Natasha V. Raikhel

Sharing publication-related data and materials: responsibilities of authorship in the life sciences.

Thomas R. Cech, (Chair), Howard Hughes Medical Institute, Chevy Chase, Maryland Sean R. Eddy, Howard Hughes Medical Institute; Washington University, St. Louis, Missouri David Eisenberg, Howard Hughes Medical Institute; University of California, Los Angeles Karen Hersey, Massachusetts Institute of Technology, Cambridge Steven H. Holtzman, Infinity Pharmaceuticals, Inc., Boston, Massachusetts George H. Poste, Health Technology Networks, Gilbertsville, Pennsylvania Natasha V. Raikhel, University of California, Riverside Richard H. Scheller, Genentech, Inc., South San Francisco, California David B. Singer, GeneSoft, Inc., South San Francisco, California Mary C. Waltham, Independent Publishing Consultant, Princeton, New Jersey

The Vegetative Vacuole Proteome of Arabidopsis thaliana Reveals Predicted and Unexpected Proteins

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.

CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway.

Plant growth and development depends on the activity of a continuously replenished pool of stem cells within the shoot apical meristem to supply cells for organogenesis. In Arabidopsis, the stem cell–specific protein CLAVATA3 (CLV3) acts cell nonautonomously to restrict the size of the stem cell population, but the hypothesis that CLV3 acts as an extracellular signaling molecule has not been tested. We used genetic and immunological assays to show that CLV3 localizes to the apoplast and that export to the extracellular space is required for its function in activating the CLV1/CLV2 receptor complex. Apoplastic localization allows CLV3 to signal from the stem cell population to the organizing center in the underlying cells.

MicroRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabidopsis

Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here, we show that the translation inhibition, but not the mRNA cleavage activity, of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild-type and the amp1 mutant for membrane-bound, but not total, polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.

Arabidopsis Argonaute 2 Regulates Innate Immunity via miRNA393*-Mediated Silencing of a Golgi-Localized SNARE Gene, MEMB12

Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNA is miR393b(∗), which targets a Golgi-localized SNARE gene, MEMB12. Pst infection downregulates MEMB12 in a miR393b(∗)-dependent manner. Loss of function of MEMB12, but not SYP61, another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein, PR1. Overexpression of miR393b(∗) resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b(∗) to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393(∗)/miR393 represent an example of a miRNA(∗)/miRNA pair that functions in immunity through two distinct AGOs: miR393(∗) through AGO2 and miR393 through AGO1.

VPEγ exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

A gene from the cellulose synthase-like C family encodes a β-1,4 glucan synthase

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the β-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a β-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

An Arabidopsis gene encoding an α-xylosyltransferase involved in xyloglucan biosynthesis

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of α-xylosyltransferase and β-glucan synthase activities. Here we report that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto β(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode α-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the 14C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an α(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an α-xylosyltransferase activity involved in xyloglucan biosynthesis.

The VTI Family of SNARE Proteins Is Necessary for Plant Viability and Mediates Different Protein Transport Pathways

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

In this work, Kyn is identified as an auxin biosynthesis inhibitor that effectively and selectively targets TAA1-like Trp aminotransferases. Moreover, it describes a previously undiscovered positive feedback loop between auxin biosynthesis and ethylene signaling pathways in roots. The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.