Effect of Photoperiod on Photosynthate Partitioning and Diurnal Rhythms in Sucrose Phosphate Synthase Activity in Leaves of Soybean (Glycine max L. [Merr.]) and Tobacco (Nicotiana tabacum L.)

Studies were conducted to identify the existence of diurnal rhythms in sucrose phosphate synthase (SPS) activity in leaves of three soybean (Glycine max L. [Merr.]) and two tobacco (Nicotiana tabacum L.) cultivars and the effect of photoperiod (15 versus 7 hours) on carbohydrate partitioning and the rhythm in enzyme activity. Acclimation of all the genotypes tested to a short day (7 hours) photoperiod resulted in increased rates of starch accumulation, whereas rates of translocation, foliar sucrose concentrations, and activities of SPS were decreased relative to plants acclimated to long days (15 hours). Under the long day photoperiod, two of the three soybean cultivars (`Ransom' and `Jupiter') and one of the two tobacco cultivars (`22NF') studied exhibited a significant diurnal rhythm in SPS activity. With the soybean cultivars, acclimation to short days reduced the activity of SPS (leaf fresh weight basis) and tended to dampen the amplitude of the rhythm. With the tobacco cultivars, photoperiod affected the shape of the SPS-activity rhythm. The mean values for SPS activity (calculated from observations made during the light period) were correlated positively with translocation rates and were correlated negatively with starch accumulation rates. Overall, the results support the postulate that SPS activity is closely associated with starch/sucrose levels in leaves, and that acclimation to changes in photoperiod may be associated with changes in the activity of SPS.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

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.     

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.     

Regulation of photosynthetic carbon metabolism in cucumber by light intensity and photosynthetic period

The effects of photosynthetic periods and light intensity on cucumber (Cucumis sativus L.) carbon exchange rates and photoassimilate partitioning were determined in relation to the activities of galactinol synthase and sucrose-phosphate synthase. Carbon assimilation and partitioning appeared to be controlled by different mechanisms. Carbon exchange rates were influenced by total photon flux density, but were nearly constant over the entire photoperiod for given photoperiod lengths. Length of the photosynthetic periods did influence photoassimilate partitioning. Assimilate export rate was decreased by more than 60% during the latter part of the short photoperiod treatment. This decrease in export rate was associated with a sharp increase in leaf starch acccumulation rate. Results were consistent with the hypothesis that starch accumulation occurs at the expense of export under short photoperiods. Galactinol synthase activities did not appear to influence the partitioning of photoassimilates between starch and transport carbohydrates. Sucrose phosphate synthase activities correlated highly with sugar formation rates (sucrose, raffinose, stachyose + assimilate export rate, r = 0.93, α = 0.007). Cucumber leaf sucrose phosphate synthase fluctuated diurnally in a similar pattern to that observed in vegetative soybean plants.     

Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations

Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. `Ransom' or `Arksoy') to determine whether sourcesink manipulations, which rapidly changed the `demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light.     

Role of sucrose-phosphate synthase in partitioning of carbon in leaves

Variations in leaf starch accumulation were observed among four species (wheat [Triticum aestivum L.], soybean [Glycine max L. Merr.], tobacco [Nicotiana tabacum L.], and red beet [Beta vulgaris L.]), nine peanut (Arachis hypogea L.) cultivars, and two specific peanut genotypes grown under different nutritional regimes. Among the genotypes tested, the activity of sucrose phosphate synthase was correlated negatively with leaf sucrose content in seven of the nine peanut cultivars as well as the two peanut cultivars grown with different mineral nutrition. The peanut cultivars differed in the effect of 10 millimolar sucrose on sucrose phosphate synthase activity in leaf extracts. Enzyme activity in crude leaf extracts was inhibited by sucrose (10-42%) in four of the cultivars tested whereas five cultivars were not. Overall, the results suggest that a correlation exists between the activity of sucrose phosphate synthase and starch/sucrose levels in leaves.     

Rhythmic growth and carbon allocation in Quercus robur. Sucrose metabolizing enzymes in leaves

The high sucrose phosphate synthase (SPS) capacity and the low soluble acid invertase activity of mature leaves of the first flush of leaves remained stable during second flush development. Conversely, fluctuations of sucrose synthase (SS) activity were in parallel with the sucrose requirement of the second flush. Sucrose synthase activity (synthesis direction) in first flush leaves could increase in 'response' to sink demand constituted by the second flush growth. Only the ptotosynthates provided by flush mature leaves were translocated for a current flush, while the starch content of these leaves remained stable. After their emergence, second flush leaves showed an increase in SPS and SS (Synthetic direction) activities. The high sucrose synthesis in second flush leaves was used for leaf expansion. When young leaves were 30% fully expanded (stage II20), SPS activity showed little change whereas SS activity declined rapidly toward and after full leaf expansion. The starch accumulation in the young leaves occured simultaneously with their expansion. Developing leaves showed a high level of acid invertase activity until maximum leaf expansion (stage II1). In first and second flush leaves, changes in acid invertase activity correlated positively with changes in reducing sugar concentrations. Alkaline invertase and sucrose synthase (cleavage direction) activities showed similar changes with low values when compared with those of acid invertase activity, especially in second flush leaves. The present results suggest that soluble acid invertase was the primary enzyme responsible for sucrose catabolism in the expanding common oak leaf.     

Diurnal Changes in Maize Leaf Photosynthesis : III. Leaf Elongation Rate in Relation to Carbohydrates and Activities of Sucrose Metabolizing Enzymes in Elongating Leaf Tissue

Maize (Zea mays L. cv. Pioneer 3184) leaf elongation rate was measured diurnally and was related to diurnal changes in the activities of sucrose metabolizing enzymes and carbohydrate content in the elongating portion of the leaf. The rate of leaf elongation was greatest at midday (1300 hours) and was coincident with the maximum assimilate export rate from the distal portion of the leaf. Leaf elongation during the light period accounted for 70% of the total observed increase in leaf length per 24 hour period. Pronounced diurnal fluctuations were observed in the activities of acid and neutral invertase and sucrose phosphate synthase. Maximum activities of sucrose phosphate synthase and acid invertase were observed at 0900 hours, after which activity declined rapidly. The activity of sucrose phosphate synthase was substantially lower than that observed in maize leaf source tissue. Neutral invertase activity was greatest at midday (1200 hours) and was correlated positively with diurnal changes in leaf elongation rate. There was no significant change in the activity of sucrose synthase over the light/dark cycle. Sucrose accumulation rate increased during a period when leaf elongation rate was maximal and beginning to decline. Maximum sucrose concentration was observed at 1500 hours, when the activities of sucrose metabolizing enzymes were low. At no time was there a significant accumulation of hexose sugars. The rate of starch accumulation increased after the maximum sucrose concentration was observed, continuing until the end of the light period. There was no delay in the onset of starch mobilization at the beginning of the dark period, and essentially all of the starch was depleted by the end of the night. Mobilization of starch in the elongating tissue at night could account for a significant proportion of the calculated increase in the tissue dry weight due to growth. Collectively, the results suggested that leaf growth may be controlled by the activities of certain sucrose metabolizing enzymes and may be coordinated with assimilate export from the distal, source portion of the leaf. Results are discussed with reference to diurnal photoassimilation and export in the distal, source portion of the leaf.     

The base of the leaf acts as a localized sink for photosynthate in mature barley leaves

The gradients in photosynthetic and carbohydrate metabolism which persist within the fully expanded second leaf of barley ( Hordeum vulgare ) were examined. Although all regions of the leaf blade were green and photosynthetically active, the basal 5 cm, representing approximately 20% of the leaf area, retained some characteristics of sink tissue. The leaf blade distal from the leaf sheath exhibited characteristics typical of source tissue; the activities of sucrolytic enzymes (invertase and sucrose synthase) were relatively low, whilst that of sucrose phosphate synthase was high. These regions of the leaf accumulated sucrose throughout the photoperiod and starch only in the second half of the photoperiod whilst hexose sugars remained low. By contrast the leaf blade proximal to the leaf sheath retained relatively high activities of sucrolytic enzymes (especially soluble, acid invertase) whilst sucrose phosphate synthase activity was low. Glucose, as well as sucrose, accumulated throughout the photoperiod. Although starch accumulated in the second half of the photoperiod, a basal level of starch was present throughout the photoperiod, by contrast with the rest of the leaf. The ¹⁴CO₂ feeding experiments indicated that a constant amount of photosynthate was partitioned towards starch in this region of the leaf irrespective of irradiance. These findings are interpreted as the base of the leaf blade acting as a localized sink for carbohydrate as a result of sucrose hydrolysis by acid invertase.     

Photosynthetic Determinants of Growth in Maize Plants: Effects of Nitrogen Nutrition on Growth, Carbon Fixation and Photochemical Features

Maize(Zea mays L.) plants were grown in a greenhouse with differentlevels of nitrate-N (2 to 20 millimolar). Nitrogen nutritionhad dramatic effects on plant growth and photosynthetic characteristicsof mature leaves. Increasing nitrogen resulted in greater biomassproduction, shoot/root ratios, and rates of leaf expansion duringthe day. The elongating zone of high-N plants had higher activities(per gram fresh weight) of sucrose synthase and neutral invertasethan low-N plants, suggesting that increased leaf growth wasrelated to a greater biochemical capacity for sucrose metabolism. Mature leaves of high-N plants had higher rates of photosynthesisand assimilate export (sucrose formation), and partitioned morecarbon into sucrose relative to starch. Increased photosyntheticrates (leaf area basis) were associated with higher levels ofribulose-l,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylaseand pyruvate, phosphate dikinase (determined immunochemically).In addition, N-nutrition affected the functional organizationof chlorophyll in the leaves. Large increases in the numberof PS I reaction centers were observed which fully accountedfor increases in leaf chlorophyll content with increasing nitratesupply. Collectively, the results suggest that increased growth of maizeplants at high light and optimal nitrogen nutrition is relatedto greater capacity for photosynthesis and translocation inmature leaves, and possibly increased capacity for sucrose metabolismin expanding leaves. (Received May 22, 1989; Accepted August 28, 1989)     

Comparative investigations on the distribution of sucrose synthase activity and invertase activity within growing, mature and old leaves of some C3 and C4 plant species

Sucrose synthase (EC 2.4.1.13) activity in young growing leaves was highest in the leaf base in eggplants ( Solanum melongena L.), cassava ( Manihot esculenta Crantz), grapevine ( Vitis vinifera L.), and in the leaf sheath of sugar cane ( Saccharum of ficinarum L.) and maize ( Zea mays L.). In addition, increasing sucrose synthase activity was measured towards the edge of growing eggplant leaves while the activity in mature leaves was highest in the midrib. The activity of acid and alkaline invertase was very low in the midrib but higher in the blade of fully expanded eggplant leaves. Highest invertase activities were found in younger growing leaves. It was concluded that in growing leaves a close relationship might exist between the activity of sucrose synthase and the import of sucrose from source leaves.
Detachment of mature eggplant leaves led to a 2- to 3-fold increase of sucrose synthase activity in blade and midrib of these leaves. In contrast, invertase activity decreased after detachment in both leaf blade and midrib. It was concluded that the rise in sucrose synthase activity might have been caused by the observed increase of sucrose concentration in detached leaves and that sucrose synthase might have an important role in the regulation of sucrose content of the conducting tissue.     

Grafting helps improve photosynthesis and carbohydrate metabolism in leaves of muskmelon

The most important quality for muskmelon (Cucumis melo L.) is their sweetness which is closely related to the soluble sugars content. Leaves are the main photosynthetic organs in plants and thus the source of sugar accumulation in fruits since sugars are translocated from leaves to fruits. The effects of grafting muskmelon on two different inter-specific (Cucurbita maxima×C. moschata) rootstocks was investigated with respect to photosynthesis and carbohydrate metabolism. Grafting Zhongmi1 muskmelon on RibenStrong (GR) or Shengzhen1 (GS) rootstocks increased chlorophyll a, chlorophyll b and chlorophyll a+b content and the leaf area in middle and late developmental stages of the plant compared to the ungrafted Zhongmi1 check (CK). Grafting enhanced the net photosynthesis rate, the stomatal conductance, concentration of intercellular CO(2) and transpiration rate. Grafting influenced carbohydrates contents by changing carbohydrate metabolic enzymes activities which was observed as an increase in acid invertase and neutral invertase activity in the functional leaves during the early and middle developmental stages compared to CK. Grafting improved sucrose phosphate synthase and stachyose synthase activities in middle and late developmental stages, thus translocation of sugars (such as sucrose, raffinose and stachyose) in GR and GS leaves were significantly enhanced. However, compared with CK, translocation of more sugars in grafted plants did not exert feedback inhibition on photosynthesis. Our results indicate that grafting muskmelon on inter-specific rootstocks enhances photosynthesis and translocation of sugars in muskmelon leaves.     

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.     

Regulation of key enzymes of sucrose biosynthesis in soybean leaves : effect of dark and light conditions and role of gibberellins and abscisic Acid

An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10⁻⁶m) and gibberellins 4 and 7 (10⁻⁵m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes.     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

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.     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.