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Phleum pratense-specific T cells of allergic rhinitis patients display a broader recognition pattern than phleum pratense-specific serum immunoglobulin E.

BACKGROUND: The role of allergen-specific CD4+ T lymphocytes in the pathophysiology of atopic disease is well established. Previous studies on allergen-specific T-cell responses have focused on the recognition of single major allergens to identify T-cell epitopes. OBJECTIVE: However, it is not clear whether immune responses to allergen extracts are exclusively targeted at major allergens or whether additional proteins are recognized. METHODS: Here we describe the Phleum pratense-specific immunoglobulin E (IgE) and T-cell responses of six allergic rhinitis patients. Reactivity was measured to size-separated fractions of a P. pratense extract as well as to the purified major allergens Phl p 1, Phl p 2/3 and Phl p 5. RESULTS: The specificity of the patients' serum IgE, measured in a fluid phase assay, was restricted to one or two of the major allergens. Even though the majority of the patients had IgE antibodies reactive with a single major allergen, one patient reacted with both Phl p 5 and with Phl p 2/3. Analysis of the T-cell repertoire with P. pratense-specific T-cell lines (TCLs) and CD4+ T-cell clones (TCCs) revealed that at least six different proteins were recognized, including the three major allergens, most notably Phl p 5. Simultaneous production of IL-5 and interferon (IFN) -gamma was detected in supernatants of the TCLs stimulated with P. pratense extract and the major allergens. CONCLUSION: These results indicate that allergic rhinitis patients have a large pool of circulating allergen-specific CD4+ T cells that recognize many different proteins in an allergenic extract, whereas only a small number of these proteins are recognized by serum IgE.

Allergens↗

A double-blind, randomized study investigating the efficacy and specificity of immunotherapy with Artemisia vulgaris or Phleum pratense/betula verrucosa.

This study investigated the specificity and efficacy of immunotherapy with Artemisia vulgaris (Artemisia) extract. We studied 25 patients with a more than two year lasting history of seasonal rhinoconjunctivitis and who had only two clinically important allergies, either to Artemisia and Betula verrucosa (Betula) or to Artemisia and Phleum pratense (Phleum). Patient selection was randomized and evaluation of results was conducted in double blind fashion. Twenty patients completed two years of specific immunotherapy. Nine patients were treated with extracts of Artemisia and 11 with extracts of either Betula (n = 3) or Phleum (n = 8). Treatment with Artemisia was followed by a significant decrease in skin sensitivity (p < 0.05) and eye sensitivity (p < 0.01) to Artemisia but not to Betula/phleum. No significant decrease was observed in medicine consumption or symptom scores. Patients treated with Betula or Phleum experienced a significant decrease in skin sensitivity to Betula or Phleum (p < 0.001), and eye sensitivity to Betula or Phleum (p < 0.05), but not to Artemisia and had significant decreases in medicine and symptom scores (p < 0.05) in Betula/phleum seasons but not in the Artemisia season. The treatment was both effective and specific with the one unexplained exception that both patient groups (Artemisia and Betula/Phleum) decreased their skin sensitivity to Artemisia.

Adolescent↗

Comparison of in-house reference extracts of Dermatophagoides pteronyssinus and Phleum pratense with the WHO International Standards.

Two of the WHO First International Standards (IS) of allergenic extracts, the corresponding to Dermatophagoides pteronyssinus (House dust mite) and phleum pratense (Thymothy grass), have been used to assess the quality of our in-house reference preparations (IHR) and to calibrate their relative potency. The qualitative comparison was achieved by SDS-PAGE, Agarose-IEF and their respective enzime immunoblottings (Western blot and immunoprinting). The band patterns obtained were fairly similar for both Phleum pratense extracts. With the exception of two basic IgE binding proteins not detected in the IEF-blotting of the International Standard. However, several differences were noted between IS and IHR Dermatophagoides pteronyssinus extracts, indicating so the higher raw material heterogeneity. The difference was specially remarkable at the level of SDS-PAGE Immunoblotting since while our IHR extract showed similar ratio of Der p I to Der p II major allergens, the IS exhibited a large preponderance of Der p I, with Der p II allergen scarcely detected by our chromogenic method. Calibration of potency was done by means of RAST inhibition, therefore allowing the expression of our extract concentrations in International Units. The higher degree of homogeneity between Phleum pratense extracts was corroborated by the comparison of slopes from the RAST-inhibition plots.

Allergens↗

Genomic selection in timothy (Phleum pratense L.): a comprehensive evaluation of prediction models, multi-trait strategies, and forward validation across Norwegian environments.

This study presents a comprehensive evaluation of genomic selection (GS) in timothy (Phleum pratense L.), comparing nine prediction models across yield and quality traits at two Norwegian locations. Forward validation with independent full-sib (FS2) families revealed a substantial generalization gap, highlighting the need for realistic accuracy assessment in polyploid forage breeding. Timothy (Phleum pratense L.) is the most important forage grass in Northern Europe, yet genomic selection has not been systematically evaluated in this hexaploid species. We assessed 889 FS2-families originating from biparental crosses among 49 cultivars/populations. The FS2-families were genotyped with 30,698 SNP markers derived from genotyping-by-sequencing (GBS) and field tested for three harvest years at a highland and a lowland continental location in Southern Norway. Nine genomic prediction models were compared for six yield traits (dry matter yield per cut and total) and six quality traits (protein, digestibility, and fiber fractions) across three cuts/year. Within-training cross-validation accuracies were moderate to high (mean r = 0.62), with Random Forest and SVR consistently outperforming GBLUP. However, forward validation using 213 independent FS2-families revealed dramatically lower accuracies (mean r = 0.16), with only 16 of 30 trait-dataset combinations reaching statistical significance (p < 0.05). Genomic heritabilities (GREML), estimated across environments, ranged from near zero for the quality traits to 0.55 for the yield traits. Multi-trait models improved accuracy by 3-5% over single-trait approaches, while FS2 families-by-environment interaction models with Random Forest achieved the highest within-training accuracy (mean r = 0.71). Marker density analysis showed accuracy plateauing at approximately 15000 SNPs. Genetic correlations among the yield component traits were estimated by multi-trait REML; correlations among the quality traits could not be estimated reliably because their genomic heritabilities were low. A multi-trait selection index identified top-performing FS2-families for further crossing recommendations. These results provide a benchmark for GS implementation in hexaploid timothy and emphasize that cross-validation substantially overestimates prediction accuracy for truly independent material.

Norway↗

Effects of sulphur dioxide (SO2) on growth and flowering of SO2-tolerant and non-tolerant genotypes of Phleum pratense.

The objective of this study was to compare the growth and interaction of clipping and sulphur dioxide (SO(2)) exposure on SO(2)-tolerant and non-tolerant genotypes of Phleum pratense at two field sites along an SO(2)-concentration gradient. Sulphur-dioxide-tolerant and non-tolerant genotypes of Phleum pratense were identified from indigenous populations that had been collected along the same SO(2)-concentration gradient in southern Alberta, Canada. Physiological differences between the two genotypes were confirmed by supplying leaves with (14)CO(2) and examining the assimilate partitioning between the genotypes. For the field experiment, clones of each genotype and seedlings grown from commercial seed were planted at two different field sites along an SO(2)-emission gradient. There were no differences in growth between the genotypes at the two field sites after the first year except that the SO(2)-tolerant clones had a greater percentage of root length colonised by vesicular-arbuscular (VA) mycorrhizal fungi. After the second growing season, there was a significant decrease in the number of inflorescences produced by plants exposed to SO(2), particularly by the non-tolerant genotype. The added stress of defoliation appeared to increase the sensitivity of flowering to SO(2), again particularly in the non-tolerant genotype. The results of the field study showed that flowering as opposed to vegetative plant growth was more sensitive to long-term low-concentration SO(2) exposure and that this sensitivity was compounded by the stress interaction of defoliation.

Journal Article↗

Isolation and characterization of simple sequence repeat markers in the hexaploid forage grass timothy (Phleum pratense L.).

To develop simple sequence repeat (SSR) markers for the hexaploid forage grass timothy ( Phleum pratense L.), we used four SSR-enriched genomic libraries to isolate 1,331 SSR-containing clones. All four libraries contained a high percentage of perfect clones, ranging from 78.1% to 91.6%. From these clones, we developed 355 SSR markers when tested from 502 SSR primer pairs. Using all 355 SSR markers we tested one screening panel consisting of eight timothy clones to detect the level of polymorphism and identify a set of loci suitable for framework mapping. The SSR markers detected 90.4% polymorphism between the parents of a pseudo-testcross F(1) population. These SSR markers will provide an ideal marker system to assist with gene targeting, QTL (quantitative trait locus) mapping, and marker-assisted selection in timothy.

Base Sequence↗

Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense).

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

Allergens↗

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.).

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.

Allergens↗

Assessment of genetic variation in timothy (Phleum pratense L.) using RAPD and UP-PCR.

DNA-based fingerprinting technologies including random amplified polymorphic DNA (RAPD) and universally primed PCR (UP-PCR), a novel method for studying genetic variation, were employed as genetic markers for assessing genetic diversity and relationships in timothy (Phleum pratense L.). This study sought to identify the genetic background of the genotypes used in timothy breeding. Thirty eight genotypes from fifteen countries were used as test materials. RAPD and UP-PCR dendrograms based on 132 (from 3 primers) and 44 highly reproducible bands, respectively, were analyzed. The electrophoretic gels showed that the PCR products were informative and polymorphic. Different geographic genotype groups were distinguished according to the combined RADP and UP-PCR results. The results demonstrate that methods based on molecular fingerprinting can be used for timothy identification.

Breeding↗

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen.

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.

Allergens↗

Quantification of photoperiodic effects on growth of Phleum pratense.

BACKGROUND AND AIMS: Accurate quantifications of plant responses to photoperiod are useful for physiological studies, in growth modelling and in other studies of environmental effects. The objective of the current work was a mathematical description of photoperiodic influence on plant morphological traits, using functions with few and common parameters related to key plant characteristics and typical response patterns. METHODS: Two latitudinal cultivars of timothy (Phleum pratense) were studied in a climate chamber experiment at 9, 12, 15, 18, 21 and 24 h photoperiods. Seedling growth was recorded by measurements of main tiller leaf tip heights every other day from the 5-6 leaf stage onwards, and as plant size and dry weight at days 37, 46, 62 and 70, i.e. at the end of experiment. The plant responses to photoperiod were described by the term PPR = (eci(PP-PPc))/(1 + e(ci+di)(PP-PPc)), where PP = photoperiod in h, PPc = photoperiod of maximum response, c = characteristic coefficient of main response interval, d = sensitivity coefficient characterizing course of function beyond the main response interval. PPR was tested on experimental data for different growth characteristics (i), e.g. size of individual leaves (Yi), identified by their sequential numbers on the main tiller (LN) using the function: Yi = Ybi + aiLN + biLNalphai (PRR). The growth course was described by the same function, replacing LN with day number of treatment exposure. KEY RESULTS AND CONCLUSIONS: The functions described with high precision (r2 > 0.97) the effect of photoperiod on growth as expressed by several plant characteristics, such as leaf area development, top and root DM production, as well as cultivar differences. Green leaf area was more sensitive to photoperiod than above-ground DM production. The southern cultivar 'Grindstad' was more sensitive than the northern one 'Engmo'. The functional relationships suggest mechanisms for plants' daylength responses and latitudinal adaptation.

Models, Biological↗

Diurnal variation in uptake and xylem contents of inorganic and assimilated N under continuous and interrupted N supply to Phleum pratense and Festuca pratensis.

Compensation by dark-period uptake of NH(4)(+) and NO(3)(-) in the grasses Phleum pratense L. and Festuca pratensis Huds. following N deprivation during the preceding light period was investigated in flowing solution culture under an artificial 10/14 h light/dark cycle. N was supplied as either NO(3)(-), NH(4)(+) or NH(4)NO(3) at 20+/-5 mmol m(-3), available continuously or only during the dark period, for 5-10 d. Intermittent N supply did not affect total daily N uptake, growth rate or net partitioning of dry matter. Net uptake and influx of NO(3)(-) varied similarly throughout the diurnal cycle when NO(3)(-) was supplied continuously, with a marginal contribution by NO(3)(-) efflux. Influx was significantly higher and efflux slightly higher following interruption of NO(3)(-) supply during the light period. Nitrate accounted for 80% of N in xylem exudate except between hours 6-9 of the light period when the amino acid concentration increased 3-fold, primarily as glutamine. Diurnal variation in relative NO(3)(-) uptake exhibited five phases of constant acceleration/deceleration, described reasonably well assuming NO(3)(-) influx was subject to metabolic co-regulation by NO(3)(-) and amino acid levels in the cytoplasmic compartment of the roots. Accordingly, influx is determined by variation in root NO(3)(-) levels throughout the dark period and the first half of the light period, but is down-regulated by increased amino acid levels during the second half of the light period. The sharp light/dark transitions affect transpiration rate and hence xylem N flux which, in turn, affect NO(3)(-) levels in the cytoplasmic compartment of the roots and the rate of NO(3)(-) assimilation in the shoot.

Circadian Rhythm↗

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

Allergens↗

Double-blind study of tolerability and antibody production of unmodified and chemically modified allergen vaccines of Phleum pratense.

BACKGROUND: The physicochemical modification of allergen extracts provides a chance for administering higher doses of allergen vaccines. OBJECTIVE: To evaluate the safety of a chemically modified (depigmented-glutaraldehyde polymerized) therapeutic vaccine of Phleum pratense administered at doses that are 10 times higher than those used in clinical practice, in comparison with conventional doses of the corresponding non-modified alum-adsorbed vaccine. MATERIALS AND METHODS: The design of the study was randomized, double-blind, parallel and included two groups of patients. Twenty-three patients were treated weekly during nine visits for the build-up phase, followed by two weekly maintenance doses (a total of 11 injections per patient). Twelve patients received a vaccine containing the standardized unmodified extract, at a maximum concentration of 308.5 mcg of freeze dried material/mL (Group A). Eleven patients received a standardized modified allergen extract (Group B). The maximum dose used was 2400 mcg/mL. Safety was evaluated recording all adverse events. Skin test results and specific antibody levels were evaluated at the beginning and at the end of the study. RESULTS: Group A patients experienced three local immediate (two clinically irrelevant and one with a diameter>5 cm) and 18 delayed reactions (15 irrelevant and three with a diameter>10 cm), while Group B experienced six local immediate and 12 delayed reactions (all clinically irrelevant). Nine Group A patients experienced 12 systemic reactions (one immediate of grade 1, one of grade 2; and one delayed of grade 1; four of grade 2 and three of grade 3), while Group B patients experienced one immediate systemic reaction of grades 1, and 1 delayed reaction of grade 1. CONCLUSIONS: The modified extract of P. pratense is safe to treat sensitive patients, even at concentrations that are 10 times higher than those regularly administered in clinical practice. The majority of the local reactions were clinically irrelevant. No systemic reactions of grade 2, 3 or 4 were reported using the modified extract.

Adult↗

Intratracheal instillation of cytoplasmic granules from Phleum pratense pollen induces IgE- and cell-mediated responses in the Brown Norway rat.

BACKGROUND: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. OBJECTIVES: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. METHODS: PCG were purified from timothy grass pollen by filtration through 5-microm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 x 10(6) PCG/rat). Rats were then challenged 4 weeks later (1.5 x 10(6) PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [(3)H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. RESULTS: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. CONCLUSIONS: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.

Administration, Inhalation↗

Mouse strain specificity of the IgE response to the major allergens of Phleum pratense.

BACKGROUND: IgE immune responses against major allergens from Phleum pratense in low- and high-responder mouse strains were compared and the influence of alum was assessed, in order to evaluate the effect of the genetic background and adjuvants on IgE reactivity in a mouse model for P. pratense allergy. METHODS: Different mouse strains and F1 offspring were sensitized with P. pratense pollen extract. Serum IgE levels, the induction of specific IgE antibodies and immediate cutaneous hypersensitivity reactions were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: All mouse strains investigated mounted an IgE response and exhibited a positive skin test to pollen extract. Differences were seen in the level of total serum IgE and in specific IgE reactivity to different major allergens of P. pratense. Notable differences were seen in IgE reactivity and immediate hypersensitivity against Phl p 1, which were only observed in SJL/j mice. The foremost influence of alum was on total IgE production levels. CONCLUSIONS: Alum is not necessary as an adjuvant to elicit IgE reactivity against the clinically relevant allergens of P. pratense, since even low-responder mouse strains mounted a hypersensitivity reaction after sensitization without the adjuvant using otherwise identical sensitization strategies. Moreover, when analyzing the allergenicity of a compound, the hypersensitivity response of different mouse strains should be considered, as implicated by the differential results obtained for IgE reactivity against Phl p 1. Lastly, a genetic component may be involved in IgE reactivity to Phl p 1.

Allergens↗

Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential.

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

Allergens↗

Graviresponse and the localization of its initiating cells in roots of Phleum pratense L.

Roots of Phleum pratense L. were photographed during both vertical growth and gravitropic bending, and positions of anticlinal rhizodermal cell walls were digitized on the physically upper and lower flanks of the root in the curvature plane. By using B-splines, arc lengths of these positions, i.e. distances along the root surface, values of curvature, and relative elemental rates of elongation were estimated. The whole graviresponse can be divided into phases according to growth-rate values: (i) an increase of rates on the upper side of the root and a decrease on the lower side during the first 1-1 1/2 h after the root has been moved from the vertical to a horizontal position, (ii) a transient equality of the rates on both sides, (iii) 2-3 h after the beginning of graviresponse, the growth gradient is inverted, and (iv) finally, after about 4 h, the growth rates of both flanks are approximately equal again. Curvature begins 15-20 min after horizontal placement of the root. During the first 2 h of graviresponse, plots of curvature versus arc length show one maximum value. After 2-2 1/2 h, two maximum values can be observed, the apical one near the root tip always keeping the same distance from the tip, the other one drifting basipetally relative to the growing tip. By evaluating photographs of high magnification, a group of six rhizodermal cells on each side of the root was identified which are the first cells showing gravitropic bending. These cells are located at the beginning of the elongation zone, enclosing the region 480-680 micrometers from the root tip. These cells might be target cells for a signal which the statenchyma, the site of graviperception, sends to the reacting zone of gravicurvature.

Gravitropism↗