1
Supporting Information Tables
2
3
4
5
6
Table Supplementary S1. Mitochondrial respiration in the light at 25ºC (Rl), intercellular CO2photocompensation point (Ci*), slope of the linear regression between the theorical electron
transport rate and the same measured with the fluorometer (a*), product of the absorptance and
the partition of photons between photosystem (αβ). Mean values (n=4-5) and standard error in
parentheses, letters correspond to homogeneous group by Duncan´s test.
WW
MWS
SWS
WW
End WS 31/02MWS
04/04
SWS
WW
RWS, new leaf
MWS
15/04-18/04
SWS
Before WS
09/02-12/02
7
Rl 25ºC
1.9 (0.2) f
1.7 (0.2) ef
1.8 (0.2) ef
1.4 (0.1) de
1.1 (0.1) bcd
0.6 (0.1) a
1.4 (0.1) de
1.3 (0.1) cd
1.1 (0.1) cd
E. dumosa
Ci* 25ºC
a
39.9 (1.1) abcd 1.15 (0.03) bc
39.5 (1.1) abcd 1.13 (0.03) bc
39.4 (1.3) abcd 1.13 (0.03) bc
36.0 (2.0) a
1.24 (0.06) c
38.9 (2.0) abcd 1.12 (0.06) abc
42.9 (2.2) abcd 0.99 (0.06) a
37.1 (1.8) abcd 1.17 (0.03) c
37.2 (1.8) abcd 1.12 (0.03) c
40.2 (2.1) abcd 1.18 (0.03) c
αβ
Rl 25ºC
0.43 (0.01) ab
2.1 (0.2) f
0.42 (0.01) ab 1.7 (0.2) ef
0.43 (0.02) ab
1.9 (0.2) f
0.43 (0.02) ab 1.2 (0.1) cd
0.41 (0.02) ab 0.8 (0.1) abc
0.40 (0.02) ab 0.6 (0.1) ab
0.43 (0.01) ab
1.3 (0.1) d
0.44 (0.01) ab 1.0 (0.1) cd
0.44 (0.02) ab 1.0 (0.1) abcd
E. pauciflora
Ci* 25ºC
a
42.2 (1.3) c
1.20 (0.03) c
43.1 (1.1) d
1.25 (0.03) c
42.6 (1.1) b
1.23 (0.03) c
37.0 (2.2) ab 1.18 (0.06) bc
37.9 (2.0) ab 1.19 (0.06) bc
41.3 (2.0) abcd 1.02 (0.06) ab
37.3 (2.1) abd 1.20 (0.03) c
37.0 (1.8) abc 1.23 (0.03) c
37.8 (1.8) acd 1.20 (0.03) c
αβ
0.44 (0.01) ab
0.44 (0.01) ab
0.43 (0.02) ab
0.43 (0.02) ab
0.44 (0.02) ab
0.39 (0.02) a
0.44 (0.01) ab
0.45 (0.01) b
0.45 (0.02) b
8
9
10
11
12
13
14
15
16
17
18
Table Supplementary S2. Parameters derived of analysis of An/Cc curves on water stressed and
rewatered plants, the latter measured in younger leaves. Maximum rate of carboxylation (Vcmax),
maximum rate of electron transport (Jmax), the mesophyll conductance to CO2 (gm), the stomatal
conductance measured at ambient CO2 (equals to 380 μmol CO2 mol air -1), the concentration of
CO2 in the place of carboxylation (Cc), the threshold for Cc limited to RuBP limitation of net
photosynthesis (Cc of Ac-Aj) and the net light and CO2 saturated photosynthesis, estimated at
ambient CO2 equal to 2000 μmol CO2 mol air -1 (An CO2 sat). The temperature dependant
variables were corrected to 25 ºC as in Sharkey et al. (2007) unless the gm that followed
Warren& Epron (2006).
Vcmax (25ºC)
(μmol CO2 m-2 s -1)
Jmax (25ºC)
(μmol e- m-2 s -1)
gm (25ºC)
(mol CO2 m-2 s -1)
gsw cut
(mol H2O m-2 s -1)
Cc (25ºC)
(μmol CO2 mol air-1)
Cc of Ac-Aj
(μmol CO2 mol air-1)
An CO2 sat
(μmol CO2 m-2 s -1)
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
E.
dumosa
pauciflora
dumosa
pauciflora
dumosa
pauciflora
dumosa
pauciflora
dumosa
pauciflora
dumosa
pauciflora
dumosa
pauciflora
WW_Old
86.49 (6.0) abc
73.75 (6.0) ab
132.9 (8.3) cde
114.8 (8.3) abcd
0.323 (0.03) ef
0.367 (0.03) f
0.382 (0.03) d
0.379 (0.03) d
218.1 (9.27) cd
239.1 (9.27) de
289.6 (19.6) d
264.9 (19.6) cd
28.29 (1.88) cd
22.87 (1.88) abc
Water stress
MWS
SWS
85.37 (6.0) abc
70.41 (6.0) ab
83.63 (6.0) abc
70.00 (6.0) a
123.5 (8.3) bcde 104.5 (8.3) ab
107.3 (8.3) abc
89.0 (8.3) a
0.216 (0.03) cd
0.114 (0.03) ab
0.198 (0.03) bcd 0.077 (0.03) a
0.128 (0.03) ab
0.045 (0.03) a
0.137 (0.03) abc
0.058 (0.03) a
148.5 (9.27) b
101.6 (9.27) a
143.9 (9.27) b
101.6 (9.27) a
247.9 (19.6) bcd 220.2 (19.6) abc
204.7 (19.6) ab
187.1 (19.6) a
26.54 (1.88) cd
19.46 (1.88) a
21.69 (1.88) ab
18.19 (1.88) a
19
1
WW_New
97.10 (5.4) c
89.01 (5.4) bc
138.1 (7.0) de
141.4 (7.0) e
0.394 (0.03) f
0.519 (0.03) g
0.467 (0.04) de
0.536 (0.04) e
226.6 (7.80) cde
246.1 (7.80) e
Rewatering
MWS
93.56 (5.4) c
82.62 (5.4) abc
135.1 (7.0) de
120.9 (7.0) bcde
0.270 (0.03) de
0.239 (0.03) cde
0.181 (0.04) bc
0.382 (0.04) d
162.6 (7.80) b
205.9 (7.80) c
SWS
100.89 (5.4) c
84.74 (5.4) abc
143.3 (7.0) e
122.9 (7.0) bcde
0.235 (0.03) cde
0.162 (0.03) abc
0.177 (0.04) bc
0.242 (0.04) c
153.5 (7.80) b
169.2 (7.80) b
261.2 (16.2) bcd 239.4 (16.2) abcd 226.5 (16.2) abc
264.5 (16.2) cd 244.6 (16.2) abcd 238.4 (16.2) abcd
29.92 (1.48) d
26.46 (1.48) bcd
28.15 (1.48) bcd
24.84 (1.48) bcd
29.73 (1.48) d
26.09 (1.48) bcd
20
21
Supporting Information Figures
22
23
24
25
26
27
28
Fig. S1. Water consumption rate (ml/h) for plants of E. dumosa (solid line) and E.
29
30
31
32
33
Fig. S2. The relationship between the maximum capacity for carboxylation (Vcmax)
34
35
36
37
38
39
40
41
42
Fig. S3. Height growth rate (cm day-1) in E. dumosa (black colour) and E. pauciflora
43
44
45
46
Fig. S4. Relation between the maximum carboxylation rate (Vcmax) and the CO2
47
48
49
50
51
52
53
54
Fig. S5. Observed carbon isotope discrimination (Δo) as a function of the intrinsic
55
56
57
Fig. S6. The relationship between the photorespiration rate at 25ºC (F) and the leaf
pauciflora (dashed line) submitted to MWS (grey colour) or SWS (black clour). Water
consumption was calculated by the daily weight loss estimated as the water added to
sustain gsw, divided by time difference between pot weights and considering 11h of
night time without evapotranspiration. Symbols represent mean values of two
consecutive days (n=5) and regression line correspond to polynomial equation of third
order.
determined using the single point method and using the whole An/Cc curve in single
replicates for both Eucalyptus species under full irrigation, at the end of the moderate
and severe water stress, and 5-6 days after rewatering. The thick line is the straight-fit
while the dashed line represents the 1:1 relationship.
(grey colour) measured in three moments of the drought-recovery cycle. Dotted pattern
are well watered plants, striped pattern are plants submitted to Moderate waters tress
and solid pattern plants of the Severe water stress treatment. Data in parentheses
represent the day of height measured and the growth rate was calculated as the
difference in height between consecutive measured dates. The first period corresponds
to WW plants, i.e. before the WS period, and covers from 02-02 to 20-02. The last
period refers to rewatering plants. Means (n=5) and standard error, letters correspond to
homogeneous group by Duncan´s test.
concentration in the chloroplasts (Cc). The thick line is the linear regression of WS
plants and the dashed line is for WW plants. E. dumosa is represented in black and E.
pauciflora in grey. Symbols are as in Fig. 1.
water use efficiency (iWUE) in individual WW, MWS and SWS seedlings of E. dumosa
(left) and E. pauciflora (right) measured one week prior to rewatering. Δi is the
discrimination predicted assuming Ci = Cc (in the absence of respiratory and
photorespiratory fractionation). The difference between Δo and the sum of
discriminations predicted for the mesophyll conductance (Δgm), photorespiration (Δf)
and mitochondrial respiration in light (Δe), represents the predicted discrimination for
stomatal conductance (Δo - Δf - Δe - Δgm).
concentration of amino acid serine (Ser) in E. dumosa (blue) and E. pauciflora (red).
Each point is the mean of 5 plants corresponding to three watered treatments (WW,
2
58
59
60
MWS and SWS measured on 6th of April, one week before rewatering) and after
rewatering in a new leaf (WW, RMWS and RSWS on 18th of April, 4 days after
rewatering).
61
62
63
64
65
Fig. S7. The mitochondrial respiration in the light at 25ºC (Rl) and the leaf
66
67
68
69
Fig. S8. Relation of the intrinsic water use efficiency (iWUE) and the fraction of
70
Fig. S9. Relation of the intrinsic water use efficiency (iWUE) and the CO2
71
72
73
concentration in the chloroplasts (Cc) in both Eucalyptus species submitted to three
watered treatments and after rewatering of droughted plants. E. dumosa was fitted in
blue line and E. pauciflora in red line. Symbols are like in Fig. 3.
74
75
76
77
78
79
Fig. S10. The relationship between the mesophyll conductance to CO2 estimated by
80
81
82
83
Fig. S11. Comparative of the estimation of the mesophyll conductance to CO2
84
85
86
87
Fig. S12. Difference between the CO2 concentration in the chloroplasts without
88
89
90
91
92
93
94
Figure S13.
concentration of sucrose (Suc). Each point is the mean of 5 plants corresponding to
three watered treatments (WW, MWS and SWS measured on 6th of April, one week
before rewatering) and after rewatering in a new leaf (WW, RMWS and RSWS on 18 th
of April, 4 days after rewatering).
photorespiration to carboxylation rate (F/Vc) and in both Eucalyptus species submitted
to three watered treatments and after rewatering of droughted plants. E. dumosa was
fitted in blue line and E. pauciflora in red line. Symbols are like in Fig. 3.
three method and the stomatal conductance to H2O (gsw). The mesophyll conductance
was estimated by the J-variable method using the apparent photocompensation CO2
point determined followed the Laisk method (Ci*) or by using a constant
photocompensation point (Γ*). Third order polynomial regressions were fitted to
individual measurements of plants of the three treatments recorded the last week of WS.
measured by the J-variable method by using equation 1, and thus using the apparent
photocompensation CO2 point previously measured (Ci*), or by using a constant
photocompensation point (Γ*). Each point represents the mean of 4-5 replicates.
accounting for a resistance in the chloroplasts (Cc) to the refixation of CO2 and by
assuming the chloroplast resistance is half of the total mesophyll resistance (Cc*, γ =
0.5). Symbols are like in Fig. 3.
The mesophyll conductance calculated after accounting for the
chloroplast resistance to the CO2 refixation by the method of Gu & Sun (2013) (gm* eq.
(A22)), following equation A22, respect to our calculation following equation A14 (gm*
eq. (A15)) against the stomatal conductance to H2O (gsw). Each symbol represents
single measurement. Those plants with imaginary solution by equation A22 are
represented as green crosses equal to 0.75 for E. dumosa or purple blades equal to 0.65
for E. pauciflora.
3
95
96
Supporting Information
Daily evapotranspiration relative to
WW plants (rel. units)
1.2
E. dum MWS R² = 0.7925
E. pau MWS R² = 0.7158
E. dum SWS
E. pau SWS R² = 0.8814
1
0.8
0.6
0.4
0.2
0
97
98
Fig. S1.
4
R² = 0.9128
Vcmax _Single Point (μmol CO2 m-2 s-1)
160
140
120
100
y = 0.9651x + 3.2278
r² = 0.7691
80
60
40
20
0
0
20
40
60
80
100
Vcmax _curve fitting (μmol CO2
99
100
Fig. S2.
101
5
120
m-2 s-1)
140
160
1.2
a
Growth rate (cmday-1)
a
a
0.9
b
0.6
b
bcd
b b
bc
bcd
bcd
0.3
d
d
b
bc
bcd
bcd
cd
0.0
Before WS
(20/02/2009)
102
103
End WS
End RWS
(14/04/2009) (04/05/2009)
Fig. S3
6
y = -2.058+582.77
r2 = 0.73
y = -1.115+342.90
140
r2 = 0.68
-2
-1
Vcmax (µmol CO2 m s )
160
120
y = 0.363+50.65
r2 = 0.53
100
80
y = 0.182+61.03
r2 = 0.46
60
40
100
150
200
-1
Cc (µmol CO2 mol air )
104
105
Fig. S4
7
250
Eucalyptus dumosa
30
Eucalyptus pauciflora
i
g
e
o
f
m
o-g -f-e
m
(‰)
20
10
0
40
80
120
160
40
iWUE (µmol CO2 mol H2O-1)
106
107
80
120
iWUE (µmol CO2 mol H2O-1)
Fig. S5
8
160
8
y = 38.985x + 2.9891
R² = 0.7794
E. dumosa
7
F (μmol CO2 m-2 s-1)
E. pauciflora
6
5
y = 41.517x + 1.7567
R² = 0.8082
4
3
2
1
0
0
0.04
0.06
[Ser] (mmol
108
109
0.02
Fig. S6
9
0.08
m-2)
0.1
0.12
1.6
Rl (μmol CO2 m-2 s-1)
1.4
1.2
1
E. dumosa WS
0.8
E. pauc WS
0.6
E. dum RW
0.4
E. pauciflora RW
0.2
E. dumosa WW
E. pauciflora WW
0
4
5
5.5
[Suc] (mmol
110
111
4.5
Fig. S7.
10
6
m-2)
6.5
7
iWUE
-1
(µmol CO2 mol H2O )
160
y = 0.07x2+7.80x-76.83
140
r2 = 0.96
120
100
y = 0.06x2+6.25x-53.75
80
r2 = 0.95
60
40
20
10
20
30
40
F/V (%)
c
112
113
Fig. S8
11
50
60
70
114
150
y = -0.76x+211.86
r2 = 0.98
120
iWUE
-1
(µmol CO2 mol H2O )
180
90
y = -0.54x+167.72
60
r2 = 0.97
30
0
100
115
116
150
200
Cc (µmol CO2 air-1)
Fig. S9
12
250
0.6
E. dumosa
(06-13 of April)
0.5
R² = 0.8626
R² = 0.885
gm (mol CO2 m-2 s-1)
0.4
R² = 0.5936
R² = 0.8541
0.3
0.2
gm J- variable Ci*
gm J-variable Γ* cte
0.1
gm isotopic + ternary
gm curve fitting
0
0
0.1
0.2
0.3
0.4
0.5
0.6
gsw (mol H2O m-2 s-1)
0.7
E. pauciflora
(06-13 of April)
gm J- variable Ci*
0.6
gm J-variable Γ* cte
gm isotopic + ternary
gm (mol CO2 m-2 s-1)
0.5
gm curve fitting
R² = 0.9647
0.4
R² = 0.9801
R² = 0.836
0.3
R² = 0.9226
0.2
0.1
0
0
117
118
119
0.1
0.2
0.3
gsw (mol H2O m-2 s-1)
Fig. S10
13
0.4
0.5
0.6
120
121
122
gm (from Ci*)/gm (from constant Γ*)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
WW E. dumosa
WS E dumosa
0.4
RWS E. dumosa
WW E. pauciflora
0.2
WS E. pauciflora
RWS E. pauciflora
0
0
123
124
125
0.1
0.2
0.3
0.4
0.5
gsw (mol H2O m-2 s-1)
Fig. S11
14
0.6
0.7
0.8
80
-1
Cc-Cc* (µmol CO2 mol air )
126
y = 5.05+1.99x-1
r2 = 0.88
60
40
y = 8.50+0.94x-1
r2 = 0.80
20
0
0.0
0.4
gsw (mol H2O m-2 s-1)
127
128
0.2
Fig. S12
15
0.6
gm* eq. (A22)/gm* eq. (A15)
1.05
1
0.95
0.9
0.85
0.8
E. dumosa
0.75
E. pauciflora
0.7
E. dumosa failed
0.65
E. pauciflora failed
0.6
0
0.4
0.6
gsw (mol H2O m-2 s-1)
129
130
0.2
Fig. S13
131
16
0.8
1