• The relationship in sugarcane (Saccharum spp.) between photosynthetic source tissue and sink material was examined through manipulation of the sink:source ratio of field-grown Saccharum spp. hybrid cv. N19 (N19).
• To enhance sink strength, all leaves, except for the third fully expanded leaf, were enclosed in 90% shade cloth for varying periods of time. Variations in sucrose, glucose and fructose concentrations were measured and the effects of shading on the leaf gas exchange and fluorescence characteristics recorded. Changes in carbon partitioning caused by shading were examined based on the uptake and translocation of fixed 14CO2.
• Following a decline in sucrose concentrations in young internodal tissue and shaded leaves, significant increases in the CO2-saturated photosynthetic rate (Jmax), carboxylation efficiency (CE) and electron transport rate were observed in unshaded leaves after 8 d of shading treatment.
• It was concluded that up-regulation of source-leaf photosynthetic capacity is correlated with a decrease in assimilate availability to acropetal culm sink tissue. Furthermore, a significant relationship was revealed between source hexose concentration and photosynthetic activity.
Although there have been limited studies on sugarcane focusing on the relationship between source and sink tissue (Marcelis, 1996; Pammenter & Allison, 2002), in various other plant species the dominant influence of sink activity on source photosynthesis and carbon partitioning has been demonstrated. In Solanum tuberosum, a high sink demand in the form of rapidly growing tubers caused increased rates of photosynthesis (Dwelle et al., 1981) and enhanced translocation of photosynthate (Moorby, 1978). Removal of the tubers led to a marked decrease in net photosynthesis as a result of the imbalance between source and sink activity (Nosberger & Humphries, 1965). Irrespective of the presence or absence of water stress conditions, plants with artificially lowered sink strength (tuber excised) accumulated carbohydrate in the leaves and displayed a considerably reduced maximum photosynthetic rate (Amax), electron transport rate (ETR) and quantum yield (Fv/Fm) (Basu et al., 1999). Cold girdling of the leaves of Citrus unshiu to reduce carbon export and defruiting also reduced rates of photosynthesis (A), and this reduction coincided with an accumulation of carbohydrate in the source leaf (Iglesias et al., 2002). Sugar accumulation in leaves also represses the expression of photosynthetic genes (Sheen, 1990). In transgenic Nicotiana tabacum leaves the expression of a yeast invertase in the cell wall resulted in increased carbohydrate content, especially soluble sugars, which gradually inhibited photosynthetic levels as sugars accumulated (Von Schaewen et al., 1990; Stitt et al., 1991). Similarly, mature leaves of Spinacia oleracea supplied with glucose through the transpiration stream lost ribulose-1.5-bisphosphate carboxylase (Rubisco) activity over a 7-d period (Krapp et al., 1991).
In sugarcane, the sucrose-accumulating processes within the maturing stem are likely to be strong sinks for photoassimilate (Marcelis, 1996). Sucrose accumulation in sugarcane culm has recently been shown to receive high priority in the allocation of assimilates (Pammenter & Allison, 2002). Coincidently, large differences in photosynthetic rates have, in the past, been reported for individual sugarcane leaves related to the age of the plant, with young plants typically assimilating at significantly higher rates than older plants (Hartt & Burr, 1967; Bull & Tovey, 1974). Gross photosynthesis has been found to be lower in 8-month-old sugarcane plants compared with 4-month-old plants, regardless of the light intensity (Allison et al., 1997). Another study reported that 3-month-old sugarcane exhibited photosynthetic rates of 45 µmol m−2 s−1 under intense illumination, while young leaves on 10-month-old plants only photosynthesized at 25 µmol m−2 s−1 (Amaya et al., 1995).
In plants, sugar status modulates and coordinates growth and development (Smeekens, 2000) and, although the regulatory role of sugar in photosynthesis and metabolism is well known, progress has only recently been made in determining the molecular mechanisms of sugar sensing and signalling (Rolland et al., 2002; Gibson, 2005). Components of sugar sensing systems that have been identified include glucose, sucrose and trehalose sensing systems. For example, hexokinase (HXK) functions as a glucose sensor that modulates gene expression, and sucrose non-fermenting 1 (Snf1)-related protein kinases (SnRKs) are known to have diverse functions in carbon metabolism and sugar signalling (Rolland et al., 2002). The details of sink regulation of photosynthetic source relations in C3 plants are only now emerging, and the picture is even less clear in the more complex C4 species, such as sugarcane. In part, regulation of C4 photosynthesis is achieved through compartmentation of the process between mesophyll and bundle sheath cells and control of metabolite transfers through a set of cell-specific organelle metabolite translocators (e.g. dicarboxylic acid transporters) together with symplastic connections (Edwards et al., 2001). Various specializations have been demonstrated for C4 leaf carbon metabolism, including bundle sheath cell-specific storage of starch for a range of species (Downton & Tregunna, 1968; Laetsch, 1971; Lunn & Furbank, 1997) and preferential localization of genes involved in sucrose biosynthesis in the mesophyll cells (Lunn & Furbank, 1999). Studies continue to 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 photosystem II (PSII) efficiency were investigated and changes in photosynthesis were explained on the basis of leaf sugar concentrations and variations in sugar partitioning based on the uptake of a 14CO2 label.
Materials and Methods
Nine- to 12-month-old field-grown plants of Saccharum (L.) spp. hybrid cv. N19 (N19) cultivated at Mount Edgecombe, KwaZulu-Natal, South Africa on a 5 × 15 m plot were used in this study, which was conducted during summer (December 2004). The plot was located on a north-east facing slope with a gradient of c. 10°.
Manipulation of sink capacity
To increase plant sink:source ratios, all leaves except the third fully expanded leaf (leaf 6) (Fig. 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 illicit 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 neighbouring plants. Treatments were applied between 1 and 14 d before the measurements and sampling, effectively rendering leaf 6 the sole light-receiving source for photosynthetic carbon assimilation for this variable period before analysis. These plants which had been shaded for variable periods of time were compared with ‘control’ plants which were not shaded.
Following shading treatments for 1, 3, 6 and 14 d, treated and unshaded plants (n = 7) were concurrently harvested. In this way, all plants were exposed to the same environmental factors immediately before harvest. To decrease the risk of potential sucrose hydrolysis, the time between harvest and processing was kept to a minimum. Stalks were retained 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 (c. 2 × 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 (−196°C). The samples were stored in 50 ml centrifuge tubes at −80°C. Before analysis, leaf and culm tissue was incubated overnight in 20 volumes of sugar extraction buffer [30 mm HEPES (pH 7.8), 6 mm MgCl2 and 70% volume/volume (v/v) ethanol] at 70°C. Extracts were centrifuged for 10 min at 23 200g and sucrose, fructose and glucose concentrations in the supernatant were 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 18.104.22.168) (Roche, Mannheim, Germany) and fructose by phosphoglucose isomerase (EC 22.214.171.124) (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.
Gas exchange and fluorescence determinations
A Li-6400 portable photosystem unit (Li-Cor Biosciences Inc., Lincoln, NB, USA) was used to measure the photosynthetic assimilation (A), transpiration rate (E), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and leaf temperature of leaf 6. Comparative measurements were performed on the day of harvest for plants that were unshaded or had previously been partially shaded for 1–14 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 1000 µmol mol−1 under a constant photosynthetically active radiation (PAR) of 2000 µmol 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 as a result of 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 µmol mol−1 to determine the efficiency of carboxylation (CE; Lawlor, 1987). The assimilation rate in the absence of stomatal limitation (Aa) was calculated as A interpolated from the response curve at Ci = 380 µmol 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; 6000 µmol m−2 s−1) was applied to determine the maximal fluorescence yield (Fm′) at varying external CO2 concentrations (0–1000 µmol mol−1). The ETR, defined as the actual flux of photons driving photosystem II (PSII), was calculated from ETR = [(Fm′ − Fs)/Fm′] (fIαleaf), where Fs is ‘steady-state’ fluorescence (at 2000 µmol 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 αleaf is leaf absorptance (0.85; Li-Cor manual). The component fluorescence variables were derived as described by Maxwell & Johnson (2000).
The influence of shading treatments on carbon allocation was measured by supplying leaf 6 of unshaded and partially shaded plants (4 and 10 d) (n = 3) with 14CO2 using a protocol modified from Hartt et al. (1963). A portion of leaf (5 × 20 cm) weighing approximately 5 g was sealed in an air-inflated polythene bag containing 50 µl of NaH14CO3 (specific activity, 55 mCi mmol−1; ICN Radiochemicals, Irvine, CA, USA) to which 1 ml of 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 (c. 10 mg) from 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 20 volumes of sugar extraction buffer [30 mm HEPES, pH 7.8, 6 mm MgCl2 and 70% (v/v) ethanol] at 70°C. The radioactivity in the 70% (v/v) alcohol-soluble component was measured with a Tri-Carb Liquid Scintillation Analyzer (Packard, Milford, MA, USA) using Ultima GoldTM XR (Packard).
Labelled sugars in the 70% alcohol-soluble component were spotted onto 10 × 20 cm silica gel plates (Merck, Darmstadt, Germany) using a semiautomatic thin layer chromatography (TLC) sample applicator (Linomat 5; CAMAG, Muttenz, Switzerland) and fractionated using a mobile phase consisting of 50% (v/v) ethyl acetate, 25% (v/v) acetic acid, and 20% filter-sterilized water for 3 h. Silica plates were dried at 70°C for 10 min, sealed in polyethylene film and exposed to high-resolution phosphor screens (Packard). After 24 h of 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).
Results were subjected to analysis of variance (ANOVA) or Student's t-tests to determine the significance of the difference between responses to treatments. When ANOVA was performed, Tukey's honestly 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.
Effect of source:sink variations on sugar concentrations
Glucose concentrations in the unshaded leaf (leaf 6) declined over the duration of the shading treatment (Fig. 2), whereas fructose concentrations remained constant until day 6, declining subsequently. Apart from a temporary increase in sucrose after 6 d, there were no changes in sucrose concentration in leaf 6. Sucrose concentrations in shaded leaf 3 decreased over the initial 24-h period, and then remained constant at 21 µmol g−1 (Fig. 2). No significant changes were observed in glucose or fructose concentrations of leaf 3 over time.
Internodes above and below leaf 6 responded differently to the shading treatment, although internode 6 had consistently higher concentrations of hexoses and sucrose than internode 4 (Fig. 2). A comparable decline in sucrose concentrations over time was seen in both internodes 4 and 6; however, this trend was stronger in internode 4. There were no significant changes in hexose concentrations in internodes 4 and 6.
Sucrose concentrations in mature internodes (internodes 8, 10 and 12) were consistently highest in internode 12, followed by internodes 10 and 8 (Fig. 2). Sucrose concentrations decreased at day 3 across all mature internodes, followed by a sharp increase at day 6. This trend was more marked in internodes closer to leaf 6. Hexose concentrations in internodes 8 and 10 also declined significantly during the course of the time treatments, while hexose concentrations in internode 12 remained constant throughout. Concentrations of hexose in mature internodes were consistently highest in internode 8, followed by internodes 10 and 12, respectively.
Partial shading effect on 14C partitioning
The 14C detected in a sample from leaf 6 immediately after the 1-h feeding period was statistically indistinguishable between the sample groups (unshaded, 4 d; partially shaded, 10 d), although variations might have been anticipated as a result of variation in total leaf size and weight (Table 1). After the 24-h chase period, the amount of 14C in leaf 6 was significantly lower in the shaded than in the unshaded treatments. The allocation of 14C-labelled assimilate to the leaf roll and leaf 3 (shaded) increased with increased duration of shading. Similarly, the amount of 14C allocated to internodes 4 and 6 also increased with increased duration of shading. In contrast, the amount of label in internode 8 was reduced by shading, while internode 10 received a small amount of label, which was not influenced by the shading treatment. The amount of 14C recovered from internode 12 was negligible (data not shown).
Table 1. Incorporation and distribution of a 14C label in field-grown sugarcane (Saccharum spp.) plants that were either unshaded or partially (leaf 6 not shaded) shaded for either 4 or 10 d
The plants were supplied with 100 µCi 14CO2 to leaf 6 followed by a 24-h chase period. The means ± standard errors (kBq g−1 FW; n = 3) are followed by letters indicating for each tissue type whether the treatments had a significant influence (P < 0.05) as determined by analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) tests.
Leaf 6 samples were taken directly after labelling.
In leaf 6, labelled sucrose was reduced after 4 d of shading. However, labelled hexose concentrations only showed a significant reduction after 10 d (Fig. 3). Shading caused a significant increase in 14C allocation to sucrose in the leaf roll, in leaf 3 and in internodes 4 and 6. In contrast, there was a reduction in allocation to sucrose in internode 8 after 4 d of shading. Changes in 14C-hexose concentrations were generally smaller than the changes in 14C-sucrose concentrations. However, shading increased 14C allocation to hexoses in leaf 3 and internode 6, but reduced allocation to 14C-hexose in internode 8.
Source leaf photosynthesis and sugar correlations
Photosynthetic gas exchange characteristics and leaf chlorophyll fluorescence activities were determined on leaf 6 of unshaded plants and plants partially shaded for 1, 3, 6 and 14 d (Fig. 4). For partially shaded plants, a striking increase in both photosynthetic assimilation (A) and electron transport rate (ETR) across all Ci values was observed over the duration of the shading. Interestingly, plants shaded for 6 d exhibited a 37% higher Jmax compared with day 3 (Fig. 4). This was associated with a significant increase in leaf sucrose concentrations between days 3 and 6 (Fig. 2). The gas exchange variables and leaf ETR variables derived from A:Ci and ETR:Ci curves increased over the duration of the shading treatment (Table 2). After 8 d, significant increases in the substrate-limited photosynthetic rate (Jmax, 42%) and carboxylation efficiency (CE, 28%) were observed in comparison to unshaded plants, while the assimilation rate (A) and assimilation rate in the absence of stomatal limitation (Aa) were 48% and 51% higher, respectively, than in unshaded plants. The ETR of leaf 6 at ambient CO2 was also elevated by 29% after 8 d.
Table 2. Variables from A:Ci curves based on gas exchange analysis and leaf fluorescence of leaf 6 from unshaded and partially (leaf 6 not shaded) shaded (2, 4 and 8 d) 12-month-old field-grown N19 sugarcane (Saccharum spp.) plants
Jmax, substrate supply-limited assimilation; Rd, dark respiration; CE, carboxylation efficiency; Ai and Aa, photosynthetic rate in the presence and absence of stomatal limitation, respectively; Gs, stomatal conductance; Ca, ambient CO2 concentration; Ci at Ca = 380, intercellular CO2 concentration at ambient CO2 concentration; ETR at Ca = 380, electron transport rate at ambient CO2 concentration.
Measurements were performed over a period of 8 d at an ambient relative humidity of 35.9 ± 0.8%[mean ± standard error (SE)] and an irradiance of 2000 µmol m−2 s−1. The shade treatment values are the mean ± SE (n = 4) and are followed by letters indicating for each tissue type whether the treatments had a significant influence (P < 0.05), as determined by analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) tests. For each measurement time-point, unshaded plants were measured as controls, but there were no significant differences between the photosynthetic parameters for the unshaded plants over time and thus the overall mean ± SE (n = 12) is presented for this group.
Jmax (µmol m−2 s−1)
22.7 ± 2.0 a
23.4 ± 1.1 a
29.7 ± 4.5 a
32.2 ± 0.7 b
Rd (µmol m−2 s−1)
3.7 ± 0.8 a
2.2 ± 0.3 a
2.6 ± 0.3 a
2.3 ± 0.4 a
CE (mol m−2 s−1)
129 ± 36.6 a
130 ± 39 a
153 ± 34 a
164.5 ± 5.4 b
Aa (µmol m−2 s−1)
18 ± 2.5 a
19.3 ± 0.7 a
21.2 ± 0.4 b
27.1 ± 0.6 b
Ai (µmol m−2 s−1)
13.8 ± 2.6 a
14.2 ± 1.1 a
16.3 ± 3.0 a
20.4 ± 0.5 b
Gs (mmol m−2 s−1)
131 ± 26 a
137 ± 10 a
119 ± 27 a
145 ± 7 a
Ci at Ca = 380 (µmol m−2 s−1)
162 ± 11.8 a
168 ± 9 a
167 ± 19 a
187 ± 17 a
ETR at Ca = 380 (µmol m−2 s−1)
55 ± 6.2 a
62 ± 3.0 a
67 ± 8.1 a
71 ± 2.0 b
A comparison between changes in sugar concentrations and photosynthetic activity variables of unshaded leaf 6 over 14 d of shading treatment further revealed a strong negative correlation between hexose concentrations in source leaf tissue and Jmax and CE (Fig. 5). This relationship was not evident for these variables with sucrose. Further analysis revealed significant relationships between sucrose and hexose concentrations, and leaf 6 photosynthesis levels in the internodal tissue sampled (Table 3 and Fig. 6). All sampled internodes produced a positive correlation between glucose and fructose concentrations. Immature sink tissue (internodes 4 and 6) was characterized by decreased sucrose concentrations which were correlated with an increase in Jmax (and CE for internode 4) over the 14-d period. The decreased hexose concentrations in internode 8 were negatively correlated with both sucrose and photosynthetic variables Jmax and CE, while the increased concentrations of sucrose in mature internodes (10 and 12) were positively correlated with changes in CE.
Table 3. Bivariate Pearson's correlation coefficients between sugar concentrations and photosynthetic variables [substrate supply-limited assimilation (Jmax) and carboxylation efficiency (CE)] of leaf 6 for field-grown N19 sugarcane (Saccharum spp.) plants either unshaded or partially shaded for between 1 and 14 d
Significance levels (P) are reported for the Pearson's correlation coefficients (in brackets).
Although sucrose concentrations in internodes followed the well-established pattern of greater sucrose concentrations in older internodes (Whitaker & Botha, 1997), the manipulation of sink strength through partial shading produced significant changes in plant sugar concentrations and assimilate partitioning. In general, young internodal tissue was characterized by low concentrations of hexose and shading treatments produced a decrease in sucrose over time. This trend was more evident in the youngest internode sampled (internode 4) than in internode 6, which may be a result of the location of internode 6 relative to leaf 6, and/or the lower rate of sucrose accumulation observed in younger internodal tissue (Whitaker & Botha, 1997). Overall, shading produced an initial (day 3) decrease in sucrose concentrations in internodes 10 and 12. Shading also resulted in a reduced sucrose concentration in shaded leaf 3 after 24 h which was sustained for the duration of the treatments. The relatively short period of time taken to reduce sucrose concentrations in both shaded leaf and internodal tissue indicated that, for sugarcane, partial shading was a practical means to evaluate the effects of changes in sink demand on the remaining unshaded source material.
Leaf 6 of unshaded plants distributed more 14C to internode 8 than to internodes 6, 4, 10 and 12 (in order of diminishing distribution). Thus, shading of the entire plant, except leaf 6, would seem likely to reduce the sugar concentrations in all other internodes more significantly than in internode 8. Label analyses of partially shaded plants further revealed a significant shift in 14C partitioning to immature culm and shaded leaf tissue. Distribution of 14C allocated to sucrose followed a similar pattern to total 14CO2 distribution, while trends for labelled sucrose and hexoses were generally comparable (Fig. 3), indicating sucrose and hexose as the two major pools for labelled assimilate.
The observed shifts in assimilate partitioning for partially shaded plants emphasize the roles of phloem loading at the source (van Bel, 1993, 2003) and unloading at the sink (Patrick, 1997; Walsh et al., 2005) as crucial links in the sink:source relationship (Kühn et al., 1999). Shading treatments produced a significant drop in the total sucrose pools of shaded leaf and immature culm tissue, while 14C analysis confirmed a shift in assimilate partitioning to these tissues, which are typically not supplied by leaf 6 (MacDonald, 2000). Plants partially shaded for 4 d showed 14C allocation patterns for treated plants before any significant change in photosynthesis in leaf 6, while plants shaded for 10 d showed distribution after adaptation of leaf 6 to changes in sink demand. The increased allocation of leaf 6-derived 14C to the leaf roll and leaf 3 of shaded plants further indicated prioritization of young leaf tissues as sinks for carbon from leaf 6. This change in typical leaf 6 partitioning patterns indicated not only a sink-strength-related response to the decreasing concentrations of sucrose measured in young shaded leaf tissue, but also a change in physiological state from source to sink. Such an event is not uncommon in infected leaves following pathogen attack, where an increase of import to the infected sites occurs (Farrar, 1992; Wright et al., 1995; Ayres et al., 1996), but has not yet been reported in shading experiments. While no overall change in sucrose was observed in leaf 6 after shading for 14 d, a decreased concentration of 14C-labelled sucrose after 24 h was evident in plants shaded for both 4 and 10 d. As partial shading did not produce any significant variation in stomatal conductance in leaf 6, reduced labelled sucrose might be indicative of increased sucrose turnover and a higher assimilate transport rate in the phloem of treated plants. This study has thus illustrated the ability of the phloem transport of sugarcane to respond to changes in environment and alter assimilate translocation patterns among various sink and source tissues. These results substantiate the role of sucrose as a signalling molecule in assimilate partitioning (Chiou & Bush, 1998); however, the signalling mechanisms that link phloematic, apoplastic and intracellular sucrose concentrations remain to be fully elucidated (Gibson, 2005).
The changes observed in sugar concentrations over time in maturing internodes (8, 10 and 12; Fig. 2) are indicative of the many complex factors influencing the overall physiological environment of the plant in shading treatments. Although partial shading produced an acropetal shift in assimilate partitioning from leaf 6 to younger internodes and young leaf tissue, the effect of this on the overall sugar content of mature internodes would be further confounded by the acclimation of leaf 6 to increased sink demand over time and the overall drop in available assimilate for the entire plant. Shading treatments would additionally influence plant water relations. Assuming that water loss from shaded leaves was reduced, this would increase water potential (Ψp) and possibly reduce the flow of nutrients to culm and leaf tissue. This could influence shaded leaf and root metabolic activities which could in turn reduce the overall demand for CH2O and consequently affect carbon accumulation in mature internodes which typically supply root tissue. As a number of factors may thus affect mature internodal tissue under the present shading treatment, more detailed study is required for accurate interpretation of the observed changes and correlations between sugar concentrations and photosynthesis.
Significant increases in photosynthetic rate, carboxylation efficiency and PSII efficiency were measured in leaf 6 over the duration of the shading treatment. A significant linear relationship was further found between the maximum photosynthetic assimilation rates (Jmax) of leaf 6 and decreasing concentrations of sucrose in immature culm tissue (internodes 4 and 6) over the partial shading time treatments. This supports evidence that decreased sucrose at the sink is a likely physiological signal to the source for increased assimilate requirements (van Bel, 2003). A similar effect has previously been observed in defoliated sugarcane plants, which resulted in increased rates of assimilation on the remaining leaf surface (Pammenter & Allison, 2002). However, the dramatic photosynthetic increase in leaf 6 observed here may have been compounded by the sustained presence and required maintenance of other leaves. Furthermore, a depletion or excess of sugars has previously been shown to, respectively, activate or repress the expression of genes for photosynthetic components and ultimately influence photosynthesis itself (Stitt, 1991; Krapp et al., 1993; Van Oosten & Besford, 1994, 1995; Basu et al., 1999). The plasticity of leaf assimilation capacity over time observed in sugarcane may thus be linked to regulation of C4 leaf metabolism at the molecular level, such as regulatory phosphorylation of phosphoenolpyruvate carboxylase (PEPc) activity (Vidal & Chollet, 1997) and/or adjustments in several other C4 photosynthetic control mechanisms (Furbank & Taylor, 1995). It is important to note that this study has ‘simulated’ an increase in plant sink strength via an increased demand for carbon from leaf 6. Thus, although it is feasible that the overall sink activity of internodal tissue may have, in fact, declined as a result of the lack of source supply, this research has provided evidence for the physiological ability of the source to adapt to increased sink requirements. The Saccharum complex is potentially capable of storing more than 25% sucrose on a fresh weight basis (Bull & Glasziou, 1963; Moore et al., 1997). As this estimate is still almost double current commercial yields (Grof & Campbell, 2001), further understanding of source regulation may assist in the eventual utilization of a greater portion of the potential sink strength of sugarcane.
Interestingly, no relationship was observed between sucrose concentrations, in either unshaded or shaded leaves, and photosynthesis in this study. These results are comparable to studies on maize (Zea mays) leaves, where changing sucrose concentrations were shown to have no significant short-term feedback inhibitory effects on the synthesis of sucrose itself in the leaf (Lunn & Furbank, 1997). Instead, a strong negative correlation was found between hexose and photosynthetic gas exchange variables Jmax and CE in unshaded leaf 6, which implicated hexoses, rather than sucrose, as possible signal factors involved in photosynthetic feedback regulation. In the past, hexoses have been shown to be inhibitors of photosynthesis (Goldschmidt & Huber, 1992). For example, the external supply of glucose (50 mm) to excised Spinacea oleracea leaves over 4 d led to inhibition of the light harvesting complex (LHC) II-encoding chlorophyll a/b binding protein (cab) genes and a 60% decrease in Rubisco content (Kilb et al., 1995). Thus, in sugarcane, a decreased leaf glucose pool could constitute a signal of increased demand from sinks. More recently, hexoses have been shown to play an important role in regulating photosynthesis and leaf development (Ehness et al., 1997; Paul & Pellny, 2003). Hexokinase has been implicated as a putative receptor (Jang et al., 1997); however, the mechanisms involved in hexokinase sensing remain contentious. It has also been demonstrated that glucose itself, and not an analogous phosphorylated metabolite, may be the primary signal that interacts with some putative receptor involved in transduction of the carbohydrate signal (Ehness et al., 1997). Progress has been made (Rolland et al., 2002), but further efforts will be required to fill in the gaps in this complex network, especially for C4 species. Compared with C3 species, relatively little is known about the control of sugar biosynthesis in the leaves of C4 plants; however, sugar-induced changes in gene expression are likely to be as important in C4 as in C3 plants in balancing sink:source interactions (Lunn & Furbank, 1999).
Although it is likely that the hexose concentrations in the leaf tissue are under strict metabolic control, it will ultimately be difficult to elucidate the actual mechanisms of hexose responses, as sugars can act by affecting osmotic potentials as well as by functioning as signal molecules (Gibson, 2005). This may be further complicated by the interactions between carbon and nitrogen concentrations in leaf developmental processes (Paul & Pellny, 2003). For instance, the application of moderate (111 mm) concentrations of glucose stimulates the senescence of Arabidopsis, but only under limited nitrogen conditions (Wingler et al., 2004). Furthermore, as most plants can synthesize sucrose when fed with hexoses, it is difficult to attribute the effects of hexoses to their direct sensing, as sucrose sensing could possibly occur. However, our correlations indicate that, although sucrose must play a key role in regulating sink assimilate partitioning, hexoses may be a more proximal component of the signalling mechanism between photosynthetic source activity and sink requirements in sugarcane. The regulatory effects of hexose sensors such as HXK are well documented in C3 plants (Rolland et al., 2002) and may provide a useful starting point for examining the control of photosynthesis in the C4 plant sugarcane.
Sugarcane, like other plants, exhibits a robust soluble carbohydrate-dependent relationship between source and sink tissue based on assimilate demand and partitioning needs. This study has provided good evidence for a sink-dependent relationship between source and sink tissues as well as an important role for sugars in this relationship. Further study will be required to substantiate the molecular basis of these correlations, specifically the observed effects of hexose on photosynthesis, as the molecular pathways involved in regulating such a relationship are not yet fully understood. Future research may include comparison of different sugarcane cultivars; however, the general patterns observed here with the model N19 hybrid are likely to pertain for other cultivars. The existence of sink regulation of source activity in sugarcane should inform biotechnological efforts to modify culm metabolism to improve sugar accumulation. The fact that sink demand limits source activity indicates that the signal feedback system reporting sink sufficiency and regulating source activity may be important loci for investigation/modification in sugarcane. We are currently attempting to elucidate which genes and enzymes in sugarcane leaves are responsive to changes in the sink:source ratio, with particular emphasis on links between photosynthesis and sugar sensing/signalling.
The authors are grateful for funding provided by the South African Sugarcane Research Institute, SA Sugar Association Trust Fund for Education and the National Research Foundation.