Emmanuel Liscum

Genetics of Aux/IAA and ARF action in plant growth and development

Dramatic advances in our understanding of auxin signal-response pathways have been made in recent years. Much of this new knowledge has come through the study of mutants in Arabidopsis thaliana. Mutations have been identified in a wide variety of auxin-response components, including auxin transporters, protein kinases and phosphatases, components of a ubiquitin-proteosome pathway, and transcriptional regulators. This review focuses on mutations that affect auxin-modulated transcription factors, in particular those in the Aux/IAA and AUXIN RESPONSE FACTOR(ARF) genes. Mutants in members of these related gene families exhibit phenotypes that indicate both unique localized functions, as well as overlapping redundant functions, throughout plant development – from embryogenesis to flowering. Effects of specific mutations on Aux/IAA and ARF protein functions at the biochemical and physiological levels will be discussed. We will also discuss potential mechanisms for interactions between auxin and light response pathways that are suggested by these mutants.

Mutations in the NPH1 Locus of Arabidopsis Disrupt the Perception of Phototropic Stimuli

The phototropic response is an important component of seedling establishment in higher plants because it orients the young seedlings for maximal photosynthetic light capture. Despite their obvious importance, little is known about the mechanisms underlying the perception and transduction of the light signals that induce phototropic curvatures. Here, we report the isolation of eight mutants of Arabidopsis that lack or have severely impaired phototropic responses. These nph (for nonphototropic hypocotyl) mutants comprise four genetic loci: nph1, nph2, nph3, and nph4. Physiological and biochemical characterization of the nph1 allele series indicated that the NPH1 locus may encode the apoprotein for a dual-chromophoric or multichromophoric holoprotein photoreceptor capable of absorbing UV-A, blue, and green light and that this photoreceptor regulates all the phototropic responses of Arabidopsis. It appears that the NPH1 protein is most likely a 120-kD plasma membrane-associated phosphoprotein because all of the nph1 mutations negatively affected the abundance of this protein. In addition, the putative NPH1 photoreceptor protein is genetically and biochemically distinct from the HY4 protein, which most likely acts as a photoreceptor for blue light-mediated hypocotyl growth inhibition. Furthermore, the NPH1 and HY4 proteins are not functionally redundant because mutations in either gene alone affect only one physiological response but not the other, thus providing strong support for the hypothesis that more than one blue light photoreceptor is required for the normal growth and development of a seedling.

The NPH4 Locus Encodes the Auxin Response Factor ARF7, a Conditional Regulator of Differential Growth in Aerial Arabidopsis Tissue

Organ bending through differential growth represents a major mechanism by which plants are able to adaptively alter their morphology in response to local changes in the environment. Two plant hormones, auxin and ethylene, have been implicated as regulators of differential growth responses; however, the mechanisms by which they elicit their effects remain largely unknown. Here, we describe isolation of the NPH4 gene of Arabidopsis, which is conditionally required for differential growth responses of aerial tissues, and we report that NPH4 encodes the auxin-regulated transcriptional activator ARF7. The phenotypes of nph4 mutants, which include multiple differential growth defects associated with reduced auxin responsiveness, including impaired auxin-induced gene expression, are consistent with the predicted loss of function of a transcriptional activator, and these phenotypes indicate that auxin-dependent changes in gene transcription are prerequisite for proper organ bending responses. Although NPH4/ARF7 appears to be a major regulator of differential growth, it is not the sole regulator because phenotypes of nph4 null mutants were suppressed by application of ethylene. This latter finding illustrates the intimate connection between auxin and ethylene in the control of growth in higher plants.

MASSUGU2 Encodes Aux/IAA19, an Auxin-Regulated Protein That Functions Together with the Transcriptional Activator NPH4/ARF7 to Regulate Differential Growth Responses of Hypocotyl and Formation of Lateral Roots in Arabidopsis thaliana

We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.

Development and mapping of SSR markers for maize.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

The Phototropin Family of Photoreceptors

The past decade has seen dramatic advances in our knowledge of plant photoreceptors and in our understanding of the signal transduction pathways that they activate ([Briggs and Olney, 2001][1]). A major part of these advances has been the identification and characterization of photoreceptors that

A Mutant Arabidopsis Heterotrimeric G-Protein β Subunit Affects Leaf, Flower, and Fruit Development

A genetic screen was performed to find new mutants with an erecta (er) phenotype and to identify genes that may function with ER, a receptor-like kinase. These mutants were named elk (for erecta-like) and were placed into five complementation groups. We positionally cloned ELK4 and determined that it encodes AGB1, a putative heterotrimeric G-protein β subunit. Therefore, elk4 was renamed agb1. agb1-1 plants express similar fruit phenotypes, as seen in er plants, but differ from er in that the stem is only slightly shorter than that in the wild type, the pedicel is slightly longer than that in the wild type, and the leaves are rounder than those in er mutants. Molecular analysis of agb1-1 indicates that it is likely a null allele. AGB1 mRNA is expressed in all tissues tested but is highest in the silique. Analysis of agb1-1 er double mutants suggests that AGB1 may function in an ER developmental pathway regulating silique width but that it functions in parallel pathways affecting silique length as well as leaf and stem development. The finding that AGB1 is involved in the control of organ shape suggests that heterotrimeric G-protein signaling is a developmental regulator in Arabidopsis.

Mutations of Arabidopsis in Potential Transduction and Response Components of the Phototropic Signaling Pathway

Four genetic loci were recently identified by mutations that affect phototropism in Arabidopsis thaliana (L.) Heyhn. seedlings. It was hypothesized that one of these loci, NPH1, encodes the apoprotein for a phototropic photoreceptor. All of the alleles at the other three mutant loci (nph2, nph3, and nph4) contained wild-type levels of the putative NPH1 protein and exhibited normal blue-light-dependent phosphorylation of the NPH1 protein. This indicated that the NPH2, NPH3, and NPH4 proteins likely function downstream of NPH1 photoactivation. We show here that, although the nph2, nph3, and nph4 mutants are all altered with respect to their phototropic responses, only the nph4 mutants are also altered in their gravitropic responsiveness. Thus, NPH2 and NPH3 appear to act as signal carriers in a phototropism-specific pathway, whereas NPH4 is required for both phototropism and gravitropism and thus may function directly in the differential growth response. Despite their altered phototropic responses in blue and green light as etiolated seedlings, the nph2 and nph4 mutants exhibited less dramatic mutant phenotypes as de-etiolated seedlings and when etiolated seedlings were irradiated with unilateral ultraviolet-A (UV-A) light. Examination of the phototropic responses of a mutant deficient in biologically active phytochromes, hy1–100, indicated that phytochrome transformation by UV-A light mediates an increase in phototropic responsiveness, accounting for the greater phototropic curvature of the nph2 and nph4 mutants to UV-A light than to blue light.

Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.

Modulation of Phototropic Responsiveness in Arabidopsis through Ubiquitination of Phototropin 1 by the CUL3-Ring E3 Ubiquitin Ligase CRL3 NPH3

This work demonstrates that the NPH3 protein of Arabidopsis represents a core component of a CULLIN3-based E3 ubiquitin ligase that targets the phototropin1 (phot1) photoreceptor for blue light–stimulated mono/multi- and polyubiquitination. In addition, it was shown that phot1 ubiquitination by this E3 complex is necessary for normal phototropic responsiveness. Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3NPH3. Under low-intensity BL, CRL3NPH3 mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3NPH3, with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.