Environmental Engineering Reference
In-Depth Information
using a modulated pulse (<0.05 µmol photons m
-2
s
-1
for 1.8s). Maximal fluorescence (Fm)
was measured after applying a saturating actinic light pulse of 8000µmol photons m
-2
s
-1
for
0.7s. Subsequently, actinic light intensity was altered to 1000µmol photons m
-2
s
-1
in leaf
cuvette and maintained for about 30min. The temperature, carbon dioxide (CO
2
)
concentration and relative humidity in the leaf cuvette depended on ambient conditions.
Stomatal conductance (Gs) and intercellular CO
2
concentration (Ci) were recorded
simultaneously with Pn. In addition, steady-state fluorescence yield (Fs) was also recorded. A
saturating actinic light pulse of 8000µmol photons m
-2
s
-1
for 0.7s was then used to produce
maximum fluorescence yield (Fm') by temporarily inhibiting photosystem II (PSII)
photochemistry. The actual photochemical efficiency of PSII (ΦPSII) proportion of open PSII
(qP) and nonphotochemical quenching (NPQ) were calculated by fluorescence parameters
determined in both light- and dark-adapted states [13].
For the measurement of carboxylation efficiency (CE), photon flux density and
temperature were set at 1000µmol photons m
-2
s
-1
and 25
℃
in the leaf cuvette, respectively.
Pn was measured under CO
2
concentrations in a sequence of 600, 500, 400, 300, 200, 150,
100, and 50µmol mol
-1
. The leaves were kept under each level of CO
2
concentration for 5min
to let leaves reach steady photosynthesis, the Pn and Ci were then recorded. The correlation
curve of Pn related to Ci was established. CE was calculated from the linear portion of the Pn-
Ci curve according to Chen et al. [14].
2.5. Water Potential (ψ
w
) and Relative Water Content (RWC)
Leaf samples were placed in a sample cup, and the cup bottom should be completely
covered. Then, water potential was measured by chilled-mirror dewpoint technique with a
WP4-T Dewpoint PotentiaMeter (USA). The RWC was measured according to Flexas et al.
[15]. FW means the fresh weight of the harvested leaves, while the SFW stands for the weight
of the leaves which were harvested and soaked in deionized water for 24h at 4
℃
Finally, the
leaves were dried completely in an oven and weighed (called dry weight, DW). RWC was
calculated as: RWC= (FW-DW) / (SFW-DW).
2.6. Proline Content
Leave samples (0.2g) were homogenized through grinding with 3ml sulphosalicylic acid
[5%(w/v)] and incubated at 100
℃
for 10min. After centrifugation at 13000g for 10min, 2ml
glacial acetic acid and 3ml ninhydrin reagent were added to the supernatant and incubated at
100
℃
for 40min. For cooling it to room temperature, 5ml toluene was added to the mixture
and the absorbance at 520nm of the toluene phase was recorded. The standard curve was
plotted according to the proline solution of known concentration.
