Athanasios Theologis

Early Genes and Auxin Action

The plant hormone IAA (or auxin) is central to the control of plant growth and development. Processes governed by auxin in concert with other plant growth regulators include development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses (Went and Thimann, 1937). At the level of cellular physiology, auxin profoundly affects turgor, elongation, division, and cell differentiation, the major driving and shaping forces in morphogenesis and oncogenesis. The molecular mechanisms of auxin action are still unknown, although it is now well established that auxin modulates membrane function and gene expression (for review, see Napier and Venis, 1995). These biochemical changes, in turn, most likely affect fundamental aspects of plant morphology and physiology. However, a causal relationship between auxin-mediated alterations in gene expression or membrane function and a particular growth process has not yet been demonstrated. Despite its critical role in plant development and the immense volume of studies on the diverse auxin effects, understanding of the molecular mechanisms of auxin action remains one of the major challenges in plant biology. The signal transduction cascades leading from auxin perception to altered gene expression or membrane function hold the key in our attempts to elucidate the primary mechanism(s) of auxin action. An array of experimental strategies has been mounted to investigate auxin signaling pathways. The combination of biochemical, molecular, and genetic approaches will allow for significant new insights into how the hormone works in molecular terms (Fig. 1). One strategy employs genetics and reverse genetics to construct transgenic plants with perturbations in auxin homeostasis and to screen for mutants with defects in auxinrelated physiology. Transgenic plants expressing altered hormone levels have already resolved some longstanding questions in plant physiology. Mutant plants defective in auxin responses will rejuvenate and stimulate research by identifying novel genes involved in hormone perception, signal transduction, and physiological responses (for review, see Hobbie and Estelle, 1994; Klee and Romano, 1994). The first significant result (to our knowledge) of this approach was the cloning of the AXR1 gene, which encodes a protein related to the ubiquitin-activating enzyme El (Leyser et al., 1993). Although AXRl is probably not a functional El homolog, it is nonetheless an exquisite example of the potential of molecular genetics to connect the unexpected. The biochemical strategy is based on the identification of auxin receptors and subsequent isolation of interacting components. The search for auxin receptors has led to the discovery of a number of soluble and membranebound proteins that bind auxin with moderate but physiologically relevant affinity. Their functional role in auxin signaling is still unclear and is a major target of current research (for review, see Jones, 1994; Napier and Venis, 1995). Auxin-regulated genes provide yet another source of molecular tools to dissect auxin action. The hormone modulates gene expression in a wide variety of plant tissues and cell types over a broad period of time (for review, see Guilfoyle, 1986; Theologis, 1986). However, early genes selectively induced as a primary response to auxin and prior to the initiation of cell growth are likely candidates to play a pivotal role in mediating growth-stimulating effects of the hormone. This review focuses on recent advances in our knowledge on early auxin-inducible gene expression and possible functions of the polypeptides encoded.

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.

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.

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 β-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.

Ethylene biosynthesis and action: a case of conservation

Ethylene is one of the simplest organic molecules with biological activity. At concentrations as low as 0.1 ppm in air, it has been shown to have dramatic effects on plant growth and development [1]. Neljubov [78] was the first to show that ethylene has three major effects in etiolated pea seedlings called the triple response: (1) diageotropic growth, (2) thickening of stem and inhibition of stem elongation, and (3) exaggeration of apical hook curvature. Since then, numerous ethylene effects have been described in light-grown plants such as sex determination in curcurbits, fruit ripening in climacteric fruits, epinastic curvature, flower senescence, and root initiation [1]. Interestingly, ethylene has also been shown to have opposite effects in some plants; for instance, it inhibits stem elongation in most dicots, whereas in some aquatic dicots and rice, it stimulates growth [1, 45, 72].

Genome-wide mapping with biallelic markers in Arabidopsis thaliana

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Rapid induction of specific mRNAs by auxin in pea epicotyl tissue

DNA sequences complementary to three indoleacetic acid (IAA)-inducible mRNAs in pea epicotyl tissue were isolated by differential plaque filter hybridization of cDNA libraries constructed in the vector lambda gt10. Clone pIAA6 hybridized to an mRNA encoding the previously identified translational product polypeptide 6 (Mr 22,000), and clone pIAA4/5 hybridized to one or two mRNAs, encoding polypeptides 4 and 5 (Mr 23,000 and 25,000, respectively). The cDNA clones were subsequently used to characterize the hormonally mediated mRNA accumulation. The induction of the mRNAs was rapid, within 15 minutes of exposure to the IAA, and specific to auxins. Anaerobiosis, heat and cold stress did not induce the mRNAs. Other plant hormones, such as gibberellic acid, kinetin, abscisic acid and ethylene were also unable to cause or interfere with the IAA-induced mRNA accumulation. The hormonally regulated mRNAs were induced at least 50 to 100-fold above control levels after two hours of treatment with IAA and the accumulation was (1) independent of protein synthesis, (2) completely abolished by alpha-amanitin, (3) not due to polyadenylylation of pre-existing RNAs, and (4) independent of IAA and fusicoccin-induced H+ secretion. The IAA-induced mRNAs returned to control levels within three hours after removal of IAA, and the hormonally regulated genes were primarily expressed in the third and second internode of the seven-day-old etiolated pea seedling. The data indicate that IAA increases the amount of specific mRNAs rather than alters the translatability of pre-existing mRNAs. Auxin-induced H+ secretion appears not to have a potential role in mediating the induction and perhaps is a consequence of the enhanced biosynthetic activity induced by the hormone. The IAA-mediated mRNA induction is the fastest known for any plant growth regulator and may represent a primary hormonal response to auxin.

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.

IAA17/AXR3: Biochemical Insight into an Auxin Mutant Phenotype

The Aux/IAA genes are rapidly and specifically induced by the plant hormone auxin. The proteins encoded by this gene family are short-lived nuclear proteins that are capable of homodimerizing and heterodimerizing. Molecular, biochemical, and genetic data suggest that these proteins are involved in auxin signaling. The pleiotropic morphological phenotype and altered auxin responses of the semidominant axr3-1 mutant of Arabidopsis result from a single amino acid change in the conserved domain II of the Aux/IAA protein IAA17. Here, we show that the biochemical effect of this gain-of-function mutation is to increase the half-life of the iaa17/axr3-1 protein by sevenfold. Intragenic mutations that suppress the iaa17/axr3-1 phenotype have been described. The iaa17/axr3-1R3 revertant contains a second site mutation in domain I and the iaa17/axr3-1R2 revertant contains a second site mutation in domain III. Transient expression assays show that the mutant forms of IAA17/AXR3 retain the ability to accumulate in the nucleus. Using the yeast two hybrid system, we show that the iaa17/axr3-1 mutation does not affect homodimerization. However, the iaa17/axr3-1 revertants counteract the increased levels of iaa17/axr3-1 protein by decreasing the capacity of the mutant protein to homodimerize. Interestingly, heterodimerization of the revertant forms of IAA17/AXR3 with IAA3/SHY2, another Aux/IAA protein, and ARF1 or ARF5/MP proteins is affected only by changes in domain III. Collectively, the results provide biochemical evidence that the revertant mutations in the IAA17/AXR3 gene affect the capacity of the encoded protein to dimerize with itself, other members of the Aux/IAA protein family, and members of the ARF protein family. By extension, these findings may provide insight into the effects of analogous mutations in other members of the Aux/IAA gene family.

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

Ethylene (C2H4) 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.