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.     

Growth-promoting Activity of an Aqueous Extract of Soybean Seeds for Some Microorganisms

The metabolic fates of the carbon skeletons of leucine, lysine, and threonine were studied in growing rats on the diets containing graded levels of protein calorie percentages (10, 20, 30, and 40PC%) by use of either gluten or zein at 4100 kcal of metabolizable energy per kg of diets. In growth experiment for 21 days, body weight gain, food intake, and body fat increased at higher PC% in the gluten diets, but rats given zein did not maintain their initial weight even at 40PC%. The concentration of plasma free lysine remained low with the zein diets, but plasma threonine increased at 10 and 20PC% in the gluten and zein diets, respectively. Plasma leucine increased as the protein level increased either dietary protein. More than 70% of ¹⁴C was incorporated into body protein 12 h after an intraperitoneal injection of labeled lysine in all groups, but little ¹⁴CO₂ was expired in rats on the gluten and zein diets. About 79% of ¹⁴C-threonine was incorporated into body protein in rats given the gluten and zein diets at 10PC%, but the values were gradually decreased with increasing the dietary protein levels. Some 40–50% of ¹⁴C-leucine was incorporated into the body protein in rats given the gluten diets, and the values for the zein diets were extensively decreased in the higher PC% groups where the expired ¹⁴CO₂ was inversely increased to a great extent. These results showed that, when a specific amino acid was limiting or deficient in the diet, the major portion of the labeled amino acid was utilized for body protein synthesis and little was oxidized to carbon dioxide, whereas the oxidative degradation of essential amino acid other than limiting one was increased and the efficiency of the amino acid utilization was relatively decreased.     

C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants

Indole-3-acetic acid (IAA) labeled with ¹³C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion monitoring. [¹³C₆]IAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of [¹³C₆]IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

Carbon-11 Reveals Opposing Roles of Auxin and Salicylic Acid in Regulating Leaf Physiology,Leaf Metabolism,and Resource Allocation Patterns that Impact Root Growth in Zea mays

Auxin (IAA) is an important regulator of plant development and root differentiation. Although recent studies indicate that salicylic acid (SA) may also be important in this context by interfering with IAA signaling, comparatively little is known about its impact on the plant’s physiology, metabolism, and growth characteristics. Using carbon-11, a short-lived radioisotope (t _1/2 = 20.4 min) administered as ¹¹CO₂ to maize plants (B73), we measured changes in these functions using SA and IAA treatments. IAA application decreased total root biomass, though it increased lateral root growth at the expense of primary root elongation. IAA-mediated inhibition of root growth was correlated with decreased ¹¹CO₂ fixation, photosystem II (PSII) efficiency, and total leaf carbon export of ¹¹C-photoassimilates and their allocation belowground. Furthermore, IAA application increased leaf starch content. On the other hand, SA application increased total root biomass, ¹¹CO₂ fixation, PSII efficiency, and leaf carbon export of ¹¹C-photoassimilates, but it decreased leaf starch content. IAA and SA induction patterns were also examined after root-herbivore attack by Diabrotica virgifera to place possible hormone crosstalk into a realistic environmental context. We found that 4 days after infestation, IAA was induced in the midzone and root tip, whereas SA was induced only in the upper proximal zone of damaged roots. We conclude that antagonistic crosstalk exists between IAA and SA which can affect the development of maize plants, particularly through alteration of the root system’s architecture, and we propose that the integration of both signals may shape the plant’s response to environmental stress.     

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.     

Interaction of indoleacetic Acid with its inositol and glucoside conjugates in Avena coleoptile curvature

Avena coleoptile curvature is promoted by indoleacetic acid (IAA) IAA-glucoside, and IAA-inositol when these substances are applied in agar to the decapitated apical end of deseeded plantlets. Absorption of [³H]IAA-inositol over a wide range of concentrations during the 20 hour period of incubation is only 20 to 50% of the applied amount, compared with 85 to 92% of uptake of the applied [³H]IAA at equimolar concentrations. The absorption of IAA-glucoside could not be readily measured. The stimulation by both IAA-conjugates is very similar to that of free IAA at low concentrations (0.2 and 0.4 micromolar), but much less at higher concentrations. The interaction of free IAA with IAA-glucoside is additive or synergistic (depending on concentration). The interaction of free IAA with IAA-inositol is an inhibition (i.e. less than additive). The simultaneous application of equimolar concentrations of free IAA does not change the chromatographic pattern of the metabolic products of [³H] IAA-inositol. One of the more polar metabolites of [³H]IAA-inositol has chromatographic characteristics similar to the major polar metabolite of free [³H]IAA on an isocratically eluted reversed phase C₁₈ high performance liquid chromatography system that separates a number of IAA sugar and amino acid conjugates from each other, and from free IAA.     

Saturable uptake of indol-3yl-acetic Acid by maize roots

The uptake of 5-[³H]indol-3yl-acetic acid (IAA^*) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K_m of 0.285 micromolar and apparent V_max 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA^* took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.     

Bound indoleacetic Acid in Avena coleoptiles

When C¹⁴ carboxyl indoleacetic acid (IAA) is transported through Avena coleoptile sections a fraction of the activity becomes bound. The nature of this bound IAA has been investigated. Upon extraction with solvents and chromatography a substance having the R_F of IAA in 4 solvents was detected. No evidence could be found for the formation of indoleacetyl conjugates. In pea stem sections subjected to a similar experimental regime good evidence was obtained for the occurrence of conjugates. When IAA was supplied exogenously to coleoptile sections floating in solutions the occurrence of conjugates was shown to be dependent on the presence of the primary leaf. In its absence no conjugates could be detected.

On grinding coleoptile sections and subsequent centrifugation at 240 × g the radioactivity was found to be in the tissue fraction as opposed to the supernatant. The radioactivity cannot be removed from the tissue by extraction with water, buffer solution or treatment with ribonuclease. It is readily removed by 10% urea, crystalline trypsin and chymotrypsin. It is therefore concluded that IAA becomes bound to a protein. Bound IAA does not appear to be able to cause growth in Avena coleoptile sections.

     

Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.)

Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [¹⁴C]IAA and [¹³C₆]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The K_m value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant.     

Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand

A total of 1035 yeast isolates, obtained from rice and sugar cane leaves, were screened primarily for indole-3-acetic acid (IAA) production. Thirteen isolates were selected, due to their IAA production ranging from 1.2 to 29.3 mg g⁻¹ DCW. These isolates were investigated for their capabilities of calcium phosphate and ZnO₃ solubilisation, and also for production of NH₃, polyamine, and siderophore. Their 1-aminocyclopropane-1-carboxylate (ACC) deaminase, catalase and fungal cell wall-degrading enzyme activities were assessed. Their antagonism against rice fungal pathogens was also evaluated. Strain identification, based on molecular taxonomy, of the thirteen yeast isolates revealed that four yeast species – i.e. Hannaella sinensis (DMKU-RP45), Cryptococcus flavus (DMKU-RE12, DMKU-RE19, DMKU-RE67, and DMKU-RP128), Rhodosporidium paludigenum (DMKU-RP301) and Torulaspora globosa (DMKU-RP31) – were capable of high IAA production. Catalase activity was detected in all yeast strains tested. The yeast R. paludigenum DMKU-RP301 was the best IAA producer, yielding 29.3 mg g⁻¹ DCW, and showed the ability to produce NH₃ and siderophore. Different levels of IAA production (7.2–9.7 mg g⁻¹ DCW) were found in four strains of C. flavus DMKU-RE12, DMKU-RE19, and DMKU-RE67, which are rice leaf endophytes, and strain DMKU-RP128, which is a rice leaf epiphyte. NH₃ production and carboxymethyl cellulase (CMCase) activity was also detected in these four strains. Antagonism to fungal plant pathogens and production of antifungal volatile compounds were exhibited in T. globosa DMKU-RP31, as well as a moderate level of IAA production (4.9 mg g⁻¹ DCW). The overall results indicated that T. globosa DMKU-RP31 might be used in two ways: enhancing plant growth and acting as a biocontrol agent. In addition, four C. flavus were also found to be strains of interest for optimal IAA production.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

Effect of plant growth promoting Bacillus subtilis and Pseudomonas fluorescens on growth and pigment composition of radish plants (Raphanus sativus) under NaCl stress

Soil salinity is one of the most severe factors limiting growth and physiological response in Raphanus sativus. In this study, the possible role of plant growth promoting bacteria (PGPB) in alleviating soil salinity stress during plant growth under greenhouse conditions was investigated. Increasing salinity in the soil decreased plant growth, photosynthetic pigments content, phytohormones contents (indole-3-acetic acid, IAA and gibberellic acid, GA₃) and mineral uptake compared to soil without salinity. Seeds inoculated with Bacillus subtilis and Pseudomonas fluorescens caused significantly increase in fresh and dry masses of roots and leaves, photosynthetic pigments, proline, total free amino acids and crude protein contents compared to noninoculated ones under salinity. The bacteria also increased phytohormones contents (IAA and GA₃) and the contents of N, P, K⁺, Ca²⁺, and Mg²⁺ but decreased ABA contents and Na⁺ and Cl^? content which may contribute in part to activation of processes involved in the alleviation of the effect of salt.     

Soil Nitrogen Status Modifies Rice Root Response to Nematode-Bacteria Interactions in the Rhizosphere

It has been hypothesized that faunal activity in the rhizosphere influences root growth via an auxin-dependent pathway. In this study, two methods were used to adjust nematode and bacterial populations within experimental soils. One is “exclusion”, where soil mixed with pig manure was placed in two bags with different mesh sizes (1mm and 5μm diameter), and then surrounded by an outer layer of unamended soil resulting in soil with a greater populations of bacterial-feeding nematodes (1mm) and a control treatment (5μm). The second method is “inoculation”, whereby autoclaved soil was inoculated with bacteria (E. coli and Pseudomonas) and Nematodes (Cephalobus and C. elegans). In order to detect the changes in the rice’s perception of auxin under different nutrient and auxin conditions in the presence of soil bacterial-feeding nematodes, responses of soil chemistry (NH₄⁺, NO₃^- and indole acetic acid (IAA)), rice root growth and the expression of an auxin responsive gene GH3-2 were measured. Results showed that, under low soil nutrient conditions (exclusion), low NO₃^- correlated with increased root branching and IAA correlated with increased root elongation and GH3-2 expression. However, under high soil nutrient conditions (inoculation), a high NH₄⁺ to NO₃^- ratio promoted an increase in root surface area and there was an additional influence of NH₄⁺ and NO₃^- on GH3-2 expression. Thus it was concluded that soil bacterial-feeding nematodes influenced soil nutritional status and soil IAA content, promoting root growth via an auxin dependent pathway that was offset by soil nitrogen status.