The role of dietary polyphenols in the moderation of the inflammatory response in early stage colorectal cancer.

Current focus in colorectal cancer (CRC) management is on reducing overall mortality by increasing the number of early-stage cancers diagnosed and treated with curative intent. Despite the success of screening programs in down-staging CRC, interval cancer rates are substantial and other strategies are desirable. Sporadic CRC is largely associated with lifestyle factors including diet. Polyphenols are phytochemicals ingested as part of a normal diet, which are abundant in plant foods including fruits/berries and vegetables. These may exert their anti-carcinogenic effects via the modulation of inflammatory pathways. Key signal transduction pathways are fundamental to the association of inflammation and disease progression including those mediated by NF-κB and STAT, PI3K and COX. Our aim was to examine the evidence for the effect of dietary polyphenols intake on tumor and host inflammatory responses to determine if polyphenols may be effective as part of a dietary intervention. There is good epidemiological evidence of a reduction in CRC risk from case-control and cohort studies assessing polyphenol intake. It would be premature to suggest a major public health intervention to promote their consumption; however, dietary change is safe and feasible, emphasizing the need for further investigation of polyphenols and CRC risk.

Foliar application of GA3 during terminal long-shoot bud development stimulates shoot apical meristem activity in Pinus sylvestris seedlings.

The effect of exogenous gibberellin (GA3) on shoot apical meristem activity in conifer vegetative buds was investigated by spraying 0 or 0.1% GA3 on the foliage of first-year Scots pine (Pinus sylvestris L.) seedlings twice weekly for 9 weeks during development of the terminal long-shoot bud. Exogenous GA3 promoted mitotic activity in the apical zone, thereby increasing both the rate and duration of cataphyll formation and giving rise to a higher and wider apical meristem. The increase in number of cataphylls increased the number of axillary meristems, which developed as short-shoot buds.

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen.

By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18 promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening.

Effects of exogenous gibberellin and auxin on shoot elongation and vegetative bud development in seedlings of Pinus sylvestris and Picea glauca.

The hormonal control of stem unit (foliar appendage and axillary structure, if present, plus subtending internode) number and length was investigated in shoots of Scots pine (Pinus sylvestris L.) and white spruce (Picea glauca (Moench) Voss). Seedlings were treated with six gibberellins (GA1, GA3, GA4, GA5, GA9 and GA20) and two auxins (indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA)) when either neoformed growth was occurring or the terminal vegetative bud was developing. Hormones were applied by drenching the shoot tip, injecting the stem or spraying the foliage. Combined results for all three application methods indicated that shoot elongation in first-year seedlings (i.e., neoformed growth) was promoted in both species by GA1, GA3, GA4 and, less obviously, by GA9. This promotion was attributable to an increase in length, rather than number, of stem units. However, the number of stem units formed during terminal bud development, as reflected in the number of needles (white spruce) or cataphylls (Scots pine) present on the shoot resulting from the terminal bud, was stimulated by GA1, GA3 and GA4 in both species and by GA9 in Scots pine. The GA-induced increase in the number of preformed stem units was associated with increased bud width in white spruce and increased bud length and resulting shoot length in Scots pine. In contrast, application of IAA or NAA either did not affect or inhibited both neoformed growth and terminal bud stem unit number, depending on the application method and concentration. We conclude that, in the Pinaceae, (1) GA stimulates the activity of both the subapical meristem during neoformed growth and the apical meristem during vegetative bud development, and (2) the early non-hydroxylation pathway, via GA9, is the major route of GA biosynthesis. The role of auxin in the control of stem unit number and length remains to be resolved.

Gravimorphism in current-year shoots of Abies balsamea: involvement of compensatory growth, indole-3-acetic acid transport and compression wood formation.

We investigated the cause of gravimorphic growth inhibition in current-year shoots of balsam fir (Abies balsamea (L.) Mill.) seedlings displaced from their normal orientation in the gravitational field. Tilting the main stem of seedlings decreased shoot elongation, cambial growth as measured by tracheid production, and leaf dry weight of the terminal shoot and the lateral shoots on the lower side of the tilted stem. Removing either the terminal shoot or all lateral shoots induced compensatory growth in the remaining shoots, but did not reduce the inhibitory effect of tilting on shoot growth. Bending the apical part of a tilted main stem to restore it to the vertical did not fully reverse the inhibition of terminal shoot growth caused by stem tilting. Stem tilting inhibited cambial activity at the base of decapitated terminal shoots treated apically with indole-3-acetic acid (IAA) and decreased the basipetal transport of a [1-(14)C]-IAA pulse. Stem tilting also induced compression wood formation on the lower side of the tilted stem. Compression wood formation was associated with increases in cambial activity and stem respiration. Stem tilting did not affect either the net photosynthetic rate in 1-year-old leaves or the xylem water potential in current-year lateral shoots. These results support the hypothesis that gravimorphic growth inhibition in a current-year shoot on a tilted stem involves reductions in (1) the shoot's capacity to export IAA, and hence to mobilize photoassimilates, and (2) the supply of photoassimilates available for import by the shoot, as a result of increased cambial sink activity associated with compression wood formation outside that shoot.

Ethylene metabolism in Scots pine (Pinus sylvestris) shoots during the year.

Ethylene evolution, concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC conjugates, activities of ACC synthase and ACC oxidase, and cambial growth as measured by tracheid production were monitored from November to July in 1-year-old shoots, and between July and September in current-year shoots, of Scots pine (Pinus sylvestris L.). Needles, buds and four stem parts (cortex, phloem, cambial region and mature xylem) were surveyed. Ethylene evolution was quantified by gas chromatography. Free ACC and bound ACC (after acidic hydrolysis of ACC conjugates) were quantified by combined gas chromatography-mass spectrometry, with [2H]ACC as an internal standard. Activities of ACC synthase and ACC oxidase were measured in crude protein extracts. We detected high activities of ACC synthase in needles and buds, but not in stem fractions except the phloem. In July, during the period of intensive shoot growth, we found ACC only in buds and needles. In contrast, ACC oxidase activity was high in stem tissues, particularly in the cambial region during the period of rapid tracheid production, but no ACC oxidase activity was detected in needles and buds. Nevertheless, needles evolved large amounts of ethylene. Ethylene was produced by all stem fractions, and the peak rate of ethylene evolution in the cambial region coincided with the period of maximal tracheid production. Conjugated ACC was present in every fraction except mature xylem. The concentration of conjugated ACC decreased when rates of tracheid production and ethylene evolution were high, suggesting that conjugated ACC may serve as a source for ACC in the cambial region. The regulation of ethylene biosynthesis in Scots pine shoots is discussed.

Increasing the sink:source balance enhances photosynthetic rate of 1-year-old balsam fir foliage by increasing allocation of mineral nutrients.

To determine the impact of altering the sink:source balance on gas exchange rates in 1-year-old foliage of balsam fir (Abies balsamea (L.) Mill.), seedlings were either debudded before the growing season began or left intact. To assess alternative explanations for the observed photosynthetic response, additional seedlings were variously root pruned, shaded, drought stressed or deprived of fertilizer in combination with debudding. Foliar gas exchange rates and carbohydrate concentrations, xylem water potential and current-year growth were measured on three occasions spanning the shoot extension period. In addition, growth, foliar concentrations of chlorophyll, nitrogen, phosphorus and potassium, chlorophyll fluorescence and photosynthetic rate at different CO2 concentrations were determined 1 month after shoot extension ceased. Debudded seedlings had higher photosynthetic rates than budded seedlings, and the difference increased as the growing season progressed. We observed no evidence that debudding increased photosynthetic rate by improving water relations or by reducing foliar carbohydrate concentrations. Debudding increased foliar concentrations of nitrogen, phosphorus, potassium and chlorophyll, as well as quantum yield and photochemical quenching as determined by chlorophyll fluorescence measurements. Therefore, we conclude that debudding increased the photosynthetic rate by increasing the allocation of nutrients to 1-year-old foliage, thereby enhancing the amount or activity, or both, of photosynthetic enzymes, as well as increasing chlorophyll concentration.

Immunological and in-vivo neurological studies on a benzoic acid-specific T cell-derived antigen-binding molecule from the serum of a toluene-sensitive patient.

T-cell-derived antigen-binding molecules (TABMs) specific for benzoic acid were isolated from the serum of a toluene-sensitive patient. The resulting purified TABMs (BA-TABMs) did not contain immunoglobulin G and were associated with the cytokine transforming growth factor-beta (TGF-beta). BA-TABMs bound to benzoic acid conjugated to human serum albumin (BA-HSA), as well as to other chemicals conjugated to human serum albumin-including dinitrophenol and oxazolone. The binding of BA-TABMs to the conjugated chemicals increased the level of detectable TGF-beta, and a similar effect was observed with the unconjugated chemicals, benzoic acid and 2,4-dinitrophenol glycine. The increase in TGF-beta was critically dependent on the ratio between BA-TABMs and the conjugated or unconjugated chemicals; the increase was optimum at intermediate concentrations and absent at low and high concentrations. The authors used an established animal model in vivo and demonstrated that TGF-beta enhanced the inflammatory response induced by the release of neuropeptides from sensory nerves; this enhancement occurred in a dose-dependent manner. The BA-TABMs also enhanced this neurogenic inflammatory response in a dose-dependent manner, and this effect was blocked by anti-TGF-beta antibody. When the authors added either BA-HSA or benzoic acid, the effect of BA-TABMs on neurogenic inflammation was further enhanced at intermediate concentrations of antigen and was unaltered or reduced at higher concentrations. TABMs specific to particular chemicals, as a result of their association with cytokines (e.g., TGF-beta), may be implicated in symptom production in chemically sensitive patients.

Detection of antigen-specific human serum proteins related to the T-cell receptor in infectious disease and in an immune response to milk proteins or chemicals.

A monoclonal IgG2 antibody, MG3C9-1 A12, was prepared by immunization of mice with human serum Cohn Fraction III proteins enriched for TCR Ca+ proteins. MG3C9-1 A12 bound to Mr 28,000, antigen-specific TCR Ca+, beta-, and TCR Ca+, beta+ serum proteins associated with TGF-beta1, 2. The IgG2 monoclonal antibody also bound to T-lymphocyte proteins but did not bind to B lymphocyte proteins, human albumin, IgM, IgG, IgA, or TGF-beta1, 2, 3 immunogenic peptides. Monoclonal MG3C9-1 A12 detected TCR-related proteins specific for filarial extract, milk proteins, or benzoic acid in the sera of individuals with chronic or asymptomatic filariasis, milk intolerance, or sensitivity to toluene, respectively. TCR-related serum proteins were also detected intracellularly in mononuclear cells in frozen sections of ileum from a patient with milk intolerance and reactive mesenteric lymph nodes from a patient with a gastric ulcer. The results suggest that antigen-specific TCR-related serum proteins may be elevated during an immune response to oral, environmental, or infectious stimuli.

Measurement of T-cell-derived antigen binding molecules and immunoglobulin G specific to Candida albicans mannan in sera of patients with recurrent vulvovaginal candidiasis.

Immunoglobulin G (IgG) and T-cell-derived antigen binding molecules (TABM) specific to whole Candida extract and to Candida-derived mannans prepared by both the cetryltrimethylammonium bromide (CTAB) and alkaline degradation (PEAT) methods were measured in the sera of women with vulvovaginal candidiasis and controls. In the patients there were significantly higher levels of IgG to both CTAB and PEAT mannans and of TABM to CTAB mannan. TABM specific to CTAB mannan was purified from the serum of a patient with a high titer of this TABM. The purified TABM bound specifically to CTAB mannan and to other yeast and mold extracts. This TABM preparation was associated with transforming growth factor beta2 (TGF-beta2), and on specific binding to mannan there was a marked increase in the level of detectable TGF-beta2. This increase in TGF-beta2 level was critically dependent on the relative concentrations of the purified TABM and mannan, being smallest when either was in excess. The TABM specific to CTAB mannan was also shown to inhibit Candida-stimulated gamma interferon production. The results suggest that CTAB mannan-specific TABM may increase susceptibility to vulvovaginal candidiasis in association with a shift in the immune response to the Th2 type.

Clinical and immunological responses in subjects sensitive to solvents.

Twenty patients proved sensitive to a 15-min exposure to 15 ppm toluene. We assessed patients neuropsychologically before and after toluene exposure, and they had impaired cognitive functioning characterized by a deterioration in short- and long-term memory and psychomotor coordination. We measured total immunoglobin G and T-cell antigen-binding molecules against an antigen prepared by conjugation of para-aminobenzoic acid to human serum albumin in 20 patients and 16 controls. There was no significant difference in the immunoglobulin G levels to the antigen in the 2 groups, but the levels of T-cell antigen-binding molecules against the para-aminobenzoic acid conjugated to human serum albumin were elevated significantly in subjects sensitive to toluene. We also found significant associations between T-cell antigen-binding molecule levels and (a) decreased performance on the STROOP (Colour Word) test, (b) a shift in focal length following toluene exposure, (c) clinical assessment of disability, and (d) longer histories of chemical exposure. The measurement of T-cell antigen-binding molecules against chemical haptens may be valuable in the assessment of patients who are sensitive to chemicals.

Production of serum immunoglobulins and T cell antigen binding molecules specific for cow's milk antigens in adults intolerant to cow's milk.

The immune response to three cow's milk antigens, beta-lactoglobulin (BLG), alpha-lactalbumin (AL), and casein (CA) was studied in 15 milk-intolerant adult patients and 11 adult controls. IgG, IgE, and IgG subclasses (IgG1, IgG2, IgG3, IgG4) and T cell-derived antigen-binding molecules (TABM) specific for each antigen were measured in both groups. In the patient group, a significant elevation of total IgG and TABM against each of the milk antigens was found as well as raised levels of IgG1 to BLG and CA, IgG4 to BLG, and IgE to CA. TABM specific for BLG were isolated by affinity for BLG and found to be Mr 28,000-46,000 polypeptides functionally and physically associated with TGF-beta1 and TGF-beta2. These results indicate a Th2-type immune response to the milk antigens in milk-intolerant individuals compared with the control group which shows a pattern typical of anergy or deletion.

Ethylene evolution, radial growth and carbohydrate concentrations in Abies balsamea shoots ringed with Ethrel.

The terminal (1-year-old) shoot of quiescent, 2-year-old Abies balsamea (L.) Mill. seedlings was either left untreated or ringed with 0, 1 or 10 mg Ethrel g(-1) lanolin. After 5 weeks of culture under environmental conditions favorable for growth, the shoots were harvested to measure ethylene evolution and carbohydrate concentrations by gas chromatography, and tracheid number and bark radial width by microscopy. In untreated shoots, the basal rate of ethylene evolution followed the order: cambial region > cortex + periderm = xylem + pith = needles. Wound-induced ethylene production was not detected until at least 4 h after excision, but was evident in all fractions 24 h after excision; the increase in wound-induced ethylene evolution followed the order: cambial region > cortex + periderm > xylem + pith > needles. Compared with untreated controls, the application of plain lanolin, which involved the removal of needles and periderm, increased bark radial width and wound-induced ethylene production by the cambial region and the cortex + periderm, but decreased cambial region concentrations of fructose, glucose and starch at the application point. At the application point, Ethrel concomitantly increased ethylene evolution from the cambial region and the cortex + periderm, tracheid number, bark radial width, and the cambial region concentrations of fructose, glucose, sucrose and starch. No effects of Ethrel treatment were detected above or below the application point, with the exception that the 10 mg g(-1) Ethrel treatment stimulated ethylene evolution and decreased starch concentration of the cambial region. The results indicate that: (1) the cambial region is the major source of endogenous ethylene in the 1-year-old shoot; (2) the magnitude of the difference in ethylene evolution between particular shoot fractions is different before and after the start of wound-induced ethylene production; (3) the Ethrel-induced increase in tracheid number and bark radial width at the application point is positively related to ethylene evolution from the cambial region and the cortex + periderm, respectively; and (4) ethylene derived from Ethrel applied laterally to a woody stem can mobilize carbohydrates to the application point.

Control of longitudinal and cambial growth by gibberellins and indole-3-acetic acid in current-year shoots of Pinus sylvestris.

We investigated the involvement of gibberellins (GAs) and indole-3-acetic acid (IAA) in the control of longitudinal and cambial growth in current-year shoots of Pinus sylvestris L. Elongating terminal shoots, located at the apex of previous-year (1-year-old) branches in the uppermost whorl on the main stem, were variously decapitated (apical 5 to 10 mm removed), defoliated (all developing needle fascicles removed) and treated with endogenous GA(4/7) or IAA, or both. Shoot length and the radial widths of xylem and phloem were measured, and the concentrations of GA(1), GA(3), GA(4), GA(9) and IAA in the stem were determined by combined gas chromatography-mass spectrometry with deuterated GAs and [(13)C(6)]-IAA as internal standards. Decapitation decreased the production of xylem and phloem and the IAA concentration, but did not alter either longitudinal growth or the concentrations of GAs. Defoliation markedly inhibited shoot elongation, as well as cambial growth, and reduced the concentrations of GA(1), GA(3), GA(4), GA(9) and IAA. Application of GA(4/7) to defoliated shoots promoted longitudinal growth and phloem production, without affecting xylem production or IAA concentration. Application of GA(4/7) and IAA together to decapitated + defoliated shoots increased shoot elongation, xylem and phloem production and IAA concentration, whereas applying either substance alone had a smaller effect or none at all. We conclude that, for elongating current-year shoots of Pinus sylvestris, (1) both the shoot apex and the developing needle fascicles are major sources of the IAA present in the stem, whereas stem GAs originate primarily in the needle fascicles, (2) GAs and IAA are required for both shoot elongation and cambial growth, and (3) GAs act directly in the control of shoot growth, rather than indirectly through affecting the IAA concentration.

Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy.

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA beta-glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wild-type and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.

Laterally applied Ethrel causes local increases in radial growth and indole-3-acetic acid concentration in Abies balsamea shoots.

The terminal (1-year-old) shoot of quiescent, two-year-old balsam fir (Abies balsamea (L.) Mill.) seedlings was ringed with lanolin containing 0, 1 or 10 mg g(-1) Ethrel, an ethylene-generating compound, and cultured for 6 weeks under environmental conditions favorable for growth. Bud break and the elongation of the current-year terminal shoot were monitored, and the subjacent previous-year terminal shoot that had been treated with Ethrel was harvested to measure stem radial growth by microscopy, shoot ethylene evolution by gas chromatography, and cambial region indole-3-acetic acid (IAA) concentration by combined gas chromatography-mass spectrometry. Compared with the lanolin controls, Ethrel at 1 and 10 mg g(-1) did not affect bud break or longitudinal growth, but stimulated tracheid production and bark increment up to about 2-fold at the application site, though not above or below it. In addition, the 1 and 10 mg g(-1) Ethrel treatments increased the cambial region IAA concentration about 3-fold and the evolution of ethylene at least 40-fold at the application site, compared with unwounded portions of both treated and control shoots. The 10 mg g(-1) Ethrel treatment also stimulated ethylene evolution about 10-fold, both above and below the application site. However, this stimulation was not associated with an elevation in cambial region IAA concentration. Similarly, the lanolin control treatment increased ethylene evolution at the application site about 10-fold, without affecting the cambial region IAA concentration. Our results suggest that the localized stimulation of radial growth in woody shoots ringed with Ethrel is mediated by an increase in IAA concentration, which in turn is induced by a threshold, abnormally high concentration of Ethrel-derived ethylene.

Interaction between indole-3-acetic acid and ethylene in the control of tracheid production in detached shoots of Abies balsamea.

We investigated the interaction between indole-3-acetic acid (IAA) and ethylene in the regulation of the seasonal periodicity of tracheid production in 1-year-old balsam fir (Abies balsamea (L.) Mill.) cuttings collected at different times during the dormant period. The cuttings were left with their buds intact or were debudded and treated either apically with IAA or 2-chloroethylphosphonic acid (Ethrel) in lanolin, laterally with IAA or Ethrel in lanolin, or basally with Ethrel, Co(2+) or Ag(+) in deionized water. The treated cuttings were then cultured for up to 5 weeks under controlled environment conditions favorable for cambial growth. No change in ethylene evolution was detected during the rest-quiescence transition, when IAA-induced tracheid production increased. The induction of cambial reactivation by IAA was associated with a rise in ethylene evolution, but there was no consistent relationship among IAA concentration, tracheid number and ethylene emission. Neither Ethrel, Co(2+) nor Ag(+) affected tracheid production when applied basally, except for 10 and 100 micro M Ethrel and 100 micro M Co(2+), which were inhibitory. In contrast, ethylene evolution was promoted by Ethrel and inhibited by Co(2+), whereas Ag(+) had no effect. Similarly, applying Ethrel apically or laterally increased ethylene evolution, but did not promote tracheid production except in the treatment in which 1 mg Ethrel g(-1) lanolin was applied laterally to cuttings treated apically with 0.1 mg IAA g(-1) lanolin, and in the treatment in which 10 mg Ethrel g(-1) lanolin was applied laterally to budded cuttings. We conclude that (1) ethylene evolution is not specifically associated with IAA-induced tracheid production, (2) ethylene does not mimic the promoting effect of IAA on tracheid production, and (3) ethylene can promote tracheid production, but only when its application results in a localized unphysiologically high concentration in the cambial region, which, in turn, induces an accumulation of IAA.

The relationship between crown size and ring width in Pinus sylvestris L. stems: dependence on indole-3-acetic acid, carbohydrates and nitrogen in the cambial region.

Indole-3-acetic acid (IAA), carbohydrates, total nitrogen and amino acids in the cambial region and bark were measured at the top (10-year-old internode) and bottom (1.3 m) of the main stem of 50-year-old Pinus sylvestris L. trees, having different rates and longitudinal gradients of annual ring width formation. The trees were sampled during the most active period of wood production (June 23, July 15) and at the end of this period (August 23). Trees with a small crown and relatively slow growth rate (S-trees) were compared with trees in the same stand that had a large crown and fast growth rate (F-trees) as a result of thinning and fertilization. The effect of bottom pruning fast-growing trees (pruned F-trees) was also investigated. The F-trees had greater wood production than the S-trees at both the stem top and bottom. The difference was larger at the stem bottom, thus the relative decrease in ring width down the stem was steeper in the S-trees. The amount of sucrose and IAA per cm(2) in cambial region tissues was higher in F-trees than in S-trees. However, the differences in annual ring width between treatments and within trees were not reflected in the concentrations (expressed per gram fresh weight) of these substances, measured either in differentiating xylem, the cambium plus phloem, or in the cambial region as a whole. The concentrations of total nitrogen and amino acids were slightly higher in the F-trees than in the S-trees. Pruning the F-trees reduced wood production, particularly at the stem bottom, inhibited the springtime increase in starch, and decreased the amounts of sucrose and IAA per cm(2) in the cambial region. However, it was evident that the concentrations of sucrose, IAA, amino acid and nitrogen in the cambial region were not related to ring width. During June and July, the concentrations of these substances were generally higher at the stem bottom in the pruned F-trees than in the F-trees. The results provide evidence that it is the activity of the cambium rather than the availability of carbohydrates that determines the allocation of wood production along the tree stem. The results also indicate that tracheid production is not directly related to the IAA concentration in the cambial region.

Transgenic Tobacco Plants Coexpressing the Agrobacterium tumefaciens iaaM and iaaH Genes Display Altered Growth and Indoleacetic Acid Metabolism.

Transgenic tobacco (Nicotiana tabacum) SR1 plants expressing the Agrobacterium tumefaciens nopaline transferred DNA iaaH gene were transformed with a 35S-iaaM construct. The transformants displayed several morphological aberrations, such as adventitious root formation on stem and leaves, dwarfism, epinastic leaf growth, increased apical dominance, and an overall retardation in development. In addition, xylem lignification was higher than in wild type. Free and conjugated indoleacetic acid (IAA) levels were quantified by gas chromatography-multiple ion monitoring-mass spectrometry in leaves and internodes of wild-type plants and two transformed lines with different phenotypes. Both transformed lines contained elevated levels of free and conjugated IAA, which was associated with increased transcription of the iaaM gene. The line with the highest IAA level also had the most altered pattern of growth and development. These IAA-overproducing plants will provide a model system for studies on IAA metabolism, IAA interactions with other phytohormones, and IAA roles in regulating plant growth and development.