Full Thesis is on Research Gate DOI:10.13140/RG.2.2.14708.63368
CHAPTER 2: MAETRIALS and METHODS
2.0 Materials and Methods
The basic methods used throughout this thesis are described below. The experimental design (replication, randomization variation in conditions and statistical analysis) of individual experiments is given in each chapter.
2.1 Plant materials and growth conditions
Seeds of Oryza sativa L spp indica variety IR64 were grown in either a high irradiance (HL, 750 µmol m-2 s-1) or a low irradiance (LL, 250 µmol m-2 s-1) environment in a growth cabinet (Conviron Ltd, Winnipeg Manitoba, Canada, model BDR 16) with a 12 h photoperiod. Irradiance was provided by 48 fluorescent bulbs (Master TL5 HO SUPER 80 39W/840 SLV) and 12 tungsten incandescent bulbs (Standard 60W A55 FR 2CT). The relative humidity (RH) and temperature were kept constant at 55% and 28˚C respectively with ambient CO2 (~ 400 ppm). The rice variety IR64 was chosen as part of the work in this thesis was based on that of Narawatthana (2013).
Rice seeds were sown in plastic dishes lined with water-soaked paper towel and sealed with perforated parafilm. After 5 days the seedlings were transferred to a hydroponics system (Figure 2.1). Each seedling was inserted into an eppendorf tube (where the bottom had been removed) to contain and support the rice seedling. The eppendorf tubes were then suspended around the edges of 3 L hydroponic tanks and the centre of which was covered with black plastic sheeting. The hydroponic tanks were filled with nutrient solution following the recipe of Narawatthana (2013) (Table 2.1). The nutrient solution was made up using deionized water and was adjusted to pH 5.9 using 10% KOH. The nutrient solution was topped up every one or two days and completely changed every seven days to maintain the optimum pH environment in the system.
Figure 2.1: Image of the rice hydroponic system (A) Eppendorf tubes with the base removed and (B) rice seedlings suspended around the edge of the hydroponic container.
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Table 2.1: Composition of the nutrient solution used in the hydroponic system (Narawatthana, 2013). Solutions 1, 2 and 3 were prepared separately before being mixed and diluted with water to the final concentration to prevent precipitation. Each mineral solution was prepared from stock solutions kept at room temperature and each preparation for hydroponic solution usage was made fresh and never kept for more than one week. BDH (BDH Merck Ltd.); Fisher (Fisher Scientific UK Ltd.); Fisons (Fisons Plc.); Fluka (Fluka-Sigma-Aldrich Co. LLC).
Solution | No. | Source | Chemical Formula | Main Elements | Molecular Mass | Final concentration (mM) |
1 | 1 | BDH | NH4NO3 | N | 80.04 | 1.4 |
2 | Fisher | NaH2PO4•2H2O | P | 156.01 | 0.6 | 0.6 |
3 | Fluka | K2SO4 | K | 174.27 | 0.5 | 0.5 |
4 | Fisher | MgSO4•7H2O | Mg, S | 246.98 | 0.8 | 0.8 |
5 | BDH | MnCl24H2O | Mn | 197.9 | 0.009 | 0.009 |
6 | Fisons | (NH4)6Mo7O24•4H2O | Mo, N | 1235.86 | 0.0001 | 0.0001 |
7 | Fisons | H3BO3 | B | 61.83 | 0.09366 | 0.09366 |
8 | Fluka | CuSO4•5H2O | Cu, S | 249.7 | 0.003675 | 0.003675 |
9 | Fluka | ZnSO4•7H2O | Zn, S | 287.56 | 0.00075 | 0.00075 |
2 | 10 | Fluka | CaCl2•2H2O | Ca | 147.02 | 0.322 |
3 | 11 | Fluka | Fe-EDTA | Fe | 367.1 | 0.02 |
2.2 Approximation of leaf developmental stage
Leaf number 5 (L5) was used as the model leaf for all the experiments conducted in this study. Since the shoot apical meristem and early plastochron stages (P1 to P4) of the rice leaf are concealed in the leaf sheaths, leaf number 3 (L3) was used as a proxy in order to make an approximation of L5 developmental stages (before P5) inside the sheaths. This approximation was based on the work and findings of Narawatthana (2013) detailed in Table 2.2.
Table 2.2:
The approximation of L5 plastochron stages based on the length of L3 blades under high and low light conditions.
High Light (750 µmol m-2 s-1) |
| Low Light (250 µmol m-2 s-1) | ||
Leaf 5 Stage | Leaf 3 Length (mm) |
| Leaf 5 Stage | Leaf 3 Length (mm) |
P1 | 4-20 |
| P1 | 10-30 |
P2 | 25-70 |
| P2 | 35-75 |
P3 | 75-110 |
| P3 | 80-160 |
P4 | 120-140 |
| P4 | 170-190 |
L5 was harvested for measurement and analysis when it had fully expanded (about 21 days after sowing (DAS) for HL and 30 DAS for LL) and the leaf collar (a joint between leaf sheath and leaf blade) was visible and bendable. Length was measured from the cut point of the leaf collar to the blade apex using a ruler. The leaf blade was scanned and area was determined from the scanned image using ImageJ version 1.48 software.
2.3 Biochemical and physiological analyses
2.3.1 Measurements of photosynthesis and stomatal conductance
Measurements of photosynthesis and stomatal conductance were made using a portable photosynthesis system (LI-6400XT from LI-COR Inc.). Irradiance was provided by an LED RGB (Red Green Blue) light source (LI-6400-02B, Li-Cor Inc.) that allows irradiance levels to be changed as required. In all experiments the irradiance was composed of 10% blue light (to promote stomatal opening) with the rest red light. The block temperature within the chamber was maintained at 28°C with ambient humidity (55% relative humidity) where the flow was set at 400 μmol s-1. CO2 was provided by a CO2 cartridge (Umarex®). A CO2 injection system allowed the CO2 to be varied by delivering a precisely controlled stream of pure CO2 into CO2-free air.
In all experiments and treatments, at least three replicates of leaf no. 5 were randomly selected and measurements were made on the middle portion of the leaf blade. All measurements were made 1 hour after the light came on in the growth chamber at 8.00 am. The clamped leaf was first left to acclimatize using the settings above at an irradiance of 2000 µmol m-2 s-1, for at least 15 min, or until the stomatal conductance readings became stable, before starting the experiments.
Two types of gas exchange measurements experiments were carried out. The first was a light response curve (assimilation versus PAR) (chapter 3) in which the CO2 was set to 1000 ppm (saturating CO2) while irradiance (PAR, in µmol m-2 s-1) was progressively decreased from 2000 to 0 irradiance as follow: 2000; 1750; 1500; 1250; 1000; 750; 500; 350; 200; 150; 100; 50; 25; 0. Once the leaf had reached steady state photosynthesis the experiment was started. The minimum wait time (the time after each irradiance level change that the system will wait, before checking stability to see if it can log the rate of photosynthesis and stomatal conductance) was set at 180s.
The second experiment was an assimilation versus intercellular CO2 response curve (A-Ci curve) (chapters 3 and 4). Irradiance was set at 2000 µmol m-2 s-1 while the CO2 concentrations (Ca) were increased from 50 to 1600 ppm in a series as follows: 50; 100; 150; 200; 250; 300; 400; 500; 600; 800; 1000; 1300; and 1600. Again, the minimum wait time after each CO2 concentration change that the system will wait before logging photosynthesis and stomatal conductance was set at 180s.
Once measurements were complete for either the light response or A-Ci curves, data were analysed to derive a number of different parameters. Curves were fitted to the data for each replicate leaf using software from Landflux.org. The Photosynthetic Light Response Curve Fitting software (version 1.0) was used to analyse the light response curves where parameter estimates were based on fitting a non-rectangular hyperbola following Marshall and Biscoe (1980) and Thornley and Johnson (1990). The A/Ci Curve Fitting software v10.0 (that provides parameter estimates based on both infinite and optimized mesophyll conductance, the later following Ethier and Livingston (2004)), was used to analyse the A-Ci curves.
The following parametres were measured or derived from the light saturation or A-Ci response curves:
1. Apparent quantum yield of photosynthesis (moles of CO2 fixed per mole of quanta (photons) absorbed.
2. Maximum net assimilation rate (Amax)
3. Light compensation point (LCP) (the point at which the rate of Rubisco carboxylation equals the rate of respiration + photorespiratory CO2 release).
4. Jmax, maximum light- and CO2-saturated electron transport rate
5. Vcmax maximum Rubisco carboxylation rate
6. gs: stomatal conductance
7. Ci: intercellular CO2
8. Rd: dark respiration
9. iWUE: Water Use Efficiency (Rate of photosynthesis/stomatal conductance)
10. Relative stomatal limitation (ls in %) was calculated based on Farquhar and Sharkey (1982)
ls =1-A400/ACi400 x 100
Where A400 is the assimilation at Ca= 400ppm while ACi400 is the assimilation when Ci = 400ppm and these values were obtained from the A-Ci curve.
2.3.2 Pigment quantification
Chlorophyll and total carotenoids were extracted and quantified using a Lambda 40 UV-VIS spectrometer (PerkinElmer Instruments Inc.) A leaf disc (disk area = 12.57 mm2) was taken from the middle region of each leaf (L5) using a using a 4.0 mm revolving punch plier supported with a paper towel underneath to prevent leaf breakage. One disk per leaf was used for each extraction. For each treatment five leaves were used (n = 5). Each leaf disk was placed in an Eppendorf tube and pigments extracted by incubating in 333 μl of 80% ethanol for 15 min at 70 °C. This treatment was repeated three times. The 3 ethanol aliquots containing extracted pigments were then combined (total = 1000 μl) in a 10 x 10 x 45 mm disposable cuvette (VWR®, Germany). In order to quantify the pigments, absorption was measured at the wavelengths of 665, 649 and 470nm. Quantification of individual pigments was determined using the following equations from Lichtenthaler and Wellburn (1983). The amount of pigments in the leaf was calculated as μg m-2 of leaf.
ca = 13.95 A665 – 6.88A649
cb = 24.96A649 – 7.32A665
cx+c =(1000A470 – 2.05ca – 114.8cb)/245
* ca (chlorophyll-a); cb (chlorophyll-b);cx+c (total carotenoids xanthophylls + carotenes)
2.3.3 Carbon isotope analysis
Carbon isotope analysis of leaf tissue was carried out to evaluate water use efficiency in rice leaves. For each treatment at least five randomly selected leaf no. 5 were used. Leaves were cut into small pieces and dried in the oven at 37°C for at least a weak. Approximately 1 mg of dry leaf was obtained and loaded into a standard weight tin capsule (6x4mm from Sercon Ltd., Cheshire, UK). Sample and capsule were rolled together and turned into small balls then dropped into a furnace at 1000°C whilst in an oxygen atmosphere. Complete combustion was guaranteed by passing the combustion products through a bed of chromium oxide at 1000°C using a helium carrier gas. The combustion was completed and any remaining sulfur was removed by a 15 cm layer of copper oxide followed by a layer of silver wool. The products were then passed through a second furnace containing copper at 600 0C in which excess oxygen was absorbed and nitrogen oxide was reduced to elemental nitrogen. Water in the system was removed by a trap containing anhydrous magnesium perchlorate. The gas stream was then passed into a gas chromatograph (GC) in which the components of interest were separated (i.e. N2 from CO2) and passed into a mass spectrometer where the isotopic species (i.e. 12CO2 and 13CO2) were ionized and separated by mass using a magnetic field. The isotopes were detected separately and from the ratio the level of 13C was calculated using ANCA GSL 20-20 Mass Spectrometer (Sercon Ltd., Cheshire, UK).
The results were reported in delta values which are a difference between sample reading and internationally used standard (delta = 0) namely Pee Dee Belemnite (PDB from Sercon Ltd., Cheshire, UK) which comes from a cretaceous marine fossil, Belemnitella americana obtained from the Peedee Formation in South Carolina, USA. This material has a higher 13C/12C ratio than nearly all other natural carbon-based substances; for convenience it is assigned a delta13C (δ13C) value of zero, giving almost all other naturally-occurring samples negative delta values. δ13C was calculated as follow:
δ13C (‰) = (13C/12C of sample) – (13C/12C of standard) x 1000 13C/12C of standard
Equation 2.1
2.4 Imaging of leaf primordia using scanning electron microscopy
In order to explore the different developmental stages of rice leaves that were hidden inside the leaf sheaths (especially stage P1 - P4), five primordia were dissected using a single edge razor blade and microlance needles (size: 25G and 30G). Rice leaf primordia were placed in glass vials in a fixative solution containing 4% v/v paraformaldehyde (Thermo Fisher Scientific Inc.), 2.5% glutaraldehyde v/v (Agar Scientific Ltd), 0.5% v/v Tween-20 (Sigma-Aldrich Co.) and 0.2M Phosphate Buffered Saline (PBS) pH 7.4 (Sigma-Aldrich Co.) for 6 hours at room temperature. The glass vials were placed on an orbital shaker (set at 2 RPM). After this the fixative was replaced with two changes of PBS for 30 minutes each. Samples were then dehydrated in an acetone (Fisher Scientific UK Ltd.) series of 5%, 10%, 25%, 40%, 55%, 70%, 85%, 95% and 2 times 100% for 1 hour each at room temperature. Samples were dried using a Polaron critical point dryer (Agar Scientific, U.K). Liquid CO2 was let into the chamber and flushed several times to replace the acetone. Samples were left to soak in the liquid CO2 inside the chamber while maintaining the pressure at 800 psi and left for about 30 minutes. The chamber was heated up using circulating warm water and once the chamber’s pressure temperature exceeded 1072 psi and 31°C respectively, CO2 was immediately drained from the chamber as gas, leaving dry samples without causing the physical destruction. Samples were then mounted onto aluminum stubs using black carbon stickers (Agar Scientific, U.K) and sputter coated with gold (Edwards S150B gold sputter coater) in an argon gas atmosphere. Images were obtained using a SEM (Philips XL-20) at an accelerating voltage of 20 kV and processed with embedded XL-20 software.
2.5 Hand sectioning of leaves for measurements of leaf anatomy
Transverse sections of rice leaves were prepared in order to study aspects of leaf anatomy such as thickness, number of mesophyll cells and the size of the vascular system. A 1 cm in length of the middle portion of the leaf blade was fixed in Carnoy’s fixative (ethanol: acetic acid (7:1 v/v), Fisher Scientific UK Ltd.) overnight at 37°C. The solution was replaced with fresh Carnoy’s fixative and vacuum infiltrated using a water vacuum pump model MZ 2C NT+AK+EK (Vacuubrand, Germany) for 20 minutes.
Styrofoam blocks were used to sandwich the leaf sample to assist even leaf sectioning using single edge razor blade (Fig. 2.5). Single staining using Toluidine Blue-O (PBS: 0.1M and pH 7.0, Sigma-Aldrich; 0.1% w/v Toluidine dye salt, Gurr, BDH Ltd.) for 15 seconds was employed for thickness and vascular system observations. For mesophyll and bulliform cell observations, leaf sections were further counterstained with Neutral-red (1% w/v, Sigma Aldrich; 50 mL UHP water; one drop of acetic acid glacial, Fisher) for three minutes at room temperature. The leaf section was then rinsed with water and mounted on a glass slide in a transverse manner. Observations of leaf anatomy were made using a Leica epifluorescence-stereo microscope (LEICA-MZFLIII) and photomicrographs were taken with SPOT RT KE slider camera (Model 7.4) attached to HRD100-NIK 1.0x high-resolution digital camera adapter (Diagnostic Instruments Inc.). Images were captured and processed with SPOT Basic software Version 4.1.
Figure 2.2: Leaf sample is sandwiched in between a Styrofoam blocks that has been partially slit. The whole sandwich was always moistened with water while pressing it with index finger. Fine sections are made with sawing motions using razor blade at almost a right angle |
2.6 Stomatal and leaf epidermal characteristics
2.6.1 Preparation of leaf tissues
One cm from the middle portion of each fully expanded leaf blade (leaf no. 5) was cut and used in all studies. Samples were placed in 24 well microtiter plates containing Carnoy’s fixative (ethanol: acetic acid (7:1 v/v)) for 2-3 days at room temperature and then bleached (25% economic bleach, Ottimo Supplies Ltd.) for another 1 day. Samples were cleared using 2-3 drops of chloral hydrate solution (10gm. chloral hydrate, Riedel-de Haen®; 2.5 mL UHP water; and 1.0 gm glycerol, Sigma-Aldrich Co.) for one hour at room temperature. Leaf sections were then placed on glass slides and mounted in 1 to 2 drops of modified Hoyer’s Solution (10gm. chloral hydrate; 1.0 gm glycerol; and 3.0 gm 20% Arabic gum solution, Minerals-Water Ltd.). Leaves were covered with square cover slips (18mm). The epidermal surface of the leaves was observed under an Olympus BX51 light microscope using Nomarski differential interference contrast (DIC) mode.
Photomicrographs were taken with an Olympus camera DP71 (12.5 megapixels) using an Olympus a U-TVO.63XC camera adapter, both mounted on the microscope. Images were captured and processed using CELL-B Version 2.7. Physical measurements of a stomatal complex and its components (Figure 2.3) were made by tracing their outline on the images using a Bamboo® Pen Tablet from Wacom Co., Ltd. ImageJ Version 1.48 software was used to quantify the leaf stomatal and epidermal characteristics described below.
2.6.2 Measurement of stomatal and leaf epidermal characteristics
Stomatal Complex Area (SCA) width (SCW) and length (SCL): A stomatal complex in rice consists of four specialized epidermal cells, a pair of guard cells being sandwiched by two subsidiary cells as indicated by the blue dotted lines (Fig. 2.3). SCA was calculated based on measured values of stomatal complex width (SCW) and length (SCL) (Fig 2.3).
Guard Cell Width (GCW): Guard cell width was measured by drawing lines at the top and bottom of the guard cell and measuring the width (Figure 2.3).
Approximation of Stomatal Pore Area (SPA): Since aperture (pore) area is a likely structure that determines the amount of CO2 that gets in to the leaf, it is essential to measure pore area to quantify stomatal performance. However with the clearing technique used (in 2.6.1), the aperture is always closed. Therefore a method was developed to approximate stomatal pore area (SPA) using a simple trigonometric calculation. In a number of stomatal studies, it has been shown that an open stomatal pore (turgid guard cells) in grass species has an elongated hexagon (e-Hex) shape (Willmer and Fricker, 1996 and Taylor et al., 2012). This is also true for rice (Fig. 2.4).
Figure 2.3: A typical structure of a stomatal complex in rice leaves. Each unit comprises four cells where a pair of dumbbell shaped guard cells is sandwiched by two subsidiary cells (SCs). SCA (stomatal complex area is indicated by the blue dotted line); SCL (stomatal complex length); SCW (stomatal complex width) SPA (stomatal pore area) GCW (guard cell width). Scale bar = 5µm |
Figure 2.4: SEM micrograph of a partially open stomatal aperture (yellow area) within a stomatal complex on a rice leaf (bar = 10 µm). The red dotted line shows an approximately elongated-hexagon shaped pore area if the aperture were to open maximally. A stomatal complex comprises a pair of guard cells (GC) being sandwiched by two subsidiary cells (SC). (Yaapar, unpublished). |
Since two parameters namely aperture length and guard cells width were measurable, these were used to make an approximation of the maximum stomatal pore area opening in rice. The assumptions aree when the stomatal pore is fully open it would occupy the guard cell width and has a perfect e-Hex shape. The e-Hex formula was derived as follows (Thanks to mathematician Yusuf Harun for the advice):
The elongated hexagon (Fig. 2.5A) was split into two identical shapes namely trapezoid. Trapezoid area was determined using the standard formula:
Area trapezoid= [(½ height i.e. : ½ (½ aw)] x [sum of bases i.e. : (Bs+Al)]
Equation 2.2
![The elongated hexagon (Fig. 2.5A) was split into two identical shapes namely trapezoid. Trapezoid area was determined using the standard formula: Area trapezoid= [(½ height i.e. : ½ (½ aw)] x [sum of bases i.e. : (Bs+Al)] Equation 2.2](https://static.wixstatic.com/media/d3313d_c4094a44bd7d478797ee4c8eadf77f07~mv2.png/v1/fill/w_616,h_103,al_c,q_85,enc_auto/d3313d_c4094a44bd7d478797ee4c8eadf77f07~mv2.png)
Figure 2.5: Description of an elongated hexagon (e-Hex) which is supposed to be the shape of a fully open stomatal pore. Two parameters are measurable (A) for any given aperture namely aperture length (Al) and width (Aw). The formula for an e-Hex area can be derived by first splitting the shape into two producing two equal trapezoids (B). Each trapezoid in turn contains two right-angled triangle whose unknown adjacent lengths can be found using Tangent function (Tan θ = opposite length/adjacent length) in trigonometry. |
Each trapezoid has two right-angled triangles (Fig. 2.5 B). This means the two angles in each right-angled triangle must be 45° each because the right angle value is always 90° (Fig. 2.6).
Figure 2.6: Description of angles for a right-angled triangle in either side of a trapezoid. Since the total angle must be 180° for any triangle, the two unknown theta (θ) values must be 45° each. |
To determine the short base (Bs) length, only one of the right-angled triangles in the trapezoid had to be dealt with. From here, the unknown adjacent length of the right-angled triangle (Tadj) was calculated using a tangent function in trigonometry:
Tan θ = Opposite length/Adjacent length
Since θ = 45° then Tan 45° = 1
Therefore, ½ Aw = Tadj
Thus, Bs = Al – Aw
Then the new derivation of the trapezoid area is:
Areatrapezoid=½ (½ Aw) x ((Al-Aw) + Al)
= ¼ Aw x (2Al - Aw)
= (2AlAw - Aw2)/4
As this was just for one trapezoid (half e-Hex) it was multiplied by 2 to obtain the area of the whole elongated hexagon i.e. the maximum area of fully open stomatal pore is:
Stomatal Pore Areamax = 2AlAw – Aw2
2
Equation 2.3
Where Al is the stomatal aperture length while Aw is the aperture width
In general people define maximum the stomatal pore area as an ellipse with major axis equal to the pore length and minor axis equal to half the pore length. This assumption has been widely accepted and employed especially when using diffusive stomatal conductance model (Franks and Farquhar (2001), Eq. 1.1). Thus for rice which has dumbbell guard cells pair that lead to an e-Hex aperture shape, the ellipsoid definition commonly used for pore area might be incorrect. The approximation method proposed here can be validated by conducting a similar experiment as the one described by Dow et al. (2013) who deliberately subjected Arabidopsis to conditions that promoted maximal stomatal opening for maximum operational (measured) stomatal conductance. They then compared this value with maximum diffusive (theoretical) conductance obtained by using Eq. 1.1. However the confirmative work was not done in this study.
Stomatal density (SD): In order to calculate stomatal density the number of stomatal complexes was counted in between two interveinal gap areas (Figure 2.6) at 400 X magnification using an Olympus BX51 light microscope. This allowed values to be normalized to express the stomatal density per mm-2 of leaf.
Leaf epidermal characteristics:
Figure 2.6 shows the epidermal features and stomatal patterning of a typical interveinal gap (IG). The percentage of files (rows of cells) containing stomata (a cell file row containing at least one stoma) to the total no. of cell files was calculated. Cell file number was obtained by manually counting all cell files in between two parallel veins (Figure. 2.6). Average file cell width was then calculated by dividing the interveinal distance by the cell file number.
Figure 2.7: Epidermal features and stomatal patterning of a typical interveinal gap (IG). DIC image of an Interveinal Gap (IG) bound by a small vein and a large vein. Each IG comprises a number of cell files where some are pure long epidermal cells (E) and some contain stomata (SF, stomatal file). |
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