Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development

Constitutive over-expression of a maize sucrose-phosphate synthase (SPS) gene in tobacco (Nicotiana tabacum) had major effects on leaf carbohydrate budgets with consequences for whole plant development. Transgenic tobacco plants flowered earlier and had greater flower numbers than wild-type plants. These changes were not linked to modified source leaf carbon assimilation or carbon export, although sucrose to starch ratios were significantly higher in leaves expressing the transgene. The youngest and oldest leaves of plants over-expressing SPS had up to 10-fold wild-type maximal extractable SPS activity, but source leaf SPS activities were only 2-3 times greater in these lines than in the wild type. In the oldest leaves, where the expression of the transgene led to the most marked enhancement in SPS activity, photosynthesis was also increased. It was concluded that these increases in the capacity for sucrose synthesis and carbon assimilation, particularly in older leaves, accelerate the whole plant development and increase the abundance of flowers without substantial changes in the overall shoot biomass.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.     

Role of sucrose-phosphate synthase in sucrose metabolism in leaves

Sucrose is formed in the cytoplasm of leaf cells from triose phosphates exported from the chloroplast. Flux control is shared among key enzymes of the pathway, one of which is sucrose-phosphate synthase (SPS). Regulation of SPS by protein phosphorylation is important in vivo and may explain diurnal changes in SPS activity and carbon partitioning. The signal transduction pathway mediating the light activation of SPS in vivo appears to involve metabolites and novel “coarse” control of the protein phosphatase that dephosphorylates and activates SPS. Regulation of the phosphorylation of SPS may provide a general mechanism whereby sucrose formation is coordinated with the rate of photosynthesis and the rate of nitrate assimilation. There are apparent differences among species in the properties of SPS that may reflect different strategies for the control of carbon partitioning. The SPS gene has recently been cloned from maize; results of preliminary studies with transgenic tomato plants expressing high levels of maize SPS support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose.     

Influence of Carbohydrates on Photosynthesis in Single, Rooted Soybean Leaves Used as a Source-Sink Model

The single rooted leaf of soybean (Glycine max L. Merr.) wasused to study source-sink relationships in photosynthesis. Whenthe leaves were kept under a regime of 10 h light (410–480µmol photons m^–2, 400–700 nm)–14 h dark,they did not expand, the increase in leaf dry weight almoststopped, and photosynthetic activity remained at a high andconstant level for 8 d while the dry weight of the roots increasedat a constant rate throughout the period. Thus, under this conditionthe leaf and the root system served as the only source and sinkorgans, respectively. When leaves grown for 7 d under this conditionwere placed under continuous light to alter the source/sinkbalance in photosynthate, the root dry weight increased at aconstant rate equal to that found under the 10 h light–14h dark condition. The leaf dry weight markedly increased andby day 5 of continuous light had increased 1.6-fold, mainlyas a consequence of accumulation of starch and sucrose, whichwere not translocated for root growth. The continuous lightcaused an abrupt decrease in the photosynthetic activity (40%of initial value by day 5). However, the activity recoveredalmost completely after a 32-h transfer to darkness. Significantnegative correlations existed between photosynthetic activityand the sucrose and starch contents in the rooted leaves placedunder continuous light. When the plants were treated with variouslight conditions, there was no significant difference (p<0.01)among the regression line slopes for photosynthetic activityon the sucrose content, but there was some deviation among thosefor the photosynthetic activity on the starch content. Thisresult suggests that sucrose accumulated in the leaf has a moredirect influence on photosynthetic activity when the source/sinkbalance was altered. (Received September 9, 1985; Accepted February 21, 1986)     

Mulberry Leaf Metabolism under High Temperature Stress

Effects of high temperature on the activity of photosynthetic enzymes and leaf proteins were studied in mulberry (Morus alba L. cv. BC2-59). A series of experiments were conducted at regular intervals (120, 240 and 360 min) to characterize changes in activities of ribulose-1,5-bisphosphate carboxylase (RuBPC) and sucrose phosphate synthase (SPS), photosystem 2 (PS 2) activity, chlorophyll (Chl), carotenoid (Car), starch, sucrose (Suc), amino acid, free proline, protein and nucleic acid contents in leaves under high temperature (40 °C) treatments. High temperature markedly reduced the activities of RuBPC and SPS in leaf extracts. Chl content and PS 2 activity in isolated chloroplasts were also affected by high temperature, particularly over 360 min treatment. Increased leaf temperature affected sugar metabolism through reductions in leaf starch content and sucrose-starch balance. While total soluble protein content decreased under heat, total amino acid content increased. Proline accumulation (1.5-fold) was noticed in high temperature-stressed leaves. A reduction in the contents of foliar nitrogen and nucleic acids (DNA and RNA) was also noticed. SDS-PAGE protein profile showed few additional proteins (68 and 85 kDa) in mulberry plants under heat stress compared to control plants. Our results clearly suggest that mulberry plants are very sensitive to high temperature with particular reference to the photosynthetic carbon metabolism.     

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO<Subscript>2</Subscript> levels

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO₂ levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO₂ levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO₂ levels.     

Growth rate,photosynthetic activity,and leaf development of Brazil pine seedlings (Araucaria angustifolia[Bert.] O. Ktze.)

Seedlings of Brazil pine, a large-seeded South American conifer, were grown in a climate chamber to investigate vertical growth pattern and the time course of leaf development. We examined shoot growth, photosynthetic performance and markers of leaf maturation such as contents of soluble sugars and activities of sucrose-phosphate synthase (SPS), neutral invertase (nI) and sucrose synthase (Susy). The daily increment of shoot length showed an optimum curve during the first 70 days after germination. The low growth rate during the first 20 days of development correlated with net CO² emission of the seedling. Analyses of leaf maturation markers in older seedlings revealed low sucrose/hexose ratios and high activities of nI and Susy in the uppermost leaves. Although the SPS/Susy ratio was low in these leaves the extractable SPS activity did not change significantly among leaves of different age. The photosynthetic light compensation points of young leaves were about 2-fold higher than those of mature leaves and their photosynthetic capacity was only 50% as high. Our results indicate that a rapid maturation of leaves of Brazil pine seedlings may reduce the respiratory loss of carbohydrates and that the mobilisation of seed storage compounds supports initial shoot growth under light-limiting conditions which may occur in the forest-grassland succession zone.     

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.     

Up-regulation of sucrose metabolizing enzymes in Oncidium goldiana grown under elevated carbon dioxide

Experiments were conducted in controlled growth chambers to evaluate how increase in CO₂ concentration affected sucrose metabolizing enzymes, especially sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13), as well as carbon metabolism and partitioning in a tropical epiphytic orchid species (Oncidium goldiana). Response of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) to elevated CO₂ was determined along with dry mass production, photosynthesis rate, chlorophyll content, total nitrogen and total soluble protein content. After 60 days of growth, there was a 80% and 150% increase in dry mass production in plants grown at 750 and 1 100 μl l^?1 CO₂, respectively, compared with those grown at ambient CO₂ (about 370 μl l^?1). A similar increase in photosynthesis rate was detected throughout the growth period when measured under growth CO₂ conditions. Concomitantly, there was a decline in leaf Rubisco activity in plants in elevated CO₂ after 10 days of growth. Over the growth period, leaf SPS and SS activities were up‐regulated by an average of 20% and 40% for plants grown at 750 and 1100 μl l^?1 CO₂, respectively. Leaf sucrose content and starch content were significantly higher throughout the growth period in plants grown at elevated CO₂ than those at ambient CO₂. The partitioning of photosynthetically fixed carbon between sucrose and starch appeared to be unaffected by the 750 μl l^?1 CO₂ treatment, but it was favored into starch under the 1 100 μl l^?1 CO₂ condition. The activities of SPS and SS in leaf extracts were closely associated with photosynthetic rates and with partitioning of carbon between starch and sucrose in leaves. The data are consistent with the hypothesis that the up‐regulation of leaf SPS and SS might be an acclimation response to optimize the utilization and export of organic carbon with the increased rate of inorganic‐carbon fixation in elevated CO₂ conditions.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

Relations between carbohydrate accumulation in leaves, sucrose phosphate synthase activity and photoassimilate transport in chilling treated maize seedlings

Sucrose and starch concentration, sucrose phosphate synthase (SPS) activity in leaves, and long distance transport were studied in maize seedlings treated with moderate chilling (14 °C/12 °C - day/night). Two inbred lines were tested: chilling-tolerant KW1074 and chilling-sensitive CM109. Seedlings were grown in phytotrone on water nutrient until the 4-th leaf appearance. The estimations were done on fully developed 2-nd leaf. Six days after the temperature was lowered, leaves of line KW 1074 plants contained 5-fold more sucrose and starch than the control ones. The same treatment of CM 109 seedlings resulted in accumulation of sucrose and starch by 2-fold and 8.5-fold, respectively. As the result of chilling-treatment, ¹⁴C assimilation rate (P_a), transport speed in the leaf blade (TS₁) and along the plant (TS_m) decreased by about 50 % in both lines. On the other hand, time necessary for radiolabel movement into the phloem loading region (AT) increased strongly, especially in chilling-sensitive line CM 109. It was also noted, that the radioactivity exported from leaves (R₁) and imported by roots (R_m) decreased in line CM 109, and increased slightly in line KW 1074. The activity of SPS extracted from leaves of both lines decreased by about 3.3 when temperature was lowered form 30°C to 10°C. There was no effect of 6 day treatment of chilling on SPS activity. Changes in sucrose and starch concentration, SPS activity as well as differences in transport parameters observed in KW1074 and CM109 seedlings at moderate low temperatures are discussed in terms of mechanism of maize chilling-sensitivity.     

Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning.

We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence. The 3509-bp cDNA encodes a 1068-amino acid polypeptide. The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli. Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product. Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase. When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism. The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves. This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.     

小麦开花后旗叶中蔗糖合成与籽粒中蔗糖降解

在小麦开花后,旗叶中蔗糖磷酸合成酶（SPS）活性在开花后14d内一直维持较高水平,蔗糖合成酶（SS）的活性在开花后14-28d较高,蔗糖的含量与SPS活性呈显著正相关,籽粒中蔗糖合成酶（SS）在开花后28d内一直维持较高的活性;与此相对应,籽粒蔗糖的含量在开花后28d内呈明显的下降趋势。而旗叶和籽粒中SS活性均与籽粒淀粉的积累速率呈极显著正相关。     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.)

Experiments were conducted with soybean (Glycine max [L.] Merr. cv `Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study.     

Regulation of leaf photosynthetic rate correlating with leaf carbohydrate status and activation state of Rubisco under a variety of photosynthetic source/sink balances

There is evidence suggesting that in plants changes in the photosynthetic source/sink balance are an important factor that regulates leaf photosynthetic rate through affects on the leaf carbohydrate status. However, to resolve the regulatory mechanism of leaf photosynthetic rate associated with photosynthetic source/sink balance, information, particularly on mutual relationships of experimental data that are linked with a variety of photosynthetic source/sink balances, seems to be still limited. Thus, a variety of manipulations altering the plant source/sink ratio were carried out with soybean plants, and the mutual relationships of various characteristics such as leaf photosynthetic rate, carbohydrate content and the source/sink ratio were analyzed in manipulated and non-manipulated control plants. The manipulations were removal of one-half or all pods, removal of one-third or two-third leaves, and shading of one-third or one-half leaves with soybean plants grown for 8 weeks under 10 h light (24 degrees C) and 14 h darkness (17 degrees C). It was shown that there were significant negative correlations between source/sink ratio (dry weight ratio of attached leaves to other all organs) and leaf photosynthetic rate; source/sink ratio and activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract; leaf carbohydrate (sucrose or starch) content and photosynthetic rate; carbohydrate (sucrose or starch) content and activation ratio of Rubisco; amount of protein-bound ribulose-1,5-bisphosphate (RuBP) in leaf extract and leaf photosynthetic rate; and the amount of protein-bound RuBP and activation ratio of Rubisco. In addition, there were significant positive correlations between source/sink ratio and leaf carbohydrate (sucrose or starch) content; source/sink ratio and the amount of protein-bound RuBP; carbohydrate (sucrose or starch) content and amount of protein-bound RuBP and the activation ratio of Rubisco and leaf photosynthetic rate. The plant water content, leaf chlorophyll and Rubisco contents were not affected significantly by the manipulations. There is a previous report in Arabidopsis thaliana that the amount of protein-bound RuBP in leaf extract correlates negatively with the activation ratio of Rubisco in the leaf extract. Therefore, the results obtained from the manipulation experiments indicate that there is a regulatory mechanism for the leaf photosynthetic rate that correlates negatively with leaf carbohydrate (sucrose and starch) status and positively with the activation state of Rubisco under a variety of photosynthetic source/sink balances.     

Effect of N-source on soybean leaf sucrose phosphate synthase, starch formation, and whole plant growth

Soybeans (Glycine max L. Merr. cv Tracy and Ransom) were grown under N₂-dependent or NO₃⁻-supplied conditions, and the partitioning of photosynthate and dry matter was characterized. Although no treatment effects on photosynthetic rates were observed, NO₃⁻-supplied plants in both cultivars had lower starch accumulation rates than N₂-dependent plants. Leaf extracts of NO₃⁻-supplied plants had higher activities of sucrose phosphate synthase (SPS) and cytoplasmic fructose-1,6-bisphosphatase (FBPase) than N₂-dependent plants. The variation in starch accumulation was correlated negatively with the activity of SPS, but not the activity of FBPase, UDP-glucose pyrophosphorylase, or ADP-glucose pyrophosphorylase. These results suggested that starch accumulation is biochemically controlled, in part, by the activity of SPS. Leaf starch content at the beginning of the photoperiod was lower in NO₃⁻-supplied plants than N₂-dependent plants in both cultivars which suggested that net starch utilization as well as accumulation was affected by N source.

Total dry matter accumulation and dry matter distribution was affected by N source in both cultivars, but the cultivars differed in how dry matter was partitioned between the shoot and root as well as within the shoot. The activity of SPS was correlated positively with total dry matter accumulation which suggested that SPS activity is related to plant growth rate. The results suggested that photosynthate partitioning is an important but not an exclusive factor which determines whole plant dry matter distribution.

     

Expression of a heterologous SnRK1 in tomato increases carbon assimilation, nitrogen uptake and modifies fruit development

SnRK1 (sucrose non-fermenting-1-related protein kinase 1) plays an important role in plant carbon metabolism and development. To understand the mechanism of carbon and nitrogen metabolism regulated by MhSnRK1 from pingyitiancha (Malus hupehensis Rehd. var. pinyiensis Jiang), two transgenic lines (T2-7 and T2-9) over expressing this gene in tomato were studied. SnRK1 activity in the leaves of 2 transgenic lines was increased by 15-16% compared with that in the wild-type. The leaf photosynthetic rate in transgenic tomatoes was higher than the wild-type. The activity of sucrose synthase breakdown and ADP-glucose pyrophosphorylase was also increased, by approximately 25-36% and 44-48%, respectively, whereas sucrose synthase synthesis and sucrose phosphate synthase activities were unchanged. The content of starch in the leaves and red-ripening fruits was higher than that of the wild-type. The transgenic fruit ripened ～10 days earlier than the wild-type. The nitrate reductase activity (mgplant?1 h?1) shows no significant difference between the transgenic plant and the wild-type, but the N-uptake efficiency and root/shoot ratio in the T2-9 line were 15% and 35% higher than that in the wild-type, respectively. These results suggest that over expressing MhSnRK1 can increase both the carbon and nitrogen assimilation rate of the plant as well as regulate the development of fruit.     

Nonstructural carbohydrate metabolism during leaf ageing in tobacco (Nicotiana tabacum)

Nonstructural carbohydrate status and activities of ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADPG pyrophosphorylase) and sucrose phosphate synthase (EC 2.4.1.14, SPS) were determined during ageing of tobacco ( Nicotiana tabacum L., cvs KY 14 and Speight G28) leaves sampled from control plants and from plants that had the apical meristem and subsequent axillary growth removed (detopped plants). Over the 30-day period shoot growth increased much more for control compared to detopped plants, but the increase in root growth was similar for both treatments. Dry matter and leaf area of the individual leaf used for enzyme and metabolite analysis were constant over time for controls but increased 5-fold for detopped plants. Ageing of control leaves was indicated by a progressive loss of chlorophyll and ribulose 1, 5-bisphosphate carboxylase (EC 4.1.1.39, Rubisco) activity; loss of these components was diminished for detopped plants. In contrast to chlorophyll and Rubisco activity, activities of ADPG pyrophosphorylase and SPS remained relatively constant over time for controls. Thus, under normal ageing conditions, changes in activities of ADPG pyrophosphorylase and SPS were not closely associated with changes in the standard senescence indicators chlorophyll and Rubisco activity. The activities of ADPG pyrophosphorylase and SPS were enhanced, relative to controls, within 6 days after applying the detopping treatment and activities remained high for the duration of the 30-day period. Detopping also led to increased concentrations of starch and sucrose, but the increases were not well correlated with changes in enzyme activities. The data indicated that the leaves of detopped plants functioned as both source leaves, with enhanced ability to synthesize carbohydrate, and sink leaves, with enhanced growth. Therefore, activities of ADPG pyrophosphorylase and SPS were more responsive to changes within an individual leaf than to changes in whole plant growth.