SE-CCC
3.3 Materials and methods
uncover new aspects of the control mechanisms involved in C4 photosynthesis (Kubien et al., 2003; von Caemmerer et al., 2005), however little is known about the unique regulatory interactions that determine assimilatory flux in C4 plants, such as sugarcane.
However, many of the controls elucidated for C3 systems also operate in C4 plants (Sheen, 2001); for example carbamylation of Rubisco by Rubisco activase has been shown to be essential for photosynthesis in the C4 dicot, Flaveria bidentis (von Caemmerer et al., 2005).
The existence of a sugar-dependent relationship between source and sink tissues in sugarcane could represent a potentially fundamental limiting factor for sucrose accumulation in the stalk and consequently play a major role in overall sucrose accumulation and crop yield. In the current study, the relationship between photosynthetic source tissue and sink material was examined through manipulation of sink demand and total sink-strength in field-grown sugarcane. To artificially increase sink-strength by manipulating the sink:source ratio, all leaves, except for the third fully expanded leaf, were enclosed in 90% shade cloth. In this way leaves that served as source were converted to sinks, producing an overall increase in plant sink-size. The effects on gas exchange characteristics and PSII efficiency were investigated and changes in photosynthesis were explained on the basis of leaf sugar levels and variations in sugar partitioning based on the uptake of a 14CO2 label.
3.3.2 Manipulation of sink capacity
To increase plant sink:source ratios, all leaves except the third fully-expanded leaf (leaf 6) (Fig. 3.1) were covered in a black sleeve constructed from 90% shade cloth. Leaf 6 was chosen as the most suitable intermediate between mature and maturing stalk tissue. Shade cloth was selected so as not to totally impede gas flow to the plant or to elicit changes in photomorphogenesis. Treated plants were selected based on similar height and stalk width, and were separated by at least two unshaded plants to negate potential shading effects of the shade cloth on neighboring plants. Treatments were applied between 1 and 14 d prior to the measurements and sampling, effectively rendering leaf 6 the sole light receiving source for photosynthetic carbon assimilation for this variable period prior to analysis. Light conditions were measured regularly throughout the experiment using a LI-6400 portable photosystem unit (LI-COR Biosciences Inc., Nebraska, USA) to ensure that leaf 6 from control and partially shaded plants received similar levels of light exposure. These plants that had been shaded for variable periods of time were compared to “control” plants that were not shaded.
3.3.3 Sugar determination
Following shading treatments for 1, 3, 6 and 14 d, treated and unshaded plants (n=7) were concurrently harvested at 12h00. In this way, all plants were exposed to the same environmental factors immediately prior to harvest. To decrease the risk of potential sucrose hydrolysis, time taken between harvest and processing was kept to a minimum.
Stalks were kept intact and internodes 4, 6, 8, 10 and 12 were excised sequentially from top to bottom. The rind was removed and the underlying tissue, spanning the core to the periphery of the entire internode, was cut into small pieces (ca. 2 x 5 mm). Leaf material representing the meristematic leaf roll (designated leaf 0), first fully-expanded leaf (designated leaf 3) and third fully expanded leaf (designated leaf 6) was sliced thinly.
Tissues were then milled in an A11 Basic Analysis Mill (IKA, Staufen, Germany) and frozen in liquid nitrogen (–196o C). The samples were stored in 50 ml centrifuge tubes at –80o C. Prior to analysis, leaf and culm tissues were incubated overnight in 20 volumes of sugar extraction buffer (30 mM HEPES [pH 7. 8], 6 mM MgCl2 and ethanol 70% [v/v]) at 70o C. Extracts were centrifuged for 10 min at 23 200 g and sucrose, fructose and
Fig. 3.1. The upper section of a sugarcane stalk showing leaves 1 to 9 and internodes 1 to 9.
Leaves are consecutively numbered and attached to the bottom of their corresponding internode. The third fully-expanded leaf (leaf 6) is indicated in brackets. Adapted from van Dillewijn (1952).
glucose concentrations in the supernatant measured by means of a spectrophotometric enzymatic coupling assay modified from Jones et al. (1977). The phosphorylation of glucose by hexokinase/glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (Roche Mannheim, Germany) and fructose by phosphoglucose isomerase (EC 5.3.1.9) (Roche) was quantified by following the reduction of NADP+ to NADPH at 340 nm (A340).
Absorbance measurements and data analysis were conducted on a Synergy HT Multi- Detection Microplate Reader (Biotek Instrument, Inc., Winooki, VT, USA) using KC4 software (Biotek Instrument, Inc), respectively.
3.3.4 Gas exchange and fluorescence determinations
A LI-6400 portable photosystem unit was used to measure photosynthetic assimilation (A), transpiration rate (E), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and leaf temperature of leaf 6 between 9h00 and 12h00. Comparative measurements were performed on the day of harvest for plants that were unshaded or had previously been partially shaded for 1 to 14 d (n=4). Partially shaded plants were further measured over a period of 2, 4 and 8 d (n=4). The latter experiment was repeated at least once to confirm results. The response of A to Ci (A:Ci) was measured by varying the external CO2 concentration from 0 to 1 000 mol mol-1 under a constant photosynthetically active radiation (PAR) of 2 000 mol m-2 s-1. An equation A=a
(
1−e(−bCi))
−c was fitted to the A:Ci data using least squares. The portion of the curve where the slope approaches zero due to limitation in the supply of substrate (ribulose-1,5-bisphosphate), which is equivalent to the CO2- and light-saturated photosynthetic rate(Jmax) (Lawlor, 1987), was calculated from this equation (a, Jmax; b, curvature parameter; c, dark respiration (Rd)).Linear regression was performed on the data between a Ci of 0 and 200 mol mol-1 to determine the efficiency of carboxylation (CE; Lawlor, 1987). The assimilation rate in the absence of stomatal limitations (Aa) was as calculated as A interpolated from the response curve at Ci = 380 mol mol-1.
Chlorophyll fluorescence was determined concurrently with A:Ci gas exchange measurements using the LI-6400-40 Leaf Chamber Fluorometer (LI-COR Biosciences Inc.). A saturating pulse of red light (0.8 s, 6 000 µmol m-2 s-1) was applied to determine the maximal fluorescence yield (Fm’) at varying external CO2 concentrations (0 - 1 000 mol mol-1). The electron transport rate (ETR), defined as the actual flux of photons
driving photosystem II (PSII) was calculated from fI leaf Fm
Fs ETR= Fm−
α
'
' , where Fs is
“steady-state” fluorescence (at 2 000 mol m-2 s-1), Fm’ is the maximal fluorescence during a saturating light flash, f is the fraction of absorbed quanta used by PSII, typically assumed to be 0.4 for C4 plant species (Edwards & Baker, 1993), I is incident photon flux density and leaf is leaf absorptance (0.85, LI-COR manual). The component fluorescence variables were derived as described by Maxwell & Johnson (2000).
3.3.5 14CO2 labelling
The influence of shading treatments on carbon allocation was measured by supplying leaf 6 of unshaded and partially shaded plants (4 d and 10 d) (n=3) with 14CO2 using a protocol modified from Hartt et al. (1963). A portion of leaf (5 x 20 cm) weighing approximately 5 g was sealed in an air-inflated polythene bag containing 50 l NaH14CO3
(specific activity, 55 mCi mmol-1, ICN Radiochemicals, Irvine, CA, USA) to which 1 ml 10% (v/v) lactic acid was added to release 14CO2. The sealed bags were then gently palpated to ensure equilibration of released 14CO2 and even distribution of uptake over the leaf surface. After 1 h, bags were removed and a leaf disc (ca. 10 mg) of the labelled region of leaf 6 was excised and stored in liquid nitrogen. The plants were harvested 24 h after 14CO2 supply and tissue samples milled in an A11 Basic Analysis Mill (IKA) and incubated overnight in twenty volumes of sugar extraction buffer (30 mM HEPES [pH 7. 8], 6 mM MgCl2 and ethanol 70% [v/v]) at 70oC. The radioactivity in the 70% (v/v) alcohol-soluble component was measured with a Tri-Carb Liquid Scintillation Analyzer (Packard, Massachusetts, USA) using Ultima GoldTM XR (Packard, Milford, MA, USA).
Labelled sugars in the 70% alcohol-soluble component were spotted onto 10 x 20 cm silica gel plates (Merck, Darmstadt, Germany) using a semi-automatic Thin Layer Chromatography (TLC) sample applicator (Linomat 5, CAMAG, Muttenz, Switzerland) and fractionated using a mobile phase consisting of 50% ethyl acetate (v/v), 25% acetic acid (v/v), and 25% filter-sterilized water for 3 h. Silica plates were dried at 70oC for 10 min, sealed in polyethylene film and exposed to high-resolution phosphor screens (Packard). After 24 h exposure, the images on the phosphor screens were captured and analysed by means of a Cyclone Storage Phosphor Screen imaging system (Packard) using Optiquant Ver. 03.10 (Packard).
3.3.6 Statistical analysis
Results were subjected to analysis of variants (ANOVA) or Student’s t tests to determine the significance of difference between responses to treatments. When ANOVA was performed, Tukey's honest significant difference (HSD) post-hoc tests were conducted to determine the differences between the individual treatments (SPSS Ver. 11.5, SPSS Inc., Chicago, IL, USA). SPSS was also used to calculate the Pearson’s correlation coefficients for correlation analyses.