New Papers

Plant Ubiquitin/26S Proteasome Papers in The Last Three Months

Updated on 10/25/2009

1: Genes Cells. 2009 Oct 22. [Epub ahead of print] Related Articles: Mammalian COP9 signalosome.

Kato JY, Yoneda-Kato N.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

The COP9 signalosome (CSN) complex is highly conserved from yeast to human. Although the plant CSN was first identified as a negative regulator of photomorphogenesis, the mammalian CSN is linked to different biological responses such as checkpoint control, signal transduction, development and the cell cycle. Frequent over-expression of the CSN subunit in a variety of human cancers suggests its involvement in cell transformation and tumorigenesis. The best-known biochemical function associated with the CSN is the control of protein stability via the ubiquitin-proteasome system through regulation of cullin-RING-E3 ubiquitin ligase activity by deneddylation, by controlling the activity of COP1 E3 ligase, or by counteracting ubiquitin-mediated degradation through a CSN-associated deubiquitinating enzyme. In addition to affecting the stability of transcription factors, the CSN may regulate gene transcription by directly associating with chromatin. This review summarizes recent findings and discusses the physiological role and the cellular function of the mammalian CSN in terms of the regulation of cell proliferation.

PMID: 19849719 [PubMed - as supplied by publisher]

2: Plant Signal Behav. 2009 Sep 10;4(9). [Epub ahead of print] Related Articles: Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants.

Stuttmann J, Parker JE, Noël LD.

Institut de Biologie Environnementale et Biotechnologie (IBEB), UMR 6191 CNRS-CEA-Université de la méditerranée Aix-Marseille II, CEN Cadarache, Saint Paul-lez-Durance, France; Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, Cologne, Germany.

The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.(1) Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization.

PMID: 19847120 [PubMed - as supplied by publisher]

3: Yi Chuan. 2009 Oct;31(10):1042-8. Related Articles

[Analysis of cDNA library of Cucumis sativus L. challenged by Pseudomonas syringae pv. Lachrymans.]

[Article in Chinese]

Liu GJ, Wang LJ, Qin ZW, Meng LB.

1. College of Horticulture, Northeast Agricultural University, Harbin 150030, China; 2. The Laboratory of Forest Genetics and Breeding and Biotechnology of Ministry of Education, Northeast Forestry University, Harbin 150040, China E-mail: liuguanjun2003@126.com.

A cDNA library was constructed from the leaves of the disease-resistant cucumber (Cucumis sativus L.) cultivar 'D0462' challenged by Pseudomonas syringae pv. Lachrymans for 48 h. The inserted fragment sizes ranged from 0.45 to 2.1 kb and the average inserted size was 1 kb. Sequencing analysis showed that 2 352 TUTs (Tentative unique transcripts), 282 contigs, and 2 070 singlets were identified in the 2 966 ESTs derived from the cDNA library. The result of the BlastX analysis indicated that there were 1 848 ESTs with known or unknown function, 504 ESTs with no significant similarity matching with any protein or DNA sequence in the databases. In this library, many defense/disease-resistant related genes, such as metallothionein, glutathione S-transferase, ubiquitin, b-1, 3-glucanase, zinc finger protein, and cysteine protease, which might participate in the plant and the pathogens, are inclued.

Publication Types:

English Abstract

PMID: 19840927 [PubMed - in process]

4: Plant Cell. 2009 Sep 30. [Epub ahead of print] Related Articles, Links: Cryptochromes, Phytochromes, and COP1 Regulate Light-Controlled Stomatal Development in Arabidopsis.

Kang CY, Lian HL, Wang FF, Huang JR, Yang HQ.

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

In Arabidopsis thaliana, the cryptochrome (CRY) blue light photoreceptors and the phytochrome (phy) red/far-red light photoreceptors mediate a variety of light responses. COP1, a RING motif-containing E3 ubiquitin ligase, acts as a key repressor of photomorphogenesis. Production of stomata, which mediate gas and water vapor exchange between plants and their environment, is regulated by light and involves phyB and COP1. Here, we show that, in the loss-of-function mutants of CRY and phyB, stomatal development is inhibited under blue and red light, respectively. In the loss-of-function mutant of phyA, stomata are barely developed under far-red light. Strikingly, in the loss-of-function mutant of either COP1 or YDA, a mitogen-activated protein kinase kinase kinase, mature stomata are developed constitutively and produced in clusters in both light and darkness. CRY, phyA, and phyB act additively to promote stomatal development. COP1 acts genetically downstream of CRY, phyA, and phyB and in parallel with the leucine-rich repeat receptor-like protein TOO MANY MOUTHS but upstream of YDA and the three basic helix-loop-helix proteins SPEECHLESS, MUTE, and FAMA, respectively. These findings suggest that light-controlled stomatal development is likely mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.

PMID: 19794114 [PubMed - as supplied by publisher]

5: Plant Cell. 2009 Sep 29. [Epub ahead of print] Related Articles, Links: Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex.

Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR.

Department of Cell and Systems Biology, University of Toronto, Toronto, Canada M5S 3B2.

In the Brassicaceae, compatible pollen-pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat-containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.

PMID: 19789280 [PubMed - as supplied by publisher]

6: Plant Physiol. 2009 Sep 23. [Epub ahead of print] Related Articles, Links: LIN, a novel type of U-box/WD40 protein, controls early infection by rhizobia in legumes.

Kiss E, Olah B, Kalo P, Morales M, Heckmann AB, Borbola A, Lozsa A, Kontar K, Middleton P, Downie JA, Oldroyd GE, Endre G.

Institute of Genetics, Biological Research Center, Szeged 6726, Hungary; Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom; Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom.

The formation of a nitrogen fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase (CCaMK) is independent of LIN, thus LIN is not necessary for nodule organogenesis. From this we infer that LIN predominantly functions during rhizobial colonization and the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the Nod factor signal transduction pathway and its function is required for correct temporal and spatial activity of the target protein(s).

PMID: 19776163 [PubMed - as supplied by publisher]

7: Plant Cell. 2009 Aug;21(8):2378-90. Epub 2009 Aug 28. Related Articles, Links: Biochemical insights on degradation of Arabidopsis della proteins gained from a cell-free assay system.

Wang F, Zhu D, Huang X, Li S, Gong Y, Yao Q, Fu X, Fan LM, Deng XW.

National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China.

The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro. Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity. Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which regulated proteolysis is a key recurrent theme.

PMID: 19717618 [PubMed - in process]
PMCID: PMC2751948 [Available on 2010/08/01]

8: Plant Cell. 2009 Aug;21(8):2220-36. Epub 2009 Aug 28. Related Articles, Links: The Arabidopsis CORONATINE INSENSITIVE1 Protein Is a Jasmonate Receptor.

Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H, Nan F, Wang Z, Xie D.

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.

Jasmonates play a number of diverse roles in plant defense and development. CORONATINE INSENSITIVE1 (COI1), an F-box protein essential for all the jasmonate responses, interacts with multiple proteins to form the SCF(COI1) E3 ubiquitin ligase complex and recruits jasmonate ZIM-domain (JAZ) proteins for degradation by the 26S proteasome. To determine which protein directly binds to jasmonoyl-isoleucine (JA-Ile)/coronatine (COR) and serves as a receptor for jasmonate, we built a high-quality structural model of COI1 and performed molecular modeling of COI1-jasmonate interactions. Our results imply that COI1 has the structural traits for binding JA-Ile or COR. The direct binding of these molecules with COI1 was further examined using a combination of molecular and biochemical approaches. First, we used the immobilized jasmonate approach to show that the COI1 protein in crude leaf extracts can bind to the jasmonate moiety of JA-Ile. Second, we employed surface plasmon resonance technology with purified COI1 and JAZ1 protein to reveal the interaction among COI1, JA-Ile, and JAZ1. Finally, we used the photoaffinity labeling technology to show the direct binding of COR with purified insect-expressed COI1. Taken together, these results demonstrate that COI1 directly binds to JA-Ile and COR and serves as a receptor for jasmonate.

PMID: 19717617 [PubMed - in process]
PMCID: PMC2751961 [Available on 2010/08/01]

9: Plant J. 2009 Aug 21. [Epub ahead of print] Related Articles, Links: CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings.

Sato T, Maekawa S, Yasuda S, Sonoda Y, Katoh E, Ichikawa T, Nakazawa M, Seki M, Shinozaki K, Matsui M, Goto DB, Ikeda A, Yamaguchi J.

Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, Kita-ku N10-W8, Sapporo 060-0810, Japan.

Summary Plants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

PMID: 19702666 [PubMed - as supplied by publisher]

10: Plasmid. 2009 Nov;62(3):191-200. Epub 2009 Aug 21.
Related Articles, Links

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter.

Masani MY, Parveez GK, Izawati AM, Lan CP, Siti Nor Akmar A.

Advanced Biotechnology and Breeding Centre (ABBC), Biological Research Division, Malaysian Palm Oil Board (MPOB), Kuala Lumpur, Malaysia. masani@mpob.gov.my

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19699761 [PubMed - in process]

11: Phytochemistry. 2009 Aug 18. [Epub ahead of print]
Related Articles, Links: The wound hormone jasmonate.

Koo AJ, Howe GA.

Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, United States.

Plant tissues are highly vulnerable to injury by herbivores, pathogens, mechanical stress, and other environmental insults. Optimal plant fitness in the face of these threats relies on complex signal transduction networks that link damage-associated signals to appropriate changes in metabolism, growth, and development. Many of these wound-induced adaptive responses are triggered by de novo synthesis of the plant hormone jasmonate (JA). Recent studies provide evidence that JA mediates systemic wound responses through distinct cell autonomous and non-autonomous pathways. In both pathways, bioactive JAs are recognized by an F-box protein-based receptor system that couples hormone binding to ubiquitin-dependent degradation of transcriptional repressor proteins. These results provide a framework for understanding how plants recognize and respond to tissue injury.

PMID: 19695649 [PubMed - as supplied by publisher]

12: Annu Rev Genet. 2009 Aug 17. [Epub ahead of print]
Related Articles, Links: Mechanism of Auxin-Regulated Gene Expression in Plants.

Chapman EJ, Estelle M.

Division of Biology, University of California, San Diego, La Jolla, CA 92093-0116; email: echapman@ucsd.edu.

Plant hormones control most aspects of the plant life cycle by regulating genome expression. Expression of auxin-responsive genes involves interactions among auxin-responsive DNA sequence elements, transcription factors and trans-acting transcriptional repressors. Transcriptional output from these auxin signaling complexes is regulated by proteasome-mediated degradation that is triggered by interaction with auxin receptor-E3 ubiquitin ligases such SCF(TIR1). Auxin signaling components are conserved throughout land plant evolution, and have proliferated and specialized to control specific developmental processes. Expected final online publication date for the Annual Review of Genetics Volume 43 is October 28, 2009. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

PMID: 19686081 [PubMed - as supplied by publisher]

13: Plant J. 2009 Aug 13. [Epub ahead of print]
Related Articles, Links: An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis.

Gou M, Su N, Zheng J, Huai J, Wu G, Zhao J, He J, Tang D, Yang S, Wang G.

State Key Laboratory of Agrobiotechnology and National Center for Plant Gene Research (Beijing), China Agricultural University, Beijing 100193, China.

Summary Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30-conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30-conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30-conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis.

PMID: 19682297 [PubMed - as supplied by publisher]

14: Planta Med. 2009 Aug 3. [Epub ahead of print]
Related Articles, Links: Apigenin, Chrysin, and Luteolin Selectively Inhibit Chymotrypsin-Like and Trypsin-Like Proteasome Catalytic Activities in Tumor Cells.

Wu YX, Fang X.

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan.

The ubiquitin-proteasome pathway has an important role in regulating apoptosis and the cell cycle. The function of proteasomes is mediated by three main catalytic activities: (1) chymotrypsin-like (CT-L), (2) trypsin-like (T-L), and (3) peptidylglutamyl peptide hydrolyzing (PGPH). Recently, proteasome inhibitors have been revealed to have an antitumor effect, and have been used to treat cancers such as multiple myeloma. Previous studies have reported that some flavonoids can inhibit proteasome activity in tumor cells. To further investigate the proteasome-inhibitory mechanism of flavonoids, we examined the effects of the plant flavonoids apigenin, chrysin, and luteolin on the three individual catalytic activities in various cancer cell lines. Using fluorogenic substrates specific for proteasome catalytic subunits, we demonstrated the subunit specificity of each flavonoid. Addition of apigenin, chrysin and luteolin inhibited CT-L and T-L catalytic activities in a dose-dependent manner, whereas their effect on PGPH catalytic activity was weak. Our study suggested that these flavonoids have a specific role in inhibition of CT-L and T-L proteasome catalytic activities. © Georg Thieme Verlag KG Stuttgart · New York.

PMID: 19653143 [PubMed - as supplied by publisher]

15: Carcinogenesis. 2009 Oct;30(10):1754-62. Epub 2009 Jul 24.
Related Articles, Links

1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic alpha,beta-unsaturated aldehyde acrolein.

Kelleher MO, McMahon M, Eggleston IM, Dixon MJ, Taguchi K, Yamamoto M, Hayes JD.

Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Scotland, UK.

Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate-cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 microM CETP eliciting a remarkable approximately 10-fold induction of NQO1. Furthermore, 50 microM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1(-/-) mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1(-/-) MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2(+/+) MEFs, but not Nrf2(-/-) MEFs, with 15 microM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the alpha,beta-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19633057 [PubMed - in process]

16: Plant Mol Biol. 2009 Oct;71(3):307-18. Epub 2009 Jul 21.
Related Articles, Links

Overexpression of the Arabidopsis anaphase promoting complex subunit CDC27a increases growth rate and organ size.

Rojas CA, Eloy NB, Lima Mde F, Rodrigues RL, Franco LO, Himanen K, Beemster GT, Hemerly AS, Ferreira PC.

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher (3)H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19629716 [PubMed - indexed for MEDLINE]

17: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13618-23. Epub 2009 Jul 20.
Related Articles, Links

The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development.

Graciet E, Walter F, Maoiléidigh DO, Pollmann S, Meyerowitz EM, Varshavsky A, Wellmer F.

Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland. graciete@tcd.ie

The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. This proteolytic system is present in all organisms examined and has been shown to have a multitude of functions in animals and fungi. In plants, however, the functional understanding of the N-end rule pathway is only beginning. The N-end rule has a hierarchic structure. Destabilizing activity of N-terminal Asp, Glu, and (oxidized) Cys requires their conjugation to Arg by an arginyl-tRNA-protein transferase (R-transferase). The resulting N-terminal Arg is recognized by the pathway's E3 ubiquitin ligases, called "N-recognins." Here, we show that the Arabidopsis R-transferases AtATE1 and AtATE2 regulate various aspects of leaf and shoot development. We also show that the previously identified N-recognin PROTEOLYSIS6 (PRT6) mediates these R-transferase-dependent activities. We further demonstrate that the arginylation branch of the N-end rule pathway plays a role in repressing the meristem-promoting BREVIPEDICELLUS (BP) gene in developing leaves. BP expression is known to be excluded from Arabidopsis leaves by the activities of the ASYMMETRIC LEAVES1 (AS1) transcription factor complex and the phytohormone auxin. Our results suggest that AtATE1 and AtATE2 act redundantly with AS1, but independently of auxin, in the control of leaf development.

Publication Types:

PMID: 19620738 [PubMed - indexed for MEDLINE]

PMCID: PMC2726413 [Available on 2010/02/11]

18: Plant Cell Physiol. 2009 Sep;50(9):1721-5. Epub 2009 Jul 15.
Related Articles, Links

The Arabidopsis 26S proteasome subunit RPN1a is required for optimal plant growth and stress responses.

Wang S, Kurepa J, Smalle JA.

Plant Physiology, Biochemistry, Molecular Biology Program, Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40546, USA.

The current literature offers contradictory results regarding the role of the proteasome subunit RPN1a in Arabidopsis development. Here we show that plants lacking RPN1a are viable and have increased cell sizes, decreased heat shock tolerance, increased oxidative stress tolerance and other phenotypes characteristic for 26S proteasome subunit mutants. These results strengthen our contention that most of the phenotypes of 26S proteasome mutants in Arabidopsis described to date reflect a general impairment in 26S proteasome function rather than a specific defect of a single subunit, and suggest that the role of the RPN1a subunit during embryogenesis needs to be reconsidered.

Publication Types:

PMID: 19605416 [PubMed - indexed for MEDLINE]

19: Plant Cell Physiol. 2009 Aug;50(8):1558-72. Epub 2009 Jul 4.
Related Articles, Links

Pollen-specific SKP1-like proteins are components of functional scf complexes and essential for lily pollen tube elongation.

Chang LC, Guo CL, Lin YS, Fu H, Wang CS, Jauh GY.

Institute of Life Science, National Defense Medical Center, Taipei, Taiwan, ROC.

The ubiquitin-proteasome pathway mediates protein degradation and is involved in diverse aspects of plant development and differentiation, including pollen tube elongation and self-incompatibility. We characterized three lily (Lilium longiflorum) SKP1-like genes, LSK1-LSK3, that are specifically expressed in late pollen developmental stages and the elongating pollen tube. The encoded peptide sequences reveal that LSK1-LSK3 share high identity with Arabidopsis ASK1 and contain a putative N-terminal CUL1- and a C-terminal F-box-interacting domain. Yeast two-hybrid and in vitro affinity binding assays revealed that the LSKs associate with lily CULLIN1. In addition, the LSK genes can functionally complement the yeast skp1 deletion mutant YDR328C. To investigate their biological functions in pollen tube elongation, an in vivo approach for green fluorescent protein (GFP)-tagged dominant-negative LSK1-LSK3 was developed. Microprojectile bombardment with N-terminally truncated LSK1-LSK3 (LSK1-LSK3Delta-GFP) significantly retarded pollen tube elongation in both in vitro germination and in vivo self- and cross-pollination after >12 h incubation. Interestingly, elongation of pollen tubes harboring overexpressed LSK2Delta-GFP and LSK3Delta-GFP was substantially inhibited within the self-pollinated styles. The elongation of most LSK2Delta-GFP-transformed pollen tubes could germinate only on the stigmatic surface of self style and showed statistically significant growth arrest as compared with control pollen tubes. Lily exhibits typical gametophytic self-incompatibility via an unknown mechanism, but LSK2 and LSK3 may be involved in this complex machinery. These results suggest critical roles for LSK1-LSK3 in regulating fundamental pollen tube elongation in vitro and in vivo and that the 26S proteasome-mediated protein pathway plays an important role in pollen tube elongation.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19578169 [PubMed - indexed for MEDLINE]

20: Biotechnol Lett. 2009 Sep;31(9):1477-83. Epub 2009 Jun 20.
Related Articles, Links

Genetic transformation of cork oak (Quercus suber L.) for herbicide resistance.

Alvarez R, Alvarez JM, Humara JM, Revilla A, Ordás RJ.

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK. ra352@cam.ac.uk

The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities of phosphinothricin acetyl transferase were detected among the lines, which carried 1-4 copies of the insert. Molecular and biochemical assays confirmed the stability and expression of the transgenes 3 months after thawing the cultures. These results demonstrate genetic engineering of herbicide tolerance in Quercus spp.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19543858 [PubMed - indexed for MEDLINE]

21: Plant Physiol. 2009 Aug;150(4):1880-901. Epub 2009 Jun 5.
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Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars.

Fan RC, Peng CC, Xu YH, Wang XF, Li Y, Shang Y, Du SY, Zhao R, Zhang XY, Zhang LY, Zhang DP.

State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China.

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 --> proline in MdSUT1 and leucine-117 --> proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19502355 [PubMed - indexed for MEDLINE]

PMCID: PMC2719124

22: Plant J. 2009 Oct;60(1):68-78. Epub 2009 May 23.
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Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit.

Sonoda Y, Sako K, Maki Y, Yamazaki N, Yamamoto H, Ikeda A, Yamaguchi J.

Faculty of Advanced Life Science, Hokkaido University, Kita-ku N10-W8, Sapporo 060-0810, Japan.

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19500299 [PubMed - in process]

23: Plant Mol Biol. 2009 Aug;70(6):693-708. Epub 2009 Jun 6.
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Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases.

Jia F, Gampala SS, Mittal A, Luo Q, Rock CD.

Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, USA. fan.jia@ttu.edu

The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.

Publication Types:

PMID: 19499346 [PubMed - indexed for MEDLINE]

PMCID: PMC2755202 [Available on 2010/08/01]

24: Plant J. 2009 Sep;59(6):974-86. Epub 2009 May 18.
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A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis.

Koo AJ, Gao X, Jones AD, Howe GA.

Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, USA.

Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F-box protein COI1 triggers the SCF(COI1)/ubiquitin-dependent degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the expression of JA-response genes. JA is required for many wound-inducible systemic defense responses, but little is known about the role of the hormone in long-distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA-Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound-induced systemic production of JA-Ile required the JA biosynthetic enzyme 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA-conjugating enzyme JAR1. Interestingly, the wound-induced synthesis of JA/JA-Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA-Ile, activates the expression of early response genes by the SCF(COI1)/JAZ pathway.

Publication Types:

PMID: 19473329 [PubMed - in process]

25: Plant J. 2009 Sep;59(5):802-13. Epub 2009 May 12.
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ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis.

Sung DY, Kim TH, Komives EA, Mendoza-Cózatl DG, Schroeder JI.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA, USA.

A forward-genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 was the strongest arsenate- and arsenite-resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knock-out mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by the overexpression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild-type (WT) Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces the enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in the WT, and this arsenic induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5, compared with WT, suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background.

Publication Types:

PMID: 19453443 [PubMed - indexed for MEDLINE]

26: Cell Mol Neurobiol. 2009 Sep;29(6-7):991-8. Epub 2009 Mar 17.
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Effect of noradrenalin and EGb 761 pretreatment on the ischemia-reperfusion injured spinal cord neurons in rabbits.

Mechírová E, Domoráková I, Danková M, Danielisová V, Burda J.

Department of Histology and Embryology, Faculty of Medicine, PJ Safárik University in Kosice, 04180 Kosice, Slovak Republic. mechir@post.sk

Short term sublethal ischemia or ischemic preconditioning gives protection to the neurons against subsequent lethal ischemic attack. This so-called ischemic tolerance can also be provided by certain drugs. We examined the effect of noradrenalin and EGb 761 on the spinal cord neurons injured by 30 min occlusion of abdominal aorta in rabbits. The animals survived 48 and 72 h. Degenerated neurons were visualized by Fluoro Jade B method, viable neurons were demonstrated immunohistochemically with NeuN and ubiquitin antibodies. The rabbits with noradrenalin administration 48 h before 30 min of ischemia and 48/72 h of reperfusion, showed significant increase of degenerated Fluoro Jade B labeled neurons. Animals of both groups were paraplegic. Rabbits pretreated 7 days with EGb 761 prior to 30 min of ischemia and with 48/72 h of reperfusion revealed significant decrease of Fluoro Jade B-positive neurons when compared with the groups with 30 min of ischemia followed by 48/72 h of reperfusion. In the NeuN sections, the number of viable neurons was moderately decreased. These animals showed no paraplegia. Ubiquitin aggregates occurred in the cytoplasm of degenerated neurons in the sections of rabbits preconditioned with noradrenalin 48 h prior to 30 min of ischemia and followed by 48 h of reperfusion while after 72 h of reperfusion, shrunk light shadows without ubiquitin reaction were visible. Our results indicate that EGb 761 could be involved in protection of spinal cord neurons against ischemic injury while effect of noradrenalin is not unambiguous.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19291391 [PubMed - indexed for MEDLINE]

27: Transgenic Res. 2009 Aug;18(4):655-60. Epub 2009 Feb 25.
Related Articles, Links

Production of taxa-4(5),11(12)-diene by transgenic Physcomitrella patens.

Anterola A, Shanle E, Perroud PF, Quatrano R.

Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901, USA. anterola@siu.edu

Taxadiene synthase gene from Taxus brevifolia was constitutively expressed in the moss Physcomitrella patens using a ubiquitin promoter to produce taxa-4(5),11(12)-diene, the precursor of the anticancer drug paclitaxel. In stable moss transformants, taxa-4(5),11(12)-diene was produced up to 0.05% fresh weight of tissue, without significantly affecting the amounts of the endogenous diterpenoids (ent-kaurene and 16-hydroxykaurane). Unlike higher plants that had been genetically modified to produce taxa-4(5),11(12)-diene, transgenic P. patens did not exhibit growth inhibition due to alteration of diterpenoid metabolic pools. Thus we propose that P. patens is a promising alternative host for the biotechnological production of paclitaxel and its precursors.

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 19241134 [PubMed - indexed for MEDLINE]

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