Specific Invertase Activity Research Articles

Effects of Dietary Inclusion of Bilberry and Walnut Leaves Powder on the Digestive Performances and Health of Tetra SL Laying Hens.

Simple SummaryIn poultry, diet composition influences growth performance, egg production, as well as digestion. In this study, the effects of dietary additives obtained from bilberry and walnut leaves powder on the digestive performances of Tetra SL hens were evaluated by histologic and morphometric analyses of the intestinal mucosa as well as by the enzymatic activity measurements of alpha-amylase, invertase, maltase, and trypsin correlated with cecum microbiota.The purpose of this study was to examine the effects of dietary inclusion of two additives at the final concentration of 0.5% bilberry (E1) and 1% walnut (E2) leaves powder in the basal diet on digestive health of hens. A total number of 90 Tetra SL hens were divided into two experimental groups (E1 and E2) and one control group (C) consisting of 30 hens each. After four weeks, 10 hens of each group were sacrificed and tissue samples and intestinal content were taken from the duodenum, jejunum, and cecum in order to perform histological, enzymatic, and microbiota analyses. In groups E1 and E2, the histological analysis showed a significant increase of villus height, resulting probably in increased absorption of nutrients in duodenum and jejunum. A decrease in the specific activity of alpha-amylase and trypsin in E1 and E2 for both duodenum and jejunum compared to the control one was also recorded. In addition, the maltase and invertase specific activity in duodenum increased, a tendency that was kept for maltase but not for invertase in jejunum. The cecal microbiota of E1 and E2 individuals was characterized by an increase of Firmicutes and Lactobacilli and a decrease of Enterobacteriaceae. In conclusion, our results indicate that bilberry and walnut leaves additives in feed may improve the health status of the poultry gastrointestinal tract.

Detection of Invertase Activity Produced by Saccharomyces cerevisiae as Source of Sucrose Degradation in Sugarcane Juice

The decline in purity of sugarcane juice used for the production of sucrose leads to huge losses in food processing chains. However, this sugar is much consumed by the population. Knowledge of the degradation origin of sucrose in the process of transformation is therefore necessary. It was more precisely a matter of assessing the quality of four sugarcane (Saccharum officinarum) juice of different varieties (R 570, R 590, Co 997 and SP 711406) by determining their physicochemical parameters and by highlighting the presence of Saccharomyces cerevisiae and invertase activity produced. These analyses were conducted in the Agrovalorization laboratory of the UFR Agroforesterie; University Jean Lorougnon Guédé, between November 2018 and February 2019. Thus, all juices are acidic (pH 5.05 ± 0.13) with average dry matter and sugar contents of 16.70 ± 1.04 and 59.03 ± 5.02, respectively, with favorable conditions for yeasts proliferation. In addition, S. cerevisiae was found in three juices (R 590, Co 997 and SP 711406) with a specific invertase activity of 0.42 10-2 IU / mg protein. This activity would be the basis of the degradation of sucrose in crude sugarcane juice. On the other hand, the juice resulting from the variety R 570 contained germ deprived of invertase activity (1.57 10-5 IU / mg of proteins). Its use would be a good source of crude materials for optimizing sugar production.

PURIFICATION OF AN EXO-INULINASE FROM BACILLUS SP. SG7

An exo-inulinase from strain Bacillus sp. SG7 was isolated and purified. A two-phase system PEG/Dextran, size-exclusion chromatography and ion-exchange chromatography were used in the purification process. The enzyme was purified to homogeneity with specific inulinase activity 18.47 U/mg protein and specific invertase activity 196.5 U/mg protein, purification fold of 10.44 and 27.4% yield. The molecular mass of the purified enzyme was estimated to be 56 000 Da. Strong inhibitors of enzyme activity are Pb, Hg, Zn and Cu ions with inhibition levels rising up to 55% for Cu and 95% for Pb. SDS totally inhibited the purified inulinase. The kinetic constants Km and Vmax for inulin as substrate were determined to be 1.0 mg/mL and 6.25 mg/mL.h, respectively. The pH optimum is at pH 7.0 and the enzyme is stable between pH 6.0 and pH 7.5, while retaining 100% of its initial activity between pH 6.5 and pH 7.0. The temperature optimum for the purified inulinase from strain Bacillus sp. SG7 was at 60°С. In the presence of inulin the purified inulinase sustains its activity at 100% for 55 minutes at 65°С. After the 70th minute the residual activity is 63% of the initial. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. The inulins from chicory (Cichorium intibus), from dahlia (Dahlia pinnata) and Jerusalem artichoke (Helianthus tuberosus) roots were hydrolysed by the purified enzyme.

Screening of Barley Resistance Against Powdery Mildew by Simultaneous High-Throughput Enzyme Activity Signature Profiling and Multispectral Imaging.

Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of H. vulgare (susceptible, mildew locus o (mlo) and Mildew locus a (Mla) resistant). Multispectral imaging of barley plants was daily conducted 0–8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with mlo and Mla based resistances 0–120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of Blumeria graminis f.sp. hordei (Bgh) conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The Mla resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening.

Sucrose hydrolysis by invertase using a membrane reactor: effect of membrane cut-off on enzyme performance

Sucrose hydrolysis by invertase [EC.3.2.1.26] produces inverted sugar syrup, an ingredient mainly used in the food industry. To properly catalyze hydrolysis, the enzyme should be reused after this reaction. It is advisable to maintain constant activity over a considerable period. Thus, sucrose hydrolysis was performed in a membrane bioreactor - a continuously stirred tank reactor coupled with an ultrafiltration membrane (UFM) which provides good diffusion and high activity per unit volume. Molecular weight cut-off for soluble invertase UFMs was up to 100kDa. This study focused on the role of UFM invertase cut-off as it is the main element in the process. We demonstrated that both the cut-off and chemical nature of the UFM affected specific invertase activity.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.

Long‐term effect of compost and inorganic fertilizer on activities of carbon‐cycle enzymes in aggregates of an intensively cultivated sandy loam

AbstractThe activities of carbon‐cycle enzymes were measured in soil and aggregates to understand compost and inorganic fertilizer amendment effects on soil organic carbon accumulation in an intensively cultivated upland field. Soil samples were collected from a long‐term field experiment with seven treatments: compost, half‐compost N plus half‐fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK and no fertilizer control. The 18‐yr continuous application of compost increased organic C content in soil and three aggregate sizes by 72–124 and 78–234%, respectively, compared with the control. Fertilization also significantly increased organic C contents in soil, macroaggregates and the silt + clay fraction, but not in microaggregates. Compost application significantly reduced the specific activities of polyphenol oxidase (activity per unit organic C) in soil and three aggregate sizes compared with control, whereas fertilization had a much weaker effect. Compost amendment also significantly lowered the specific activities of invertase in macroaggregates and the silt + clay fraction, and this effect was more pronounced than the addition of fertilizer NPK. In contrast, inorganic fertilizer and compost application significantly increased the specific activities of cellobiohydrolase in soil, macroaggregates and microaggregates (but not in the silt + clay fraction), and xylosidase in microaggregates. The application of fertilizer NPK had a more pronounced effect than compost. We considered that the increase in organic C in compost‐amended soil was therefore probably associated with the accumulation of lignocellulose and sucrose in macroaggregates, lignocellulose and hemicellulose in microaggregates and lignin (its derivative) and nonstructural carbohydrates in the silt + clay fraction. However, the application of fertilizer NPK enhanced organic C probably due to an increase in the content of lignin (its derivative) and sucrose in macroaggregates and the silt + clay fraction. Therefore, the application of compost with high lignocellulose should be effective to increase soil organic C in the North China Plain.

PRODUCTION OF FRUCTOSE RICH SYRUPS USING INVERTASE FROM DATE PALM FRUITS

ABSTRACT The invertase activity of eight varieties of date palm from the Tunisian oasis of Gabes and Jerid was extracted with distilled water and concentrated by ammonium sulfate precipitation at 80% saturation. The determination of invertase specific activity in these extracts showed that it was variable and higher in fruit from Gabes varieties (from 0.23 to 1.38 U/mg). Additionally, zymogram analysis showed the presence of at least two isoforms for tested extracts. The invertase extracted from Korkobbi variety has been more characterized. It presented an optimal temperature, optimum pH, apparent Km, apparent Vmax and catalytic efficiency of 45C, 3.5–4, 8.4 mM, 17.3 µmol/min/mL and 2.05 mM/µmol/min/mL, respectively. This invertase was mixed with an aqueous extract rich in sucrose prepared from the Deglet Nour variety. A high-fructose syrup was obtained after only 30 min of incubation. This syrup produced with a very low cost could be used as food additive in the food industry. PRACTICAL APPLICATIONS In Tunisia, discarded dates witnessed a remarkable increase during the last years and the amounts reached an average of 50,000 tons per year. Among these abandoned varieties are those of the Gabes oasis and a higher quantity of Deglet Nour varieties that are removed by sorting. In this context, our work focuses on valorizing these discarded dates by the implementation of a process aimed at the production of low-cost fructose-rich syrups destined to the agro-alimentary industry.

Immobilization of Invertase on Mesoporous Silicas to Obtain Hyper Active Biocatalysts

Comparative studies of invertase immobilized on two mesoporous cellular foams (MCF) and two supports of SBA-15 type with different pore sizes, using three highly recommended methods of protein bonding showed that activity of aminated MCF-based biocatalysts is significantly higher than that of SBA-15 counterparts, with the same textural properties. Specific activities of invertase covalently attached via GLA spacer was about 73–111% of a native enzyme. A unique microenvironment of functionalized cage-like pores of aminated MCFs is deemed to be responsible for this ultra high activity of invertase.

Effects of Pichia pastoris INO1 expression in Schizosaccharomyces pombe on phosphatidylinositol (PI) synthesis and expression of INV+ encoding invertase

In this study, Schizosaccharomyces pombe, a natural inositol auxotroph, was transformed with the two plasmids, pADH-INO and pSPIN-22, which contain a Pichia pastoris INO1 gene encoding inositol-phosphate synthase. The two transformants named Sch. p944 and Sch. p1025 were obtained, respectively. The results show that the plasmids in the cells could replicate autonomously and confer inositol prototrophy to S. pombe, but the rate of inositol synthesis and the amount of inositol synthesized in Sch. p944 were faster and more than those in Sch. p1025. Plasmid loss experiments show that the rate of plasmid loss in Sch. p944 was lower than that in Sch. p1025. More mRNA encoding inositol synthase in Sch. p944 was detected than that in Sch. p1025. We found that the highest specific invertase activity occurred when the Sch. p944 was grown in the synthetic medium containing 1.0% (w/v) glucose, whereas repression of invertase secretion occurred when Sch. p1025 was cultivated in the same medium containing glucose more than 0.2% (w/v). It was also found that more mRNA encoding secreted invertase and more phosphatidylinositol (PI) and less phosphatidylserine (PS) contents existed in Sch. p944 than those in Sch. p1025. The results show that the increase in produced inositol in the cells could lead to the increase of PI and the decrease of PS. These results also mean that PI may be involved in derepression of invertase secretion at transcriptional level.

Inositol and phosphatidylinositol-mediated invertase secretion in Schizosaccharomyces pombe

In this study, the effects of inositol addition on invertase secretion, biosynthesis of mRNA encoding secreted invertase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe were examined. We found that specific invertase activity was at its maximum when the concentration of added inositol reached 800 μg/100 ml in the synthetic medium containing 2.0% (w/v) sucrose. However, cell growth was steadily increased as inositol concentrations were increased from 0 to 1000 μg/100 ml. When the concentration of added inositol was 100 μg/100 ml in the synthetic medium, repression of invertase secretion occurred at sucrose concentration higher than 0.5% (w/v). However, when S. pombe was cultured in the synthetic medium containing 800 μg/100 ml, repression of invertase secretion occurred only at sucrose concentrations above 2.0% (w/v). Less residual reducing sugar was left in the 800 μg/100 ml inositol-containing medium than in the 100 μg/100 ml inositol-containing medium as sucrose concentration was increased. More mRNA encoding secreted invertase was detected in cells grown in the 800 μg/100 ml inositol-containing medium (2.0% sucrose) than in those grown in the 100 μg/100 ml inositol-containing medium. These results demonstrate that higher concentration of added inositol in the synthetic medium could derepress invertase secretion and INV + gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 800 μg/100 ml of inositol was higher than that of the yeast cells grown in the same medium with 100 μg/100 ml of inositol. This means that PI may be involved in derepression of invertase secretion.

Changes in Invertase Activities and Reducing Sugar Content in Sweetpotato Stored at Different Temperatures

Cured sweetpotato roots were stored at different temperatures (4.5, 15.6, and 24 degrees C) for 7 weeks and assayed for invertase activities and reducing sugar levels during two separate years. Invertase activities and reducing sugar concentration significantly increased in the roots kept at low temperature. Of the three types of invertases assayed, acid invertase specific activity was the highest. Acid invertase was the most influential in determining reducing sugar levels in stored sweetpotato. Cultivar differences were found in invertase specific activities and reducing sugar concentration. Reducing sugar content was highly correlated to acid and total invertase activity, regardless of cultivar.

An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection.

An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative "fur 1" mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur 1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur 1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system.

Fructan and fructan‐metabolizing enzymes in the growth zone of barley leaves

SUMMARYTo study the role of fructan in leaf development of barley seedlings, fructan contents and the activities of enzymes involved in fructan metabolism were measured in segments of the third leaf at the stage of maximal growth. Fructans amounted to more than 10% of the d. wt in the growth zone at the base of the developing leaf and decreased sharply in the zone above. The specific activities of sucrose: sucrose 1‐fructosyltransferase (1‐SST) and sucrose: fructan 6‐fructosyltransferase (6‐SFT), two key enzymes in fructan synthesis, also peaked in the growth zone but decreased more slowly in the zone above. By contrast, the specific activities of invertase and fructan exohydrolase (FEH) were low in the growth zone at the leaf base and had their peak just above it. The specific activity of fructan: fructan 1‐fructosyltransferase (1‐FFT) was approx. constant over the whole leaf. These results indicate that transitory accumulation of fructans in the growth zone plays a key role in the development of barley leaves.

Fructan and fructan‐metabolizing enzymes in the growth zone of barley leaves

To study the role of fructan in leaf development of barley seedlings, fructan contents and the activities of enzymes involved in fructan metabolism were measured in segments of the third leaf at the stage of maximal growth. Fructans amounted to more than 10% of the d. wt in the growth zone at the base of the developing leaf and decreased sharply in the zone above. The specific activities of sucrose∶sucrose 1‐fructosyltransferase (1‐SST) and sucrose∶fructan 6‐fructosyltransferase (6‐SFT), two key enzymes in fructan synthesis, also peaked in the growth zone but decreased more slowly in the zone above. By contrast, the specific activities of invertase and fructan exohydrolase (FEH) were low in the growth zone at the leaf base and had their peak just above it. The specific activity of fructan∶fructan 1‐fructosyltransferase (1‐FFT) was approx. constant over the whole leaf. These results indicate that transitory accumulation of fructans in the growth zone plays a key role in the development of barley leaves.

Effect of pH, aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O2 L-1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L-1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attaining S. cerevisiae cells suitable for invertase production were: temperature = 30 degrees C; pH = 5.0; DO = 3.3 mg O2 L-1; (S) = 0.5 g L-1 and sucrose added into the fermenter according to the equations: (V-Vo) = t2/16 or (V - Vo) = (Vf - Vo).(e0.6t-1)/10.

Fermentation kinetics of recombinant yeast in batch and fed‐batch cultures

Fed-batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned-gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed-batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy-efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.

An immobilized whole yeast cell biocatalyst for enzymatic sucrose hydrolysis

A novel granular immobilized biocatalyst with invertase activity was prepared by mutual covalent bonding of native bakers' yeast cells Saccharomyces cerevisiae with polyethyleneimine and glutaraldehyde without the use of any solid carrier. The specific invertase activity of the biocatalyst was 300–1500 Ug −1. The optimum pH value for the invertase activity was 4.6. The half-life of the invertase activity was 500–1000 h at reaction temperatures of 60–75°C. The activation energy of sucrose hydrolysis equaled 36.2 ± 5 kJ kmol −1 at reaction temperatures below 65°C. The enzyme reaction was inhibited by substrate at concentrations above 1.0–1.5 mol l −1, depending on the particle size. The biocatalyst was used for batch sucrose hydrolysis in laboratory-scale reactors at substrate concentrations of 10–70% (w/w), pH 4.6, and temperatures of 60–75°C. The specific productivity of the biocatalyst was 3–10 h −1. The total biocatalyst productivity over the period of its lifetime was 2000–10,000 kg of sucrose hydrolysed per kilogram of the biocatalyst. The mechanical stability of the biocatalyst particles was quite satisfactory even during long-term operation in stirred reactors. The biocatalyst was stored at temperature +4°C for 1 year without appreciable loss of the invertase activity. The results of the biocatalyst exploration confirmed its applicability for enzymatic hydrolysis of even highly concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

The effect of pretreatment on the invertase activity of gel-entrapped yeast biomass

Gel-entrapped, non-viable yeast biomass with specific invertase activity has been produced by two different pretreatment protocols: a short-time thermal treatment and a brief contact with concentrated ethanol solutions. Four yeast strains were most promising:K. fragilis L-293,C. utilis L-282,S. cerevisiae L-170 and L-209. Of these, the ethanol-tolerant L-282 and the ethanol-tolerant and heat-resistant L-170 gave the most active gel-entrapped biocatalysts: around 2 mg of reducing sugars produced per mg dry yeast per min.

Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenylacetic acid

Phenylacetic acid (PAA) significantly stimulated the elongation of isolated Phaseolus vulgaris internodal segments and prevented the decline in acid invertase specific activity observed in segments incubated in the absence of growth substances. Unlike IAA, which stimulated both elongation and invertase activity over a very wide range of concentrations (<10-4 - 1 mol.m-3; optimum 10-2 mol.m-3), the response to PAA was restricted to a much narrower range of concentrations (3 × 10-2 - 1 mol.m-3; optimum ca. 1–2 × 10-1mol.m-3). At the optimum concentration of PAA, the stimulation of both responses was about 63–75% of that induced by the optimum concentration of IAA. The differences in the concentration range and magnitude of the responses to IAA and PAA were not due to differences in uptake of the two compounds. The stimulation of elongation by both compounds was prevented by 3.6 × 10-2mol.m-3 cycloheximide (CH), and acid invertase activites were greatly reduced compared with samples treated with growth substances alone. A saturating concentration of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA) slightly promoted the growth of control segments, probably by reducing the loss of residual endogenous auxin to the incubation medium. The elongation induced by PAA at its optimum concentration was considerably greater than the elongation induced by NPA, indicating that PAA did not cause growth by preventing the loss of endogenous auxin from the segments. Elongation responses to combinations of IAA and PAA suggested that the compounds were acting additively and that they were affecting growth by the same mechanism.