Auxin Action in a Cell-Free System

The plant hormone auxin regulates diverse aspects of plant growth and development [1]. Despite its importance, the mechanisms of auxin action remain poorly understood. In particular, the identities of the auxin receptor and other signaling proteins are unknown. Recent studies have shown that auxin acts by promoting the degradation of a family of transcriptional regulators called the Aux/IAA proteins 2, 3 and 4. These proteins interact with another large family of plant-specific transcription factors called Auxin Response Factors (ARF) and negatively regulate their activity [5]. Auxin stimulates Aux/IAA degradation by promoting the interaction between a ubiquitin protein ligase (E3) called SCFTIR1 and the Aux/IAA protein [2]. In this report, we demonstrate that auxin promotes the interaction between the Aux/IAA proteins and SCFTIR1 in a soluble extract free of membranes, indicating that this auxin response is mediated by a soluble receptor. In addition, we show that the response is not dependent on protein phosphorylation or dephosphorylation but rather is prevented by an inhibitor of peptidyl-prolyl isomerases

Molecular cloning and characterization of nucleoside diphosphate kinase in cultured sugarcane cells

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995.Includes bibliographical references (leaves 104-124).Microfiche.xii, 124 leaves, bound photos. 29 cmA low molecular weight autophosphorylating protein (pp18) in cultured sugarcane cells was identified and characterized as nucleoside diphosphate (NDP) kinase. The purified NDP kinase separated into five isoforms of 16.5kD (NDP kinase I) and two isoforms of 18kD (NDP kinase IT) on two dimensional IEF/SDS-PAGE. The apparent K, values of the purified enzyme containing both size classes were 2.3mM and 0.2mM for ATP and GDP, respectively. Twenty one positive clones were isolated by screening a λgt11 cDNA library derived from cultured sugarcane cells with spinach NDP kinase I cDNA probe, and four were sequenced. The cDNA pSCNDK8 contained a 447 bp coding region (149 amino acids), and 58 bp and 238 bp 5' and 3' flanking sequences respectively. This cDNA hybridized to a 0.91kb mRNA. The deduced amino acid sequence of sugarcane NDP kinase cDNA clone showed over 60% sequence identity to many eukaryotic NDP kinases. The cDNA pSCNDK8 showed a frame shift resulting in 55 amino acid residues at the carboxyl terminus with no homology to NDP kinases. The sugarcane NDP kinase showed approximately a 3 to 4-fold enhancement in in situ autophosphorylation and 1.5 to 2 fold increase in NDP kinase activity in response to HS at 40-42°C for 2h. However, NDP kinase protein or mRNA levels did not show an increase during HS. The synthesis and phosphorylation of NDP kinase appears to be developmentally and heat shock regulated. NDP kinase levels were highest and showed a greater enhancement in autophosphorylation in response to HS in cells in fresh culture. In contrast to cultured sugarcane cells, mRNAs for NDP kinase I enhanced at least 2-fold in response to a 2 h HS at 40°C in young sugarcane shoots

The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis

The r elated-to- ub iquitin (RUB) protein is post-translationally conjugated to the cullin subunit of the SCF (SKP1, Cullin, F-box) class of ubiquitin protein ligases. Although the precise biochemical function of RUB modification is unclear, studies indicate that the modification is important for SCF function. In Arabidopsis , RUB modification of CUL1 is required for normal function of SCF TIR1 , an E3 required for response to the plant hormone auxin. In this report we show that an Arabidopsis protein called RCE1 functions as a RUB-conjugating enzyme in vivo . A mutation in the RCE1 gene results in a phenotype like that of the axr1 mutant. Most strikingly, plants deficient in both RCE1 and AXR1 have an embryonic phenotype similar to mp and bdl mutants, previously shown to be deficient in auxin signaling. Based on these results, we suggest that the RUB-conjugation pathway is required for auxin-dependent pattern formation in the developing embryo. In addition, we show that RCE1 interacts directly with the RING protein RBX1 and is present in a stable complex with SCF. We propose that RBX1 functions as an E3 for RUB modification of CUL1

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCF(TIR1) complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCF(TIR1) function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCF(TIR1) substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCF(TIR1) function, suggesting that cycles of RUB conjugation and removal are important for SCF activity

The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE Proteins Are Nucleoporins with an Important Role in Hormone Signaling and Development

Nucleocytoplasmic transport of macromolecules is regulated by a large multisubunit complex called the nuclear pore complex (NPC). Although this complex is well characterized in animals and fungi, there is relatively little information on the NPC in plants. The suppressor of auxin resistance1 (sar1) and sar3 mutants were identified as suppressors of the auxin-resistant1 (axr1) mutant. Molecular characterization of these genes reveals that they encode proteins with similarity to vertebrate nucleoporins, subunits of the NPC. Furthermore, a SAR3–green fluorescent protein fusion protein localizes to the nuclear membrane, indicating that SAR1 and SAR3 are Arabidopsis thaliana nucleoporins. Plants deficient in either protein exhibit pleiotropic growth defects that are further accentuated in sar1 sar3 double mutants. Both sar1 and sar3 mutations affect the localization of the transcriptional repressor AXR3/INDOLE ACETIC ACID17, providing a likely explanation for suppression of the phenotype conferred by axr1. In addition, sar1 sar3 plants accumulate polyadenylated RNA within the nucleus, indicating that SAR1 and SAR3 are required for mRNA export. Our results demonstrate the important role of the plant NPC in hormone signaling and development