Chapter 4: Results and discussion: Influence of sulphur dioxide on the photosynthetic capacity of the C

77

Chapter 4:

Results and discussion: Influence of sulphur dioxide on the photosynthetic

capacity of the C

crop, Brassica napus.

4.1.

Water status

The plant water status of Brassica napus test plants was evaluated by determining the relative water content (RWC) in leaf tissues for WW and DS treatments. Samples were collected of plants treated with elevated SO2 concentrations (50, 100 and 200 ppb) after a period of 5 weeks’ fumigation and

compared to the control treatments CFWW (carbon filtered well watered) and CFDS (carbon filtered drought stress) (Figure 4.1). The 50WW and 100WW treatments showed significant reductions of -5,72% and -10,53% (p ≤ 0.05), and a reduction of 8,65% in the 200WW treatment. Drought stressed test plants displayed reductions only in the 100DS treatment (-9,08%) when compared to the CFDS control, with the 50DS and 200DS displaying increases in RWC namely 0,01% and 2,70%, respectively. When RWC values of CFDS control test plants were compared to that of CFWW test plants, it was clear that the drought stress alone brought about a reduction in the water status of

Brassica napus test plants, though changes in RWC values were not significant. This occurrence of

a decline in RWC in DS treatments confirms the successful application of drought as co-stress.

4.2. Visual effects on SO

treated plants

Injury symptoms were visible as small chlorotic, interveinal lesions on leaves after 23 days of fumigation with SO2. The occurrence of chlorotic lesions on leaf laminas correspond to the increase

in SO2 concentration, i.e., the most affected test plants having larger necrotic areas in the 200WW &

200DS treatments. In Figure 4.2 symptoms are shown for test plants in the 50WW (a) and 200WW (b) treatments, illustrating that the severity of the symptoms strongly intensified at the higher SO2

level. Due to the acute stress imposed in the 200WW treatment, the destructive nature of SO2 and

oxidative damage by sulphite molecules resulted in cell death, seen as the necrotic leaf areas. It is important to note that the changes in gas exchange and fluorescence parameters were detected even before visual damage occurred. Note that the physiological effects are discussed later in chapter 6.

78

SO₂ fum igtion level (ppb)

0 ppb 50 ppb 100 ppb 200 ppb R e la ti v e w a te r c o n te n t (% ) 65 70 75 80 85 90 95 W W DS

*

*

Figure 4.2 a: After 23 days’ fumigation with SO2 at 50 ppb visual symptoms became evident as

interveinal chlorotic regions on older Brassica napus leaf laminas. b: At more acute exposure (200 ppb SO2) and/or prolonged exposure, necrotic lesions started to form.

a b

Figure 4.1: Relative water content in WW and DS Brassica napus exposed to different SO2

79 SO₂ concentration (ppb) 0 50 100 200 C h lo ro p h y ll c o n te n t in d e x 30 32 34 36 38 40 42 44 46 48 50 WW DS

4.3.

Chlorophyll content

Leaf pigment content provides valuable information about the physiological status of plants (Gitelson & Merzlyak, 2004). Even though a reduction of 8.14% in the chlorophyll content index could be seen in DS Brassica napus plants exposed to 50 ppb SO2 after 4 weeks’ exposure relative

to the control plants (CFDS) (Figure 4.3), all other treatments displayed an increase in chlorophyll content index, relative to CFWW and CFDS. The reason for this phenomenon could be due to a compensatory effect. Previous studies demonstrated the reduction in chlorophyll content of soybean plants due to the exposure to SO2 (Liu et al., 2009). In this study, however, an increase occurred in

the chlorophyll content index in plants exposed to elevated SO2 concentrations. The increased

chlorophyll content found might result from leaf shrinkage leading to seemingly higher chlorophyll content per unit leaf area (Linke, 2012).

Figure 4.3: Chlorophyll content index in the leaves of Brassica napus plants. Control plants were exposed to carbon filtered air and the treated plants were exposed to different SO2

concentrations namely 50, 100 and 200 ppb for 4 weeks, respectively. Each value represents the mean (±SE) of 4 measurements.

80

4.4. Photosynthetic gas exchange

The effect of different SO2 concentrations (50, 100 and 200 ppb) on the photosynthetic capacity of

Brassica napus test plants were evaluated after 3 weeks’ exposure by analysing the A:Ci response

curves (Figure 4.4 a for WW plants and in Figure 4.4 b for DS treatments). From the A: Ci response curves the different gas exchange parameters were calculated and shown in Table 4.1 for WW plants and Table 4.2 for DS plants. The accuracy of the measurements is evident from the good fit of the demand function on the data points.

The response parameters derived from the A: Ci curves shown in Table 4.1 revealed that at atmospheric CO2 concentration (370 µmol.mol-1) the CO2 assimilation rate (A370) decreased in all

treatments (50WW, 100WW and 200WW) compared to the control test plants (CFWW), namely 17.30%, 43.11% and 26.85%; respectively. These decreases could be attributed to: (i) The concomitant decrease in the apparent carboxylation efficiency (CE) of 14.58%, 56.99% (p < 0.05) and 33.23%, respectively, indicating that the limitation on photosynthesis was partially due to a decrease in Rubisco activity. (ii) The decrease in the maximum rate of CO2 assimilation (Jmax)

namely 2.1%, 32.95% and 12.79%, respectively. The decrease in Jmax represents a SO2-induced

inhibition in the regeneration capacity of RuBP and a decrease in the maximum electron transport rate (Farquhar & Sharkey, 1982). (iii) The SO2-induced increase in the compensation point namely

3.67%, 6.04% and 19.08%, respectively. These changes all strongly point at biochemical limitation of the photosynthetic capacity. In addition (iv) a decrease in the stomatal conductance occurred (Gs370), namely 28.46%, 53.62% (p ≤ 0.05) and 37.97%, respectively. This phenomenon

corresponds to (v) the increase in the calculated % stomatal limitation (ℓ) for the 50WW and 100WW treatments (16.92 % and 106.86 %, respectively), whereas the values for the 200WW treatment decreased with 38.06 %. (vi) The decrease in transpiration rate (E) namely 226 %, 237 % (p ≤ 0.03) and 180%, which corresponds to the decrease in A370. (vii) Increases occurred in the

WUE for 50WW, 100WW and 200WW namely 55.67%, 4.26% and 3.9 (50DS, 100DS and 200DS) respectively. Though the changes in stomatal conductance, % ℓ and E indicate a stomatal limitation on photosynthetic capacity, the data suggest that the limitation on the photosynthetic capacity is

81

Intercellular CO

concentration (µmol mol

)

0 200 400 600 800 1000 1200

C

O

a

s

s

im

il

a

ti

o

n

r

a

te

(

µ

m

o

l

C

O

m

s

)

strongly due to mesophyll (biochemical) limitation and that stomatal limitation played a secondary role.

Figure 4.4 a: The carbon dioxide assimilation rate (A) as function of intercellular CO2

concentration of intact leaves of WW Brassica napus plants exposed to CF air and different SO2 fumigation levels (50, 100 and 200 ppb, respectively) after 3 weeks’ exposure. Each

value represents the mean (± SE) of 4 measurements. The supply functions [A =

g

-Ci)] corresponding to the demand functions [A = CE (Ca – Γ)] are drawn by simply joining

the value of Ci = Ca (= 370 ppm) on the abscissa to the point giving A370 at this value of Ca

82

Figure 4.4 b: The carbon dioxide assimilation rate (A) as function of intercellular CO2

concentration of intact leaves of DS Brassica napus plants exposed to CF air and different SO2 fumigation levels (50, 100 and 200 ppb) respectively after 3 weeks’ exposure. Each value

represents the mean (± SE) of 4 measurements. The supply functions [A =

g

corresponding to the demand functions [A = CE (Ca – Γ)] are drawn by simply joining the

value of Ci = Ca (= µmol.mol-1) on the abscissa to the point giving A370 at this value of Ca

(Pammenter, 1989).

Intercellular CO

concentration (µmol mol

)

0 200 400 600 800 1000 1200

C

O

a

s

s

im

il

a

ti

o

n

r

a

te

(

µ

m

o

l

C

O

m

s

)

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Drought (DS) as co-stress to SO2 stress, resulted in changes in the CO2 response relationship

compared to the WW treatments. The parameters derived from the A: Ci curves (Figure 4.4 b) are shown in Table 4.2. The A370 values showed a stimulatory effect in 50DS and 200DS test plants

(14.44% and 2.53%), while a reduction of 10.65% occurred in the 100DS treatment. These changes could be attributed to: (i) A decrease in the CE namely 10.46%, 15.69% and 2.94% a (50, 100 and 200 ppb), respectively, indicating that photosynthesis was partially limited by a decrease in Rubisco activity. (ii) The regeneration capacity of RuBP and the maximum electron transport rate, represented by Jmax (Farquhar & Sharkey, 1982), displayed an initial increase at 50DS namely 1.09

%, followed by a slight reduction (0.2%) in 100DS and an increase of 6.44% in 200DS. (iii) A reduction in the CO2 compensation point in the 50DS and 200DS treatments, namely 15.27% and

9.37 %, but an increase in the 100DS treatment, namely 3.03 %. The CO2 compensation point

correlated with the trend observed in the A370 values. (iv) An increase in the Gs370 namely 2.22%

and 22.72% (50DS and 200DS treatments) and a decrease of 10.21% in the 100DS treatment. (v) The % ℓ on photosynthesis calculated that corresponded with the stomatal conductance. With the increase in Gs370, a decrease in % ℓ occurred in the 50DS and 200DS treatments namely 20.18% and

28.83%. With the decrease in Gs370 a concomitant increase occurred in % ℓ in the 100DS treatment,

namely 14.81%. (vi) The E corresponded with A370 values namely increases of 2.49% and 16.87%

in 50DS and 200DS, but a decrease in the 100DS treatment (8.03%). (vii) The decreases in the WUE namely 2.79%, 7.24% and 19.33% at 50DS, 100DS and 200DS occurred, respectively.

Data of the plants subjected to a combination of drought stress and elevated SO2 suggest that the

limitation of the photosynthetic capacity only occurred in the 100DS treatment. The changes in stomatal conductance, % ℓ and E, point at some stomatal limitation on the photosynthetic capacity. The decreases mentioned in (i), (ii) and (iii) are indications of constraints on the biochemical pathways during photosynthesis. Reductions in these parameters indicate that there was a degree of biochemical limitations imposed on the photosynthetic capability of 100DS test plants. The Jmax

value, for example, did not decrease as markedly as in the WW plants, which is an indication that even though drought stress resulted in an initial stomatal response to the additional stress, the most pronounced contribution to the decline in photosynthetic gas exchange in drought stress treatments

84

was due to mesophyll limitation. According to our gas exchange data, drought stress ameliorated the inhibitory effect of SO2 in Brassica napus to some extent.

ESEM micrographs (Figure 4.5 a (WW) & b (DS)) of the leaf surface of test plants exposed to different SO2 treatments revealed the influence of SO2 on the functioning of the stomata, resulting in

stomatal limitation of photosynthesis. The stomatal pores of the plants in the CFWW control chambers were wide open with the adjacent epidermal cells turgid and the structure thereof intact. However, with increasing SO2 concentration, the aperture of the stomatal pores of 50WW, 100WW

and 200WW became increasingly smaller (reduced stomatal opening of 49.26%, 5.75% and 59.94%, respectively). The reduction in stomatal apperture was quantified according to the weighing method described in Chapter 3 (p. 72) and presented as a percentage increase or decrease, relative to the controls (CFWW and CFDS) (Figure 4.6). Plants respond to the absorbed pollutant dose, i.e. the amount of pollutant found in the substomatal space. The rate at which a pollutant is absorbed by plant surfaces is determined by various components, one of which is the stomatal aperture. It is widely accepted that reduced stomatal aperture results in decreased pollutant damage to the plant. Less SO2 will enter the substomatal space with a smaller stomatal aperture. The effects of the

increase in SO2 concentration are evident in the 200WW treatment where the epidermal cells

bordering the stomata showed signs of damage in structure and loss of turgidity. This phenomenon corresponded to the decrease in A370 values, but because of the large decrease in the E relative to the

control, the stomatal opening of the corresponding micrographs did however not match the WUE in WW treatments. The effect at SO2 treatments of 50, 100 and 200 ppb, combined with DS is shown

in Figure 4.5 b. The effect of DS alone (CFDS) compared with the well watered control treatment (CFWW) already showed a significant reduction in the stomatal opening (29.8%; p ≤ 0.01). The SO2

drought treatments showed the same tendency of increasing closure of the stomatal opening with increasing SO2 concentrations. Compared to the CFDS treatment, 50DS and 100DS showed

significant reductions in stomatal apperture (42.52% (p ≤ 0.03); 39.43% (p ≤ 0.01)) and in the 200DS treatment a large reduction of 46.88 % occurred (Figure 4.6). This decrease corresponded to the reduction of A370 and E in the 100DS treatment as well as the reduced WUE for all the DS

85

Table 4.1: Mean values of photosynthetic parameters after 3 weeks’ fumigation with SO2 in

WW Brassica napus plants. The values in brackets represent the standard error (± standard error). The assimilation rate under atmospheric conditions is denoted as A370; CE =

carboxylation efficiency; Γ = CO2 compensation concentration; Jmax = rate of CO2 assimilation

at saturated levels of CO2 (the RuBP regeneration capacity); A0 = assimilation rate at the

point where stomatal limitation is artificially eliminated by raising the internal CO2

concentration (Ci) to the atmospheric CO2 concentration by increasing the external CO2

concentration (p ≤ 0.01 = **; p ≤ 0.05 = *). CFWW 50WW 100WW 200WW E (mmol.m-2.s-1) 5.63 (0.76) 3.37 (1.02) 3.26 (0.45*) 3.83 (0.80) A370 (µmol.m-2.s-1) 15.17 (2.82) 12.55 (2.38) 8.63 (2.08) 11.1 (3.30) Ci370 (µmol.mol-1) 288 (16.87) 279 (8.96) 267.33 (31.29) 286.33 (17.70) A0 (µmol.m-2.s-1) 19.3 (3.29) 16.35 (2.49) 11.6 (3.22) 13.18 (3.41) Gs370 (µmol.m-2.s-1) 386.5 (81.58) 204.5 (72.1) 179.25 (7.38*) 239.75 (63.14) CE (mol.m-2.s-1) 0.09 (0.007) 0.08 (0.01) 0.04 (0.009*) 0.06 (0.01) Jmax (µmol.m-2.s-1) 26.2 (4.38) 25.65 (2.85) 17.56 (3.77) 22.85 (4.54) Γ (µmol.mol-1) 81.1 (8.429) 84.07 (5.75) 88.66 (8.38) 96.57 (9.39) ℓ (%) 21.28 (3.93) 24.88 (4.58) 44.02 (14.29) 13.18 (6.09) WUE (µmol.mmol.-1) 2.82 (0.44) 4.39 (1.09) 2.94 (0.59) 2.93 (0.48)

86

Figure 4.5 a: The SEM micrographs of stomata on the abaxial surface of leaves of WW

Brassica napus after 39 days’ SO2 fumigation. (a) = CFWW; (b) = 50WW; (c) = 100WW and

(d) = 200WW.

a

b

c

d

87

Figure 4.5 b: The SEM micrographs of stomata on the abaxial surface of leaves of DS Brassica

napus after 39 days’ SO2 fumigation. (a) = CFDS; (b) = 50DS; (c) = 100DS and (d) = 200DS.

d c b a (2)

a

b

c

d

88

SO2 concentration (ppb)

R

e

la

ti

v

e

%

s

to

m

a

ta

l

a

p

e

rt

u

re

Figure 4.6: The stomatal opening of Brassica napus test plants expressed as a percentage of the control plants of the respective water regimes (CFWW and CFDS). For the CFDS treatment, the % stomatal opening was expressed as a % of the CFWW control plants in order to evaluate the effect of drought alone would have on the stomatal opening.

89

Table 4.2: The combined SO2 and drought induced effect on photosynthetic gas exchange

parameters of Brassica napus test plants after 3 weeks’ fumigation. The value in brackets represents the standard error (±SE) of 4 measurements (p ≤ 0.01 = ***; p ≤ 0.05 = *).

CFDS 50DS 100DS 200DS E (mmol.m-2.s-1) 4.01 (1.16) 4.11 (0.52) 3.69 (0.82) 4.69 (0.87) A370 (µmol.m-2.s-1) 13.85 (3.30) 15.85 (0.11) 12.38 (2.47) 14.2 (3.14) Ci370 (µmol.mol-1) 256 (15.42) 267.5 (9.5) 265.75 (13.57) 277.33 (6.64) A0 (µmol.m-2.s-1) 17.8 (3.42) 19.85 (0.31) 17.18 (2.64) 18.77 (3.29) Gs370 (µmol.m-2.s-1) 247.25 (85.27) 252.75 (44.98) 222 (64.14) 303.5 (77.86) CE (mol.m-2.s-1) 0.096 (0.02) 0.086 (0.006) 0.081 (0.01) 0.093 (0.01) Jmax (µmol.m-2.s-1) 25.25 (4.61) 25.95 (0.81) 25.2 (3.02) 23.37 (5.57) Γ (µmol.mol-1) 86.75 (7.46) 73.25 (0.37) 89.37 (9.61) 78.62 (6.32) ℓ (%) 25.21 20.13 28.95 17.94 WUE (µmol.mmol.-1) 3.68 (0.27) 3.58 (0.42) 3.41 (0.29) 2.97 (0.10)

90

4.5. The effect of SO

fumigation on PSII structure and function assessed by

fast phase chlorophyll a fluorescence kinetics (JIP test)

The leaves of Brassica napus test plants were dark-adapted before chlorophyll a fluorescence transients were recorded after 3 weeks’ fumigation with SO2. Whole plants were dark-adapted for at

least one hour to ensure that all reactions centers were fully open for primary photochemistry. The average fluorescence transients for WW and DS test plants are shown on a logarithmic time scale from 10 µs up to 1 s (Figure 4.7. a and Figure 4.7. b), respectively). These transients show the typical OJIP fluorescence rise following excitation of the dark-adapted leaves of test plants with a saturated light pulse. A rapid initial rise in fluorescence intensity from O (Fo = at 50 µs) to the

intermediate step, J, at 2 ms occurred. During this phase mainly single turn-over events occur with respect to QA reduction. A further rise in fluorescence to the second intermediate step, I at 30 ms

followed, after which a final fluorescence band (P) at 300 ms to 1 s could be seen. Note the low Fo

value which indicates that the reaction centers in leaves were fully open i.e. oxidised. The decrease in FP (=FM) was positively correlated with the SO2 concentration of the treatments. In both WW and

DS treatments, elevated SO2 concentrations (100WW, 200WW, 100DS and 200DS) yielded lower

maximal fluorescence (FP), which indicated a diminished PSII capacity for electron transport from

the water-splitting system towards PSI (Perreault et al., 2011). To compare the differences between the treatments more clearly, the raw average fast phase fluorescence transients were normalised between 50µs (Fo) and 2ms (FJ). This normalisation elucidated the SO2-induced effects in the single

turnover phase (Fo – FJ) and the multiple turnover phase (FJ – FP) of PSII function in test plants

treated with SO2 alone (Figure 4.8 a) or in combination with drought (Figure 4.8 b). In both WW

and DS treatments a decrease in fluorescence at the maximum level of fluorescence (FP) was evident

when compared to the control treatments (CFWW and CFDS). Note that the DS test plants, displayed greater differences between the treatments. The treatments yielding the lowest FP values in

both WW and DS treatments were the 100WW and 100DS treatments, respectively. This reduction in the multiple turn-over events (J-P phase) of PSII function is strongly associated with the dark reactions in the electron transport chain (Strasser et al., 1999).

91

Time (ms)

Fl

uor

e

s

c

e

nc

e

a

Figure 4.7: Raw average chlorophyll a fluorescence transients of well watered (a) and drought stress (b) treated Brassica napus plants. Plants were exposed to carbon filtered air and different SO2 fumigation levels (50, 100 and 200 ppb) for 3 weeks. Each value represents the mean (±SE) of 4 measurements.

b

92

Figure 4.8: Chlorophyll a fluorescence transients of (a) WW and (b) DS Brassica napus plants, normalised between Fo (0.01 ms) and FJ (2 ms). Plants were exposed to carbon filtered air and different SO2 fumigation levels (50, 100 and 200 ppb) for 3 weeks. Each value represents the mean (±SE) of 4 measurements. a

a

b

Time (ms)

F

lu

o

re

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e

n

c

e

Time (ms)

F

lu

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rescen

ce

93

Figure 4.9: Specific energy fluxes (per RC) and phenomenological energy fluxes (per cross section) through PSII including the efficiencies of the partial processes of primary photosynthesis, namely absorption (RC/ABS = PIRC), trapping (FV/FM = φPo = PITR),

electron transport (ψo = PIET) and reduction of end electron acceptors (δRo = PIRE), as well

as the PIABS.total for (a) WW and (b) DS Brassica napus test plants after 3 weeks’ fumigation

with SO2. The 0 ppb treatment served as the control. Each value represents a mean (± SE)

of 4 measurements. (a) PIABS,total PITR PIET PIRE PIRC (b) PIABS,total PITR PIET PIRE PIRC

94

Strauss et al. (2006) stressed the importance of using more than one parameter to evaluate the effect of an environmental stressor on the PSII function, i.e. not just using Fv/Fm (

φ

present investigation JIP-test parameters obtained by analysing the fluorescence transients (Strasser

et al., 1999) were presented in a multi-parametric radar plot to illustrate the photosynthetic

behaviour of Brassica napus plants treated with elevated SO2 at two water regimes (WW: Figure

4.9 a; and DS: Figure 4.9 b) after 3 weeks’ fumigation. The values were expressed relative to that

of the control treatments (CFWW and CFDS, respectively). The control treatments were thus used as reference point (dark green line) for the treatments (50, 100 and 200 ppb). The performance index (PIABS.total) calculated, is based on the fast phase chlorophyll fluorescence rise. This

multi-parametric function takes into account all partial processes of primary photochemistry, namely absorption of light energy (ABS), trapping of excitation energy (TR), conversion of excitation energy to electron transport (ET) and reduction of end electron acceptors (RE). It is a powerful non-intrusive measure (index) of plant response to environmental stress (Ripley et al., 2004; Govindjee, 2004).

Changes in the PIABS.total are clearly visible in the radar plots for WW (Figure 4.9 a) test plants after

a 3 weeks fumigation period with SO2, namely an increase in 50WW (24.15%) and decreases in

100WW and 200WW treatments occurred (16.79% and 10.57%, respectively). The different partial processes of primary photochemistry included in the radar plot shed light on the cause of the changes in the respective PIABS.total values (see chapter 3 for the explanation of the partial processes

of PIABS.total). Decreases in the partial processes compared to the CFWW were as follows: the PIRC

(the number of active reaction centers per total absorption, sometimes described as RC/ABS) displayed increases in 50WW and 100WW (1.85 and 0.86%; respectively) and a reduction in 200WW of 1.03%; reductions in PITR occurred namely 0.31 %, 1.7 % and 2 % for 50WW, 100WW

and 200WW, respectively; PIET displayed a 7.53% increase in 50WW, 4.48 % decrease in 100WW

and 0.49 % increase in 200WW (i.e. no change). The PIRE displayed an increase in 50WW (13.39

%) and decreases of 9.55% and 7.82% in 100WW and 200WW, respectively. The data showed that the treatment most influenced by SO2 was the well watered test plants exposed to 100 ppb SO2

(100WW) and that the greatest contributor to the decrease in PIABS.total was the decline in the

efficiency to reduce end electron acceptors (PIRE). Very small decreases in the apparent antenna size

95

and a 0.89% increase (highly significant p ≤ 0.01) in 200WW test plants. The maximum yield of primary photochemistry (TRo/ABS = φPo = FV/FM) displayed negligible decreases upon exposure to

elevated SO2 treatments (50WW, 100WW and 200WW) namely 0.21%, 0.29% and 0.35%,

respectively, indicating that the latter parameter which is often used, is not a sensitive indicator for SO2 stress in this experiment. The TRo/RC which represents the maximum trapping flux, decreased

with 1.46 % and 1.05 % in 50WW and 100WW treatments, but increased only slightly by 0.52% in 200WW, indicating the lack of response to SO2 with respect to the latter parameters in WW plants.

Changes relative to the control plants (CFWW) were also evident in the phenomenological electron transport flux, ETo/CS (electron transport per excited cross section per active measured leaf area),

namely an increase in 50WW (1.04%) and highly significant (p ≤ 0.01) decreases of 8.35% and 6.09% in 100WW and 200WW, respectively. Decreases in the ETo/CS point at a decrease in the

electron transport per measured cross section due to inactivation of the reaction center complexes. According to Strasser et al. (1999) this may be due to the inactivation of the oxygen evolving system. The same trend was evident in the RC/CS, namely a 0.25% increase in 50WW, a highly significant (p ≤ 0.01) decrease in 100WW (5.93%) and a highly significant (p ≤ 0.01) decrease in 200WW (6.36%). The reduction in RC/CS in 100WW and 200WW reflects a decrease in the density of active reaction centers in plants of these two treatments. All fluorescence parameters indicated a stimulatory response in the 50WW treatment and an inhibitory response in the 100WW and 200WW.

For the SO2-drought combination treatments an SO2-induced reduction in the PIABS.total was evident

in all the treatments relative to the CFDS control plants (Figure 4.10 b). The values displayed a 12.45% decline in 50DS treatments, a highly significant (p ≤ 0.01) reduction in the 100DS treatment (61.05%) and a reduction of 44.2% in the 200DS treated plants. Although reductions in the partial processes PIRC, PITR and PIET contributed to the decline in PIABS.total, the largest contribution to the

reduction in the PIABS,total was however due to the reduction in PIRE. The reductions in PIRE were as

follows: a decline in 50DS with 3.18%; 100DS declined significantly with 28.6% (p ≤ 0.01) and a reduction of 22.85% in 200DS plants. The decline in PIRE indicates a reduction in the efficiency of

reduction of end electron acceptors. The number of active reaction centers per total absorption (PIRC

and sometimes described as RC/ABS) of the test plants that received elevated SO2 concentrations

(50, 100 and 200 ppb), decreased significantly (p ≤ 0.05) with 5.39%, and shighly significantly (p ≤ 0.01) with 15.88% and 10.1%, respectively. Data on the PITR displayed reductions in all treatments

96

(50DS, 100DS and 200DS) namely 1.85%, and highly significantly (p ≤ 0.01) with 12.86% and 7.45%, respectively. For PIET an increase of 0.22% occurred in 50DS, but highly significant

decreases occurred in 100DS (26.47%; p ≤ 0.01) and 200DS (12.28%; p ≤ 0.01), respectively. An increase in the antenna size (ABS/RC) occurred (6.08 % (p ≤ 0.05) for 50DS, 23.72% (p ≤ 0.01) for 100DS and 12.37% (p ≤ 0.01) for 200DS). The maximum yield of primary photochemistry (TRo/ABS = φPo = FV/FM) decreased slightly in all the treatments exposed to SO2, namely 0.38%,

2.77% (p ≤ 0.01) and 1.30% (p ≤ 0.01) for the 50DS, 100DS and 200DS treatments, respectively. This once again illustrated the low sensitivity of the latter parameter in this instance. Decreased photochemistry is commonly reported to occur at severe drought stress (Flexas & Medrano, 2002). The TR/RC, which represents the maximum trapping flux, increased with 5.58% (p ≤ 0.05), 18.93 % (p ≤ 0.01) and 10.75 % (p ≤ 0.01), respectively. The increases in antenna size and trapping flux could according to Strasser et al. (2004) be due to a response to compensate for the inactivation of actively absorbing reaction centres which may be due to a fraction of RCs that is inactivated (i.e. when they are transformed to non-QA- reducing centres), or that the functional antenna, i.e., the

antenna that supplies excitation energy to active RCs, has increased in size. An increase of 6.52% and 3.1% occurred in the electron transport per cross section (ETo/CS) in the 50DS and 200DS

treatments, but however decreased with 0.55% in the 100DS treatment. A very small increase of 0.39% in the density of the reaction centers (RC/CS) was displayed for the 50DS treatment, as well as significant decreases in 100DS (p ≤ 0.05) and 200DS of 5.69% and 2.93%, respectively.

For further in depth analysis of the O-J-I-P fluorescence transients, the difference in relative variable fluorescence were calculated to present ΔV curves (expressed as V = f(t)), i.e. the subtraction of the fluorescence values of the controls’ transients from the transients of the respective treatments. This was done for fluorescence values normalised between Fo and FJ (VOJ = (Ft-Fo)/(FJ-Fo), ΔVOJ = (VOJ

treatment - VOJ control); normalised between FJ and FP (VJP = (Ft-FJ) / (FP-FJ), ΔVJP = VJP treatment – VJP control); normalised between Fo and FP (VOP = (Ft-Fo) / (FP-Fo), ΔVOP = (VOPtreatment - VOPcontrol);

normalised between FI and FP (VIP = (Ft-FI) / (FP-FI), ΔVJP = VIP treatment – VIP control), respectively

(Stirbet & Govindjee, 2011; Heyneke et al., 2011). This made the comparison of transients possible in WW (Figure 4.10 a) and DS (Figure 4.10 b) test plants exposed to elevated SO2 concentrations.

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biochemical nature of the constraints on the photosynthetic apparatus. With the VOJ normalisation

for WW treatments (Figure 4.11 a), no ∆V bands appeared, whereas a prominent ∆VK (300 µs)

band occurred in DS plants (Figure 4.11 b). A ∆VK band is associated with inhibition of the PSII

water-splitting system as a consequence of slow supply of electrons to the reaction centre (an inhibition on the donor side of PSII) (Yusuf et al., 2010; Pollastrini et al., 2011). In both WW and DS treatments, SO2-induced positive ΔVI bands (30 ms), reflecting the inhibition of the reduction of

end electron acceptors beyond PSI such as NADP+, were evident (Yusuf et al., 2010). Note that the DS treated plants were more affected, hence the larger ΔVI band at 30 ms.

Figure 4.10 a: Difference in variable chlorophyll a fluorescence transients normalised between F0 (50 µs) and FP (300 ms): [VOP = (Ft – Fo)/(FP – Fo), ∆VOJ = VOP (treatment) – VOP

(control)] of WW Brassica napus plants exposed to different SO2 concentrations for 3 weeks,

relative to the control plants exposed to carbon filtered air. The CFWW treatment served as the control (dark green line). Each plot represents a mean (± SE) of 4 plants measured.

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Figure 4.10 b: Difference in variable chlorophyll a fluorescence transients normalised between F0 (50 µs) and FP (300 ms): [VOP = (Ft – Fo)/(FP – Fo), ∆VOJ = VOP (treatment) – VOP

(control)] of DS Brassica napus plants exposed to different SO2 concentrations for 3 weeks,

relative to the control plants exposed to carbon filtered air. The CFDS treatment served as the control (dark green line). Each plot represents a mean (± SE) of 4 plants measured.

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Figure 4.11 a: Difference in variable chlorophyll a fluorescence transients of WW Brassica

napus plants exposed to different SO2 concentrations for 3 weeks, relative to the control

plants exposed to carbon filtered air. The chlorophyll a fluorescence transients were normalised between F0 (50 µs) and FJ (2 ms) [VOJ = (Ft – Fo)/(FJ – Fo), ∆VOJ = VOJ (treatment) –

VOJ (control)] and fluorescence transients normalised between FJ and FP [VJP = (Ft – FJ)/(FP –

FJ), ∆VJP = VJP (treatment) – VJP (control)]. The CFWW treatment served as the control (dark

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Figure 4.11 b: Difference in variable chlorophyll a fluorescence transients of DS Brassica

napus plants exposed to different SO2 concentrations for 3 weeks, relative to the control

plants exposed to carbon filtered air. The chlorophyll a fluorescence transients was normalised between F0 (50 µs) and FJ (2 ms) [VOJ = (Ft – Fo)/(FJ – Fo), ∆VOJ = VOJ (treatment) –

VOJ (control)] and fluorescence transients normalised between FJ and FP [VJP = (Ft – FJ)/(FP –

FJ), ∆VJM = VJP (treatment) – VJP (control)]. The CFDS treatment served as the control (dark

101

In order to interpret the I-P phase, two different normalisation procedures were used, namely: (i) The average relative variable fluorescence transients were normalised at Fo and at FI, VIP = (Ft – Fo)

/ (FI – Fo) and plotted on a linear time scale from 30 ms (I) to 300 ms (P), hence the notation

‘IP-phase (WW: Figure 4.12 a and DS: Figure 4.13 a). (ii) The average relative variable fluorescence were normalised between Fo and FI, VOI = (Ft – Fo) / (FI – Fo) and the part VOI ≥ 1was plotted on a

logarithmic time scale from 30 ms (I) to 300 ms (P) (Figures 4.12 b and Figure 4.13 b).

In the graph with a linear time scale (Figure 4.12 a and 4.13 a) the half-time for reduction of end-electron acceptors is indicated with a horizontal dashed line. The rate constant is the inverse of the half time, i.e., 1/half-time. This rate constant is an indication of the rate of reduction of end electron acceptors. A concentration dependent decline occurred in the rate at which end electron acceptors were being reduced in WW plants, namely a very small decline in 50WW (0.01 %), a 6.9% decline in 100 WW and in 200WW plants an 8.53% decline occurred compared to CFWW control test plants. In the DS treatments, an increase in 50DS (6.49%) plants were displayed compared with the CFDS plants, with reductions in both 100DS and 200DS treatments (2.6% and 7.65%), respectively. Here the SO2-induced decrease in the rate constant suggests the reduced activity of FNR and a

consequent lowered reducing potential for reduction of NADP+. The logarithmic time scale curves (Figures 4.12 b and Figure 4.13 b) represent the pool-size of the amount of active end electron acceptors available for reduction. These graphs revealed that a SO2-induced decrease in the pool size

of end electron acceptors occurred for both WW and DS treatments, and that the most affected plants were those exposed to 100 ppb (reductions of 2.2% in WW and 11.75% in DS treatments).

102 Time (ms) 50 100 150 200 250 300 N o rm a li s e d f lu o re s c e n c e ( VIP ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 CFWW 50WW 100WW 200WW

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Figure 4.12 a: The IP-phase of the O-J-I-P induction curve with the maximum amplitude fixed at unity (normalised at 30 ms and 300 ms) indicating the SO2 impact on the reduction

rate of the end electron acceptor pool for WW Brassica napus.

Figure 4.12 b: The average fast phase chlorophyll fluorescence transients of WW leaves of

Brassica napus plants after 3 weeks’ exposure to filtered air (CF) and different SO2

concentrations (50, 100 and 200 ppb respectively) normalised between FO (50 µs) and FI (30

103 Time (ms) 50 100 150 200 250 300 N o rm a li s e d f lu o re s c e n c e ( VIP ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CFDS 50DS 100DS 200DS

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Figure 4.13 b: Average fast phase chlorophyll fluorescence transients of DS leaves of Brassica

napus plants after 3 weeks’ exposure to filtered air (CF) and different SO2 concentrations (50,

100 and 200 ppb, respectively) normalised between FO (50 µs) and FI (30 ms), plotted above 1 and 30 ms and on a logarithmic time scale.

Figure 4.13 a: IP-phase of the O-J-I-P induction curve with the maximum amplitude fixed at unity (normalised at 30 ms and 300 ms) indicating the SO2 impact on the reduction rate of the end electron acceptor pool for DS Brassica napus.

104

4.6. Shoot mass

The detrimental effect of SO2 on photosynthetic performance (Pfanz et al., 1998) could not be more

evident than the cumulative effect on the carbon accumulation in the shoot biomass. In previous studies done on the effect of SO2 on Blackgram, a significant decline in plant length (up to 34%) and

biomass (up to 38% reduction) occurred at an SO2 concentration of 200 ppb (Singh & Khan, 2006).

In our experiment, compared to the CFWW control plants, all test plants subjected to elevated SO2

concentrations showed a decrease in dried shoot biomass (Figure 4.14). Test plants in the WW treatment showed a highly significant (p ≤ 0,01) decrease of 29,43% in 50WW test plants and in the 100WW and 200WW treatments highly significant (p ≤ 0,01) reductions of 16,64% and 30,12%, respectively. In the DS water regime, test plants subjected to elevated SO2 concentrations also

showed a reduction in shoot biomass (50DS: 6,65% reduction; 100DS: 8,41% reduction) and at the highest level of fumigation (200DS) a highly significant (p ≤ 0,01) decrease (24,33%) was displayed. Before the shoot biomass of plants were dried and the average mass determined, photos of the different treatments were taken to illustrate the effect of elevated SO2 concentrations on the

above ground growth of Brassica napus test plants (Figure 4.15). In both WW and DS treatments a clear SO2-dependent decline in plant length and shoot biomass occurred.

105 SO₂ concentration levels (ppb) Carbon filtered 50 100 200 S h o o t m a s s ( g ) 0 20 40 60 80 100 120 140 WW DS ** ** ** *** ***

Figure 4.14: Shoot biomass of WW and DS Brassica napus test plants exposed to different concentrations of SO2 enriched, carbon filtered air (50, 100 and 200 ppb SO2) for 6 weeks

106

Figure 4.15: The effect of SO2 on the carbon accumulation in WW (a) and DS (b) treated

Brassica napus plants after 6 weeks’ fumigation.

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CF 50 100 200

b