Publications by authors named "Marcela Rojas-Pierce"

18 Publications

  • Page 1 of 1

A whole-cell electron tomography model of vacuole biogenesis in Arabidopsis root cells.

Nat Plants 2019 01 17;5(1):95-105. Epub 2018 Dec 17.

School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

Plant vacuoles are dynamic organelles that play essential roles in regulating growth and development. Two distinct models of vacuole biogenesis have been proposed: separate vacuoles are formed by the fusion of endosomes, or the single interconnected vacuole is derived from the endoplasmic reticulum. These two models are based on studies of two-dimensional (2D) transmission electron microscopy and 3D confocal imaging, respectively. Here, we performed 3D electron tomography at nanometre resolution to illustrate vacuole biogenesis in Arabidopsis root cells. The whole-cell electron tomography analysis first identified unique small vacuoles (SVs; 400-1,000 nm in diameter) as nascent vacuoles in early developmental cortical cells. These SVs contained intraluminal vesicles and were mainly derived/matured from multivesicular body (MVB) fusion. The whole-cell vacuole models and statistical analysis on wild-type root cells of different vacuole developmental stages demonstrated that central vacuoles were derived from MVB-to-SV transition and subsequent fusions of SVs. Further electron tomography analysis on mutants defective in MVB formation/maturation or vacuole fusion demonstrated that central vacuole formation required functional MVBs and membrane fusion machineries.
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http://dx.doi.org/10.1038/s41477-018-0328-1DOI Listing
January 2019

Phosphoinositides control the localization of HOPS subunit VPS41, which together with VPS33 mediates vacuole fusion in plants.

Proc Natl Acad Sci U S A 2018 08 13;115(35):E8305-E8314. Epub 2018 Aug 13.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695;

The vacuole is an essential organelle in plant cells, and its dynamic nature is important for plant growth and development. Homotypic membrane fusion is required for vacuole biogenesis, pollen germination, stomata opening, and gravity perception. Known components of the vacuole fusion machinery in eukaryotes include SNARE proteins, Rab GTPases, phosphoinositides, and the homotypic fusion and vacuolar protein sorting (HOPS) tethering complex. HOPS function is not well characterized in plants, but roles in embryogenesis and pollen tube elongation have been reported. Here, we show that HOPS subunits VPS33 and VPS41 accumulate in late endosomes and that VPS41, but not VPS33, accumulates in the tonoplast via a wortmannin-sensitive process. VPS41 and VPS33 proteins bind to liposomes, but this binding is inhibited by phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P, which implicates a nonconserved mechanism for HOPS recruitment in plants. Inducible knockdown of VPS41 resulted in dramatic vacuole fragmentation phenotypes and demonstrated a critical role for HOPS in vacuole fusion. Furthermore, we provide evidence for genetic interactions between VPS41 and VTI11 SNARE that regulate vacuole fusion, and the requirement of a functional SNARE complex for normal VPS41 and VPS33 localization. Finally, we provide evidence to support VPS33 and SYP22 at the initial stage for HOPS-SNARE interactions, which is similar to other eukaryotes. These results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants.
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http://dx.doi.org/10.1073/pnas.1807763115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126739PMC
August 2018

Vacuolar trafficking and biogenesis: a maturation in the field.

Curr Opin Plant Biol 2017 12 1;40:77-81. Epub 2017 Sep 1.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, United States. Electronic address:

The vacuole is a prominent organelle that is essential for plant viability. The vacuole size, and its role in ion homeostasis, protein degradation and storage, place significant demands for trafficking of vacuolar cargo along the endomembrane system. Recent studies indicate that sorting of vacuolar cargo initiates at the ER and Golgi, but not the trans-Golgi network/early endosome, as previously thought. Furthermore, maturation of the trans-Golgi network into pre-vacuolar compartments seems to contribute to a major route for plant vacuolar traffic that works by bulk flow and ends with membrane fusion between the pre-vacuolar compartment and the tonoplast. Here we summarize recent evidence that indicates conserved and plant-specific mechanisms involved in sorting and trafficking of proteins to this major organelle.
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http://dx.doi.org/10.1016/j.pbi.2017.08.005DOI Listing
December 2017

Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of Upland cotton (Gossypium hirsutum L.).

Proc Natl Acad Sci U S A 2017 01 20;114(1):E57-E66. Epub 2016 Dec 20.

Department of Crop and Soil Sciences, North Carolina State University, Raleigh, NC 27695-7620;

Leaf shape varies spectacularly among plants. Leaves are the primary source of photoassimilate in crop plants, and understanding the genetic basis of variation in leaf morphology is critical to improving agricultural productivity. Leaf shape played a unique role in cotton improvement, as breeders have selected for entire and lobed leaf morphs resulting from a single locus, okra (l-D), which is responsible for the major leaf shapes in cotton. The l-D locus is not only of agricultural importance in cotton, but through pioneering chimeric and morphometric studies, it has contributed to fundamental knowledge about leaf development. Here we show that an HD-Zip transcription factor homologous to the LATE MERISTEM IDENTITY1 (LMI1) gene of Arabidopsis is the causal gene underlying the l-D locus. The classical okra leaf shape allele has a 133-bp tandem duplication in the promoter, correlated with elevated expression, whereas an 8-bp deletion in the third exon of the presumed wild-type normal allele causes a frame-shifted and truncated coding sequence. Our results indicate that subokra is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele and that normal is a derived mutant allele that came to predominate and define the leaf shape of cultivated cotton. Virus-induced gene silencing (VIGS) of the LMI1-like gene in an okra variety was sufficient to induce normal leaf formation. The developmental changes in leaves conferred by this gene are associated with a photosynthetic transcriptomic signature, substantiating its use by breeders to produce a superior cotton ideotype.
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http://dx.doi.org/10.1073/pnas.1613593114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224360PMC
January 2017

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls.

J Exp Bot 2016 12 5;67(22):6459-6472. Epub 2016 Nov 5.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 μm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.
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http://dx.doi.org/10.1093/jxb/erw418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181587PMC
December 2016

REGULATOR OF BULB BIOGENESIS1 (RBB1) Is Involved in Vacuole Bulb Formation in Arabidopsis.

PLoS One 2015 27;10(4):e0125621. Epub 2015 Apr 27.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

Vacuoles are dynamic compartments with constant fluctuations and transient structures such as trans-vacuolar strands and bulbs. Bulbs are highly dynamic spherical structures inside vacuoles that are formed by multiple layers of membranes and are continuous with the main tonoplast. We recently carried out a screen for mutants with abnormal trafficking to the vacuole or aberrant vacuole morphology. We characterized regulator of bulb biogenesis1-1 (rbb1-1), a mutant in Arabidopsis that contains increased numbers of bulbs when compared to the parental control. rbb1-1 mutants also contain fewer transvacuolar strands than the parental control, and we propose the hypothesis that the formation of transvacuolar strands and bulbs is functionally related. We propose that the bulbs may function transiently to accommodate membranes and proteins when transvacuolar strands fail to elongate. We show that RBB1 corresponds to a very large protein of unknown function that is specific to plants, is present in the cytosol, and may associate with cellular membranes. RBB1 is involved in the regulation of vacuole morphology and may be involved in the establishment or stability of trans-vacuolar strands and bulbs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411111PMC
April 2016

Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles.

Plant Signal Behav 2014 ;9(10):e972113

a Department of Plant and Microbial Biology ; North Carolina State University ; Raleigh , NC USA.

Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells.
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http://dx.doi.org/10.4161/psb.29783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623060PMC
August 2015

PLASTID MOVEMENT IMPAIRED1 mediates ABA sensitivity during germination and implicates ABA in light-mediated Chloroplast movements.

Plant Physiol Biochem 2014 Oct 1;83:185-93. Epub 2014 Aug 1.

Department of Botany and Plant Sciences and the Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA.

The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development, including seed development, germination and responses to water-deficit stress. A complex ABA signaling network integrates environmental signals including water availability and light intensity and quality to fine-tune the response to a changing environment. To further define the regulatory pathways that control water-deficit and ABA responses, we carried out a gene-trap tagging screen for water-deficit-regulated genes in Arabidopsis thaliana. This screen identified PLASTID MOVEMENT IMPAIRED1 (PMI1), a gene involved in blue-light-induced chloroplast movement, as functioning in ABA-response pathways. We provide evidence that PMI1 is involved in the regulation of seed germination by ABA, acting upstream of the intersection between ABA and low-glucose signaling pathways. Furthermore, PMI1 participates in the regulation of ABA accumulation during periods of water deficit at the seedling stage. The combined phenotypes of pmi1 mutants in chloroplast movement and ABA responses indicate that ABA signaling may modulate chloroplast motility. This result was further supported by the detection of altered chloroplast movements in the ABA mutants aba1-6, aba2-1 and abi1-1.
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http://dx.doi.org/10.1016/j.plaphy.2014.07.014DOI Listing
October 2014

Homotypic vacuole fusion requires VTI11 and is regulated by phosphoinositides.

Mol Plant 2014 Jun 25;7(6):1026-1040. Epub 2014 Feb 25.

Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA. Electronic address:

Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vti11 mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase (PI3K). We provide evidence that Phosphatidylinositol 3-Phosphate (PtdIns(3)P) regulates vacuole fusion in vti11 mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.
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http://dx.doi.org/10.1093/mp/ssu019DOI Listing
June 2014

Targeting of tonoplast proteins to the vacuole.

Plant Sci 2013 Oct 18;211:132-6. Epub 2013 Jul 18.

Department of Plant Biology, North Carolina State University, Raleigh, NC 27695, United States.

Vacuoles are essential for plant growth and development, and are dynamic compartments that require constant deposition of integral membrane proteins. These membrane proteins carry out many critical functions of the vacuole such as transporting ions and metabolites for vacuolar storage. Understanding the mechanisms for targeting proteins to the vacuolar membrane, or tonoplast, is important for developing novel applications for biotechnology. The mechanisms to target tonoplast proteins to the vacuole are quite complex. Multiple routes, including both Golgi-dependent and Golgi-independent mechanisms, have been implicated in tonoplast protein trafficking. A few endomembrane proteins that regulate this traffic at the level of the endoplasmic reticulum, the pre-vacuolar compartment and the tonoplast are now known. Recent reports indicate that the Golgi-dependent and independent pathways may merge at the level of the pre-vacuolar compartment. Finally, the small GTP-binding protein Rab7 and the SNARE protein SYP21 have been implicated in the traffic of tonoplast proteins from the pre-vacuolar compartment to the tonoplast. With multiple cargo proteins being analyzed under a variety of experimental systems, a clearer picture for targeting mechanisms for tonoplast proteins is starting to emerge.
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http://dx.doi.org/10.1016/j.plantsci.2013.07.005DOI Listing
October 2013

A small molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in Arabidopsis.

PLoS One 2012 5;7(9):e44735. Epub 2012 Sep 5.

Department of Plant Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044735PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434187PMC
February 2013

Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects plant nuclear lipids and nuclear functions.

Plant Physiol Biochem 2012 Aug 17;57:32-44. Epub 2012 May 17.

Department of Genetics, North Carolina State University, Raleigh, NC, USA.

In order to characterize the effects of increasing phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P(2)) on nuclear function, we expressed the human phosphatidylinositol (4)-phosphate 5-kinase (HsPIP5K) 1α in Nicotiana tabacum (NT) cells. The HsPIP5K-expressing (HK) cells had altered nuclear lipids and nuclear functions. HK cell nuclei had 2-fold increased PIP5K activity and increased steady state PtdIns(4,5)P(2). HK nuclear lipid classes showed significant changes compared to NT (wild type) nuclear lipid classes including increased phosphatidylserine (PtdSer) and phosphatidylcholine (PtdCho) and decreased lysolipids. Lipids isolated from protoplast plasma membranes (PM) were also analyzed and compared with nuclear lipids. The lipid profiles revealed similarities and differences in the plasma membrane and nuclei from the NT and transgenic HK cell lines. A notable characteristic of nuclear lipids from both cell types is that PtdIns accounts for a higher mol% of total lipids compared to that of the protoplast PM lipids. The lipid molecular species composition of each lipid class was also analyzed for nuclei and protoplast PM samples. To determine whether expression of HsPIP5K1α affected plant nuclear functions, we compared DNA replication, histone 3 lysine 9 acetylation (H3K9ac) and phosphorylation of the retinoblastoma protein (pRb) in NT and HK cells. The HK cells had a measurable decrease in DNA replication, histone H3K9 acetylation and pRB phosphorylation.
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http://dx.doi.org/10.1016/j.plaphy.2012.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601448PMC
August 2012

Preparation of methyl ester precursors of biologically active agents.

Biotechniques 2008 Mar;44(3):377-84

Department of Chemistry, University of California, Riverside, CA 92521, USA.

This method enables scientists to easily convert biologically active carboxylic acids into their methyl esters ("pro-drugs" generally having improved ability to penetrate cell membranes) using only equipment commonly found in a biology laboratory. An ion-exchange resin is used to convert the acid into its salt, which is thereby sequestered on the resin. The addition of methyl iodide converts the salt to the ester, which has no affinity for the resin and is readily eluted. Evaporation of the liquid phase provides the pure methyl ester. The preparation in good chemical yields of methyl esters of bioactive agents in excellent purity and 10-20 mg quantities can be achieved using this method. The method can be completed in 1 day.
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http://dx.doi.org/10.2144/000112704DOI Listing
March 2008

Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin.

Chem Biol 2007 Dec;14(12):1366-76

Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA 92521, USA.

ATP-binding cassette (ABC) transporters have been implicated in a multitude of biological pathways. In plants, some ABC transporters are involved in the polar transport of the plant hormone auxin and the gravitropic response. We previously identified Gravacin as a potent inhibitor of gravitropism in Arabidopsis thaliana. We demonstrate that P-glycoprotein19 (PGP19) is a target for Gravacin and participates in its inhibition of gravitropism. Gravacin inhibited the auxin transport activity of PGP19 and PGP19-PIN complexes. Furthermore, we identified E1174 as an important residue for PGP19 activity and its ability to form active transport complexes with PIN1. Gravacin is an auxin transport inhibitor that inhibits PGPs, particularly PGP19, which can be used to further dissect the role of PGP19 without the inhibition of other auxin transporters, namely PIN proteins.
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http://dx.doi.org/10.1016/j.chembiol.2007.10.014DOI Listing
December 2007

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole.

Proc Natl Acad Sci U S A 2007 Nov 14;104(47):18801-6. Epub 2007 Nov 14.

Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA.

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for "modified traffic to the vacuole") mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative delta-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.
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http://dx.doi.org/10.1073/pnas.0708236104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2141857PMC
November 2007

The power of chemical genomics to study the link between endomembrane system components and the gravitropic response.

Proc Natl Acad Sci U S A 2005 Mar 16;102(13):4902-7. Epub 2005 Mar 16.

Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521,USA.

Chemical genomics is a powerful approach to dissect processes that may be intractable using conventional genetics because of gene lethality or redundancy. Recently, a link has been established between endomembrane trafficking and gravitropism. To understand this link, we screened a library of 10,000 diverse chemicals for compounds that affected the gravitropism of Arabidopsis seedlings positively or negatively. Sixty-nine of 219 compounds from the primary screen were retested, and 34 of these were confirmed to inhibit or enhance gravitropism. Four of the 34 compounds were found to cause aberrant endomembrane morphologies. The chemicals affected gravitropism and vacuole morphology in concert in a tissue-specific manner, underscoring the link between endomembranes and gravitropism. One of the chemicals (5403629) was structurally similar to the synthetic auxin 2,4-dichlorophenoxy acetate, whereas the other three chemicals were unique in their structures. An in vivo functional assay using the reporter beta-glucuronidase under the auxin-inducible DR5 promoter confirmed that the unique compounds were not auxins. Interestingly, one of the unique chemicals (5850247) appeared to decrease the responsiveness to auxin in roots, whereas another (5271050) was similar to pyocyanin, a bacterial metabolite that has been suggested to target the endomembranes of yeast. These reagents will be valuable for dissecting endomembrane trafficking and gravitropism and for cognate target identification.
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http://dx.doi.org/10.1073/pnas.0500222102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC555711PMC
March 2005

Gene and enhancer traps for gene discovery.

Methods Mol Biol 2003 ;236:221-40

Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California-Riverside, Riverside, CA, USA.

Gene traps and enhancer traps provide a valuable tool for gene discovery. With this system, genes can be identified based solely on the expression pattern of an inserted reporter gene. The use of a reporter gene, such as beta-glucuoronidase (GUS), provides a very sensitive assay for the identification of tissue- and cell-type specific expression patterns. In this chapter, protocols for examining and documenting GUS reporter gene activity in individual lines are described. Methods for the amplification of sequences flanking transposant insertions and subsequent molecular and genetic characterization of individual insertions are provided.
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http://dx.doi.org/10.1385/1-59259-413-1:221DOI Listing
February 2004
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