A Quantitative Estimation of Alkali-labile Indole-3-Acetic Acid Compounds in Dormant and Germinating Maize Kernels

An estimate has been made of the quantities of alkali-labile esters of indoleacetic acid (IAA) in kernels of sweet corn (Zea mays). The amount is between 70 to 90 mg of IAA per kilogram of dry kernels. About one-half of the IAA is present as high molecular weight esters and the remaining one-half as esters of myo-inositol. Free IAA, which may have existed in the kernels, or may have resulted from ester hydrolysis during isolation or storage, amounts to between 1 to 10% of the esterified IAA. Five newly observed low molecular weight indoleacetyl compounds are described and their chromatographic behavior reported. The total IAA content of corn kernels and intact seedlings decreases during germination, declining to about 10% of the original content during 96 hr of germination. Difficulties in obtaining quantitative results and the possible physiological significance of these results is discussed.     

Concentration and Metabolic Turnover of Indoles in Germinating Kernels of Zea mays L

The amounts and rates of metabolic turnover of the indolylic compounds in germinating kernels of sweet corn were determined. Knowledge of pool size and rate of pool turnover has permitted: (a) identification of indole-3-acetyl-myo-inositol as the major chemical form for transport of indole-3-acetic acid (IAA) from endosperm to shoot; (b) demonstration that the free IAA of the endosperm is turning over rapidly with a half-life of 3.2 hours; (c) identification of esters of IAA as the immediate precursors of IAA in the endosperm and shoot; (d) demonstration that neither tryptophan nor tryptamine is a major precursor of IAA for the seed or shoot; (e) identification of IAA-myo-inositol glycosides as precursors of IAA-myo-inositol.     

Movement of Indole-3-acetic Acid and Tryptophan-derived Indole-3-acetic Acid from the Endosperm to the Shoot of Zea mays L

The structures and the concentrations of all of the indolylic compounds that occur in the endosperm of the seeds of corn (Zea mays L.) are known. Thus, it should be possible to determine which, if any, of the indolylic compounds of the endosperm can be transported to the seedling in significant amounts and thus help identify the seed-auxin precursor of Cholodny (1935. Planta 23:289-312) and Skoog (1937. J. Gen. Physiol. 20:311-334). Of interest is the transport of tryptophan, indole-3-acetic acid (IAA), and the esters of IAA, which comprise 95% of the IAA compounds of the seed. We have shown that: (a) IAA can move from the endosperm to the shoot; (b) the rate of movement of IAA from endosperm to shoot is that of simple diffusion; (c) 98% of the transported IAA is converted into compounds other than IAA, or IAA esters, en route; (d) some of the IAA that has moved into the shoot has been esterified; (e) labeled tryptophan applied to the endosperm can be found as labeled IAA in the shoot; and (f) with certain assumptions concerning IAA turnover, the rate of movement of IAA and tryptophan-derived IAA from the endosperm to shoot is inadequate for shoot growth or to maintain IAA levels in the shoot.     

Structure of indole-3-acetic acid myoinositol esters and pentamethyl-myoinositols

GC-MS properties of three isomeric esters of indole-3-acetic acid and myoinositol, three esters of indole-3-acectic acid and myoinositol arabinoside and three esters of indole-3-acetic acid and myoinositol galactoside are presented. MS fragmentation patterns for the four possible pentamethyl myoinositols are also shown. These data indicated that the arabinose, and galactose of the glycosides were in the pyranose form and that C-1 of the sugar was linked to the 5 hydroxyl of myoinositol. Homologies in fragmentation patterns for the esters and the glycoside esters, together with knowledge of the properties of 2-O-indole-3-acetyl-myoinositol, permitted identification of one of the arabinosides as 5-O-l-arabinopyranosyl-2-O-indole-3-acetyl-myoinositol and one of the galactosides as 5-O-d- galactopyranosyl-2-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the arabinoside were then, most likely, the two mixtures of diastereoisomers 1 d- and 1 l-5-O-l-arabinopryranosyl-1-O-indole-3-acetyl myoinositol and 1 d- and 1 l-5-O-l-arabinopyranosyl-4-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the galactoside would then be the 1 d and 1 l-5-O-d-galactopyranosyl-1-O-indole-3-acetyl-myoinositol and 1 d- and 1 l-5-O-d- galactopyranosyl-4-O-indoleacetyl-myoinositol.     

Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose : II. Metabolic Characteristics of the Enzyme

The first enzyme-catalyzed reaction leading from indole-3-acetic acid (IAA) to the myo-inositol esters of IAA is the synthesis of indole-3-acetyl-1-O-β-d-glucose from uridine-5′-diphosphoglucose (UDPG) and IAA. The reaction is catalyzed by the enzyme, UDPG-indol-3-ylacetyl glucosyl transferase (IAA-glucose-synthase). This work reports methods for the assay of the enzyme and for the extraction and partial purification of the enzyme from kernels of Zea mays sweet corn. The enzyme has an apparent molecular weight of 46,500 an isoelectric point of 5.5, and its pH optimum lies between 7.3 and 7.6. The enzyme is stable to storage at zero degrees but loses activity during column chromatographic procedures which can be restored only fractionally by addition of column eluates. The data suggest either multiple unknown cofactors or conformational changes leading to activity loss.     

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues

Exogenous [¹⁴C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [¹⁴C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.     

Isomerization of 1-O-Indol-3-Ylacetyl-β-d-Glucose : Enzymatic Hydrolysis of 1-O, 4-O, and 6-O-Indol-3-YlacetyL-β-d-Glucose and the Enzymatic Synthesis of Indole-3-Acetyl Glycerol by a Hormone Metabolizing Complex

The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-β-d-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzymecatalyzed hydrolysis of 4-O- and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.     

Western blot analysis of cereal grain prolamins using an antibody to carboxyl-linked indoleacetic Acid

A monoclonal antibody raised against carboxyl-linked IAA was used in Western blot analysis of storage proteins from kernels of Avena sativa, Pennisetum americanum, Sorghum bicolor, and Zea mays. IAA or an IAA-like molecule is associated with the ethanolsoluble protein fraction of the seed. Western blotting of commercial zein, the major storage protein of maize, along with physicochemical evidence reported by Leverone et al. ([1991] Plant Physiol, 96: 1070-1075) indicated that IAA is linked with this prolamin. Results suggest that an IAA-prolamin association may be widespread throughout the Poaceae.     

UDP-glucose: Indoleacetic acid glucosyl transferase and indoleacetyl-glucose: Myo-inositol indoleacetyl transferase

We have demonstrated the in vitro enzymatic synthesis of an ester of indole-3-acetic acid (IAA) and glucose and of IAA and myo-inositol by the following reaction sequence: lt]o| li]1) IAA + UDPG ? IAA-glucose +UDP li]2) IAA-glucose +myo-inositol → IAA-itmyo-inositol +glucose The enzymes were partially purified from extracts of immature kernels of Zea mays sweet corn and the two activities separated on a Sephadex G-150 column. Products were characterized, primarily, by comparison of their 70 eV mass spectra with those of authentic synthetic standards. To our knowledge this is the first example of enzymatically catalyzed acylation by a 1-O-acylsugar.     

The Isolation and Characterization of d-Glucose 6-Phosphate Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cell Cultures: Its Occurrence in Plants

A soluble enzyme system from suspension cultures of Acer pseudoplatanus L. converts d-glucose 6-phosphate to myoinositol. A Mg²⁺-dependent phosphatase, present in the crude extract, hydrolyzes the product of the cyclization, myoinositol monophosphate, to free myoinositol. Further purification of the enzyme system by precipitation with (NH₄)₂SO₄ followed by diethylaminoethyl cellulose chromatography eliminates the phosphatase and makes it necessary to add alkaline phosphatase to the reaction mixture in order to assay for free myoinositol. Gel filtration on Sephadex G-200 increases the specific activity of the cycloaldolase to 8.8 × 10⁻⁴ units per milligram protein (1 unit = 1 micromole of myoinositol formed per minute). The cycloaldolase has an absolute requirement for nicotinamide adenine dinucleotide and a maximum activity at pH 8 with 0.1 mm nicotinamide adenine dinucleotide. The reaction rate is linear for 2.5 hours when d-glucose 6-phosphate is below 4 mm and has a K_m of 1.77 mm. The diethylaminoethyl cellulose-purified enzyme is stable for 6 to 8 weeks in the frozen state.     

Concentration of Indole-3-acetic Acid and Its Derivatives in Plants

Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oat, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. Three conclusions were drawn: (a) all plant tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grains examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are discussed.     

Reduction by a model of NAD(P)H: XI. Stereochemistry of a lactate dehydrogenase-model reaction

Stereochemistry of the biomimetic reduction of α-keto esters with NAD(P)H-model compounds has been investigated. The model compound with the R-configuration reduces the α-keto esters to the (R)-α-hydroxy esters, whereas (S)-α-hydroxy esters are afforded by the reduction with the S-configurational model compounds. It has been concluded that pro-R and -S hydrogens of the model compounds with R- and S-configuration, respectively, contribute predominantly to the reduction.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene

Tissues transformed with the isopentenyl transferase (ipt) gene cloned from the T-DNA region of the Ti plasmid or with the ipt gene placed under the control of the cauliflower mosaic virus 35S promoter (35S-ipt) were analyzed for auxin and cytokinin. Free and total indole-3-acetic acid (IAA) levels in 35S-ipt-transformed Nicotiana tabacum and cucumber cells were reduced by 12 to 78% in comparison to untransformed tissues. In contrast, free IAA concentrations in 35S-ipt-transformed Nicotiana plumbaginifolia were almost three times those of untransformed tissues, while total IAA levels were not significantly affected. Cytokinin levels in these antibodies were elevated an average of 300-fold resulting in a 24- to over 2000-fold increase in the cytokinin-to-auxin ratios. High cytokinin-to-auxin ratios correlated with the shooty phenotype of transformed tissues propagated in vitro in the absence of added growth hormones. We conclude that increased endogenous cytokinin-to-auxin ratios can induce cells to undergo morphogenesis and that elevated cytokinin levels can also induce auxin-autonomous growth of transformed tissues without causing a corresponding increase in endogenous IAA levels.     

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.     

Myo-Inositol Esters of Indole-3-acetic Acid as Seed Auxin Precursors of Zea mays L

Indole-3-acetyl-myo-inositol esters constitute 30% of the low molecular weight derivatives of indole-3-acetic acid (IAA) in seeds of Zea mays. [¹⁴C]Indole-3-acetyl-myo-inositol was applied to a cut in the endosperm of the seed and found to be transported from endosperm to shoot at 400 times the rate of transport of free IAA. The rate of transport of indole-3-acetyl-myo-inositol from endosperm to shoot was 6.3 picomoles per shoot per hour and thus adequate to serve as the seed auxin precursor for the free IAA diffusing downward from the shoot tip. Indole-3-acetyl-myo-inositol is the first seed auxin precursor to be identified.     

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.     

Indole-3-acetyl-myo-inositol in kernels of Oryza sativa

IAA-myo-inositol was isolated from kernels of Oryza sativa and characterized by its chromatographic properties and its mass spectral fragmentation pattern. This is the first demonstration of the occurrence of a myo-inositol ester of IAA in a plant other than Zea mays.     

The Conversion of d-Glucose-6-C to Cell Wall Polysaccharide Material in Zea mays in Presence of High Endogenous Levels of Myoinositol

When corn (Zea mays) roots are supplied with high concentrations of unlabeled myoinositol, the conversion of d-glucose-6-¹⁴C to cell wall galacturonic acid is significantly reduced compared to controls, although its incorporation into cell wall glucosyl units remains unchanged. This suggests that, in order to be converted to uronic acid, radiolabel from glucose must first pass through the internal myoinositol pool of the roots.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.