The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.plant hormone | metabolism

Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes, few genes that actually regulate the transduction of the ABA signal into a cellular response have been identified. In an attempt to determine negative regulators of ABA signaling, we identified mutants, designated enhanced response to ABA3 ( era3 ), that increased the sensitivity of the seed to ABA. Biochemical and molecular analyses demonstrated that era3 mutants overaccumulate ABA, suggesting that era3 is a negative regulator of ABA synthesis. Subsequent genetic analysis of era3 alleles, however, showed that these are new alleles at the ETHYLENE INSENSITIVE2 locus. Other mutants defective in their response to ethylene also showed altered ABA sensitivity; from these results, we conclude that ethylene appears to be a negative regulator of ABA action during germination. In contrast, the ethylene response pathway positively regulates some aspects of ABA action that involve root growth in the absence of ethylene. We discuss the response of plants to ethylene and ABA in the context of how these two hormones could influence the same growth responses. INTRODUCTIONAbscisic acid (ABA) modulates a wide variety of plant processes ranging from seed dormancy to leaf-water relations (reviewed in Zeevaart and Creelman, 1988). Over the past few years, mutational analysis of the ABA response in Arabidopsis has begun to uncover genes regulating the sensitivity of a plant cell to the hormone. Basically, genetic screens fall into two categories: screens that identify mutations conferring an ABA-insensitive phenotype and screens that identify mutations enhancing ABA sensitivity (Bonetta and McCourt, 1998;Leung and Giraudat, 1998). To date, studies of these mutants have led to the identification of two protein phosphatases (ABI1 [for ABA-insensitive] and ABI2), a protein farnesyltransferase (ERA1 [for enhanced response to ABA]), and two transcription factors (ABI3 and ABI4) involved in ABA action.Recently, however, careful phenotypic analysis has determined that several of the hormone response mutants have altered sensitivities to more than one hormone. Mutations in the AXR2 gene of Arabidopsis, for example, confer crossresistance to ABA, ethylene, and auxin . However, because only dominant axr2 alleles exist and the molecular mechanism of AXR2 is not known, it is difficult to draw conclusions regarding the ability of this gene to confer ABA sensitivity. Mutations in the SAX1 gene, which is involved in brassinosteroid biosynthesis, confer increased ABA sensitivity to the seed, suggesting that the synthesis of one hormone can affect the sensitivity of the plant to other hormones (Ephritikhine et al., 1999).Recently, the ETHYLENE-INSENSITIVE2 ( EIN2 ) gene of Arabidopsis has been shown to be involved in multiple hormone responses, including the responses to ABA (Alonso et al., 1999). Originally identified as a loss-of-function mutation that confers a strong insensitivity to exogenous ethylene, molecular dissection of this gene has separa...

Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes, few genes that actually regulate the transduction of the ABA signal into a cellular response have been identified. In an attempt to determine negative regulators of ABA signaling, we identified mutants, designated enhanced response to ABA3 (era3), that increased the sensitivity of the seed to ABA. Biochemical and molecular analyses demonstrated that era3 mutants overaccumulate ABA, suggesting that era3 is a negative regulator of ABA synthesis. Subsequent genetic analysis of era3 alleles, however, showed that these are new alleles at the ETHYLENE INSENSITIVE2 locus. Other mutants defective in their response to ethylene also showed altered ABA sensitivity; from these results, we conclude that ethylene appears to be a negative regulator of ABA action during germination. In contrast, the ethylene response pathway positively regulates some aspects of ABA action that involve root growth in the absence of ethylene. We discuss the response of plants to ethylene and ABA in the context of how these two hormones could influence the same growth responses.

The discovery of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate pathway, 1-deoxy-d-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The ␤-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.In higher plants isoprenoids are derived from isopentenyl diphosphate (IPP) and synthesized in at least two different compartments, the cytoplasm and the chloroplast. For a long time it was assumed that IPP was synthesized exclusively by the mevalonate pathway in all organisms (Spurgeon and Porter, 1981; Goldstein and Brown, 1990). However, independent studies have demonstrated that in eubacteria, green algae, and plants, IPP is also synthesized by a non-mevalonate pathway designated as the 2-Cmethyl-d-erythritol-4-P (MEP) pathway (for review, see Rohmer, 1998Rohmer, , 1999Lichtenthaler, 1999). Thus in plants cytosolic IPP is synthesized by the mevalonate pathway and plastidic IPP is synthesized by the MEP pathway (Lichtenthaler, 1999). In the MEP pathway IPP is synthesized from pyruvate and glyceraldehyde-3-P via novel intermediates (Rohmer et al

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA 1 , the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA 1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3␤-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3␤-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3␤-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA 1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/ KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16a-hydroxykaurene).

RGA (for repressor of ga1-3 ) and SPINDLY ( SPY ) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3 . We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)-RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved. INTRODUCTIONGibberellins (GAs) are members of a large family of diterpenoid compounds, some of which are plant growth regulators that control such diverse processes as seed germination, stem growth, and flower development. Although the GA biosynthetic pathway has been elucidated (reviewed in Lange, 1998;Hedden and Proebsting, 1999;Hedden and Phillips, 2000;Yamaguchi and Kamiya, 2000), much less is known about its signal transduction pathway in plants. Recent molecular and pharmacological studies in cereal aleurone showed that Ca 2 ϩ , calmodulin, cyclic GMP, heterotrimeric G proteins, GAMYB, and protein kinases may play a role in GA signaling (reviewed in Bethke and Jones, 1998;Lovegrove and Hooley, 2000). Isolation of GA response mutants and molecular cloning of corresponding genes in Arabidopsis also have identified several novel components of the GA signal transduction pathway (reviewed in Thornton et al., 1999;Sun, 2000). The putative repressors include SPINDLY ( SPY ;Jacobsen et al., 1996), RGA (for repressor of ga1-3 ; Silverstone et al., 1998), GAI (for GA insensitive; Peng et al., 1997), and SHORT INTERNODES ( SHI ;Fridborg et al., 1999), and the potential activators are SLEEPY ( SLY ;Steber et al., 1998) and PICKLE ( PKL ; Ogas et al., 1999).SPY was identified originally because spy mutations allowed the seed to germinate in the presence of the GA biosynthesis inhibitor paclobutrazol (PAC; Jacobsen and Olszewski, 1993). The defect in the SPY function also was able to partially suppress the phenotype of the GA biosynthetic mutant ga1-3 , which is a nongerminating, male-sterile, extreme dwarf (Silverstone et al., 1997). Sequence analysis of SPY and in vitro enzyme assays using the recombinant SPY protein suggest that SPY probably is a Ser/Thr O -linked N -acetylglucosamine transferase (OGT; Thornton et al., 1999).We identified RG...

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.