Indole-3-acetic acid metabolism in Lemna gibba undergoes dynamic changes in response to growth temperature

Auxin is the mobile signal controlling the rate of growth and specific aspects of the development of plants. It has been known for over a century that auxins act as the messenger linking plant development to specific environmental changes. An often overlooked aspect of how this is accomplished is the effect of the environment on metabolism of the major plant auxin, indole-3-acetic acid (IAA). We have studied the metabolism of IAA in relation to one environmental variable, growth temperature. The model system used was an inbred line of the aquatic monocot Lemna gibba G-3, 3F7-11 grown at temperatures ranging from 5 degrees C to 35 degrees C. IAA levels, the rate of IAA turnover, and the patterns of label incorporation from IAA precursors were measured using stable isotope-mass spectrometric techniques and were evaluated relative to growth at the experimental temperatures. IAA levels exhibited unusually high variability in plants grown at 15 degrees C and 20 degrees C. Turnover rates were quite rapid throughout the range of experimental temperatures except at 25 degrees C, where IAA turnover was notably slower. These results suggest that a transition occurred over these temperatures for some aspect of IAA metabolism. Analysis of [(15)N]anthranilate and [(2)H(5)]tryptophan (Trp) incorporation into IAA showed that Trp-dependent biosynthesis predominated at 15 degrees C; however, Trp-independent biosynthesis of IAA was the major route to IAA at 30 degrees C. The effects of growth temperature on auxin levels have been reported previously, but no prior studies correlated these effects with which pathway becomes the primary one for IAA production.     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

A 5-methyltryptophan resistant rice mutant,MTR1, selected in tissue culture

Summary Cell lines resistant to tryptophan analogue 5-methyltryptophan (5MT) were selected in seed-derived calli of Oryza sativa L. var. Norin 8. Plants were regenerated (R₁ from one selected callus line (MTR1). In three out of the six R₁ plants, 5MT resistance was inherited in the R₂ and R₃ generations as a dominant nuclear mutation. Segregation ratios in the progeny of heterozygous plants were 11. Morphological and fertility variation seen in some of the R₂ plants were not correlated with 5-methyltryptophan resistance. Resistance in the MTR1 callus was due to the accumulation of high levels of free tryptophan (87-fold) that was associated with an increase in free phenylalanine content (9-fold). The leaves of resistant plants also contained elevated levels of free tryptophan and phenylalanine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - 5MT D,L-5-methyltryptophan - phe phenylalanine - trp tryptophan - tyr tyrosine     

Indole-3-acetic acid biosynthesis in Lemna gibba studied using stable isotope labeled anthranilate and tryptophan

IAA biosynthesis in many plants, including Lemna gibba, has been shown to involve at least two different pathways, one from tryptophan and a tryptophan-independent route. To study the kinetics of IAA biosynthesis in Lemna, we simultaneously measured the incorporation of label from [15N]-anthranilate and [2H5]-tryptophan into IAA by Lemna plants in short term feeding studies. The data show that label from anthranilate rapidly goes into IAA and tryptophan. Labeling of the IAA pool by [15N]-anthranilate slightly precedes labeling of the tryptophan pool, confirming that more than one route to IAA exists in these plants. Longer term feeding studies (5–25 h) suggest that exogenous tryptophan is used preferentially to label IAA as compared to tryptophan made by the plant. This is indicated by the fact that the IAA pool was more enriched than the tryptophan pool in [2H5]-label, but less enriched than the tryptophan pool in [15N] (which comes about by de novo synthesis of tryptophan from [15N]-anthranilate by the plant).     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Metabolic profiling of tryptophan-overproducing rice calli that express a feedback-insensitive alpha subunit of anthranilate synthase

The profile of aromatic metabolites in calli was compared between wild-type rice (Oryza sativa cv. Nipponbare) and tryptophan-overproducing transgenic rice lines that express a gene (OASA1D) for a feedback-insensitive alpha subunit of anthranilate synthase. Metabolic profiling by high-performance liquid chromatography coupled with photodiode array detection of ultraviolet absorbance revealed a total of 71 peaks in both wild-type and transgenic calli. Only a limited effect on the pattern of major aromatic compounds was observed in tryptophan-accumulating transgenic rice lines, with the exception of an approximately 80-fold increase in the amount of tryptophan. Expression of OASA1D induced relatively small changes in several minor metabolites. One of the minor metabolites whose abundance was increased by OASA1D expression was purified and identified as a previously unknown indole-alkaloid glucoside. The levels of free and conjugated forms of indole-3-acetic acid (IAA), a plant hormone derived from the tryptophan biosynthetic pathway, were determined separately by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The amounts of both free IAA and its conjugates were increased in the transgenic calli, suggesting that the activity of anthranilate synthase or the concentration of tryptophan (or both) is an important determinant of IAA biosynthesis.     

Phenol-oxidizing peroxidases contribute to the protection of plants from ultraviolet radiation stress.

We have studied the mechanism of UV protection in two duckweed species (Lemnaceae) by exploiting the UV sensitivity of photosystem II as an in situ sensor for radiation stress. A UV-tolerant Spirodela punctata G.F.W. Meyer ecotype had significantly higher indole-3-acetic acid (IAA) levels than a UV-sensitive ecotype. Parallel work on Lemna gibba mutants suggested that UV tolerance is linked to IAA degradation rather than to levels of free or conjugated IAA. This linkage is consistent with a role for class III phenolic peroxidases, which have been implicated both in the degradation of IAA and the cross-linking of various UV-absorbing phenolics. Biochemical analysis revealed increased activity of a specific peroxidase isozyme in both UV-tolerant duckweed lines. The hypothesis that peroxidases play a role in UV protection was tested in a direct manner using genetically modified tobacco (Nicotiana sylvestris). It was found that increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin. We conclude that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.     

Epiphytic microorganisms and IAA synthesis

Summary Epiphytic microorganisms present on cotton plants synthesized 3-indoleacetic acid (IAA) from tryptophan. Microorganisms from the root zone synthesized 3 times the amount of IAA when compared with the shoot zone and the root zone contained a much higher number of microorganisms. IAA-synthesizing activity was eliminated when the tissues were treated with a weak solution of mercuric chloride. Various tests on the possible accumulation of IAA from external sources showed that IAA synthesized outside the plant does not accumulate in the plant. Although epiphytic microorganisms synthesize IAA in large amounts, they do not influence the IAA content of the plant due to (1) lack of available tryptophan, (2) destruction of the auxin by the microflora, and (3) the polar movement of the auxin.     

The Control of Apical Bud Growth and Senescence by Auxin and Gibberellin in Genetic Lines of Peas

Pea (Pisum sativum L.) lines G2 (dwarf) and NGB1769 (tall) (Sn Hr) produce flowers and fruit under long (LD) or short (SD) days, but senesce only under LD. Endogenous gibberellin (GA) levels were inversely correlated with photoperiod (over 9-18 h) and senescence: GA20 was 3-fold and GA1 was 10- to 11-fold higher in flowering SD G2 shoots, and the vegetative tissues within the SD apical bud contained 4-fold higher levels of GA20, as compared with the LD tissues. Prefloral G2 plants under both photoperiods had GA1 and GA20 levels similar to the flowering plants under LD. Levels of indole-3-acetic acid (IAA) were similar in G2 shoots in LD or SD; SD apical bud vegetative tissues had a slightly higher IAA content. Young floral buds from LD plants had twice as much IAA as under SD. In NGB1769 shoots GA1 decreased after flower initiation only under LD, which correlated with the decreased growth potential. We suggest that the higher GA1 content of G2 and NGB1769 plants under SD conditions is responsible for the extended vegetative growth and continued meristematic activity in the shoot apex. This and the increased IAA level of LD floral buds may play a role in the regulation of nutrient partitioning, since more photosynthate partitions of reproductive tissue under LD conditions, and the rate of reproductive development in LD peas is faster than under SD.     

Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [¹⁵N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[¹⁵N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[¹⁵N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants.     

Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I₅₀. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

Comparative investigations of IAA metabolism in suspension cultures and plant organs fromBeta vulgaris (sugar beet) andChenopodium album L. (common lamb’s quarters)

Exogenoualy applicated indol-3-ylaeetic acid (IAA) is metabolized mainly to IAA aspartate in intact plants and plant segments and to IAA glucose in suspension cultures fromBeta vulgaris andChenopodium album. Main metabolic product of D-tryptophan is N-malonyltryptophan in both suspension cultures and hypocotyl segments of both species. The turnover rate of L-tryptophan to IAA is comparatively low (0.1 %); inBeta the turnover rate is higher than inChenopodium. In sugar beets phenmedipham leads to a decrease in the IAA. biosynthesis rate in suspension cultures of both plant species. There is, however, an increase in the IAA content in all intact plants. The metabolic activity is substantially higher in suspension cultures than in intact plants and plant segments.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan.

The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS), which is normally subject to feedback inhibition by tryptophan. Three independent trp5 mutants defective in the Arabidopsis thaliana AS alpha subunit structural gene ASA1 were identified by selection for resistance to the herbicidal compound 6-methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspartate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration causing 50% inhibition of AS activity in the trp5 mutant increased nearly 3-fold, the apparent Km for chorismate decreased by approximately 50%, and the apparent Vmax increased 60%. As a consequence of altered AS kinetic properties, the trp5 mutants accumulated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in trp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. thaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryptophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.     

Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.     

Leptin levels in lines of mice developed by long-term divergent selection on fat content

The mouse lines were developed by long-term selection for fatness, after which the fat line (F) had about a 5-fold (23% vs 4%) higher fat percentage than the lean (L) line at 14 weeks; but the lines differed little in fat-free body weight. To assess the contribution of genetic changes in leptin hormone level to the selection response, plasma leptin levels were assayed in these lines in generation 60 and in an unselected control (C) from the same base population. With access to food prior to assay, the F, C and L lines had 16.5, 0.91 and 0.26 ng/ml leptin, respectively. In fasted animals these levels were much lower: 2.98, 0.171 and 0.0087 ng/ml, respectively. Thus the leptin levels differ greatly between the lines, with the fattest mice showing the highest level: almost 20 times higher than the control and 60-300 times higher than the L line. These correlated selection effects are an order of magnitude greater than the direct selection response, and believed to be much larger than seen for any hormonal or other trait. Correlations between leptin level and fat amount were high (over 0.86) in fed or fasted animals of the F line, indicative of leptin resistance.     

Inbred lines of mice derived from long-term growth selected lines: unique resources for mapping growth genes

Lines of mice selected for many generations for high or low growth in several laboratories around the world have been collected, and from these, inbred lines are being developed by recurrent full-sib mating in Edinburgh. There are seven high selected lines and four low lines (each low line is from the same base population as one of the high lines), and the histories of each are summarized. Mean body weight of males at 70 days of age in the Edinburgh laboratory in the heaviest inbred line (77 g) is 4.8-fold higher than in the lightest line (16 g), and 1.9-fold higher than in the least extreme high line (41 g). Litter size, food intake, and fat content also differ substantially. These inbred extreme selected lines are a uniquely valuable resource for QTL or gene mapping, candidate gene identification, and elucidation of epistatic effects. Received: 19 February 2001 / Accepted: 3 May 2001