene-emitting plants should show greater net CO2 exchange and lower oxidative stress at low soil water availability. To our knowledge, this is the first time that the drought responses of isoprene-emitting and non-isoprene-emitting plants (in the same genetic background) have been compared.

Materials and Methods

Plant material

Tobacco (Nicotiana tabacum L. cv Samsun NN) does not normally emit isoprene. To obtain an appropriately controlled experimental system in the same genetic background, transgenic tobacco plants bearing a full-length isoprene synthase gene isolated from Populus alba (GenBank EF638224) and placed under the control of the constitutive cauliflower mosaic virus 35S promoter were previously generated (Vickers et al., 2009a,b, 2011). A number of independent, single-locus, isoprene-emitting homozygous and non-emitting azygous transgenic lines were generated by segregation of primary transgenic lines after fertilization. Three lines with differing isoprene emission rates (named L22 (low emitter), L6 (mid/high emitter) and L32 (high emitter)) were used in the current study. Third-generation transgenic plants were used to ensure that epigenetic transformation- and tissue culture-mediated stress responses had been ameliorated (Molinier et al., 2006). Biochemical, physiological and morphological traits of these plants, and the response of isoprene to internal (circadian) and external (light and temperature) controls have been previously described (Vickers et al., 2009a,b, 2011). All aspects of biochemistry, physiology and morphology tested were similar to those of the non-emitting azygous controls under non-stress conditions.

Growth conditions

Plants were grown in 1-m3 climate-controlled PTFE (polytetrafluoroethylene)-lined growth chambers as previously described (Stokes et al., 1993). Each chamber was supplied with air from the building compressed air supply at a rate equal to approx. 1 complete air change per minute. A photosynthetic photon flux density of 500 ± 50 μmol photons m−2 s−1 at leaf height was provided for 14 h per day by four lamps (Powerstar HQI-BT, 600 W/D daylight; OSRAM, Munich, Germany). Day : night chamber air temperature was maintained at 24.5°C : 20.5°C ± 1.0°C with a relative humidity (RH) of 40 : 60 ± 10% and 385 ± 5 ppm CO2. Tobacco seeds for each line were germinated in covered seed trays filled with Levington M3 potting mix (Everris Ltd, Suffolk, UK). One week after sowing, 12 homozygous and 12 azygous plants from each line were transferred to 3-l pots filled with John Innes No. 2 potting mix (J. Arthur Bowers, Lincoln, UK). Lines (L32, L6 and L22), genotypes (emitter or non-emitter) and treatments (well-watered or deficit irrigated) were randomized between chambers. The tops of the pots were covered with black tape to reduce evaporative losses from the substrate. All plants were kept well watered (replacing 100% of water transpired daily) for 4 wk after potting out. Deficit irrigation treatments (replacing 50% of water transpired on a daily basis) began at the start of the fifth week.

Sampling

All measurements were taken before watering on the youngest fully expanded leaf (node number 4 counting from the plant base for week 1, when plants were 5 wk old, then increasing one node per week, to ensure sampled leaves were of a similar developmental stage). Isoprene emission rate, gas exchange, estimated operating and maximum quantum efficiencies and the efficiency factor of PSII photochemistry in light-adapted leaves (Fq′/Fm′, Fv′/Fm′ and Fq′/Fv′), substrate gravimetric water content (GWC) and whole-plant transpiration were measured before, and 3 and 7 d after, applying deficit irrigation. Leaf samples (at the same node) were taken immediately after the nondestructive sampling at each of these times for biochemical analysis of hydrogen peroxide (H2O2) and TBARS (thiobarbituric acid reactions), and to assess leaf water and osmotic potential. No plant was sampled more than once with a minimum of three replicates (one leaf per plant) taken for each variable. At the end of the treatment, all plants were harvested for measurement of shoot fresh and dry weights.

Isoprene

Concomitant with gas-exchange measurements, isoprene emission rate from each plant was analysed by proton transfer reaction mass spectroscopy (PTR-MS; Ionicon GmbH, Innsbruck, Austria). Measurements commenced 2 h after lights came on. Isoprene concentration was determined in the air exiting an open path gas exchange system (LI-6400; Li-Cor Inc., Lincoln, NE, USA) with an integrated fluorescence chamber head (LI-6400–40 leaf chamber fluorometer) via a PTR-MS connected to the cuvette via PTFE tubing (¼ inch outside diameter (OD)). The air leaving the cuvette head-space was sampled for isoprene at a flow rate of 100 ml min−1. Normalized sensitivities and isoprene volume mixing ratios (VMRs) were calculated as described by Taipale et al. (2008) using 700 ppb isoprene in nitrogen (Linde, Tunstall, UK) diluted in oxygen-free nitrogen (British Oxygen Company (BOC) gases). Protonated isoprene was detected by the PTR-MS at its molecular mass plus 1 (i.e. M + H+ = 69) using a dwell time of 5 s. After signal stabilization (c. 5 min), ten data points for one leaf per plant were averaged to give a mean value.

Leaf gas exchange and chlorophyll fluorescence

Leaf gas exchange measurements were coupled with chlorophyll a fluorescence measurements (Fq′/Fm′, Fv′/Fm′ and Fq′/Fv′) by the LI-6400 integrated fluorescence chamber head. All gas exchange measurements were corrected for diffusion of CO2 and water according to Rodeghiero et al. (2007). One leaf per plant was clamped inside the cuvette and left to stabilize for 5 min. The environmental conditions inside the cuvette were set to match chamber conditions described above and maintained by the internal controls of the LI-6400. The airflow to the cuvette was kept at 350 μmol s−1 to maintain positive pressure. Intrinsic water use efficiency (iWUE) of leaf gas exchange was calculated from the gas exchange data as A/E, where A is the carbon assimilated through photosynthesis and E is the amount of water lost via transpiration. Gas exchange measurements were taken five times per leaf (every 10 s), then averaged to give one measurement per plant.

Leaf water relations

Leaf water and osmotic potential were measured using thermocouple psychrometers (Wescor C-52 chambers; Wescor Inc., Logan, UT, USA) coupled to a microvoltmeter (HR33-T). Leaf discs (8 mm diameter) were punched from leaves, placed on clean sample holders and then immediately wrapped in foil until all leaf discs had been collected. They were then unwrapped and transferred to the psychrometers to incubate for 2.5 h. Once leaf water potentials had been measured, the discs were individually re-wrapped in foil and submerged in liquid N2 for 20 s. After thawing they were returned to the psychrometers for a further 30 min, after which osmotic potential was measured. Voltage readings were converted to potentials (MPa) based on calibration with salt solutions of known osmotic potential as previously described (Martin-Vertedor & Dodd, 2011). Leaf turgor was calculated as the sum of the two potentials.

Water use and substrate water content

Shoot biomass was measured before starting the irrigation treatments by destructively harvesting three plants of each line and genotype. Shoots were dried at 60°C for 7 d to remove all water (complete water loss was ascertained by re-weighing samples). Pots (including plants) were weighed before starting deficit irrigation and then at the same time every 2 d during treatment. Whole-plant water use was calculated as the initial pot and plant weight plus irrigation minus the final pot and plant weight (maximum plant fresh weight gain over time averaged < 0.8% of the combined weight of the pot and substrate). Whole-plant WUE was then calculated as shoot biomass (g) divided by the water used (l). Substrate gravimetric water content was calculated as the weight of water in the substrate divided by the weight of dry substrate. This was converted to a soil matric potential based on a previously determined moisture release curve for that substrate (Dodd et al., 2010).

Abscisic acid

Fresh leaf tissue was snap-frozen in liquid N2, freeze-dried for 36 h, and then ground to a fine powder and extracted in distilled water on a shaker overnight at −4°C. The concentration of foliar ABA was determined by radioimmunoassay using the monoclonal antibody AFRCMAC 252 (Quarrie et al., 1988). A spike dilution test (Jones, 1987) on aqueous extracts of tobacco leaves revealed a low level of interfering immunoreactive compounds.

Indicators of oxidative stress

Hydrogen peroxide (H2O2) concentrations were determined as previously described (Ryan et al., 2009). In brief, frozen leaf material (100 mg) was homogenized in an ice bath with 1 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12 000 g for 30 min and 0.4 ml of the supernatant was added to 0.4 ml of 10 mM potassium phosphate buffer (pH 7.0) and 0.8 ml of 1 M potassium iodide (KI). The absorbance of supernatant was read at 360 nm. The coloured reaction product of H2O2 with KI develops within 25 min and is stable for at least 2 h. The content of H2O2 was calculated against a calibration curve using H2O2 standards.

The degree of lipid peroxidation of the leaf tissue was determined by malondiadehyde (MDA) accumulation, assayed using the TBARS method (Heath & Packer, 1968). Approximately 40 mg of frozen leaf material was homogenized in 1 ml of 0.1% (w/v) TCA. The homogenate was then centrifuged at 4°C at 12 000 g for 30 min. The supernatant (0.5 ml) was mixed with 1 ml of 20% TCA containing 0.5% (w/v) thiobarbituate acid (TBA) and the mixture was heated in a water bath for 30 min at 95°C. The reaction was stopped in an ice bath and the mixture was then centrifuged at 10 000 g for a further 5 min. The absorbance of the supernatant was read at 532 and 600 nm to correct for nonspecific turbidity. To calculate the content of lipid peroxides, an absorption coefficient of 155 mM cm−1 was used.

Statistical analysis

General linear model (GLM) with ANOVA and post hoc Tukey and Waller–Duncan tests were used to identify significant differences in main effects, and to determine all two- and three-way interactions between irrigation treatments, lines and genotypes (IBM spss v20 statistical package; IBM Corp., New York, USA). Paired t-tests determined whether there were significant differences between genotypes or treatments within a line after 3 and 7 d for each variable. Two replicate experiments were performed: one using all three lines and one using the high (L32) and low (L22) emitting lines only. Data were checked for significant differences between experiments before combining, resulting in 12 replicates pre-treatment, and three to eight replicates, depending on variable, line and genotype, thereafter.

Results

Foliar isoprene emission and gas exchange decrease as soil water content declines

No isoprene was detected in azygous controls (data not shown). In L32, the highest emitting line, isoprene emission declined in the well-watered plants after the first 3 d (= 0.022). This may have been attributable to increasing leaf age, as isoprene emission declines after full leaf expansion (Vickers et al., 2011). There was no further reduction after 7 d. By contrast, isoprene emission of the well-watered plants of L6 and L22 did not significantly change over time (Fig. 1a–c). Three days of deficit irrigation decreased isoprene emission by 73–82% compared with well-watered plants (Fig. 1a–c; L32, = 0.006; L6, = 0.003; and L22, = 0.031), before any significant decline in leaf water potential or leaf turgor (Fig. 3). All three lines also showed significantly reduced isoprene emission after 7 d compared with well-watered controls on the same day (= 0.05, = 0.001 and < 0.001 for L32, L6 and L22, respectively; Table 1).

Table 1. General linear model (GLM) results for the main effects for treatment (T), line (L) and genotype (G) plus all two- and three-way interactions after 7 d of subjecting tobacco (Nicotiana tabacum) plants to deficit irrigation
Model TermSignificance of effect of model term on variable of interest
IER A g s ΨleafTurgorΨsoilDWWUWUEABAH2O2TBARSFv/Fm
  1. Significantly different genotypic (emitter versus non-emitter) responses to treatment for each variable are highlighted in bold in row 4. Significant differences are shown: *, = 0.05; **, = 0.001; ***, < 0.001; ns, no significant difference; –, no data.

  2. IER, isoprene emission rate; A, carbon assimilation; gs, stomatal conductance; Ψleaf, leaf water potential; Turgor, leaf turgor; Ψsoil, substrate gravimetric water content; DW, shoot dry weight; WU, water use; WUE, whole-plant water use efficiency; ABA, foliar abscisic acid content; H2O2, hydrogen peroxide content; TBARS, thiobarbituric acid reactive substances; Fv′/Fm′, photosystem II efficiency of light-adapted leaf.

T *** *** *** *** *** *** *** *** ** *** *** *** ***
Lns *** *** * ns *** ns ** ns *** *** ns ***
Gnsnsns ** nsnsns ** ns * ns **
T x G * nsns ** ns * nsnsns *** ** ***
T x L * nsnsns *** * nsns * nsnsnsns
G x L ** * ns * ** ** nsnsnsnsnsns
T x L x G *** * nsns * * nsnsns ** ns *
Figure 1.

Isoprene emission rate of well-watered (closed bars) and deficit irrigated (open bars) isoprene-emitting tobacco (Nicotiana tabacum) plants of lines 32 (a), 6 (b) and 22 (c) (high, medium and low emitters, respectively) (= 3 plants ± 1 SE). Carbon assimilation (A) and stomatal conductance (gs) for each line are shown in panels (d) to (i) (= 6 ± 1 SE for L32 and L22; = 3–4 ± 1 SE for L6). Transpiration (E) followed the same pattern as gs and is therefore not shown here. Individual symbols ± 1 SE represent well-watered emitting (closed circles), well-watered non-emitting (closed triangles), deficit irrigated emitting (open circles) and deficit irrigated non-emitting (open triangles) tobacco plants, respectively, and are means of 10 (isoprene) or five (gas exchange) technical replicates per leaf (one per plant), averaged over three plants. Significant differences between treatments are shown: *, = 0.05; **, = 0.001; ***, < 0.001. Shaded areas indicate the well-watered control period before the start of deficit irrigation (DI) treatments.

Compared with well-watered plants, deficit irrigation decreased the carbon assimilation (A) and stomatal conductance (gs) of L32 and L22 in both isoprene-emitting and non-emitting plants after 3 d (data not available for L6 on day 3), and these variables decreased even further in all three lines after 7 d (Fig. 1d–i). Although no significant differences in A, E or gs were found when comparing genotypes (+/− isoprene synthase) under deficit irrigation, deficit irrigated isoprene-emitting plants had a tendency to decrease values of A and gs to a greater extent than deficit irrigated non-emitting plants (significant = 0.003 treatment × genotype interaction for A; Table 1).

However, plotting A versus E (for all plants across all lines) revealed a significantly higher (P = 0.005) intrinsic WUE of emitting plants under mild to moderate drought stress (Fig. 2). Thus, there was a genotypic difference in the responses of emitters and non-emitters to treatment over time.

Figure 2.

Relationship between carbon assimilation (A) and transpiration (E) (the slope of the fitted curves gives the intrinsic water use efficiency) of isoprene-emitting (solid line) and non-emitting (dashed line) tobacco (Nicotiana tabacum) plants. Dotted lines indicate ± 95% confidence interval for individual measurements from different leaves.

There were no significant differences in leaf water potential (Ψleaf), leaf turgor or GWC in well-watered plants over time (Fig. 3). Three days of deficit irrigation did not significantly reduce these variables compared with the well-watered plants. After 7 d of deficit irrigation, Ψleaf (Fig. 3a–c), leaf turgor (Fig. 3d–f) and GWC (Fig. 3g–i) were all significantly reduced compared with the values of the well-watered controls. Only L6 showed a significant genotypic difference, with deficit irrigated isoprene-emitting plants having a significantly higher leaf turgor than deficit irrigated non-emitting plants (= 0.011) despite similar GWC (Fig. 3). Generally, isoprene emission did not affect leaf water relations or the rate of soil drying.

Figure 3.

Leaf water potential (ψleaf) (a–c), leaf turgor (d–f) and substrate gravimetric water content (GWC) (g–i) of isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants. Individual symbols ± 1 SD represent well-watered emitting (closed circles), well-watered non-emitting (closed triangles), deficit irrigated emitting (open circles) and deficit irrigated non-emitting (open triangles) tobacco plants, respectively (= 3). Significant differences between treatments are shown: *, = 0.05; **, = 0.001; ***, < 0.001. Shaded areas indicate the well-watered period before deficit irrigation (DI).

Isoprene-emitting lines have higher whole-plant water use efficiency under drought but lower shoot biomass

All lines and genotypes had similar shoot biomass under well-watered conditions (Fig. 4a). This contrasted with the L22 non-emitting plants, which had significantly lower shoot biomass as a result of unusually poor growth compared with previous studies with this line (Vickers et al., 2009a,b, 2011). Because of this, the results for L22 were investigated separately from, as well as in combination with, the results for L32 and L6 to evaluate any influence of this poor growth on the statistical analysis.

Figure 4.

Shoot biomass (a), water use (b) and whole-plant water use efficiency (WUE) (c) of well-watered and deficit irrigated tobacco (Nicotiana tabacum) plants. Bars ± 1 SE represent well-watered (closed bars) and deficit irrigated (open bars) plants. Each line is split into isoprene-emitting (E) and non-emitting (NE; see (c) horizontal axis). Different letters indicate significant differences between genotypes, lines and treatments. = 4 for L32 well-watered and = 8 for L32 deficit irrigated plants, respectively, and = 3 for L6 and L22 well-watered and deficit irrigated plants.

Deficit irrigation significantly reduced shoot biomass compared with well-watered plants in all lines and genotypes (< 0.001; Fig. 4a, Table 1). Moreover, there was a significant genotypic difference in shoot growth inhibition caused by deficit irrigation when comparing genotypes (+/− isoprene; = 0.024; Fig. 4a), with emitting lines showing a greater reduction in shoot biomass under deficit irrigation compared with their well-watered controls (51–64% reduction; < 0.001 for each line) than the non-emitters (39% (< 0.001) and 42% (= 0.006) for L32 and L6 non-emitters, respectively). Despite the poor shoot growth of the L22 non-emitting control plants, the decreased growth of L22 non-emitting plants subjected to deficit irrigation was also significant (a reduction of 27% (= 0.019) compared with well-watered plants) although less severe than for the other non-emitting lines. Thus, deficit irrigation decreased the growth of isoprene-emitting plants more than that of non-emitting plants.

Under deficit irrigation, the emitting plants of L32 and L6 had higher whole-plant water WUE (= 0.045 and = 0.021, respectively) than their corresponding non-emitting plants under the same treatment (Fig. 4c). This was despite similar shoot biomass, GWC (Fig. 3g–i) and water use (which decreased by c. 60% in all lines under deficit irrigation) when comparing genotypes within the same treatment (Fig. 4b). WUE was also higher in emitting plants than non-emitting plants in L22, but the effect was not statistically significant because of the high variability in this line (Fig. 4c). Thus, although the whole-plant WUE of isoprene-emitting tobacco was higher than that of non-emitting plants (Fig. 4c), this was not attributable to any significant genotypic differences in shoot biomass or water use under the same treatment (Fig. 4a).

Deficit irrigation increases foliar ABA content

Although isoprene emission did not significantly change the foliar ABA concentration of well-watered genotypes of L6 and L22, well-watered L32 emitters had a significantly lower ABA concentration than non-emitting plants under the same treatment (Fig. 5, = 0.011), despite similar stomatal conductance. Well-watered L22 emitting and non-emitting plants had significantly lower ABA content than emitting and non-emitting L6 plants and L32 non-emitting plants under the same treatment.

Figure 5.

Foliar abscisic acid (ABA) concentration after 7 d of deficit irrigation. Bars ± 1 SE represent mean foliar ABA concentration in well-watered (closed bars) and deficit irrigated (open bars) tobacco (Nicotiana tabacum) plants (= 3). Each pair is split into isoprene-emitting (E) and non-emitting (NE). Different letters indicate significant differences between genotypes, lines and treatments.

Seven days of deficit irrigation significantly increased leaf ABA concentration in all lines and genotypes compared with well-watered plants (Fig. 5, Table 1), concomitantly with decreased stomatal conductance (Fig. 1) and independently of isoprene emission. Under deficit irrigation, L22 did not increase foliar ABA to the same extent as L32 or L6, possibly because of the slightly (but not significantly) higher Ψleaf and GWC (Fig. 2) of this line. There was no correlation between isoprene emission rate and foliar ABA concentration measured in the same leaves (data not shown).

Drought does not significantly increase ROS or TBARS in isoprene-emitting plants

Although there was some variation in the concentration of H2O2 (an indicator of oxidative stress) when comparing all well-watered lines, the concentrations of TBARS (an indicator of oxidative damage) were similar (Fig. 6, Table 1). Relative to well-watered plants, deficit irrigation increased concentrations of H2O2 and TBARS in all emitting and non-emitting lines (effect of treatment < 0.001 for both H2O2 and TBARS; Fig. 6, Table 1). However, the relative increases were much lower in the emitting plants of L32 and L6 (18% and 9% for H2O2 and 10% and 19% for TBARS, respectively; no significant increase) than in the non-emitting plants of these lines (39% for H2O2 in L32 (< 0.001), 31% for H2O2 in L6 (= 0.002), 37% for TBARS in L32 (= 0.015), and 47% for TBARS in L6 (= 0.002)).

Figure 6.

Hydrogen peroxide (H2O2) (a–c) and thiobarbituric acid reactive substances (TBARS) (d–f) after 7 d of treatment. Bars ± 1 SE represent mean (= 3) H2O2 or TBARS concentrations in well-watered (closed bars) and deficit irrigated (open bars) tobacco (Nicotiana tabacum) plants. Different letters indicate significant differences between genotypes, lines and treatments. Isoprene-emitting plants showed no increase in either parameter, with the exception of the lowest emitting line (L22). All non-emitting plants showed significant increases under deficit irrigation compared with well-watered plants.

By contrast, the small increase in H2O2 of the isoprene-emitting deficit irrigated plants of L22, the lowest emitting line, was statistically significant compared with the well-watered plants (increase relative to controls = 36%; = 0.034) (Fig. 6c). However, the concomitant increase in TBARS in this line was not statistically significant (19%; Fig. 6f). Similar to the non-emitting plants of L32 and L6, the deficit irrigated non-emitting plants of L22 showed significant increases in both H2O2 and TBARS (49% (< 0.001) and 30% (= 0.041), respectively) compared with well-watered plants (Fig. 6). Thus, emitting plants showed no significant differences when comparing irrigation treatments, but deficit irrigation significantly increased H2O2 and TBARS in non-emitting plants (interaction of treatment × genotype < 0.001 and = 0.006 for H2O2 and TBARS, respectively; Table 1).

Isoprene maintains photosystem II efficiency under drought

There were no significant differences in the estimated operating efficiency (Fq′/Fm′), maximum efficiency (Fv′/Fm′) or efficiency factor (Fq′/Fv′) of PSII photochemistry in light-adapted plants after 7 d when comparing well-watered emitting and non-emitting plants (Fig. 7). Deficit irrigation reduced Fq′/Fm′ of the non-isoprene-emitting plants of L32 and L6, and of the isoprene-emitting plants of L6 compared with well-watered controls (< 0.001, = 0.014 and < 0.001, respectively; Fig. 7a), concomitant with lower PSII maximum efficiency in these plants (Fig. 7b). Although the deficit irrigated non-emitting plants of L22 also showed a decrease in operating efficiency compared with well-watered controls, it was not significant. In general, isoprene emission significantly altered the response of PSII to drought (< 0.001; Table 1).

Figure 7.

Estimate of photosystem II (PSII) operating efficiency (a), maximum efficiency (b) and the PSII efficiency factor (c). Bars ± 1 SE represent well-watered (closed bars; = 3) and deficit irrigated (open bars; = 5–6) tobacco (Nicotiana tabacum) plants. Each pair is split into isoprene-emitting (E) and non-emitting (NE). Letters indicate significant differences between genotypes, lines and treatments.

By contrast, L32 and L22 showed no significant difference in Fq′/Fm′, Fq′/Fv′ or Fq′/Fv′ when comparing emitting to non-emitting plants, or when comparing well-watered emitting plants to deficit irrigated emitting plants (Fig. 7). Again, L6 was an exception as a significant decrease was observed for both emitting and non-emitting deficit irrigated plants (< 0.001). This indicates a down-regulation of PSII through suppression of the maximum efficiency of this photosystem under deficit irrigation, the magnitude of which is reduced in the isoprene-emitting plants of L32 and L22, resulting in a significant genotypic difference and genotype × treatment interaction (Table 1).

Discussion

The enhanced tolerance to oxidative stress of wild-type isoprene-emitting plants such as poplar (populus sp.), relative to those in which isoprene emission has been suppressed, has been studied and demonstrated several times in different plant species (see review by Sharkey et al., 2007 and references therein). However, only a few studies have directly compared the physiological responses of transgenic isoprene-emitting and transgenic non-emitting plants of the same species under stress (Behnke et al., 2007, 2012; Loivamaeki et al., 2007; Vickers et al., 2009a,b). This study utilized a novel transgenic tobacco tool (Vickers et al., 2009a,b) to investigate the response of isoprene to drought and its effect on plant growth. Although isoprene protects the photosynthetic apparatus under conditions of water deficit (Figs 3, 7), probably through reducing damage to membranes (Fig. 6), isoprene-emitting plants also grow less (by up to 24%; Fig. 4) under short-term water deficits (7 d).

Under the deficit irrigation treatment imposed (which decreased Ψleaf by 0.5 MPa on average; Fig. 2), isoprene emission decreased before leaf wilting, concomitant with decreased carbon assimilation and stomatal conductance (Fig. 1). Previous studies found that carbon assimilation was highly sensitive to moderate drought stress, while isoprene emission was less so (Bruggemann & Schnitzler, 2002; Brilli et al., 2007; Fortunati et al., 2008), resulting in a significantly greater percentage loss of carbon under stressed conditions via isoprene emission (Centritto et al., 2011). Carbon assimilation rates of Quercus pubescens and Quercus robur decreased to nearly zero at Ψleaf below −2.5 and −1.3 MPa, respectively (Bruggemann & Schnitzler, 2002), while isoprene emission only declined at severe water deficit (−3.5 and −2 MPa for the two species, respectively). In a similar experiment, isoprene emission under mild drought stress (volumetric soil water content decreased from 0.27 to 0.13 m3 m−3 in the top 30 cm of soil over 3 months) was measured in a model ecosystem (the Biosphere 2 laboratory) (Pegoraro et al., 2006). Although gross isoprene production did not change, ecosystem gross primary productivity (carbon fixation) decreased by 32%, thus doubling the carbon lost as isoprene from c. 1 to 2%. While both isoprene emission and carbon assimilation are decreased by drought stress, a similar relative increase in carbon lost as isoprene under stress was found here. Combining all three lines, on average 0.3% of the carbon assimilated under well-watered conditions was lost as isoprene (calculated on a carbon only basis). After 3 d of deficit irrigation, this decreased to an average of 0.1%, and after 7 d increased to 1.0%. The increased investment of carbon in isoprene suggests that the emission of isoprene under drought stress is in some way advantageous to the plant and conveys some benefit or protection under these conditions (Bruggemann & Schnitzler, 2002).

Isoprene has recently been shown to enhance the thermostability of thylakoid membranes in transgenic Arabidopsis under heat stress (Velikova et al., 2011), and in both transgenic tobacco and transgenic Arabidopsis, decreased H2O2 and ROS are observed in isoprene-emitting plants under oxidative and thermal stress (Vickers et al., 2009a,b; Velikova et al., 2012). Under soil water potentials down to −0.57 MPa, wild-type (non-isoprene-emitting) drought tolerant tobacco plants had enhanced membrane stability compared with drought-sensitive cultivars (Riga & Vartanian, 1999). Enhanced membrane stability of the drought-tolerant plants delayed and reduced membrane damage, which occurred earlier and more severely in drought-sensitive tobacco cultivars (Riga & Vartanian, 1999). Furthermore, it has been suggested that plants with an increased oxidative capacity are better equipped to deal with and respond to decreased soil and leaf water potentials (Miller et al., 2010; Suzuki et al., 2012). Our work supports both suggestions. Non-isoprene-emitting plants have increased ROS content and also increased damage to cellular membranes (Fig. 6, Table 1). Coupled with significant reductions in the operating efficiency of PSII (Fq′/Fm′) driven by reductions in the maximum efficiency (Fv′/Fm′) (Fig. 7a,b), this indicates that these plants may take longer to recover from a period of water deficit and are considered to be more drought sensitive.

The isoprene-emitting tobacco plants showed no increase in ROS content or damage to membrane structures (Fig. 6) and maintained Fq′/Fm′ and Fv′/Fm′ under drought (Fig. 7a,b). As there was no difference in the efficiency factor of PSII in these genotypes (Fig. 7c), it suggests that isoprene had no effect on photochemical quenching and therefore did not protect these genotypes by acting as an additional sink for excess energy. We conclude that the contrasting genotypic responses in all three tobacco lines shows that the isoprene-emitting plants are more drought tolerant at the leaf level compared with the non-emitting plants (Table 1). Furthermore, this supports the theory that isoprene improves the oxidative capacity of these plants by decreasing membrane damage and/or enhancing membrane stability, but not by acting as a sink for excess energy under these drought conditions.

The deficit irrigated isoprene-emitting genotypes of L32 and L6 (high and mid/high isoprene-emitting lines) also had lower carbon assimilation and more strongly reduced stomatal conductance than non-emitting plants after 3 d of treatment (Fig. 1). Although this appeared to be driven by lower leaf water status in L6, in L32 both Ψleaf and GWC of the emitting and non-emitting plants were similar (Fig. 3). As expected, increased foliar ABA concentration was correlated with lower stomatal conductance in deficit irrigated plants after 7 d (Figs 1 g–i, 5). However, enhanced stomatal closure of isoprene-emitting genotypes was not caused by increased foliar ABA concentration, as this did not differ between genotypes within the same line (Table 1). In previous studies, a positive relationship between short-term changes (< 2 h) in isoprene and ABA was proposed to be attributable to isoprene and ABA sharing a common biosynthetic pathway (Barta & Loreto, 2006). However, over longer time periods (such as those used in the current study), isoprene emissions were uncoupled from ABA concentrations, and thus isoprene emission was not correlated with foliar ABA concentration after 7 d of drought (Figs 1a–c, 5).

Under drought and other oxidative stresses, retrograde (organelle to nucleus) signals initiated by the increased ROS and antioxidant activity are necessary to optimize cell functions and activate appropriate responses such as stomatal closure (Miller et al., 2010; Suzuki et al., 2012). As the enhanced stomatal closure in isoprene-emitting tobacco plants was not attributable to increased ABA concentration, we propose that it may be attributable to their enhanced antioxidant capacity resulting from the ability to emit isoprene. However, the mechanism(s) causing enhanced stomatal closure in isoprene-emitting plants requires further study to better understand potential impacts on photosynthesis and plant yields.

Tobacco plants initiate a sequence of adaptive mechanisms in response to decreasing soil water content (Riga & Vartanian, 1999) and it is pertinent to understand whether isoprene emission fundamentally alters these adaptive responses. Tobacco plants initially utilize osmotic adjustment to maintain plant turgor and rapidly decrease stomatal conductance under soil water potentials of −0.01 to −0.19 MPa. Consistent with this, leaf turgor did not significantly decline until the seventh day of soil drying (equivalent to substrate matric potentials of −0.6 to −0.75 MPa) and there were no significant genotypic differences in leaf turgor after 3 and 7 d of soil water deficit in this study (Fig. 3, Table 1). This suggests that, while isoprene did not significantly alter the ability of tobacco leaves to osmotically adjust to decreased soil water status, it did enhance initial stomatal closure (particularly in L32 tobacco plants under mild to moderate water deficit compared with non-emitting plants) to reduce transpirational water loss (Fig. 1h–i). This ultimately led to higher intrinsic WUE (the ratio of carbon assimilation to water transpired; Fig. 3) and whole-plant WUE (the ratio of shoot biomass to water used; Fig. 4c).

Despite isoprene emission enhancing WUE, isoprene-emitting plants also showed a greater reduction in shoot biomass (up to 24%) than non-emitting plants (Fig. 4a, Table 1). This suggests that, while isoprene emission may be advantageous under drought stress at the leaf level by enhancing early responses to drought (such as stomatal closure to reduce water consumption under short-term suboptimal watering conditions), prolonged drought can cause significant yield losses in isoprene-emitting plants. One explanation for the reduced yield may be the diversion of carbon and energy (20 ATP and 14 NADPH molecules per isoprene molecule) away from other necessary compounds including photosynthetic components (chlorophyll and plastoquinone), accessory pigments with antioxidant behaviour (carotenoids) and phytohormones (including ABA, gibberellins and strigolactone) that are all produced via the same chloroplastic pathway as isoprene in leaves. However, it should be noted that isoprene emission is regulated through a balance of isoprene synthase activity and availability of the DMADP substrate (Wolfertz et al., 2004; Rasulov et al., 2009, 2010; Wiberley et al., 2009; Vickers et al., 2010) with the former being more important over longer time-scales (developmentally/environmentally) and the latter being more important over short (minutes/hours) time-scales (Sharkey, 2013). Differential regulation (in particular of isoprene synthase over the longer term, and also species-specific variation in methylerythritol phosphate (MEP) pathway activity under drought stress) might influence the ultimate effect of isoprene production in naturally emitting plants compared with the transgenic system here. This might improve the overall carbon balance, reducing the cost to the plant.

Although in our work an average of 1% of carbon was lost as isoprene under drought, in wild-type isoprene-emitting species it can be as much as 15% under stress conditions (1000 μmol m−2 s−1 photosynthetically active radiation (PAR) and 40°C) (Sharkey & Yeh, 2001). This is a result of the increased carbon loss via isoprene emission relative to photosynthesis, and the enhanced suppression of carbon assimilation (up to 47% after 3 d and 89% after 7 d of soil drying) compared with non-emitting plants. We therefore hypothesize that, while isoprene emission is advantageous under short-term drought, it is disadvantageous under long-term, mild drought stresses. However, we presume that potential yield penalties associated with isoprene emission under drought stress are counteracted by the benefits of isoprene as an oxidative stress protectant (Loreto et al., 2001; Velikova et al., 2004; Penuelas & Munne-Bosch, 2005; Vickers et al., 2009a,b; Behnke et al., 2012) and/or specific protectant of the photosynthetic apparatus (Sharkey & Singsaas, 1995; Sharkey et al., 2001; Velikova et al., 2012) under those conditions. Poplar (Populus × canescens) trees in which isoprene emission had been knocked down by RNA interference technology showed a 6.9% increase in net growth and a reduced carbon loss of 2.2% of the total gross primary productivity compared with wild-type isoprene-emitting poplar over two growing seasons (Behnke et al., 2012). However, the transgenic poplar also showed increased susceptibility to herbivore attack (Behnke et al., 2012) and high temperature stress (Behnke et al., 2007), compared with the wild type. Thus, overall, isoprene provides a selective advantage for emitting plants under drought and other stresses. Future studies should examine the physiological mechanism(s) causing this.

In conclusion, the ability to emit isoprene allowed all three transgenic tobacco genotypes to maintain photosynthesis (at an equivalent stomatal conductance) under mild to moderate drought stress better than their corresponding non-emitting controls. While it is difficult to disentangle the exact mechanistic explanation for protection by isoprene (membrane stabilization and/or direct antioxidant behaviour) because of the interconnection between lipid and aqueous phase oxidative statuses (Vickers et al., 2009a,b), it is clear that the photosynthetic apparatus was protected (Fig. 7), and that fewer ROSs were produced, resulting in lower membrane damage (Fig. 6). Similarly, any genetic modification that enhances the antioxidant system or improves the stability of cellular membranes under stress may enhance drought tolerance (Begcy et al., 2011; Hussain et al., 2013). However, these isoprene-conveyed advantages at the leaf level (Figs 6, 7), despite enhancing intrinsic (Fig. 3) and whole-plant (Fig. 4c) WUE, also reduced shoot biomass (by up to 24%) after 7 d of soil water deficit. As climate models project increased aridity over many parts of the globe (Dai, 2011), plants that emit isoprene, despite their enhanced antioxidant capacity, may show yield reductions under prolonged drought stress compared with non-emitting species.

Acknowledgements

The authors would like to thank the NERC (studentship award NER/S/A/2005/13680) and the Biotechnology and Biological Sciences Research Council (award BBS/B/12172) for funding this work.

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