An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [¹⁴C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [¹⁴C]Trp nor [¹⁴C]serine substituted for [¹⁴C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.     

Low-fluence red light increases the transport and biosynthesis of auxin

In plants, light is an important environmental signal that induces photomorphogenesis and interacts with endogenous signals, including hormones. We found that light increased polar auxin transport in dark-grown Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) hypocotyls. In tomato, this increase was induced by low-fluence red or blue light followed by 1 d of darkness. It was reduced in phyA, phyB1, and phyB2 tomato mutants and was reversed by far-red light applied immediately after the red or blue light exposure, suggesting that phytochrome is involved in this response. We further found that the free indole-3-acetic acid (IAA) level in hypocotyl regions below the hook was increased by red light, while the level of conjugated IAA was unchanged. Analysis of IAA synthesized from [13C]indole or [13C]tryptophan (Trp) revealed that both Trp-dependent and Trp-independent IAA biosynthesis were increased by low-fluence red light in the top section (meristem, cotyledons, and hook), and the Trp-independent pathway appears to become the primary route for IAA biosynthesis after red light exposure. IAA biosynthesis in tissues below the top section was not affected by red light, suggesting that the increase of free IAA in this region was due to increased transport of IAA from above. Our study provides a comprehensive view of light effects on the transport and biosynthesis of IAA, showing that red light increases both IAA biosynthesis in the top section and polar auxin transport in hypocotyls, leading to unchanged free IAA levels in the top section and increased free IAA levels in the lower hypocotyl regions.     

Auxin biosynthesis in maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.     

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

The membrane interface dictates different anchor roles for "inner pair" and "outer pair" tryptophan indole rings in gramicidin A channels

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

Starch biosynthesis and intermediary metabolism in maize kernels. Quantitative analysis of metabolite flux by nuclear magnetic resonance

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-(13)C(6)]Glc, [U-(13)C(12)]sucrose, or [1,2-(13)C(2)]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-(13)C(2)]Acetate was not incorporated into starch. [U-(13)C(6)]Glc or [U-(13)C(12)]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-(13)C(3)]- and [4,5,6-(13)C(3)]-isotopomers, whereas the [U-(13)C(6)]-, [3,4,5,6-(13)C(4)]-, [1,2-(13)C(2)]-, [5,6-(13)C(2)], [3-(13)C(1)], and [4-(13)C(1)]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-(13)C(2)]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose phosphate pathway.     

Metabolism and Synthesis of Indole-3-Acetic Acid (IAA) in Zea mays (Levels of IAA during Kernel Development and the Use of in Vitro Endosperm Systems for Studying IAA Biosynthesis)

Kernels of Zea mays on an intact plant accumulate indole-3-acetic acid (IAA) at the rate of 190 ng g-1 fresh weight h-1. Of the IAA synthesized, 97% is in the esterified form and less than 3% remains as the free acid. The site of biosynthesis of the IAA, whether synthesized in the leaf and transported to the kernel, or in the kernel and remaining in the kernel, has not been established. In an attempt to determine the locus of synthesis, we grew isolated kernels on agar media not containing tryptophan or other possible aromatic precursors of IAA and observed IAA synthesis of 99 ng g-1 fresh weight h-1, approximately 52% of the in situ rate. Thus, the kernel contains all of the enzymes required for de novo aromatic biosynthesis of IAA and its ester conjugates. Furthermore, endosperm cells in suspension culture, grown on hormone-free media and in the absence of aromatic precursors, are able to synthesize IAA at a rate of 9.2 ng g-1 fresh weight h-1, or 4.8% of the in situ rate. This finding establishes that all of the enzymes of IAA biosynthesis occur in the endosperm and that the endosperm is a site of IAA biosynthesis. Isolated endosperm, prepared from developing kernels, synthesized IAA from labeled anthranilate at a rate of 8.6 ng g-1 fresh weight h-1, or 4.5% of the in situ rate. Frozen endosperm preparations maintained the ability to synthesize labeled IAA from labeled anthranilate. The identity of the synthesized IAA was established by mass spectral analysis. We suggest that endosperm preparations of Z. mays are suitable for study of the mechanism(s) of IAA biosynthesis because they (a) have high rates of synthesis; (b) show stability to freezing, enabling enzyme storage; (c) provide a system with a known rate of in situ synthesis; and (d) are available in large amounts for use as an enzyme source.     

Biosynthesis of Indole-3-Acetic Acid from L-Tryptophan in Coleoptile Tips of Maize (Zea mays L.)

Coleoptile tips (about 2.5 mm in length) were excised from 3-day-olddark-adapted maize (Zea mays L.) seedlings and incubated indarkness in potassium phosphate buffer that contained ¹⁴C-L-tryptophan(Trp). Subsequent analysis by gas chromatography-mass spectrometryindicated that a significant portion of endogenous indole-3-aceticacid (IAA) had been labeled with ¹⁴C. About 8% of the IAA thatdiffused from the tissue into the medium during incubation from0.5 to 2 h was labeled, and 12% of the IAA extracted from thetissue after a 2-h incubation was labeled. On the other hand,30% of the Trp extracted from the tissue after a 2-h incubationwas ¹⁴C-Trp, which was more than those determined for IAA. Sincethe experiments were carried out under the non-steady-stateconditions in which the tissue content of ¹⁴C-Trp increasedwith time, and since the extracted Trp included the ¹⁴C-Trpin the apoplastic space, it seemed that synthesis de novo fromTrp was the major means by which IAA was produced in the coleoptiletip. The conversion of Trp to IAA was not detected in sub-apicalsegments (5–7.5 mm from the top) that were incubated similarly,an indication that synthesis of IAA occurs specifically in thetip region. When intact seedlings were irradiated with a pulseof red light 2 h before excision of tips and the applicationof ¹⁴C-Trp, the amounts of extractable and diffusible IAA werereduced by 40–60% without a change in the rate of ¹⁴Cincorporation. This result indicated that the production ofIAA from Trp is controlled by a red-light signal. (Received May 15, 1995; Accepted September 1, 1995)     

On the question of β-indolyl-acetic acid synthesis from indole without a tryptophan intermediate

Summary Experiments with sterile grown maize coleoptiles were carried out to decide whether or not a biosynthetic path for -indolyl-acetic acid (IAA) from indole exists without tryptophan occurring as an intermediate. -Indolyl-acrylic acid as a tryptophan synthetase inhibitor significantly reduces the yield of [³H]tryptophan obtained from [³H]indole while the reduction in the [³H]IAA yield is considerably less pronounced. This, however, indicates only a non-linear relationship between the tryptophan concentration and the IAA yield and not the sought path. Moreover, double labelling combined with isotope competition methods in experiments with [³H]indole and L-[¹⁴C]serin show that all IAA synthesized from [³H]indole is produced on a path involving the synthesis of tryptophan as an intermediate.Abbreviation IAA -indolyl-acetc acid     

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.     

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.     

活性氧对外源IAA诱导的ACC合酶活性的影响

本文试图从活性氧的角度阐明外源IAA诱导ACC合酶活性的机制.绿豆(Phaseolus radiatus L.)幼苗的乙烯产生及ACC合酶活性从萌发的第5天开始上升,到第10天达到高峰,接着下降.10 μmol/L的外源IAA能明显促进绿豆幼苗乙烯的产生及ACC合酶的活性,同时也促进了超氧阴离子自由基(O(-)/(*)2)、过氧化氢(H2O2)的产生.显示外源IAA诱导的ACC合酶的活性与其诱导的活性氧的产生具有某种相关性.外源O(-)/(*)2处理能明显提高绿豆幼苗的乙烯产生速率及ACC合酶的活性,而外源H2O2无论对乙烯产生或ACC合酶的活性均没有明显的作用.外加O(-)/(*)2的清除剂SOD对绿豆幼苗乙烯的产生及ACC合酶活性的提高有一定的抑制作用,而外源过氧化氢酶却没有明显的作用.为此我们可以得出结论:外源IAA诱导的绿豆幼苗ACC合酶活性的提高可能是由于其诱导的O(-)/(*)2产生的升高引起的,这可能也是高等植物中调控乙烯生物合成的机制之一;而IAA诱导的H2O2产率的升高并不是其诱导ACC合酶活性升高的原因.     

Surface-enhanced Raman difference between bombesin and its modified analogues on the colloidal and electrochemically roughen silver surfaces

In this article, surface-enhanced Raman scattering (SERS) spectra of bombesin (BN) and its six modified analogues ([D-Phe(12)]BN, [Tyr(4)]BN, [Tyr(4),D-Phe(12)]BN, [D-Phe(12),Leu(14)]BN, [Leu(13)-(R)-Leu(14)]BN, and [Lys(3)]BN) on a colloidal silver surface are reported and compared with SERS spectra of these species immobilized onto an ellectrochemically roughen silver electrode. Changes in enhancement and wavenumber of proper bands upon adsorption on different silver surfaces are consistent with BN and its analogues adsorption primarily through Trp(8). Slightly different adsorption states of these molecules are observed depending upon natural amino acids substitution. For example, the indole ring in all the peptides interacts with silver nanoparticles in a edge-on orientation. It is additionally coordinated to the silver through the N(1)--H bond for all the peptides, except [Phe(12)]BN. This is in contrary to the results obtained for the silver roughen electrode that show direct but not strong N(1)--H/Ag interaction for all peptides except [D-Phe(12),Leu(14)]BN and [Leu(13)-(R)-Leu(14)]BN. For BN only C==O is not involved in the chemical coordination with the colloidal surface. [Lys(3)]BN and BN also adsorb with the C--N bond of NH(2) group normal and horizontal, respectively, to the colloidal surface, whereas C--NH(2) in other peptides is tilted to this surface. Also, the Trp(8) --CH(2)-- moiety of only [Tyr(4)]BN, [Lys(3)]BN, and [Tyr(4),D-Phe(12)]BN coordinates to Ag, whereas the Phe(12) ring of [Phe(12)]BN, [Tyr(4),D-Phe(12)]BN, and [D-Phe(12),Leu(14)]BN assists in the peptides binding only on the colloidal silver.     

An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).     

The effect of light on metabolism of IAA in maize seedlings

Carbon 14-labelled indole-3-acetic acid (IAA) was fed to segments of shoots of Zea mays seedlings grown in light or dark to find the effect of light on IAA metabolism. The seedling parts coleoptile, with enclosed leaf, and mesocotyl were also used to examine differences in IAA metabolism between tissue types. The rate of metabolite formation as a function of time ranging from 1 to 12 hours was determined. Light did not significantly influence the amount of IAA taken up, but significantly increased its rate of metabolism and greatly increased the content of amide conjugates formed. There were also differences in metabolism depending on tissue type. In all tissues, IAA was metabolized mainly into six compounds. Four were tentatively identified as IAA-glucose (IAGlc), IAA-myo-inositol} (IAInos), indole acetamide (IAAm) and IAA-aspartic acid (IAAsp). 1-O-IAA-D-glucose (1-O-IAGlc) was the first conjugate formed and, except for mesocotyls in the light, it was the most abundant conjugate in maize tissue. In mesocotyl tissue the conversion of IAA into IAAsp was greatly stimulated by light, and the biosynthesis of IAAsp exceeded that of IAGlc. Since light strongly inhibited the growth of the mesocotyl, it is possible that the stimulation of IAAsp synthesis by light causes depletion of free IAA with resultant inhibition of mesocotyl growth.     

粪产碱菌的Tn5转座诱变及吲哚乙酸生物合成特性的研究

粪产碱菌(Alcaligenes faecalis)A1501的吲哚乙酸(IAA)合成需要外源色氨酸参与。在不含色氨酸的限制性培养基中,A1501能良好生长,但不能合成IAA,表明在A1501中存在一条依赖于色氨酸的IAA合成途径。A1501的IAA合成具有菌体密度依赖特性。采用Tn5转座诱变技术构建A1501的突变库,从3500多株Tn5转染子中分离到一株色氨酸营养缺陷型突变株AT63。该Tn5突变株在不含色氨酸的限制性培养基上不能生长,但仍能进行IAA的生物合成,每毫升菌体密度等于10的突变株菌体的IAA合成量为224μg。对突变株AT63的研究表明在A1501中至少存在两条IAA合成途径：一条以色氨酸为合成前体,另一条以吲哚-3-磷酸甘油为前体。Southern杂交结果表明突变株中Tn5插入位点可能位于编码色氨酸合成酶基因上。     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Tissue-Specific Expression of the β-Subunit of Tryptophan Synthase in Camptotheca acuminata, an Indole Alkaloid-Producing Plant

Camptothecin is an anticancer drug produced by the monoterpene indole alkaloid pathway in Camptotheca acuminata. As part of an investigation of the camptothecin biosynthetic pathway, we have cloned and characterized a gene from C. acuminata encoding the beta-subunit of tryptophan (Trp) synthase (TSB). In C. acuminata TSB provides Trp for both protein synthesis and indole alkaloid production and therefore represents a junction between primary and secondary metabolism. TSB mRNA and protein were detected in all C. acuminata organs examined, and their abundance paralleled that of camptothecin. Within each shoot organ, TSB was most abundant in vascular tissues. Within the root, however, TSB expression was most abundant in the outer cortex. TSB has been localized to chloroplasts in Arabidopsis, but there was little expression of TSB in C. acuminata tissues where the predominant plastids were photosynthetically competent chloroplasts. Expression of the promoter from the C. acuminata TSB gene in transgenic tobacco plants paralleled expression of the native gene in C. acuminata in all organs except roots. TSB is also highly expressed in C. acuminata during early seedling development at a stage corresponding to peak accumulation of camptothecin, consistent with the idea that Trp biosynthesis and the secondary indole alkaloid pathway are coordinately regulated.