Structural, biochemical, and phylogenetic analyses suggest that indole-3-acetic acid methyltransferase is an evolutionarily ancient member of the SABATH family

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 A resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.     

The possible action mechanisms of indole-3-acetic acid methyl ester in <Emphasis Type="Italic">Arabidopsis</Emphasis>

We previously reported that Arabidopsis indole-3-acetic acid (IAA)-methyltransferase-1 (IAMT1) catalyzes the conversion of IAA, an essential phytohormone, to methyl-IAA (MeIAA) and that IAMT1 plays an important role in leaf development. Here, we present the possible mechanisms of action of MeIAA in Arabidopsis. We showed that MeIAA was more potent than IAA in the inhibition of hypocotyl elongation and that MeIAA and naphthalene-acetic acid (NAA), but not IAA, rescued the hypocotyl gravitropic defects in dark-grown aux1. However, MeIAA was less potent than IAA in the inhibition of primary root elongation in light-grown seedlings, and could not rescue the agravitropic root phenotype of aux1. MeIAA had a stronger capacity to induce lateral roots than both IAA and NAA and rescued the defective lateral root phenotype of aux1 seedlings. However, its capacity to induce root hairs was weaker than IAA and NAA and did not rescue the defective root hair phenotype of aux1 seedlings. These data indicate that MeIAA is an inactive form of IAA. The different sensitivities to MeIAA among different organs probably resulted from different expression localization and capacities of a putative MeIAA esterase to convert MeIAA to IAA.     

An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.     

Three polyphenol oxidases from hybrid poplar are differentially expressed during development and after wounding and elicitor treatment

The expression of three polyphenol oxidase (PPO; EC 1.10.3.1) genes was investigated in hybrid poplar ( Populus trichocarpa  ×  P. deltoides ). PtdPPO1 was previously isolated as a wound- and herbivore-inducible PPO (Constabel et al. Plant Physiol 124: 285–295, 2000), whereas PtdPPO2 and PtdPPO3 are two novel hybrid poplar PPO genes isolated from stem and root tissue, respectively. Sequence analysis revealed that the three PPOs were 60–66% identical at the amino acid level. Using gene-specific probes, the expression patterns of the three PPOs was investigated in various organs at different developmental stages. Under normal growing conditions, PtdPPO1 mRNA was absent from all organs tested, while PtdPPO2 was found to be expressed in mid-veins, petioles, stems and roots. PtdPPO3 was expressed only in roots. PtdPPO1 and PtdPPO2 were induced by mechanical wounding and methyl jasmonate, but in different tissues. Overall, the expression patterns suggest that the three PPO genes may have specialized physiological functions in hybrid poplar.     

Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase

5'-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe-4S](2+) center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5. Reduction of the [4Fe-4S](2+) center in the presence of this analog gives a 10K [4Fe-4S](1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the [4Fe-4S](2+) center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe-4S](2+) center. The signal shape of the low-temperature EPR signal for the [4Fe-4S](1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.     

Organ secificity of rat sodium- and potassium-activated adenosine triphosphatase.

We compared several Na,K-ATPase preparations from various organs of the rat. The brain Na,K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethylmaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain MaI-enzyme was at 7.4 to 7.5 at 37 degrees D. The Lubrol extract of this brain enzyme preparation showed a lower optimum oH of 6.6. When the Lubrol extract of the brain was fractionated wtih (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH 7.5 leads to 7.3). The brain enzyme (K 1/2 = 0.9 microM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (I 1/2 = 100 microM) in the presence of 120 mM Na+ and 10 mM K+. In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K4 on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely agai,st N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We divided rat Na,K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.     

Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus

S-Adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the carboxyl group of benzoic acid to make the volatile ester methyl benzoate, one of the most abundant scent compounds of snapdragon, Antirrhinum majus. The enzyme was purified from upper and lower petal lobes of 5- to 10-day-old snapdragon flowers using DE53 anion exchange, Phenyl-Sepharose 6FF, and Mono-Q chromatography. The purified protein has a pH optimum of 7.5 and is highly specific for benzoic acid, with no activity toward several other naturally occurring substrates such as salicylic acid, cinnamic acid, and their derivatives. The molecular mass values for native and denatured protein were 100 and 49 kDa, respectively, suggesting that the active enzyme is a homodimer. The addition of monovalent cations K+ and NH4+ stimulates BAMT activity by a factor of 2, whereas the addition of Fe2+ and Cu2+ has a strong inhibitory effect. Plant-purified BAMT has Km values of 28 microM and 1.1 mM for SAM and benzoic acid, respectively (87 microM and 1.6 mM, respectively, for plant BAMT expressed in Escherichia coli). Product inhibition studies showed competitive inhibition between SAM and S-adenosyl-L-homocysteine (SAH), with a Ki of 7 microM, and noncompetitive inhibition between benzoic acid and SAH, with a Ki of 14 microM.     

Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants.

Acquisition as well as translocation of phosphate are essential processes for plant growth. In many plants, phosphate uptake by roots and distribution within the plant are presumed to occur via a phosphate/proton cotransport mechanism. Here, we describe the isolation of two cDNAs, StPT1 and StPT2, from potato (Solanum tuberosum) that show homology to the phosphate/proton cotransporter PHO84 from the yeast Saccharomyces cerevisiae. The predicted products of both cDNAs share 35% identity with the PHO84 sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass was calculated to be 59 kD for the StPT1 protein and 58 kD for the StPT2 protein. When expressed in a PHO84-deficient yeast strain, MB192, both cDNAs complemented the mutant. Uptake of radioactive orthophosphate by the yeast mutant expressing either StPT1 or StPT2 was dependent on pH and reduced in the presence of uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. The K(m) for Pi uptake of the StPT1 and StPT2 proteins was determined to be 280 and 130 microM, respectively. StPT1 is expressed in roots, tubers, and source leaves as well as in floral organs. Deprivation of nitrogen, phosphorus, potassium, and sulfur changed spatial expression as well as the expression level of StPT1. StPT2 expression was detected mainly in root organs when plants were deprived of Pi and to a lesser extent under sulfur deprivation conditions. No expression was found under optimized nutrition conditions or when other macronutrients were lacking.     

Biochemical characterization of two differentially expressed polyphenol oxidases from hybrid poplar

Two polyphenol oxidase isoforms with distinct expression patterns were identified in hybrid poplar (Populus trichocarpaxP. deltoides). PPO-1, corresponding to the previously cloned PtdPPO (Constabel et al., Plant Physiol. 124: 285-295) was primarily leaf tissue-specific and detected only after wounding. PPO-2 was expressed constitutively in all tissue types tested except mature leaves, with highest expression in very young leaves and conducting tissues such as roots, stems and petioles. These two PPO isoforms were partially purified from hybrid poplar by ammonium sulfate fractionation followed by hydrophobic interaction chromatography. They were found to differ in stability, pH optimum, and activation by SDS. Tests with common phenolic substrates showed that PPO-1 had a broader substrate specificity than PPO-2. The distinct enzymatic properties and expression patterns of these two PPO isoforms suggest that they may have different physiological functions in hybrid poplar.     

Two poplar methyl salicylate esterases display comparable biochemical properties but divergent expression patterns

Two genes encoding proteins of 98% sequence identity that are highly homologous to tobacco methyl salicylate (MeSA) esterase (SABP2) were identified and cloned from poplar. Proteins encoded by these two genes displayed specific esterase activities towards MeSA to produce salicylic acid, and are named PtSABP2-1 and PtSABP2-2, respectively. Recombinant PtSABP2-1 and PtSABP2-2 exhibited apparent Km values of 68.2 ± 3.8 μM and 24.6 ± 1 μM with MeSA, respectively. Structural modeling using the three-dimensional structure of tobacco SABP2 as a template indicated that the active sites of PtSABP2-1 and PtSABP2-2 were highly similar to that of tobacco SABP2. Under normal growing conditions, PtSABP2-1 showed the highest level of expression in leaves and PtSABP2-2 was most highly expressed in roots. In leaf tissues of poplar plants under stress conditions, the expression of PtSABP2-1 was significantly down-regulated by two stress factors, whereas the expression of PtSABP2-2 was significantly up-regulated by four stress factors. The plausible mechanisms leading to these two highly homologous MeSA esterase genes involved in divergent biological processes in poplar are discussed.     

Inactive methyl indole-3-acetic acid ester can be hydrolyzed and activated by several esterases belonging to the AtMES esterase family of Arabidopsis

The plant hormone auxin (indole-3-acetic acid [IAA]) is found both free and conjugated to a variety of carbohydrates, amino acids, and peptides. We have recently shown that IAA could be converted to its methyl ester (MeIAA) by the Arabidopsis (Arabidopsis thaliana) enzyme IAA carboxyl methyltransferase 1. However, the presence and function of MeIAA in vivo remains unclear. Recently, it has been shown that the tobacco (Nicotiana tabacum) protein SABP2 (salicylic acid binding protein 2) hydrolyzes methyl salicylate to salicylic acid. There are 20 homologs of SABP2 in the genome of Arabidopsis, which we have named AtMES (for methyl esterases). We tested 15 of the proteins encoded by these genes in biochemical assays with various substrates and identified several candidate MeIAA esterases that could hydrolyze MeIAA. MeIAA, like IAA, exerts inhibitory activity on the growth of wild-type roots when applied exogenously. However, the roots of Arabidopsis plants carrying T-DNA insertions in the putative MeIAA esterase gene AtMES17 (At3g10870) displayed significantly decreased sensitivity to MeIAA compared with wild-type roots while remaining as sensitive to free IAA as wild-type roots. Incubating seedlings in the presence of [(14)C]MeIAA for 30 min revealed that mes17 mutants hydrolyzed only 40% of the [(14)C]MeIAA taken up by plants, whereas wild-type plants hydrolyzed 100% of absorbed [(14)C]MeIAA. Roots of Arabidopsis plants overexpressing AtMES17 showed increased sensitivity to MeIAA but not to IAA. Additionally, mes17 plants have longer hypocotyls and display increased expression of the auxin-responsive DR5:beta-glucuronidase reporter gene, suggesting a perturbation in IAA homeostasis and/or transport. mes17-1/axr1-3 double mutant plants have the same phenotype as axr1-3, suggesting MES17 acts upstream of AXR1. The protein encoded by AtMES17 had a K(m) value of 13 microm and a K(cat) value of 0.18 s(-1) for MeIAA. AtMES17 was expressed at the highest levels in shoot apex, stem, and root of Arabidopsis. Our results demonstrate that MeIAA is an inactive form of IAA, and the manifestations of MeIAA in vivo activity are due to the action of free IAA that is generated from MeIAA upon hydrolysis by one or more plant esterases.     

Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana: pectin-Ca interaction may play an important role in proton rhizotoxicity

The viability of Arabidopsis thaliana (strain Landsberg) roots exposed to a low pH (4.5 or 4.7) solution that contained 100 microM CaCl(2) was examined by staining with fluorescein diacetate-propidium iodide. The elongation zone of growing roots lost viability within 1-2 h following exposure to low pH, but non-growing roots showed no damage under the same treatment. Low-pH damage in growing roots was irreversible after 1 h incubation at pH 4.5 as judged by regrowth in growing medium at pH 5.6. Growing lateral roots also lost viability in the same treatment, whereas non-growing lateral roots remained viable during and after the treatment. The low-pH damage was ameliorated by the simultaneous application of calcium, indicating the involvement of a calcium-requiring process in overcoming proton toxicity. At pH 5.0, growing roots required 25 microM of calcium to maintain elongation, and at pH 4.8 and pH 4.5 more than 250 microM and 750 microM, respectively. The low-pH damage was ameliorated by divalent cations in the order of Ba2+, approximately Sr2+>/=Ca2+>Mg2+. The monovalent cation K+ showed no ameliorative effect, but borate showed a strong ameliorative effect with Ca2+. These results indicate that the primary target of proton toxicity may be linked to a disturbance of the stability in the pectic polysaccharide network, where calcium plays a key role in plant roots.     

Ectopic expression of soybean GmKNT1 in Arabidopsis results in altered leaf morphology and flower identity

Plant morphology is specified by leaves and flowers, and the shoot apical meristem (SAM) defines the architecture of plant leaves and flowers. Here, we reported the characterization of a soybean KNOX gene GmKNT1, which was highly homologous to Arabidopsis STM. The GmKNT1 was strongly expressed in roots, flowers and developing seeds. Its expression could be induced by IAA, ABA and JA, but inhibited by GA or cytokinin. Staining of the transgenic plants overexpressing GmKNT1-GUS fusion protein revealed that the GmKNT1 was mainly expressed at lobe region, SAM of young leaves, sepal and carpel, not in seed and mature leaves. Scanning electron micros- copy (SEM) disclosed multiple changes in morphology of the epidermal cells and stigma. The transgenic Arabidopsis plants overexpress- ing the GmKNT1 showed small and lobed leaves, shortened internodes and small clustered inflorescence. The lobed leaves might result from the function of the meristems located at the boundary of the leaf. Compared with wild type plants, transgenic plants had higher ex- pression of the SAM-related genes including the CUP, WUS, CUC1, KNAT2 and KNAT6. These results indicated that the GmKNT1 could affect multiple aspects of plant growth and development by regulation of downstream genes expression.     

Stem fasciated, a recessive mutation in sunflower (Helianthus annuus), alters plant morphology and auxin level

BACKGROUND AND AIMS: Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus). METHODS: F1, F2 and F3 generations were obtained through reciprocal crosses between stf and normal plants. For the genetic analysis, a chi2 test was used. Phenotypic observations were made in field-grown and potted plants. A histological analysis of SAM, hypocotyl, epicotyl, stem and root apical meristem was also conducted. To evaluate the level of endogenous indole-3-acetic acid (IAA), a capillary gas chromatography-mass spectrometry-selected ion monitoring analysis was performed. KEY RESULTS: stf is controlled by a single nuclear recessive gene. stf plants are characterized by a dramatically increased number of leaves and vascular bundles in the stem, as well as by a shortened plastochron and an altered phyllotaxis pattern. By histological analysis, it was demonstrated that the stf phenotype is related to an enlarged vegetative SAM. Microscopy analysis of the mutant's apex also revealed an abnormal enlargement of nuclei in both central and peripheral zones and a disorganized distribution of cells in the L2 layer of the central zone. The stf mutant showed a high endogenous free IAA level, whereas auxin perception appeared normal. CONCLUSIONS: The observed phenotype and the high level of auxin detected in stf plants suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both SAM arrangement and hormonal homeostasis.     

Levels of Indol-3yl-acetic acid (IAA), IAA-oxidase and Peroxidase in Developing Cucumber Seedlings

The level of indol-3yl-acetic acid (IAA) in the cotyledons of cucumber seedlings increases in the period 4 to 11 days after germination. In hypocotyls and roots the IAA level decreases. IAA-oxidase activity of homogenates of cotyledons, hypocotyls and roots decreases with age. The soluble fraction of all three organs contains an IAA-oxidase, which may be allosteric. It is suggested that this IAA oxidase limits the upper level of IAA in the cell. The cell wall fraction of the three organs contains IAA-oxidases with conventional kinetics. The function of this IAA oxidase is probably to degrade exogenous IAA.     

A protocol for shoot multiplication from foliar meristem of Vanda spathulata (L.) Spreng

Leaf explants collected from flowering plants of Vanda spathulata were cultured in Mitra medium with combinations of 6-benzyladenine (BA; 13.2-88.8 microM) and indole-3-acetic acid (IAA; 0.0 -85.6 microM). Combination of BA (66.6 microM) and IAA (28.5 microM) induced maximum shoots (17.33) from foliar meristems (leaf base). BA individually did not induce caulogenesis in leaf explants. For optimized multiplication, BA:IAA (2:1 microM) was essential at 22.2- 88.8 microM of BA. Re-cultured leaf explants produced lesser number of shoots compared to original explants and were nearly equal at combinations of 22.2-44.4 microM of BA and 5.7-28.5 microM of IAA. Rooting of shoots (> 95%) occurred in medium containing banana pulp (75 gl(-1)) and IAA (5.7 microM) within 3-9 weeks. Plantlets with 2-5 roots of 2-5 cm length established easily in community pots at 80-90% rates without hardening.     

Reduction of indole‐3‐acetic acid methyltransferase activity compensates for high‐temperature male sterility in Arabidopsis

High temperature is a general stress factor that causes a decrease in crop yield. It has been shown that auxin application reduces the male sterility caused by exposure to higher temperatures. However, widespread application of a hormone with vast effects on plant physiology may be discouraged in many cases. Therefore, the generation of new plant varieties that locally enhance auxin in reproductive organs may represent an alternative strategy. We have explored the possibility of increasing indole‐3‐acetic acid (IAA) in ovaries by reducing IAA methyltransferase1 (IAMT1) activity in Arabidopsis thaliana. The iamt1 mutant showed increased auxin signalling in funiculi, which correlated with a higher growth rate of wild‐type pollen in contact with mutant ovaries and premature ovule fertilization. While the production of seeds per fruit was similar in the wild type and the mutant at 20 °C, exposure to 29 °C caused a more severe decrease in fertility in the wild type than in the mutant. Loss of IAMT1 activity was also associated with the production of more nodes after flowering and higher tolerance of the shoot apical meristem to higher temperatures. As a consequence, the productivity of the iamt1 mutant under higher temperatures was more than double of that of the wild type, with almost no apparent trade‐off.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.