Differential Expression of Genes in the Leaves of Sugarcane in Response to Sugar Accumulation

In C₄ sugarcane (Saccharum spp. hybrids), photosynthetic activity has been shown to be regulated by the demand for carbon from sink tissues. There is evidence, from other plant species, that sink-limitation of photosynthesis is facilitated by sugar-signaling mechanisms in the leaf that affect photosynthesis through regulation of gene expression. In this work, we manipulated leaf sugar levels by cold-girdling leaves (5°C) for 80 h to examine the mechanisms whereby leaf sugar accumulation affects photosynthetic activity and assess whether signaling mechanisms reported for other species operate in sugarcane. During this time, sucrose and hexose concentrations above the girdle increased by 77% and 81%, respectively. Conversely, leaf photosynthetic activity (A) and electron transport rates (ETR) decreased by 66% and 54%, respectively. Quantitative expression profiling by means of an Affymetrix GeneChip Sugarcane Genome Array was used to identify genes responsive to cold-girdling (56 h). A number of genes (74) involved in primary and secondary metabolic pathways were identified as being differentially expressed. Decreased expression of genes related to photosynthesis and increased expression of genes involved in assimilate partitioning, cell wall synthesis, phosphate metabolism and stress were observed. Furthermore four probe sets homologous to trehalose 6-phosphate phosphatase (TPP; EC 5.3.1.1) and trehalose 6-phosphate synthase (TPS; EC 2.4.1.15) were up- and down-regulated, respectively, indicating a possible role for trehalose 6-phosphate (T6P) as a putative sugar-sensor in sugarcane leaves.     

Changes in photosynthetic rates and gene expression of leaves during a source-sink perturbation in sugarcane

BACKGROUND AND AIMS: In crops other than sugarcane there is good evidence that the size and activity of carbon sinks influence source activity via sugar-related regulation of the enzymes of photosynthesis, an effect that is partly mediated through coarse regulation of gene expression. METHODS: In the current study, leaf shading treatments were used to perturb the source-sink balance in 12-month-old Saccharum spp. hybrid 'N19' (N19) by restricting source activity to a single mature leaf. Changes in leaf photosynthetic gas exchange variables and leaf and culm sugar concentrations were subsequently measured over a 14 d period. In addition, the changes in leaf gene response to the source-sink perturbation were measured by reverse northern hybridization analysis of an array of 128 expressed sequence tags (ESTs) related to photosynthetic and carbohydrate metabolism. KEY RESULTS: Sucrose concentrations in immature culm tissue declined significantly over the duration of the shading treatment, while a 57 and 88% increase in the assimilation rate (A) and electron transport rate (ETR), respectively, was observed in the source leaf. Several genes (27) in the leaf displayed a >2-fold change in expression level, including the upregulation of several genes associated with C(4) photosynthesis, mitochondrial metabolism and sugar transport. Changes in gene expression levels of several genes, including Rubisco (EC 4.1.1.39) and hexokinase (HXK; EC 2.7.1.1), correlated with changes in photosynthesis and tissue sugar concentrations that occurred subsequent to the source-sink perturbation. CONCLUSIONS: These results are consistent with the notion that sink demand may limit source activity through a kinase-mediated sugar signalling mechanism that correlates to a decrease in source hexose concentrations, which, in turn, correlate with increased expression of genes involved in photosynthesis and metabolite transport. The signal feedback system reporting sink sufficiency and regulating source activity may be a potentially valuable target for future genetic manipulation to increase sugarcane sucrose yield.     

Effects of carbohydrate accumulation on photosynthesis differ between sink and source leaves of Phaseolus vulgaris L

Accumulation of non-structural carbohydrate in leaves represses photosynthesis. However, the extent of repression should be different between sink leaves (sugar consumers) and source leaves (sugar exporters). We investigated the effects of carbohydrate accumulation on photosynthesis in the primary leaves of bean (Phaseolus vulgaris L.) during leaf expansion. To increase the carbohydrate content of the leaves, we supplied 20 mM sucrose solution to the roots for 5 d (sugar treatment). Plants supplied only with water and nutrients were used as controls. The carbohydrate contents, which are the sum of glucose, sucrose and starch, of the sugar-treated leaves were 1.5-3 times of those of the control leaves at all developmental stages. In the young sink leaves, the photosynthetic rate at saturating light and at an ambient CO2 concentration (A360) did not differ between the sugar-treated and control leaves. The A360 of sugar-treated source leaves gradually decreased relative to the control source leaves with leaf expansion. The initial slope of the A-Ci (CO2 concentration in the intercellular space) curve, and the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content per leaf area showed trends similar to that of A360. Differences in Amax between the treatments were slightly smaller than those in A360. These results indicate that the effect of carbohydrate accumulation on photosynthesis is significant in the source leaves, but not in the young sink leaves, and that the decrease in Rubisco content was the main cause of the carbohydrate repression of photosynthesis.     

Sink strength regulates photosynthesis in sugarcane

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.     

Expression analysis of genes associated with sucrose accumulation in sugarcane under normal and GA<Subscript>3</Subscript>-induced source–sink perturbed conditions

Sugarcane accumulates high amount of sucrose, thus making it one of the important cash crops worldwide. The final destination of sucrose accumulation in sugarcane is sink tissue, i.e., stalk, supplied by the source, i.e., leaf, to fulfill the need of plant growth, respiration, storage, and other metabolic activities. Signals between sink and source tissues regulate sucrose accumulation in sink and possibly the negative feedback from the sink restrains further accumulation in the stalk. However, perturbation of this negative feedback may help to improve sugar yield. This can be achieved by the application of GA₃ (Gibberellic acid), a plant growth regulator, known to excite physiological responses and modify the source–sink metabolism through their effect on photosynthesis, which in turn improves sink strength by redistribution of the photoassimilates. In the present study, GA₃ applied canes showed prominent increase in invertase activity, at early stage of the application, to provide hexoses. This in turn helped increase the internodal length and cane capacity for additional accumulation of sucrose, thereby increasing sink strength. At maturity, sucrose% and brix% were found higher in middle and top portions of the GA₃-applied canes. Expression analysis of various sucrose metabolising genes viz., sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase, neutral invertase, and cell wall invertase (CWI) was carried out at different growth stages, using quantitative RT-PCR. CWI, which plays key role in phloem unloading in sink tissues, exhibited higher expression in GA₃ samples at the elongation stage which decreased with maturity, whereas both SuSy and SPS, involved in regulation of sucrose accumulation, showed a variable level of expression. Thus, GA₃ application on cane may improve the sucrose content in stalk and thus assuage maneuvering source–sink dynamics in sugarcane.     

Sugar repression of photosynthesis: the role of carbohydrates in signalling nitrogen deficiency through source:sink imbalance

The aim of this work was to examine whether carbohydrates are involved in signalling N deficiency through source:sink imbalance. Photosynthetic metabolism in tobacco was studied over 8 d during the withdrawal of N from previously N-sufficient plants in which the source:sink ratio was manipulated by shading leaves on some of the plants. In N-sufficient plants over this time-scale, there was a small decline in photosynthetic rate, Rubisco protein and amino acid content, with a larger decrease in carbohydrate content. Withdrawal of N from the growing medium induced a large decrease in the rate of photosynthesis (35% reduction after 8 d under the growing conditions, with a reduction also apparent at high and low measuring CO₂), which was caused by a large decrease in the amount of Rubisco protein (62% after 8 d) and Rubisco activity. Higher amounts; of hexoses preceded the loss of photosynthetic activity and sucrose and starch accumulation. Reduction of the sourcersink ratio by shading prevented the loss of photosynthetic activity and the increase in hexoses and other carbohydrates. These data indicate that the reduction of photosynthesis that accompanies N deficiency in intact plants has the characteristics of sugar repression of photosynthesis observed in model systems, but that the accumulation of hexose prior to the decline in photosynthesis is small. The possibility that sugar repression of photosynthesis under physiological conditions depends more crucially on the C:N status of leaves than the carbohydrate status alone is discussed.     

The effect of sulphite on the regulation of photosynthetic sucrose synthesis in poplar leaves

Sulphite at concentrations from 0.05 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bart. ex Marsh) leaves via the transpiration stream. The rate of CO2 fixation and partitioning of newly fixed carbon between sucrose and starch were measured and compared with the contents of selected phosphorylated intermediates, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and the activation of sucrose-phosphate synthase (SPS). Supplying leaves with 0.5 mM sulphite led to an increase in the sucrose/starch partitioning ratio without altering the rate of ¹⁴CO2 fixation. The increase in sucrose synthesis compared to starch synthesis was accompanied by relatively small changes of 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate (Fru1,6BP), hexose phosphates (hexose-)), uridine 5'-diphosphoglucose (UDPGlc), an accumulation of triose phosphates (triose-P), an activation of SPS, and decreased Fru2,6BP contents. Supplying leaves with 1.0 mM sulphite decreased ¹⁴CO2 assimilation and increased partitioning of fixed carbon into starch. A selective inhibition of sucrose synthesis was accompanied by an accumulation of triose-P, Fru1,6BP, hexose-P, and a decrease of PGA contents. There was also a large increase of Fru2,6BP contents and a decline in the activation of SPS. It could be argued that sulphite affects the allocation of photosynthetic carbon to sucrose and that sulphite can inhibit photosynthesis via a selective inhibition of sucrose synthesis.     

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source–sink relationships, and has substantial potential for enhanced food and biofuel production.     

Regulation of photosynthesis through source: sink imbalance in citrus is mediated by carbohydrate content in leaves

In citrus, the occurrence of a sink effect on photosynthesis (A) is controversial. Leaf carbohydrates and photosynthetic rates in field-grown trees of Satsuma mandarin ( Citrus unshiu [Mak.] Marc.) cv. Okitsu, were measured to elucidate whether or not the demand for photoassimilates regulates A. The data indicated that the source-sink imbalances induced by different treatments altered both soluble (sucrose, glucose and fructose) and insoluble carbohydrates in leaves, as well as photosynthetic rates. In general, girdling and defruiting increased starch and reduced photosynthesis, whereas source-limiting conditions imposed through partial defoliations had the opposite effect. These results are compatible with the assumption that a lack of sink activity leads to carbohydrate accumulation and feedback inhibition of A, and vice versa. Further evidence supporting a source-sink effect on A was provided by measurements of the dry matter:leaf area ratio, since defoliations, for example, increased this ratio. The in vivo sucrose supplementation to plants with different source:sink ratios (control, defoliated, girdled and defruited plants) increased carbohydrates and reduced photosynthesis. This suggests that sugars may have, per se, the potential to repress photosynthetic rates in intact plants with active sinks. Based on these results we propose that sugar accumulation in citrus leaves causes a feedback inhibition of A.     

Sugar preferences and digestive efficiency in an opportunistic avian nectarivore, the Dark-capped Bulbul Pycnonotus tricolor

It has recently been recognized that flowers pollinated by generalist opportunistic nectarivores tend to have different nectar properties to those pollinated by specialist nectarivores (including both hummingbirds and specialist passerines). While renewed interest in specialist avian nectarivore sugar preferences and digestive physiology has helped explain the concentrated sucrose-dominated nectar of plants they feed on, there has been little progress in understanding why generalist or occasional nectar-feeding birds tend to be associated with flowers that have dilute hexose-dominated nectar. We examined sugar preferences and assimilation efficiencies over a range of concentrations, and concentration preferences, in Dark-capped Bulbuls Pycnonotus tricolor, one of the more common occasional avian nectarivores in southern Africa. Dark-capped Bulbuls showed significant preference for hexose sugar solutions, irrespective of concentration, when given a choice between hexose and sucrose solutions in equicaloric pair-wise choice tests conducted at five different concentrations (5–25%). This contrasts with results from specialist nectarivore groups which generally show a significant concentration-dependant switch in preference from hexose at low concentrations to sucrose at high concentrations for equicaloric solutions. In addition, Dark-capped Bulbuls showed an unusual lack of preference for solutions of higher sugar concentration when simultaneously offered four solutions varying in concentration from 10 to 25%. Dark-capped bulbuls also showed a unique effect of concentration on sugar assimilation efficiency, assimilating relatively more energy on 5% diets than on 25% diets. Although able to assimilate sucrose effectively, assimilation rates of hexose sugars were marginally higher. These results shed new light on pollination systems involving occasional nectarivores and, in particular, help to explain the prevalence of low concentration hexose-dominated nectars in flowers pollinated by these birds.     

Carbon Partitioning and Growth of a Starchless Mutant of Nicotiana sylvestris

We have further characterized the photosynthetic carbohydrate metabolism and growth of a starchless mutant (NS 458) of Nicotiana sylvestris that is deficient in plastid phosphoglucomutase (Hanson KR, McHale NA [1988] Plant Physiol 88: 838-844). In general, the mutant had only slightly lower rates of photosynthesis under ambient conditions than the wild type. However, accumulation of soluble sugars (primarily hexose sugars) in source leaves of the mutant compensated for only about half of the carbon stored as starch in the wild type. Therefore, the export rate was slightly higher in the mutant relative to the wild type. Starch in the wild type and soluble sugars in the mutant were used to support plant growth at night. Growth of the mutant was progressively restricted, relative to wild type, when plants were grown under shortened photoperiods. When grown under short days, leaf expansion of the mutant was greater during the day, but was restricted at night relative to wild-type leaves, which expanded primarily at night. We postulate that restricted growth of the mutant on short days is the result of several factors, including slightly lower net photosynthesis and inability to synthesize starch in both source and sink tissues for use at night. In short-term experiments, increased “sink demand” on a source leaf (by shading all other source leaves) had no immediate effect on starch accumulation during the photoperiod in the wild type or on soluble sugar accumulation in the mutant. These results would be consistent with a transport limitation in N. sylvestris such that not all of the additional carbon flux into sucrose in the mutant can be exported from the leaf. Consequently, the mutant accumulates hexose sugars during the photoperiod, apparently as the result of sucrose hydrolysis within the vacuole by acid invertase.     

Sucrose Hydrolysis in Relation to Phloem Translocation in Beta vulgaris

Asymmetrically labeled sucrose, ¹⁴C(fructosyl)sucrose, was used to determine whether sucrose undergoes extracellular hydrolysis during phloem translocation in the sugar beet, Beta vulgaris. In addition, the metabolism of various sugars accumulated and translocated was determined in various regious of the plant. These processes were studied in detached regions as well as in the intact, translocating plant in the source leaf, along the translocation path, and in a rapidly growing sink leaf and storage beet. The data show that, unlike sucrose accumulation into the sink tissue of sugarcane, sucrose is neither hydrolzyed prior to phloem loading or during transit, nor is it extracellularly hydrolyzed during accumulation into sink leaves or the storage beet.     

Leaf Phosphate Status,Photosynthesis, and Carbon Partitioning in Sugar Beet (IV. Changes with Time Following Increased Supply of Phosphate to Low-Phosphate Plants)

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).     

不同供镁水平对两菊芋品种幼苗生物量、光合和叶绿素荧光特性的影响

采用砂培方式研究了不同供镁水平对"南芋1号(Nanyu No.1)"和"青芋2号(Qingyu No.2)"幼苗生物量、光合特性和叶绿素荧光特性的影响。结果表明:缺镁使菊芋叶片叶绿素含量显著降低,干物质积累减少,净光合速率、蒸腾速率和光合电子传递速率显著下降、CO2同化受抑制,同时Fv/Fm、ΦPSII、qP和qN也显著下降,加重了叶片受到光抑制的程度;随着供镁水平的增加,菊芋的干物质积累,叶绿素含量,光合参数和各系列荧光参数均呈现先增加后降低的趋势;南芋1号和青芋2号在镁离子浓度分别为3和6mmol.L-1,叶片的净光合速率、Fo、Fm、Fo’、Fm’、Fv/Fm、ΦPSII、ETR达到最高值,叶绿素a、b及总叶绿素含量也最多,干物质积累量最大;南芋1号的各系列荧光参数、光合参数、叶绿素含量和干物质积累量均显著高于青芋2号。     

Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

Ammonia accumulation and inhibition of photosynthesis in methionine sulfoximine treated spinach

Ammonia accumulation and photosynthetic rate inhibition took place when spinach leaf tissue was supplied with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. This effect was observed in the absence of significant inorganic nitrogen reduction or an exogenous source of ammonia. Both the time lag prior to the initial photosynthetic rate decrease and the rate of that decrease depend on the O₂ and MSO concentrations supplied to the leaf tissue. However, the total rate of ammonia accumulation was similar at both 20% and 2.2% O₂. The decline in photosynthetic rate was not caused by stomatal closure but may be a result of ammonia toxicity. The data point out the importance of glutamine synthetase in preventing the poisoning of leaf metabolism by ammonia generated internally through processes not involved in net nitrogen assimilation. The rapidity of the action of MSO in suppressing photosynthesis was unexpected and should not be overlooked in interpreting data from other experiments involving that inhibitor. MSO shows promise as a tool for investigating C-N flow, particularly during photorespiration.     

The mitochondrial CMSII mutation of Nicotiana sylvestris impairs adjustment of photosynthetic carbon assimilation to higher growth irradiance

The CMSII mutant of Nicotiana sylvestris, which lacks a functional mitochondrial complex I, was used to investigate chloroplast-mitochondria interactions in light acclimation of photosynthetic carbon assimilation. CMSII and wild-type (WT) plants were grown at 80 micromol m(-2) s(-1) photosynthetic active radiation (PAR; 80) and 350 micromol m(-2) s(-1) PAR (350). Carbon assimilation at saturating PFD was markedly higher in WT 350 leaves as compared with WT 80 leaves, but was similar in CMS 80 and CMS 350 leaves, suggesting that the mutant is unable to adjust photosynthesis to higher growth irradiance. WT 350 leaves showed several general characteristic light acclimation responses [increases in leaf specific area (LSA), total chlorophyll content, and chlorophyll a/b ratio, and a higher light compensation point]. In contrast, a similar chlorophyll content and chlorophyll a/b ratio were measured for both CMS 80 and CMS 350 leaves, while LSA and the light compensation point acclimated as in the WT. The failure of CMSII to adjust photosynthesis to growth PFD did not result from lower quantum efficiency of PSII, lower whole-chain electron transport rates (ETRs), or lower ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and sucrose phosphate synthase (SPS) capacities. Excess ETR not used for carbon assimilation was even higher in CMS 350 than in WT 350. Since photochemical fluorescence quenching and the initial activity of NADP malate dehydrogenase (NADP-MDH) were identical in WT 350 and CMS 350 leaves but the activation state of NADP-MDH was different, redox signals from primary ETR are not involved in the signal transduction of light acclimation, while a contribution of stromal redox state cannot be excluded. When mature plants were transferred between 350 and 80 conditions, the mutant showed acclimatory tendencies, although adjustments were not as rapid or as marked as in the WT, and the response of the initial activities of Rubisco and NADP-MDH was impaired or altered. Initial activities of Rubisco and SPS at limiting concentration were also affected in CMS 350 as compared with WT plants when compared at growth irradiance or after in situ activation at 1000 micromol m(-2) s(-1) PAR. The data demonstrate that chloroplast-mitochondria interactions are important in light acclimation, and modulation of the activation state of key photosynthetic enzymes could be an important mechanism in this cross-talk.