Approaches to understanding auxin action

The effects of auxin on plant growth and development have been studied for decades, but the molecular mechanisms of auxin action remain unknown. These mechanisms have primarily been investigated by characterization of auxin physiology mutants and analysis of auxin-binding proteins and auxin-regulated genes. These efforts are now converging, since some mutants have recently been shown to have altered expression of specific auxin-binding proteins and auxin-regulated genes. The features of these proteins and genes are providing the first tantalizing clues to the organization of auxin signal transduction pathways.     

Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression

In Arabidopsis, SHY2 encodes IAA3, a member of the auxin-induced Aux/IAA family. Gain-of-function mutations in SHY2/IAA3 cause enlarged cotyledons, short hypocotyls, and altered auxin-regulated root development. Here we show that the gain-of-function mutation shy2-2 decreases both the induction and repression of auxin-regulated genes, suggesting that SHY2/IAA3 acts as a negative regulator in auxin signaling. shy2-2 affects auxin induction of many previously characterized primary response genes, implying that it might repress primary auxin responses. In addition, shy2-2 also affects expression of multiple auxin-nonresponsive genes. Light regulates expression of SHY2/IAA3, suggesting a possible link between light and auxin response pathways.     

A role for the anaphase promoting complex in hormone regulation

To increase our knowledge of anaphase promoting complex (APC/C) function during plant development, we characterized an Arabidopsis thaliana T-DNA-insertion line where the T-DNA fell within the 5′ regulatory region of the APC10 gene. The insert disrupted endogenous expression, resulting in overexpression of APC10 mRNA from the T-DNA- internal CaMV 35S promoter, and increased APC10 protein. Overexpression of APC10 produced phenotypes resembling those of known auxin and ethylene mutants, and increased expression of two tested auxin-regulated genes, small auxin up RNA (SAUR) 15 and SAUR24. Taken together, our data suggests that elevated APC10 likely mimics auxin and ethylene sensitive phenotypes, expanding our understanding of proteolytic processes in hormone regulation of plant development.     

Auxin-regulated gene expression

During the 1960s a wide range of studies provided an information base that led to the suggestion that auxin-regulated cell processes--especially cell elongation--may be mediated by auxin-regulated gene expression. Indirect evidence from our work, based on the influence of inhibitors of RNA synthesis (e.g. actinomycin D) and of protein synthesis (e.g. cycloheximide) on auxin-induced cell elongation, coupled with correlations of the influence of auxin on RNA synthesis and cell elongation, provided the basis for this suggestion. With the availability of techniques for DNA-DNA and DNA-RNA hybridization, mRNA isolation-translation, in vitro 2D gel analysis of the translation products, and ultimately the cloning by recombinant DNA technologies of genomic DNA and copy DNAs (cDNAs) made to poly(A)+ mRNAs, we and others have provided direct evidence for the influence of auxin on the expression of a few genes (i.e. poly(A)+ RNA levels). Our laboratory has provided evidence for auxin's both down-regulating and up-regulating the level of a few poly(A)+ mRNAs out of a population of about 4 X 10(4) sequences that are not significantly affected by auxin. In our studies on auxin-regulated cell elongation, two cDNA clones (pJCW1 and pJCW2) were isolated which corresponded to poly(A)+ mRNAs that responded during growth transitions in a way consistent with a potential role of their protein products in cell elongation. These mRNAs are most abundant in the elongating zone of the soybean hypocotyl. Upon excision and incubation in the absence of auxin, these mRNAs deplete in concert with a decreasing rate of cell elongation. Addition of auxin to the medium results in both increased levels of these mRNAs and enhanced rates of cell elongation. These mRNAs do not deplete if auxin is added to the medium at the onset of excised incubation, and cell elongation rates remain high. We have isolated and sequenced genomic clones that are homologous to these cDNAs. Of the two genes sequenced, both genes are members of small multigene families. There are regions of high amino acid homology even though the nucleotide sequences are sufficiently different in these regions for cross-hybridization of the clones not to be observed. More recently others, especially Guilfoyle's laboratory, have shown that auxin selectively and rapidly influences the level of certain mRNAs and proteins. We have worked on other gene systems such as ribosomal proteins and possible cell wall proteins that are responsive to auxin; again the nature of regulation of expression of these genes is not known.(ABSTRACT TRUNCATED AT 400 WORDS)     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

A proteomics study of auxin effects in Arabidopsis thaliana

Many phytohormones regulate plant growth and development through modulating protein degradation. In this study, a proteome study based on multidimensional non-gel shotgun approach was performed to analyze the auxin-induced protein degradation via ubiquitin-proteasome pathway of Arabidopsis thaliana, with the emphasis to study the overall protein changes after auxin treatment (1 nM or 1 μM indole-3-acetic acid for 6, 12, or 24 h). More than a thousand proteins were detected by using label-free shotgun method, and 386 increased proteins and 370 decreased ones were identified after indole-3-acetic acid treatment. By using the auxin receptor-deficient mutant, tir1-1, as control, comparative analysis revealed that 69 and 79 proteins were significantly decreased and increased, respectively. Detailed analysis showed that among the altered proteins, some were previously reported to be associated with auxin regulation and others are potentially involved in mediating the auxin effects on specific cellular and physiological processes by regulating photosynthesis, chloroplast development, cytoskeleton, and intracellular signaling. Our results demonstrated that label-free shotgun proteomics is a powerful tool for large-scale protein identification and the analysis of the proteomic profiling of auxin-regulated biological processes will provide informative clues of underlying mechanisms of auxin effects. These results will help to expand the understanding of how auxin regulates plant growth and development via protein degradation.