Odyssey of auxin.

The history of plant biology is inexorably intertwined with the conception and discovery of auxin, followed by the many decades of research to comprehend its action during growth and development. Growth responses to auxin are complex and require the coordination of auxin production, transport, and perception. In this overview of past auxin research, we limit our discourse to the mechanism of auxin action. We attempt to trace the almost epic voyage from the birth of the hormonal concept in plants to the recent crystallographic studies that resolved the TIR1-auxin receptor complex, the first structural model of a plant hormone receptor. The century-long endeavor is a beautiful illustration of the power of scientific reasoning and human intuition, but it also brings to light the fact that decisive progress is made when new technologies emerge and disciplines unite.

A combinatorial interplay among the 1-aminocyclopropane-1-carboxylate isoforms regulates ethylene biosynthesis in Arabidopsis thaliana.

Ethylene (C(2)H(4)) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC, the precursor of ethylene. To understand the function of this important enzyme, we analyzed the entire family of nine ACS isoforms (ACS1, ACS2, ACS4-9, and ACS11) encoded in the Arabidopsis genome. Our analysis reveals that members of this protein family share an essential function, because individual ACS genes are not essential for Arabidopsis viability, whereas elimination of the entire gene family results in embryonic lethality. Phenotypic characterization of single and multiple mutants unmasks unique but overlapping functions of the various ACS members in plant developmental events, including multiple growth characteristics, flowering time, response to gravity, disease resistance, and ethylene production. Ethylene acts as a repressor of flowering by regulating the transcription of the FLOWERING LOCUS C. Each single and high order mutant has a characteristic molecular phenotype with unique and overlapping gene expression patterns. The expression of several genes involved in light perception and signaling is altered in the high order mutants. These results, together with the in planta ACS interaction map, suggest that ethylene-mediated processes are orchestrated by a combinatorial interplay among ACS isoforms that determines the relative ratio of homo- and heterodimers (active or inactive) in a spatial and temporal manner. These subunit isoforms comprise a combinatorial code that is a central regulator of ethylene production during plant development. The lethality of the null ACS mutant contrasts with the viability of null mutations in key components of the ethylene signaling apparatus, strongly supporting the view that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.

The basic helix-loop-helix transcription factor PIF5 acts on ethylene biosynthesis and phytochrome signaling by distinct mechanisms.

PHYTOCHROME-INTERACTING FACTOR5 (PIF5), a basic helix-loop-helix transcription factor, interacts specifically with the photoactivated form of phytochrome B (phyB). Here, we report that dark-grown Arabidopsis thaliana seedlings overexpressing PIF5 (PIF5-OX) exhibit exaggerated apical hooks and short hypocotyls, reminiscent of the triple response induced by elevated ethylene levels, whereas pif5 mutants fail to maintain tight hooks like those of wild-type seedlings. Silver ions, an ethylene receptor blocker, rescued the triple-response phenotype, and we show that PIF5-OX seedlings express enhanced levels of key ethylene biosynthesis enzymes and produce elevated ethylene levels. Exposure of PIF5-OX seedlings to prolonged continuous red light (Rc) promotes hypocotyl elongation relative to dark controls, the reciprocal of the Rc-imposed hypocotyl inhibition displayed by wild-type seedlings. In contrast with this PIF5-OX hyposensitivity to Rc, pif5 mutant seedlings are hypersensitive relative to wild-type seedlings. We show that this contrast is due to reciprocal changes in phyB protein levels in prolonged Rc. Compared with wild-type seedlings, PIF5-OX seedlings have reduced, whereas pif5 mutants have increased, phyB (and phyC) levels in Rc. The phyB degradation in the overexpressors depends on a functional phyB binding motif in PIF5 and involves the 26S proteasome pathway. Our data thus indicate that overexpressed PIF5 causes altered ethylene levels, which promote the triple response in darkness, whereas in the light, the interaction of photoactivated phyB with PIF5 causes degradation of the photoreceptor protein. The evidence suggests that endogenous PIF5 negatively regulates phyB-imposed hypocotyl inhibition in prolonged Rc by reducing photoreceptor abundance, and thereby photosensory capacity, rather than functioning as a signaling intermediate.

ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis.

Lateral root formation in Arabidopsis thaliana is regulated by two related AUXIN RESPONSE FACTORs, ARF7 and ARF19, which are transcriptional activators of early auxin response genes. The arf7 arf19 double knockout mutant is severely impaired in lateral root formation. Target-gene analysis in arf7 arf19 transgenic plants harboring inducible forms of ARF7 and ARF19 revealed that ARF7 and ARF19 directly regulate the auxin-mediated transcription of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18 (LBD16/ASL18) and/or LBD29/ASL16 in roots. Overexpression of LBD16/ASL18 and LBD29/ASL16 induces lateral root formation in the absence of ARF7 and ARF19. These LBD/ASL proteins are localized in the nucleus, and dominant repression of LBD16/ASL18 activity inhibits lateral root formation and auxin-mediated gene expression, strongly suggesting that these LBD/ASLs function downstream of ARF7- and ARF19-dependent auxin signaling in lateral root formation. Our results reveal that ARFs regulate lateral root formation via direct activation of LBD/ASLs in Arabidopsis.

Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana.

Auxin regulates various aspects of plant growth and development. The AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode short-lived transcriptional repressors that are targeted by the TRANSPORT INHIBITOR RESPONSE1/AUXIN RECEPTOR F-BOX proteins. The Aux/IAA proteins regulate auxin-mediated gene expression by interacting with members of the AUXIN RESPONSE FACTOR protein family. Aux/IAA function is poorly understood; herein, we report the identification and characterization of insertion mutants in 12 of the 29 Aux/IAA family members. The mutants show no visible developmental defects compared with the wild type. Double or triple mutants of closely related Aux/IAA genes, such as iaa8-1 iaa9-1 or iaa5-1 iaa6-1 iaa19-1, also exhibit wild-type phenotypes. Global gene expression analysis reveals that the molecular phenotypes of auxin-treated and untreated light-grown seedlings are unaffected in the iaa17-6 and iaa5-1 iaa6-1 iaa19-1 mutants. By contrast, similar analysis with the gain-of-function axr3-1/iaa17-1 mutant seedlings reveals dramatic changes in basal and auxin-induced gene expression compared with the wild type. Expression of several type-A ARABIDOPSIS RESPONSE REGULATOR genes and a number of genes involved in cell wall biosynthesis and degradation is repressed in axr3-1/iaa17-1. The data suggest extensive functional redundancy among Aux/IAA gene family members and that enhanced stability of the AXR3/IAA17 protein severely alters the molecular phenotype, resulting in developmental defects.

Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis.

Auxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylem-adjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.

AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator.

AUXIN RESPONSE FACTORS (ARFs) regulate auxin-mediated transcriptional activation/repression. They are encoded by a gene family in Arabidopsis, and each member is thought to play a central role in various auxin-mediated developmental processes. We have characterized three arf2 mutant alleles, arf2-6, arf2-7 and arf2-8. The mutants exhibit pleiotropic developmental phenotypes, including large, dark green rosette leaves, delayed flowering, thick and long inflorescence, abnormal flower morphology and sterility in early formed flowers, large organ size and delayed senescence and abscission, compared with wild-type plants. In addition, arf2 mutant seedlings have elongated hypocotyls with enlarged cotyledons under various light conditions. The transcription of ACS2, ACS6 and ACS8 genes is impaired in the developing siliques of arf2-6. The phenotypes of all three alleles are similar to those of the loss-of-function mutants obtained by RNA interference or co-suppression. There is no significant effect of the mutation on global auxin-regulated gene expression in young seedlings, suggesting that ARF2 does not participate in auxin signaling at that particular developmental stage of the plant life cycle. Because ARF2 is thought to function as a transcriptional repressor, the prospect arises that its pleiotropic effects may be mediated by negatively modulating the transcription of downstream genes in signaling pathways that are involved in cell growth and senescence.

Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19.

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members.

1-Aminocyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, and ACS11) homodimers and one nonfunctional (ACS1) homodimer. Transgenic Arabidopsis lines were constructed expressing the beta-glucuronidase (GUS) and green fluorescence protein (GFP) reporter genes from the promoter of each of the gene family members to determine their patterns of expression during plant development. All genes, except ACS9, are expressed in 5-d-old etiolated or light-grown seedlings yielding distinct patterns of GUS staining. ACS9 expression is detected later in development. Unique and overlapping expression patterns were detected for all the family members in various organs of adult plants. ACS11 is uniquely expressed in the trichomes of sepals and ACS1 in the replum. Overlapping expression was observed in hypocotyl, roots, various parts of the flower (sepals, pedicle, style, etc.) and in the stigmatic and abscission zones of the silique. Exogenous indole-3-acetic acid (IAA) enhances the constitutive expression of ACS2, 4, 5, 6, 7, 8, and 11 in the root. Wounding of hypocotyl tissue inhibits the constitutive expression of ACS1 and ACS5 and induces the expression of ACS2, 4, 6, 7, 8, and 11. Inducers of ethylene production such as cold, heat, anaerobiosis, and Li(+) ions enhance or suppress the expression of various members of the gene family in the root of light-grown seedlings. Examination of GUS expression in transverse sections of cotyledons reveals that all ACS genes, except ACS9, are expressed in the epidermis cell layer, guard cells, and vascular tissue. Similar analysis with root tip tissue treated with IAA reveals unique and overlapping expression patterns in the various cell types of the lateral root cap, cell division, and cell expansion zones. IAA inducibility is gene-specific and cell type-dependent across the root tip zone. This limited comparative exploration of ACS gene family expression reveals constitutive spatial and temporal expression patterns of all gene family members throughout the growth period examined. The unique and overlapping gene activity pattern detected reveals a combinatorial code of spatio-temporal coexpression among the various gene family members during plant development. This raises the prospect that functional ACS heterodimers may be formed in planta.

Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis.

Auxin modulates diverse plant developmental pathways through direct transcriptional regulation and cooperative signaling with other plant hormones. Genetic and biochemical approaches have clarified several aspects of the auxin-regulated networks; however, the mechanisms of perception and subsequent signaling events remain largely uncharacterized. To elucidate unidentified intermediates, we have developed a high-throughput screen for identifying small molecule inhibitors of auxin signaling in Arabidopsis. Analysis of 10,000 compounds revealed several potent lead structures that abrogate transcription of an auxin-inducible reporter gene. Three compounds were found to interfere with auxin-regulated proteolysis of an auxin/indole-3-acetic acid transcription factor, and two impart phenotypes indicative of an altered auxin response, including impaired root development. Microarray analysis was used to demonstrate the mechanistic similarities of the two most potent molecules. This strategy promises to yield powerful tools for the discovery of unidentified components of the auxin-signaling networks and the study of auxin's participation in various stages of plant development.

Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family.

The pyridoxal phosphate-dependent enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS; EC 4.4.1.14), catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, ACS11) and one nonfunctional (ACS1) homodimers. Because the enzyme is a homodimer with shared active sites, the question arises whether the various polypeptides can form functional heterodimers. Intermolecular complementation experiments in Escherichia coli by coexpressing the K278A and Y92A mutants of different polypeptides show that all of them have the capacity to heterodimerize. However, functional heterodimers are formed only among gene family members that belong to one or the other of the two phylogenetic branches. ACS7 is an exception to this rule, which forms functional heterodimers with some members of both branches when it provides the wt K278 residue. ACS1, the nonfunctional polypeptide as a homodimer, can also form functional heterodimers with members of its phylogenetic branch when its partners provide the wt K278 residue. The ACS gene family products can potentially form 45 homo- and heterodimers of which 25 are functional. Bimolecular fluorescence complementation and biochemical coaffinity purification assays show that the inactivity of certain heterodimers is not due to the absence of heterodimerization but rather to structural restraint(s) that prevents the shared active sites from being functional. We propose that functional heterodimerization enhances the isozyme diversity of the ACS gene family and provides physiological versatility by being able to operate in a broad gradient of S-adenosylmethionine concentration in various cells/tissues during plant growth and development. Nonfunctional heterodimerization may also play a regulatory role during the plant life cycle.

Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family.

1-Amino-cyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) is the key enzyme in the ethylene biosynthetic pathway in plants. The completion of the Arabidopsis genome sequence revealed the presence of twelve putative ACS genes, ACS1-12, dispersed among five chromosomes. ACS1-5 have been previously characterized. However, ACS1 is enzymatically inactive whereas ACS3 is a pseudogene. Complementation analysis with the Escherichia coli aminotransferase mutant DL39 shows that ACS10 and 12 encode aminotransferases. The remaining eight genes are authentic ACS genes and together with ACS1 constitute the Arabidopsis ACS gene family. All genes, except ACS3, are transcriptionally active and differentially expressed during Arabidopsis growth and development. IAA induces all ACS genes, except ACS7 and ACS9; CHX enhances the expression of all functional ACS genes. The ACS genes were expressed in E. coli, purified to homogeneity by affinity chromatography, and biochemically characterized. The quality of the recombinant proteins was verified by N-terminal amino acid sequence and MALDI-TOF mass spectrometry. The analysis shows that all ACS isozymes function as dimers and have an optimum pH, ranging between 7.3 and 8.2. Their Km values for AdoMet range from 8.3 to 45 microm, whereas their kcat values vary from 0.19 to 4.82 s-1 per monomer. Their Ki values for AVG and sinefungin vary from 0.019 to 0.80 microm and 0.15 to 12 microm, respectively. The results indicate that the Arabidopsis ACS isozymes are biochemically distinct. It is proposed that biochemically diverse ACS isozymes function in unique cellular environments for the biosynthesis of C2H4, permitting the signaling molecule to exert its unique effects in a tissue- or cell-specific fashion.