Chapter 5: Results and discussion: Influence of sulphur dioxide on the photosynthetic capacity of the C

107

Chapter 5:

Results and discussion: Influence of sulphur dioxide on the photosynthetic

capacity of the C4 crop, Zea mays.

5.1. Water status

Water content and water potential have been widely used to quantify the water deficits in leaf tissues. The Ψsoil determines the Ψplant and Ψleaf. The two water treatments of this experiment

namely well watered (WW; ~80% of field capacity) and drought stressed (DS; ~20% of field capacity) were obtained by placing 4 fiber glass wicks in WW pots and one fibre glass wick in DS pots (see Chapter 3: Materials and Methods). The effectiveness of the two treatments after a period of 10 weeks is illustrated by the differences in the ΨMid-day and the relative water content

in leaf tissue measured in the WW and DS treatments (Figure 5.1 and Figure 5.2).

5.1.1. Relative water content

The relative water content (RWC) can be related to several leaf physiological variables, such as leaf turgor, growth, stomatal conductance, transpiration, photosynthesis and respiration (Kramer & Boyer, 1995). Leaf water content is a useful indicator of plant water balance, since it expresses the relative amount of water present in the plant tissues (Yamasaki & Dillenburg, 1999). The RWC of the WW treatments on the average was about 94% opposed to an average of 73 % in the DS treatments. Compared to the WW treatments, the CFDS plants showed a highly significantly (p ≤ 0.01) lower RWC of 22.18%, a highly significant decrease (p ≤ 0.01) of 25.72% in 50DS, a significant (p ≤ 0.03) decrease of 13.58% in 100DS and in the 200DS treatment a highly significant decrease of 15.98% (p ≤ 0.01) occurred (Figure 5.1). In the DS treatment, elevated SO2 concentrations did not correlate with a decrease in RWC for all the treatments, but instead

showed a decrease of 4.44% in 50DS and increases of 8.21% and 6.02% for the 100DS and 200DS treatments relative to the control (CFDS), respectively.

108 SO2 concentration 0 ppb 50 ppb 100 ppb 200 ppb P re -d a w n l e a f w a te r p o te n ta l (M P a ) -25 -20 -15 -10 -5 0 WW DS SO₂ concentrations 0 ppb 50 ppb 100 ppb 200 ppb M id -d a y l e a f w a te r p o te n ti a l (M P a ) -25 -20 -15 -10 -5 0 SO₂ concentration CF 50 100 200 R e la ti v e w a te r c o n te n t (% ) 40 50 60 70 80 90 100 WW DS

Figure 5.1: Relative water content in well watered and drought stressed maize exposed to different SO2 concentrations for 8 weeks.

Figure 5.2: The pre-dawn (ΨPD) and mid-day (ΨMD) leaf water potential of WW and DS Zea mays plants after a period of 10 weeks’ fumigation with SO2.

109

5.1.2. Leaf water potential

There were only very small differences in ΨPD (pre-dawn water potential) noticed between the

WW and DS plants in the different SO2-treatments at 10 weeks’ fumigation. However, when the

ΨMD (mid-day water potential) of DS plants were compared with WW test plants, marked

differences were evident. The CFDS, 50DS, 100DS and 200DS treatments all showed reductions in the ΨMD compared to the WW treatments, namely 22.18%, 25.72%, 13.58% and 15.98%

(Figure 5.2). Even though the reductions were not statistically significant, the values reflect the successful application of the DS treatment.

5.2. Visual effects on SO

2

treated plants

According to Jones (1985) the major demonstrable effect that emissions of SO2 by utilities have

had on vegetation has been visible foliar injury. In the present study injury symptoms were already visible as small chlorotic streaks on leaves after 3 weeks of fumigation with SO2. The

intensity of chlorotic lesions on leaf laminas correlated with the SO2 concentration. The most

affected leaf material occurred in plants treated at 200 ppb SO2. Figure 5.3 displays the

appearance of leaves of Zea mays plants after 9 weeks’ fumigation with SO2 and a control plant

which only received CF air. Foliar injury, as demonstrated in Figure 5.3, could ultimately lead to reductions in growth, yield or crop quality (Jones, 1985).

5.3. Chlorophyll content index

The chlorophyll content index (CCI) of a plant directly correlates with the concentration of chlorophyll molecules in the photosystems. A reduction or increase in CCI values would then be an indication of the amount of chlorophyll molecules within the leaf of a test plant that can actively capture and absorb energy from the sun, which then will be converted to sugars and energy for the plant’s use. There have been many reported cases of reductions in the chlorophyll content with increasing pollutant concentrations (Liu et al., 2009). A correlation may exist between the CCI of a plant and the number of active chlorophyll RCs (ABS/RC).

110

The chlorophyll content in maize test plants showed an increase from the 1st week of fumigation to the 4th week of fumigation in both WW and DS treatments. The CCI then decreased to values lower than those recorded in the 1st week of fumigation, indicative of the destructive nature of SO2 on the chlorophyll pigments. When the CCI values of test plants exposed to different SO2

concentrations were compared to the corresponding CFWW values, a highly significant (p ≤ 0.01) decrease of 22.04% in 50WW, a significant (p ≤ 0.05) decrease of 11.97% in 100WW and a 7.67% decrease in 200WW test plants (Figure 5.4. a) was evident after 1 week’s fumigation. After 4 weeks’ fumigation, there was an increase in the CCI values (0.59%, 1.34% at 50WW and 100WW and highly significantly with 10.34% (p ≤ 0.01) at 200WW, respectively). Waldhoff et

al. (2002) used, among others, the chlorophyll concentration in Symmeria paniculata as an

indication for flood adaptation. They confirmed the existence of a positive correlation between

Figure 5.3: Visible SO2 symptoms in Zea mays after 9 weeks’ fumigation at 200 ppb in open-top

111

the chlorophyll concentration and leaf age. It is typical to find higher chlorophyll concentrations per leaf area sampled in mature leaves, compared to younger leaves. This finding corresponded with our data which displayed an increase in chlorophyll content in the leaves of the test plants after 4 weeks’ fumigation with SO2 (5 weeks’ growth). In the 7th week of fumigation a decrease

of 11.05% (50WW) occurred, followed by a 4.67 % increase (100WW), and again a highly significant decrease occurred (p ≤ 0.01) in the 200WW treatment (52.42%). The destruction of chlorophyll pigment could be due to the very reactive HSO3- and SO32- anions derived from SO2

molecules (Takahama et al., 1992; Ghisi et al., 1990), which in turn is associated with the development of injury symptoms in leaves (Figure 5.3).

When the effect of SO2 on the CCI of test plants with drought as co-stressor was compared to

their control plants (CFDS) after the first week of fumigation (Figure 5.4. b), a highly significant (p ≤ 0.01) decrease in the 50DS treatment (14.85 %), a significant (p ≤ 0.05) increase in 100DS (10.45 %) and an increase in the 200DS test plants of 6.63% were evident. After the 4th week of fumigation a significant (p ≤ 0.05) decrease of 6.74% (50DS), increase of 7.08 % (100DS) and a decrease of 5.51% in the 200DS test plants occurred. After 7 weeks’ fumigation, decreases occurred in all SO2 treatments, namely a highly significant (p ≤ 0.01) decrease in 50DS

(28.70%), 14.01% in 100DS and a highly significant (p ≤ 0.01) decrease of 16.80 % in the 200DS treatment. The increases in CCI in some of the treatments in the 1st week of fumigation (100DS and 200D) and in the 4th week of fumigation (100DS) may be explained by a shrinking effect. The total leaf area per amount chlorophyll is less for these specific drought stress treatments, compared to the other treatments. This leads to a higher proportion of chlorophyll measured for the same designated leaf area. The greater chlorophyll content in the mentioned drought stress treatments could have been attributed to the lower RWC and more negative water potential in the leaves of DS treatments (Figures 5.1 & 5.2). The treatments subjected to the shrinking effect, therefore yields seemingly higher chlorophyll content values per leaf area. This phenomenon will be discussed in chapter 6.

112 Time (weeks) 0 1 2 3 4 5 6 7 8 C hl or ophy ll c ont e nt i nde x 10 20 30 40 50 CFDS 50DS 100DS 200DS Time (weeks) 0 1 2 3 4 5 6 7 8 Ch lo ro p h y ll c o n te n t in d e x 10 20 30 40 50 CFWW 50WW 100WW 200WW

Figure 5.4: The Chlorophyll content index of WW (a) and DS (b) maize plants exposed to different SO2 concentrations (0, 50, 100 and 200 ppb) over a period of 7 weeks. Each data point

represents a mean of 4 plants (±SE). a

113

5.4.

Photosynthetic gas exchange

The effect of different SO2 concentrations on the photosynthetic capacity of WW Zea mays test

plants was evaluated by analysing the A: Ci response curves (Figure 5.5 a for WW plants and with drought as co-stress (DS) in Figure 5.5 b. From the A:Ci curves the different gas exchange parameters were calculated and shown in Table 5.1 for WW plants and Table 5.2 for DS plants. The A: Ci curves indicated that 7 weeks’ exposure to SO2 enriched air leads to decreases in the

CO2 assimilation rate for both the WW and DS treatments. The accuracy of the measurements is

evident from the good fit of the demand function on the data points. The effect on the CO2

assimilation rate (A370) proved to be less prominent in the DS test plants opposed to the WW

treatments.

The response parameters derived from the A: Ci curves that are depicted in Table 5.1 revealed that after 7 weeks’ fumigation at atmospheric CO2 concentration (370 µmol.mol-1) the CO2

assimilation rate (A370) showed a slight increase (i.e. stimulatory effect) in both the 50WW and

100WW treatments (3.12% and 12.89%, respectively), followed by a decrease of 24.27% in the 200WW treatment compared to the control test plants (CFWW). Interestingly, the general trend of the A: Ci curves showed that WW treatments produced lower A370 values than DS treatments,

with the exception of 200DS. This phenomenon is discussed in chapter 6. The slight increase in A370 at 50WW and 100WW and decrease in 200WW could be attributed to: (i) An increase in the

apparent carboxylation efficiency (CE) in 50WW and 100WW (39.74% and 34.62%) and the 20.51% decrease in 200WW treatments, indicating that the limitation on photosynthesis in the 200WW treatment is partially due to the decrease in PEPc activity (Watling et al., 2000). (ii) A decrease in the maximum rate of CO2 assimilation (Jmax) in 50WW, 100WW and 200WW

namely 3.83%, 3.16% and 31.11%, respectively. The decrease in Jmax represents a SO2-induced

inhibition in the regeneration capacity of phosphoenolpyruvate (PEP) and a decrease in the maximum electron transport rate (Von Caemmerer et al., 1999; Watling et al., 2000). (iii) The SO2-induced increase in the compensation point (Γ) in all WW treatments namely 27.02%,

3.73% and 29.5%, respectively. These changes all indicate that the limitation on photosynthetic capacity was due to a limitation in the biochemical processes of photosynthesis. (iv) In addition decreases occurred in the stomatal conductance (Gs370) in the 50WW and 100WW treatments,

114

namely 4.78% and 1.39%, but an increase of 2.31% in the 200WW treatments occurred which correlated with the values calculated for A370. (v) The calculated% stomatal limitation (% ℓ) for

all SO2 treatments, i.e. 50WW, 100WW and 200WW decreased by 6.12%, 60.76% and 16.68%,

respectively. (vi) The corresponding transpiration rate (E) showed increases in 50WW and 100WW (2.76% and 12.97%), but a slight decrease in 200WW (0.69%, i.e. no change). (vii) The WUE of all treatments decreased, namely 1.16%, 4.96% and 24.4%, respectively. The gas exchange data on WW treatments suggest that the limitation on photosynthetic capacity was primarily due to a limitation in the biochemical processes of photosynthesis and was less affected by stomatal limitation.

The CO2 response parameters derived from the A: Ci curves of the SO2-drought treatments (DS)

(Figure 5.5 b) are shown in Table 5.2. The A370 values revealed reductions in all treatments

(50DS, 100DS and 200DS), namely 21.44%, 18.24% and a statistically significant large decrease (p ≤ 0.05) of 60.54%. Reductions in A370 could be attributed to: (i) A decrease in the apparent

carboxylation efficiency of PEPc (CE) for all WW treatments namely 53.3%; 39.55% and 47.06%, respectively. This was an indication that a measure of limitation on photosynthesis occurred partially due to a decrease in PEPc activity. (ii) A decrease in the maximum rate of CO2

assimilation (Jmax) namely 16.11%, 12.92% and 53.76% (p ≤ 0.05) at 50DS, 100DS and 200DS,

respectively. The decrease in Jmax represents SO2-induced inhibition in the regeneration capacity

of phosphoenolpyruvate (PEP) and a decrease in the maximum electron transport rate (Von Caemmerer et al., 1999). (iii) An initial decrease in the compensation point (Γ) of 20.96% in 50DS treatments, and an increase of 3.93% and 97.97% in 100DS and 200DS, respectively. The above mentioned changes in DS treatments strongly point at a biochemical limitation on the photosynthetic capacity. (iv) Decreases also occurred in the stomatal conductance (Gs370) namely

10.26% and 32.11% for 50DS and 200DS, respectively, while no changes occurred in the 100DS treatment (Figure 5.5b). Note the substantial decrease in the slope of the supply function (i.e. stomatal conductance) for the 200DS treatment. This phenomenon will be discussed in chapter 6. (v) The calculated % stomatal limitation (ℓ) decreased with 20.17% and 28.83% in 50DS and 200 DS, respectively, and decreased with 28.83% in 200DS. (vi) The transpiration rate (E) showed decreases of 7.36% and 28.46% for 50DS and 200DS, respectively, and a slight indication of an increase in 100DS (1%). (vii) The WUE displayed small increases namely

115

5.49% and 12.56% for the 50DS and 100DS treatments, respectively, and a decrease of 22.71 % in the 200DS treatment. The data of the DS treatments, as was the case in the WW treatments, suggest that the induced limitation on photosynthetic capacity was primarily due to a limitation in the biochemical pathways of photosynthesis, while stomatal limitation played a secondary limiting role.

The main difference between the SO2-WW and SO2-DS treatments was that the drought stress

treatments showed a greater stomatal response to increased SO2 concentrations than WW

116

Intercellular CO

₂

concentration (µmol mol

-1

)

0 200 400 600 800 1000 1200

C

O

2

a

s

s

im

il

a

ti

o

n

ra

te

(

µm

o

l

C

O

2

m

-1

s

-2

)

-5 0 5 10 15 20 25 200WW 100WW CFWW 50WW a

Figure 5.5 a: The carbon dioxide assimilation rate (A) as function of intercellular CO2

concentration (Ci) of intact leaves of WW Zea mays plants exposed to CF air and

different SO2 fumigation levels (50, 100 and 200 ppb, respectively) after 7 weeks’

exposure. Each value represents the mean (± SE) of 4 measurements. The supply function [A = gCO2 (Ca-Ci)] corresponding to the demand function [A = CE (Ca – Γ)] is

drawn by simply joining the value of Ci = Ca (= 370 ppm) on the abscissa to the point giving A370 at this value of Ca (Pammenter, 1989).

117

Table 5.1: Mean values of photosynthetic parameters after 7 weeks’ fumigation with SO2 in

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

carboxylation efficiency; the CO2 compensation concentration is denoted as Γ; the Jmax

represents the rate of CO2 assimilation at saturated levels of CO2 (the RuBP regeneration

capacity); A0 is the rate of assimilation at the point where stomatal limitation is artificially

eliminated by raising the internal CO2 concentration (Ci) to atmospheric CO2

concentration by increasing the external CO2 concentration (p ≤ 0.01 = **; p ≤ 0.05 = *).

CF air 50 ppb 100 ppb 200 ppb E (mmol.m-2.s-1) 2.467 (0.449) 2.535 (0.435) 2.787 (0.268) 2.45 (0.258) A370 (µmol.m -2 .s-1) 12.875 (1.392) 13.275 (1.326) 14.325 (0.895) 9.75 (0.686) Ci370 (µmol.mol-1) 225 (18.876) 210.5 (23.203) 203.75 (21.414) 274.75 (11.183) A0 (µmol.m-2.s-1) 13.987 (0.793) 13.887 (1.088) 14.325 (0.727) 9.95 (0.686**) Gs370 (µmol.m-2.s-1) 162 (38.731) 154.25 (33.981) 159.75 (20.846) 165.75 (9.809) CE (mol.m-2.s-1) 0.156 (0.036) 0.218 (0.037) 0.218 (0.005) 0.124 (0.016) Jmax (µmol.m-2.s-1) 15.025 (0.779) 14.45 (1.158) 14.55 (0.411) 10.35 (0.780*) Γ (μmol.mol-1) 16.1 (2.811) 20.45 (1.098) 16.7 (1.382) 20.85 (2.772) ℓ (%) 12.62 (6.15) 11.84 (1.88) 4.95 (2.39) 10.51 (9.13) WUE (µmol.mmol.-1) 5.508 (0.520) 5.444 (0.377) 5.235 (0.352) 4.164 (0.572)

118

Intercellular CO

₂

concentration (µmol mol

-1

)

0 200 400 600 800 1000 1200

C

O

2

a

s

s

im

il

a

ti

o

n

r

a

te

(

µ

m

o

l

C

O

2

m

-1

s

-2

)

-5 0 5 10 15 20 25

Figure 5.5 b: The carbon dioxide assimilation rate (A) as function of intercellular CO2

concentration (Ci) of intact leaves of DS Zea mays plants exposed to CF air and different

SO2 fumigation levels (50, 100 and 200 ppb, respectively) after 7 weeks’ exposure. Each

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

g

CO2 (Ca

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

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

Ca (Pammenter, 1989). (b) 200WW 100WW CFWW 50WW

119

Table 5.2: Mean values of photosynthetic parameters after 7 weeks’ fumigation with SO2 in

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

efficiency; the CO2 compensation concentration is denoted as Γ; the Jmax represents the rate of

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

assimilation at the point where stomatal limitation is artificially eliminated by raising the internal CO2 concentration (Ci) to atmospheric CO2 concentration by increasing the external

CO2 concentration (p ≤ 0.01 = ***; p ≤ 0.03 = **; p ≤ 0.05 = *). CF air 50 ppb 100 ppb 200 ppb E (mmol.m-2.s-1) 2.105 (0.572) 1.95 (0.348) 2.126 (0.381) 1.505 (0.809) A370 (µmol.m-2.s-1) 18.5 (0.907) 14.533 (2.795) 15.125 (1.793) 7.3 (3.606) * Ci370 (µmol.mol-1) 173.666 (18.205) 167.333 (2.403) 147.75 (19.375) 187.5 (35.427) A0 (µmol.m-2.s-1) 18.85 (0.7815) 15.566 (2.085) 16.2 (1.805) 7.65 (3.353)** Gs370 (µmol.m-2.s-1) 130 (40.329) 116.66 (26.672) 130 (30.534) 88.25 (55.714) CE (mol.m-2.s-1) 0.390 (0.044) 0.182 (0.069) 0.236 (0.045) 0.13 (0.103) Jmax (µmol.m-2.s-1) 19.4 (0.787) 16.967 (3.22) 16.8 (2.425) 8.875 (3.613)* Γ ( µ mol.mol-1 ) 18.45 (3.205) 14.583 (2.381) 19.175 (1.516) 36.05 (14.004) ℓ (%) 1.4266 (1.08) 23.1 (23.09) 24.04 (11.04) 14.34 (7.54) WUE (µmol.mmol.-1) 7.055 (0.335) 7.442 (0.396) 7.94 (0.581) 5.453 (0.608)

120

5.5.

The effect of air pollutants on PSII structure and function assessed by

fast chlorophyll a fluorescence kinetics (JIP test)

The intact leaves of Zea mays test plants were dark-adapted prior to chlorophyll a fluorescence measurements to ensure that all reaction centers were fully open (oxidised) for primary photochemistry. The average raw fluorescence transients are displayed on a logarithmic time scale from 10 µs up to 1 s, for WW (Figure 5.6 a) and DS (Figure 5.6 b) SO2 treatments. Upon

excitation of dark-adapted leaves of test plants with a saturated light pulse, a rapid initial rise in fluorescence intensity from O (Fo) to the intermediate step, J, at 2 ms is evident. Within this

phase mainly single turn-over events occur with respect to QA reduction. From FJ, a further rise

in fluorescence to the second intermediate step, I, at 30 ms followed, after which a final fluorescence band (P) at approximately 300 ms to 1 s could be seen. The phase FJIP represents

the multiple turn-over redox reactions in the OJIP transients (Strasser et al., 1999). The shape of the curves of the different treatments exhibits this typical OJIP fluorescence rise and can be well distinguished from one another. The steps O, J, I, and P are clearly visible. A SO2-concentration

dependant decrease in the FP values is displayed (Figure 5.6 a). Also the DS treatments indicated

that elevated SO2 concentrations brought about changes in the different steps in the OJIP

transients (Figure 5.6 b). To explain these deviations from the control, the curves were further analysed by normalising the raw average fluorescence transients between 50µs (F0) and 2ms (FJ)

revealing the effects of SO2 on the single turn-over (F0 to FJ) and the multiple turn-over (FJ to FP)

events of PSII. This was done for WW (Figure 5.7 a) and DS (Figure 5.7 b) test plants treated with SO2. The double normalisation revealed that most of the SO2-induced changes possibly

occurred in the multiple turn-over events (FJ-FP) of PSII which are strongly influenced by the

dark reactions in the electron transport chain (Strasser et al., 1999). The WW test plants showed greater deviations from the controls than the DS treatments in this respect, already suggesting that SO2-induced inhibition in the electron transport chain of WW treatments would be more

121 Time (ms) 0.01 0.1 1 10 100 1000 F lu o re s c e n c e 0 200 400 600 800 1000 1200 1400 1600 1800 CFWW 50WW 100WW 200WW O J I P Time (ms) 0.01 0.1 1 10 100 1000 F lu o re s c e n c e 0 200 400 600 800 1000 1200 1400 1600 1800 CFDS 50 ppb 100 ppb 200 ppb

Figure 5.6: Raw average chlorophyll a fluorescence transients of WW (a) and DS (b) treated Zea

mays plants. Plants were exposed to carbon filtered air and different SO2 fumigation levels (50,

100 and 200 ppb) for 7 weeks. Each value represents the mean (±SE) of 4 measurements. a b O J I P

122 Time (ms) 0.01 0.1 1 10 100 1000 N o rm a li s e d f lu o re s c e n c e ( VO J ) 0.0 0.5 1.0 1.5 2.0 2.5 CFWW 50WW 100WW 200WW 2 Time (ms) 0.01 0.1 1 10 100 1000 N o rm a li s e d f lu o re s c e n c e ( VO J ) 0.0 0.5 1.0 1.5 2.0 2.5 CFDS 50 ppb 100 ppb 200 ppb 2

Figure 5.7: Chlorophyll a fluorescence transients of (a) WW and (b) DS Zea mays plants, normalised between F0 (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 7 weeks. Each value represents the

mean (±SE) of 4 measurements.

a b O J I P O J I P

123

The effect of SO2 concentration on the photosynthetic performance index (PIABS.total) of WW

(Figure 5.8 a) and drought stressed (Figure 5.8 b) Zea mays is shown for 1, 3, 5, 6 and 7 weeks’ fumigation periods. The performance index (PIABS.total) 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). In the first week of fumigation, the PIABS.total of the 50WW and 100WW

treatments decreased with 6.49% and 2.96%, respectively, but however increased in 200WW with 10.73%. Data of week 3, 5 and 6 did not show any significant changes, though reductions in PIABS.total was displayed at week 5 in 50WW (4.45%) and 200WW (4.68%). In the 7th week of

fumigation with SO2, marked reductions in PIABS.total occurred in all the treatments, namely

3.54%, 15.4% (p ≤ 0.03) and a highly significant (p ≤ 0.01) reduction of 62.67% in the 200WW treatment. In the DS treatments, a SO2-induced reduction in the PIABS.total occurred at all weeks

measured. All treatments showed significant (p ≤ 0.05) reductions compared to the CFDS treatment, with the exception of 100DS in week 5, and 50DS and 100DS in week 7, showing small reductions. The greatest reductions in PIABS.total always occurred in the 200DS treatments.

Selected structural and functional JIP-test parameters obtained by analysing the fluorescence transients (Strasser et al., 2004 ) are presented in a multi-parametric radar plot to illustrate the photosynthetic behaviour of WW and DS Zea mays plants treated with elevated SO2 (WW: Figure 5.9 a and DS: Figure 5.9 b). The values are expressed relative to the relevant control

treatments (CFWW and CFDS). These control treatments were thus used as reference point (dark green line) for the other treatments (50, 100 and 200 ppb). As already mentioned, the PIABS.total

reflects the relative contribution of the partial processes of primary photochemistry (i.e. PIABS.total

= PIRC · PITR · PIET · PIRE). In the radar plot for the WW treatments the concentration dependent

inhibition of these partial processes is clearly evident. The contribution of the different partial processes of primary photochemistry shown in the radar plot shed light on the possible reasons for the decrease in PIABS.total. All partial processes showed SO2-induced decreases relative to the

CFWW treatment, with the exception of PIRE, the latter which represents the reduction of end

124

Figure 5.8 a: The performance index (PIABS.tot) of WW test plants measured at 1, 3, 5, 6 and

7 weeks’ exposure to filtered air (CF) elevated SO2 levels (50, 100 and 200 ppb).

Time (weeks)

1 3 5 6 7

P

I

AB S .t o ta l

a

s

%

o

f

th

e

c

o

n

tr

o

l

40 60 80 100 120 CFWW 50WW 100WW 200WW

125

Figure 5.8 b: The performance index (PIABS.tot) of DS test plants measured at 1, 3, 5, 6 and 7

weeks’ exposure to filtered air (CF) elevated SO2 levels (50, 100 and 200 ppb).

Even though slight reductions occurred in the first three different partial processes (PIRC, PITR,

PIET), the largest contribution to the reduction in the PIABS.total was due to the reduction in PIRE.

The reductions in PIRE for all DS treatments relative to the control (CFDS) were as follows:

5.69%, a highly significant reduction of 13.08% (p ≤ 0.01) and a highly significant reduction of 21.06% (p ≤ 0.01) for 50DS, 100DS and 200DS treatments, respectively. 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 is sometimes described as RC/ABS) of the test

Time (weeks)

1 3 5 6 7

P

I

AB S .t o ta l

a

s

%

o

f

th

e

c

o

n

tr

o

l

40 60 80 100 120 CFDS 50DS 100DS 200DS

126

plants treated with elevated SO2 concentrations (50WW, 100WW and 200WW) decreased by

2.29%, 3.71% and significantly (p ≤ 0.01) with 25.75%, respectively. A simultaneous increase in the apparent antenna size (ABS/RC) relative to the control (CFWW) occurred, namely 2.76%, 5.05% and highly significantly (p ≤ 0.01) with 41.37% at 50WW, 100WW and 200WW, respectively. The maximum yield of primary photochemistry (TRo/ABS = FV/FM = φPo)

decreased for 50WW, 100WW and 200WW with 0.28%, 3.22% and significantly by 13.09% (p ≤ 0.01), respectively. The TRo/RC, which represents the maximum trapping flux, corresponded

to the increase in TRo/ABS and also increased with 2.36%, 1.74% in 50WW and 100WW and

significantly by 20.07% in 200WW. The increases in antenna size and trapping flux could have been due to a compensation strategy to offset the decrease in density of active reaction centres, the latter which may have been due to a fraction of RCs that was inactivated (i.e. when they are transformed to non-QA- reducing centres), or that the functional antenna supplying excitation

energy to active RCs, has increased in size (Strasser et al., 2004). A decrease was also evident in the phenomenological electron transport flux (ET/CS) namely 3.26%, 2.49% and 14.56%, for the different treatments, respectively. A decrease also occurred in the RC/CS (2.81%, 1.72% and significantly with 12.5% (p ≤ 0.01), respectively. The latter reduction reflects a decrease in the density of active reaction centers.

For the DS treatments, the PIRC of the test plants that received elevated SO2 concentrations

(50DS, 100DS and 200DS), decreased slightly with 1.84%, 2.7% and 3.22%, respectively. A concomitant increase in the antenna size (ABS/RC) occurred namely 2%, 5.23% and 4.64% for 50DS, 100DS and 200DS treatments, respectively. The maximum yield of primary photochemistry (TRo/ABS) decreased in all the DS treatments namely significantly with 0.24%

(p ≤ 0.05), 2.12% and 2.04% at 50DS, 100DS and 200DS, respectively. The maximum trapping flux (TRo/RC) increased in all DS treatments with 1.75%, 2.15 % and 2.03%, respectively. A

decrease occurred in the ET/CS of 3.75% and 1.77% for the 50DS and 200DS treatments, but increased slightly with 0.77% in the 100DS treatment. The RC/CS displayed only small decreases relative to the control plants (CFDS), namely a highly significant (p ≤ 0.01) decrease of 4.46%, 0.76% and 1.21% for 50DS, 100DS and 200DS treatments, respectively. These reductions reflect a decrease in the density of active reaction centers.

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Figure 5.9: The impact of SO2 on (a) WW and (b) DS Z.mays test plants, evaluated by the

behaviour pattern of structural and functional parameters of PSII, derived by the JIP-test from the chlorophyll a fluorescence transients exhibited by dark adapted leaves. In each case the parameters shown are expressed relative to the control (CF).

a

PIABS.total PITR PIET PIRE PIRC

b

PIABS.total PITR PIET PIRE PIRC

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Figure 5.10: Effect of different SO2 concentrations on WW Zea mays plants (after 7 weeks’

fumigation) on the difference in variable fluorescence:

(a) Difference in variable chlorophyll a fluorescence transients normalised between F0 and FP

[VOP = (Ft – Fo)/(FP – Fo), ∆VOP = VOP (treatment) – VOP (control)].

(b) Difference in variable chlorophyll a fluorescence transients normalised between F0 and FJ

[VOJ = (Ft – Fo)/(FJ – Fo), ∆VOJ = VOJ (treatment) – VOJ (control)] and fluorescence transients

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Figure 5.11: Effect of different SO2 concentrations on DS Zea mays plants (after 7 weeks’

fumigation) on the difference in variable fluorescence:

(a) Difference in variable chlorophyll a fluorescence transients normalised between F0 and FP

[VOP = (Ft – Fo)/(FP – Fo), ∆VOP = VOP (treatment) – VOP (control)].

(b) Difference in variable chlorophyll a fluorescence transients 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)].

130 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 30 300 Time (ms)

(b)

‘Km’

For in depth analysis of the O-J-I-P fluorescence transients the three normalisation procedures were used namely the relative variable fluorescence, Vt = (Ft – Fo)/(FM – Fo), was obtained by a

double normalisation of the fluorescence induction curve, i.e. normalisation between Fo and FP:

VOP = (Ft – Fo)/(FP – Fo), ∆VOP = VOP (treatment) – VOP (control); normalisation between F0 and FJ: VOJ

= (Ft – Fo)/(FJ – Fo), ∆VOJ = VOJ (treatment) – VOJ (control); normalised between FJ and FP: VJP = (Ft –

FJ)/(FP – FJ), ∆VJM = VJP (treatment) – VJP (control). This made the comparison of transients possible

which were measured for WW (Figure 5.10) and DS (Figure 5.11) plants exposed to 50, 100 and 200 ppb SO2, respectively. Hidden bands were revealed by computing the difference of the

variable fluorescence transients relative to the control (∆Vt = Vtreatment – Vcontrol). This

information aided in further elucidation of data previously presented. In the well watered treatments, a prominent ∆VK band appeared (300 µs) in the 200WW treatment (Figure 5.10 b)

indicating an inhibition on the donor side of PSII (OEC) (Yusuf et al., 2010), whereas no ∆VK

-band appeared in the corresponding drought stress treatments (Figure 5.11 b). In the drought stress treatments, SO2 concentration dependant positive ΔVI bands (30ms) were evident,

reflecting inhibition of the reduction of end electron acceptors beyond PSI such as NADP+ (Figure 5.11 b).

Figure 5.12: Effect of different SO2 concentrations on WW (a) and DS (b) Zea mays plants

after 7 weeks’ fumigation on the difference in variable chlorophyll a fluorescence transients normalised between FI and FP [VIP = (Ft – FI)/(FP – FI), ∆VIP = VIP (treatment) – VIP

(control)].

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

plotted on a linear time scale from 30 ms (I) to 300 ms (P), hence the notation the ‘IP-phase’ (Figure 5.13 a and Figure 5.14 a); and (ii) The average relative variable fluorescence was normalised between Fo and FI (VOI = (Ft – Fo) / (FI – Fo)) and the area VOI ≥ 1were plotted on a

logarithmic time scale from 30 ms (I) to 300 ms (P) (Figure 5.13 b and Figure 5.14 b).

The half-times on the curves plotted on a linear time axis (Figure 5.13 a and Figure 5.14 a) are indicated by a horizontal dashed line and the rate constant (which is an indication of the rate of reduction of end electron acceptors) is the inverse of the half time, i.e., 1/half-time. A decline occurred in the rate at which end electron acceptors were being reduced in plants treated with 100WW (14.93 %) and 200WW (14.93 %) relative to CFWW test plants. In the DS treatments, similar results were obtained, i.e., reductions occurred in the 100DS (13.04 %) and 200DS (8.26 %) treatments compared to CFDS control plants. The vertical change in the amplitude of the normalised fluorescence curves in the range of 100 ms (Figure 5.13 a and Figure 5.14 a) is presented more clearly by Figure 5.12 a and b. The difference in relative variable fluorescence, ∆VIP, corresponds to a change in the slope of the IP-phase measured at the relative variable

fluorescence VIP = 0.5 rise (30 to 300 ms) (Figure 5.13 a and Figure 5.14 a) indicating a change

in the original speed or fractional percentage in the IP-phase. The amplitude at 100 ms represents an apparent Km value for the enzyme reactions which are involved in the IP-phase. The VIP

kinetics can be considered as Michaelis-Menten type light driven red-ox reactions such as [PQ][PC-RC1-fd]n-[NADP] with multiple reaction centres (Schansker et al., 2003). This is

chacteristic of, among others, the enzyme ferredoxin-NADP oxidoreductase which enables the reduction of the end electron acceptor, NADP+. Here the SO2-induced increase in the amplitude

suggests the reduced activity of FNR and a consequent lowered reducing potential of NADP+. At 100 and 200 ppb SO2 the overall rate constant (1/half-time) for reduction of end electron

acceptors were lower, i.e. the reduction rate was lower. Figure 5.13 b and Figure 5.14 b represent the pool-size of the amount of active electron end acceptors available for reduction. A SO2-induced decrease in the pool size of end electron acceptors occurred in both WW and DS

132 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 V IP 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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Rate constant = 1/t Time (ms) 100 1000 R e la ti v e v a ri a b le f lu o re s c e n c e VO I 1.00 1.05 1.10 1.15 1.20 1.25 CFWW 50WW 100WW 200WW

Figure 5.13 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 Z. mays after 7 weeks’ exposure. Also see Figure 5.12 a.

Figure 5.13 b: The average fast phase chlorophyll fluorescence transients of WW leaves of

Zea mays plants after 7 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), and the part

VOI ≥ 1 plotted (30 – 300 ms). The maximum amplitude of the rise reflects the size of the pool

of end electron acceptors at the PSI acceptor side (Yusuf et al., 2010).

133 Time (ms) 50 100 150 200 250 300 N o rm a li s e d fl u o re s c e n c e (V IP ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 CFDS 50DS 100DS 200DS (a) Rate constant = 1/t

Figure 5.14 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 DS Z. mays after 7 weeks’ exposure. Also see Figure 5.12 b. Time (ms) 100 1000 N o rm a li s e d f lu o re s c e n c e ( VIO ) 1.00 1.05 1.10 1.15 1.20 1.25 1.30 CFDS 50DS 100DS 200DS

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Figure 5.14 b: The average fast phase chlorophyll fluorescence transients of DS leaves of Zea

mays plants after 7 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), and the part VOI

≥ 1 plotted (30 – 300 ms). The maximum amplitude of the rise reflects the size of the pool of end electron acceptors at the PSI acceptor side (Yusuf et al., 2010).

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5.6.

Hydrogen peroxide content

The extent of oxidative stress can be determined by the amount of hydrogen peroxide (H2O2)

present in a cell (Apel & Hirt, 2004). The general trend for the WW (Figure 5.15 a) and DS (Figure 5.15 b) of the Zea mays test plants was that the H2O2 content decreased in the leaves

over a time period of 9 weeks in response to elevated SO2 concentrations (50, 100 and 200 ppb).

At 3 weeks’ fumigation with SO2 leaf material was sampled. All WW treatments at this stage

displayed increases in H2O2 content compared to the CFWW control treatment, namely increases

of 38.75%, a highly significant increase (p ≤ 0.01) of 55.07% and 27.11% for 50WW, 100WW and 200WW treatments occurred, respectively. The increased accumulation of H2O2 resulted in

cellular injury which was presented as necrotic lesions on the leaf laminas (Figure 5.3) (Liu et

al., 2009). After 3 weeks’ fumigation with SO2, both the 50WW and 100WW treatments showed

a decrease in H2O2 content of 31.43% (p ≤ 0.03) and 37.6%, compared to the CFWW control

treatments. This could have been due to the regulation of scavenging mechanisms in test plants where the increased POD activity continually reduced H2O2 (Liu et al., 2009). The increased

H2O2 content in 200WW at 6 weeks’ fumigation with SO2 indicate that there was still an amount

of oxidative stress present in the mesophyll cells of these test plants that could not effectively be removed by means of a scavenging mechanism. Decreases in H2O2 content occurred in all WW

treatments at sampling after 9 weeks’ fumigation with SO2, namely 34.61%, 37.92% for 50WW

and 100WW treatments and a highly significant (p ≤ 0.01) decline of 43.73% in 200WW the treatment, respectively. The overall decrease in H2O2 content after 9 weeks’ fumigation with

elevated SO2 displayed the effectiveness of the scavenging enzymes to remove H2O2 from

mesophyll cells, possibly due to adaptation of test plants to prevent the accumulation of H2O2.

In the DS treatments, leaf samples were collected after 3 weeks’ fumigation with SO2 which

displayed a decreases in H2O2 content in the 50DS treatment (7.81%), an increase in 100DS and

a highly significant decrease in 200DS test plants (68.25%; p ≤ 0.01) compared to the CFDS control treatments (Figure 5.12 b). After 6 weeks’ fumigation with SO2 a significant increase

was evident in 50DS and 200DS test plants, namely 37.74% and 27.23%, respectively, but decreased with 34.04% in the 100DS treatment. A decline in H2O2 content occurred in all leaf

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47.33%, with significant reductions in 100DS (p ≤ 0.01) and 200DS (p ≤ 0.01) treatments, respectively. Overall, a decline in H2O2 content was detected for all treatments after 9 weeks’

fumigation with SO2 when compared to samples taken after 3 weeks’ fumigation with SO2. The

data thus suggest that the test plants adjusted to the stress imposed by elevated SO2 by increasing

the overall POD enzyme activity over 9 weeks (Figure 5.16).

5.7. Antioxidant enzyme: Guaiacol peroxidase

In several studies work has been done on the activation of an antioxidant scavenging system which limits the damage that oxidative stress causes to plants. Most of the studies focussed on the major scavenging enzymes such as ascorbate peroxidase (APX) and superoxide dismutase (SOD) (Rakwal et al., 2003; Singh & Khan, 2006; Liu et al., 2009). In our study the effect of SO2 fumigation and the interaction with drought stress on peroxidase activity was investigated.

In the WW treatment (Figure 5.16 a) a concentration dependent increase in POD activity occurred at one week, two weeks and three weeks after onset of fumigation, relative to CFWW control test plants. With time an increase in POD activity also occurred in WW and DS treatments (sampled at 2 and 3 weeks after fumigation) when compared to the 1st week of sampling. The increased POD activity indicates the continuing ability to reduce H2O2 (Liu et al.,

2009). At one week after onset of fumigation a strong SO2 concentration dependent increase in

POD activity was displayed (Figure 5.16 a), namely 0.7%, 100.1% and 359.26% increase in 50WW, 100WW and 200WW treatments, respectively, relative to the control (CFWW). After 2 weeks’ fumigation with SO2 a 16.38% reduction in POD activity occurred in the 100WW

treatment, but increased in the 50WW and 200WW treatments relative to the control (CFWW), namely 7.03% and 24.63%, respectively. After 3 week’s fumigation with SO2 the 50WW

treatments showed a reduction of 6.58% in POD activity, while increases were evident in 100WW and 200WW treatments namely 33% and 25.88%, respectively.

With the DS treatment (Figure 5.16 b), similar results were obtained as in the WW treatment. At T1, a 25% reduction occurred in the 50DS treatment, while increases in both 100DS and 200DS

occurred relative to the control (CFDS), namely 10.36% and 66.53%, respectively. At T2 a

decline in POD activity occurred in the 50DS and 100DS treatments relative to the control (CFDS), namely 20.93% and 29.65%. A slight increase in the 200DS of 2.24% was however

136 Time (weeks) 3 6 9 H2 O2 c o n te n t (m M ) 0 5 10 15 20 CFDS 50DS 100DS 200DS Time (weeks) 3 6 9 H2 O2 c o n te n t ( m M ) 0 5 10 15 20 CFWW 50WW 100WW 200WW

evident. After 3 weeks’ fumigation with SO2 a 6.97% decrease occurred in the 50DS treatment,

while increases of 15.64% and 38.79% occurred in 100DS and 200DS treatments relative to the control (CFDS), respectively.

Figure 5.15: The average H2O2 content in leaf samples of well watered (WW) (a) and drought

stress (DS) (b) Zea mays plants at 3, 6 and 9 weeks after onset of fumigation with SO2 (0, 50, 100,

200 ppb). Each value represents a mean of 4 plants (±SE). a

137 Time (weeks) 3 6 9 P e rox ida s e a c ti v it y (nm ol t e tr a gua ia c ol c onc e nt ra ti on. m in -1 .m g pr ot e in -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 CFDS 50DS 100DS 200DS Time (weeks) 3 6 9 P e rox ida s e a c ti v it y (nm ol t e tr a gua ia c ol c onc e nt ra ti on. m in -1 .m g pr ot e in -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 CFWW 50WW 100WW 200WW

Figure 5.16: Changes in POD activity of WW (a) and DS (b) Zea mays test plants exposed to 50, 100 and 200 ppb SO2 over a period of 9 weeks.

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5.8. Growth and yield

Garcia-Huidobro et al. (2001) classified cereal crops including maize as sensitive to SO2 as air

pollutant. Adverse effects, including significant reductions in plant height, biomass and yield have been documented by Deepak & Agrawal (1999). They found that exposure to 60 ppb SO2

for 8 hours per day for 100 days resulted in up to 20% reductions in plant yield, as well as declines in growth, biomass, foliar starch and protein content in soybean cultivars. In our study the effect of SO2 on test plants were ultimately expressed as a loss in plant height and yield (cob

mass). To evaluate the detrimental effect of SO2 on the growth of maize plants, WW and DS test

plants were measured from the base to the flag leaf just before harvesting at 12 weeks’ fumigation with SO2 and compared with the control treatments, CFWW and CFDS, respectively

(Figure 5.17 a and b). Literature states that the impact of SO2 on plant functioning appears to be

paradoxal, since in addition to its toxicity, the absorbed SO2 may also be used as a nutrient.

Foliarly absorbed SO2 may directly or after its oxidation to form SO42-, enter the S-assimilatory

pathway and be reduced to sulphide, incorporated to cysteine and subsequently organic Sulphur compounds and utilized as Sulphur nutrient (De Kok & Tausz, 2001; Tausz et al., 2003; Yang et

al., 2006). Plants could thus benefit from SO2 exposure since it may contribute to the plant’s

nutrition, thus exposure may not always result in drastic biomass or yield reduction, especially when the SO42- supply to the root is limited (Yang, et al., 2006). In our study, WW treated test

plants showed a very small reduction in shoot growth (0.2%) in the 50WW treatment followed by further reductions of 4.61% and 4.49% in the 100WW and 200WW treatments, respectively (Figure 5.17 a).

In the case where drought was additionally introduced as a co-stressor, only a slight reduction in shoot length occurred in the 50DS treatment, namely of 0.55%. This was followed by greater decreases in both 100DS and 200DS treatments, namely 10.89% and 12.71% compared to the CFDS control plant (Figure 5.17 b). Though no significant reductions in the length of test plants were detected when the SO2 concentration-effect was evaluated in well watered and drought

stressed plants, the single effect of drought stress alone indicated a highly significant reduction (p ≤ 0.01) of 21.85% when the shoot length of CFDS plants were compared to those of CFWW plants.

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At the time of harvest (i.e. 12 weeks after commencement of fumigation) the cob mass was determined to evaluate the cumulative effect of SO2 on Zea mays plants after an entire growth

season. The pictures of WW (Figure 5.18 a) and DS (Figure 5.18 b) clearly indicate a SO2

induced reduction in cob size even at the lowest SO2 concentration used. When compared to the

CFWW controls all test plants exposed to elevated SO2 concentrations displayed decreases in

yield, namely 4.24% (50WW), a significant reduction of 12.92% (p ≤ 0.03) in 100WW test plants and a highly significant decline (9.64%; p ≤ 0.01) in the 200WW treatment. When combined with DS treatment, SO2-induced decreases in yield were evident, though not

statistically significant, namely 4.49%, 0.34% and 15.43% for 50DS, 100DS and 200DS treatments compared to the control (CFDS).

140 SO2 concentration (ppb) 0 50 100 200 G row th (m ) 1.6 1.8 2.0 2.2 2.4 SO2 concentration (ppb) a 200 50 0 100 SO₂ fumigation level (ppb) 0 50 100 200 G ro w th ( m ) 1.6 1.8 2.0 2.2 2.4 SO₂ fumigation level (ppb) b -12.71 % 200 50 0 100

Figure 5.17 : The growth from the base to flag leaf for WW (a) and DS (b) Zea mays plants fumigated with different SO2 levels (50, 100 and 200 ppb) are shown after an entire growth season

of 12 weeks. Each value represents the mean ( SE) of 4 measurements.

141 SO₂ concentration (ppb) 0 50 100 200 M ass (g ) 100 120 140 160 180 200 -15.4 % SO2 concentration (ppb) (b) SO₂ concentration (ppb) 0 50 100 200 M a s s ( g ) 100 120 140 160 180 200 SO2 concentration (ppb) -9.6% (a)

Figure 5.18: The cob mass was determined for WW (a) and DS (b) treatments measured at a 12.5% moisture regime after harvest (12 weeks) show the correlation in the reduction of cob size to the increase in SO2 concentration. Each value represents the mean (±SE) of 4 measurements.