Chapter 6: Bottle Gourd [Lagenaria siceraria (Molina) Standl.] Response to Water Stress
6.3 Data collection
6.3.1 Soil moisture content monitoring
Volumetric soil water content in the upper 6-10 cm of soil on a percentage by volume were monitored using a soil moisture probe (Type ML2X attached to HH2 moisture meter, Delta devices, Cambridge, England). Ten pots for stressed and well-watered treatment were monitored for changes in soil water content.
6.3.2 Measurements of gas exchange parameters and chlorophyll fluorescence parameters
Leaf gas exchange and chlorophyll fluorescence were measured simultaneously using the LI-6400 XT Portable Photosynthesis System (Licor Bioscience, Inc. Lincoln, Nebraska, USA) fitted with an infrared gas analyzer attached to a leaf chamber fluorometer (LCF) (6400-40B, 2 cm2 leaf area, Licor Bioscience, Inc. Lincoln, Nebraska, USA). Leaf temperature was maintained at 25°C, 400 µmol mol-1 of external leaf CO2 concentration (Ca) and artificial saturating photosynthetic active radiation (PAR) was fixed at 1000 μmol m-2 s-1, using a red-blue LED light source built into the leaf chamber fluorimeter (LCF). CO2 was removed from external air using soda lime and mixed with pure liquid CO2 to control leaf air CO2 concentration in the sensor head. Flow rate was maintained at 500 µml and relative humidity maintained at 43%. The leaf-to-air vapor pressure deficit in the cuvette was maintained at 1.7 kPa to prevent stomatal closure due to the low air humidity effect. Measurements were taken between 08h00 to 11h00 h on the third half-fully expanded leaf from the tip of the plant by clamping the leaf inside the sensor head. Measurements were made from three independent plants for each genotype under non-stressed and water-stressed
−2 −1
photosynthetic rate/ net CO2 assimilation rate (A, µmol CO2m−2 s−1), transpiration rate (T, mmol H2O m−2 s−1), intercellular CO2 concentration (Ci, µmol. mol m-1) and ratio of intercellular and atmospheric CO2 (Ci/Ca) concentrations. Ratio of net CO2 assimilation rate and intercellular CO2
concentration (A/Ci) was calculated according to Dong et al. (2016). Two types of water-use efficiencies were calculated: Intrinsic water use efficiency (WUEi) calculated as the ratio of A and gs (Martin and Ruiz-Torres, 1992; Osmond et al., 1999) and instantaneous water-use efficiency (WUEins), calculated as the ratio of A and T (Anyia and Herzog, 2004; de Santana et al., 2015).
Chlorophyll fluorescence parameters such as minimal fluorescence (Fo’) of light-adapted leaves and maximum florescence (Fm’) were recorded by providing a saturating flash intensity of 1300 µmol m−2 s−1 and flash duration of 0.9 s using the LCF. The steady-state (Fs) fluorescence was also determined in light-adapted leaves under steady-state photosynthesis. The variable fluorescence was calculated in light adapted-leaves as Fv’ = Fm’- Fo’ whereas change in fluorescence was calculated as ΔF = Fm’ - Fs. Based on the measured chlorophyll fluorescence parameters some photochemical variables were calculated according to Maxwell and Johnson (2000) and Genty et al. (1989): The Fv’/Fm’, PSII maximum efficiency which estimates the maximum quantum efficiency of PSII under light conditions (equation 1), quantum yield of PSII (Ф PSII) (equation 2), where Fs is “steady-state” fluorescence, photochemical quenching (qP) (equation 3) and non-photochemical quenching (qN) (equation 4), which measures heat dissipation of absorbed light energy. Electron transport rate (ETR) which measures the actual flux of photons (µ molm−2 s−1) driving PSII relatively to photosystem I (PS I), were calculated according to equation 5, where 0.5 is the fraction of absorbed light energy that is used by PS II, 0.84 as the fractional light absorption (PPFD) by the leaf (Baker et al., 2007). The LI-6400 XT portable photosynthesis system automatically calculates these parameters. Relative measure of electron transport to oxygen molecules was calculated by the ratio of ETR/A (equation 6) (Flexas et al., 2002). The alternative electron sinks (AES) was estimated by the relationship between the effective quantum efficiency of PS II and the quantum efficiency of CO2 assimilation (A) (equation 7) (Ribeiro et al., 2004).
Fv’/Fm’ = (Fm’– Fo’)/Fm’ Equation 1
Ф PSII = (Fm’ – Fs)/Fm’ = ∆F/Fm’ Equation 2
qP = (Fm’– Fs)/(Fm’ – Fo’) Equation 3
qN = 1 – (Fm’ – Fo’)/(Fm-Fo) Equation 4
ETR = Ф PSII x PPFD x 0.5 x 0.84 Equation 5
Electron transport to O2 molecules = ETR/A Equation 6
AES = ∆F/ Ф CO2; whereФ CO2 = A/PPFD x 0.84 Equation 7 6.3.3 Determination of cucurbitacins
Cucurbitacins content was determined using a method described by Davidovich-Rikanati et al.
(2015) with some modifications. Leaves were destructively harvested, freeze-dried and ground to powder. Leaf samples (0.2 g) were homogenized with 10 ml 80% methanol (MeOH), vortexed for 30s using a homogenizer (ULTRA-TURRAX, IKA® T25 digital, Staufen, Germany). The mixture was manually shaken using IKA® (ks 130, Staufen, Germany) for 60 min, repeating the vortex every 10 min. Debris and particles were discarded by centrifugation (5000×g) for 5 min; 1ml of each sample was then filtered through Acrodisc® syringe filters with GHP membrane, 13 mm×
0.2 μm (PALL, USA), and transferred to vials for high pressure liquid pressure liquid chromatography mass spectra (HPLC-MS) analysis. Cucurbitacin analysis was performed using a Shimadzu LCMS-2020 HPLC equipped with a Shim-pack GIST 3um C18-HP 4, 6 x 150 mm column with 0.01% aqueous formic acid B: CAN 20-40% C at 20 min, 80% C at 40 min, 90% at 50.5 min, held for 4.5 min, 20% C at 55.5 min held for 4.5 min (run time 60 min, aqc time 55 min).
Flow rate was maintained at 0.5 ml/min at 40°C. Cucurbitacin identification was done by comparison of retention time and exact mass spectrum of purchased Cucurbitacins E and I standards (Sigma-Aldrich) and detected at 210 nm, with a resolution of 4nm. Quantification of
cucurbitacin E and I in samples was done using an external calibration curve on a dry weight basis, identity and purity based on retention time, peak area, UV spectra and chromatographs with authentic standards. Cucurbitacin analysis was performed at the Mass Spectroscopy laboratory, School of Chemistry, University of KwaZulu-Natal, South Africa.
6.4 Data analysis
Data on cucurbitacin content, leaf gas exchange and chlorophyll fluorescence parameters were subjected to analysis of variance using GenStat version 14th Edition (Payne et al., 2011). Mean values recorded among landraces were compared using the least significant difference (LSD) test procedure at 5% level of significance. Correlation analysis was performed to describe the pattern of association between cucurbitacin content with leaf gas exchange and chlorophyll fluorescence parameters using SPSS 16.0 (SPSS, 2007). Significance tests of the correlation coefficients were determined using the Student’s t test (Snedecor and Cochran, 1989). Principle component analysis (PCA) based on the correlation matrix was performed using SPSS 16.0. The bi-plot analysis was then used to describe and group bottle gourd genotypes for their level of drought tolerance according to Singh and Raja-Reddy (2011).