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Point-mutation Lines with Disrupted Vacuoles and High-throughput Confocal Microscopy

A functioning vacuole and intact vesicular trafficking system are necessary for plant cell viability and function. Perturbation of the trafficking machinery often impedes vital cell processes such as cytokinesis, the response to plant hormones and the development of tissue specificity. We also found that many of the genes that encode proteins that mediate endomembrane trafficking are either single-copy genes or members of gene families, which complicates assigning functions to individual proteins. Unfortunately, the knockout mutations of many of these genes are often either embryonic (Rojo et al., 2001) [Full Text PDF] or gamethopytic lethal (Sanderfoot et al., 2001) or have no obvious abnormalities under normal conditions (Zheng et al., 2002 [Full Text PDF]; Surpin et al., 2003) [Full Text PDF].

Figure 3.

Figure 4.   Using a mutagenized transgenic line expressing a tonoplast localized protein fused to GFP we were able to screen for vacuolar biogenesis mutants using confocal microscopy (Figure 3). We isolated four groups of mutants with tonoplast-localized green fluorescent protein (GFP) fusion proteins that exhibit defective or modified vacuoles (Figure 4) To perform this screen we developed a high-throughput confocal microscopy with specialized culture plates for germinating and growing seedlings (Avila et al., 2003) [Full Text PDF ]. We are in the process of cloning some of the corresponding genes and studying the biology of these interesting mutants. One of the interesting observations of these studies is that the endomembrane systems of the shoots and roots is uncoupled, a factor that will have to be taken into account when designing future studies (Avila et al., 2003 [Full Text PDF] ; Surpin and Raikhel, 2004) [Full Text PDF ]. Although we used EMS to mutagenize plants to generate point mutations, approximately 50% of the vacuolar mutants did not survive again pointing to the key role of the endomembrane system in plant development.

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Proteomic Analysis of the Arabidopsis Thaliana Vegetative Vacuole

As mentioned before, vacuoles are essential organelles for plant life. In order to better understand vacuolar 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 subjected either to 2-dimensional liquid chromatography/tandem mass spectrometry or 1-D SDS PAGE followed by nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). A tonoplast-enriched fraction was also analyzed separately by 1-D SDS PAGE followed by nano-LC MS/MS. Cumulatively, a total of 381 proteins were identified from these analyses. An analysis of the identified proteins and their roles in vacuole function and biogenesis is underway in my lab. We are using similar approaches for other mutants that affect endomembrane and/or vacuolar biogenesis. For example, precursor protease vesicles (PPVs) are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We found that the PPV-localized vacuolar processing enzyme-γ (VPEγ) is critical for maturation of the vacuolar protease AtCPY. We also showed biochemical and functional evidence that VPEγ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpeγ mutants but not in wildtype plants, suggesting a unique role of VPEγ in protein processing and degradation in Arabidopsis (Rojo et al., 2003) [Full Text PDF]. We continue an in-depth proteomics analysis of the vacuolar content of these vpeγ mutants.

Figure 5. Carter C, Pan S, Zouhar J, Avila EL, Girke T, and Raikhel NV (2004) The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16:3285-3303. A) Isolation and processing of vacuolar proteins. Vacuoles were purified from A. thaliana ecotype Columbia rosette leaf tissue and subjected to 1-D SDS PAGE. The resulting gel was stained with Coomassie blue, cut into slices, digested with trypsin and processed for nano-liquid chromatography tandem mass spectrometric analyses (nano-LC MS/MS). B) Mass spectrometry output. The upper panel represents a parent peptide ion. The monoisotopic peak (labeled with an asterisk) was selected for ESI fragmentation analysis shown in the lower panel and was identified as a peptide derived from a vacuolar invertase (AtFruct4).

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Genetic Approach to Identify Components of the CTPP Pathway

Secretory proteins are sorted to plant cell vacuoles or are retained within the secretory pathway by mechanisms that require specific targeting information contained within thestructure of the protein. It has been demonstrated that secretory proteins lacking such information follow a default pathway and are secreted. Most soluble proteins are transported through the secretory system via a series of transport vesicles that bud from one compartment and fuse specifically with the next. Two of these sorting signals, an N-terminal propeptide (NTPP) and a C-terminal propeptide (CTPP) are directed to the vacuole by distinct pathways (Figure 6). We have characterized several components of the machinery involved in the sorting of NTPP-type cargo, including a trans-Golgi network (TGN)-localized cargo receptor and many SNARE components involved in vesicular traffic between the TGN and the prevacuolar compartment of the model plant Arabidopsis (Figure 7); Zheng et al., 1999; [Full Text PDF] Ahmed et al., 2000 [Full Text PDF]; Bassham and Raikhel, 2000 [Full Text PDF ]; Sanderfoot et al., 2000 [Full Text PDF], 2001 [Full Text PDF] ). However, we believe that the CTPP pathway is unique to plants. We are using genetic approaches to identify components that are either unique to the CTPP pathway or used by the NTPP- and CTPP pathways simultaneously.

Figure 6.

Figure 7. The VTI Family of v-SNAREs. The A. thaliana VTI family of v-SNAREs contains two members, VTI11 and VTI12, which are expressed in detectable amounts. VTI11 forms a SNARE complex at the pre-vacuolar compartment (PVC) with members of the SYP2 and SYP5 families of t-SNAREs, and VTI12 forms a complex on the trans-Golgi network (TGN) with members of the SYP4 and SYP6 families of t-SNAREs. Both VTI11 and VTI12 can substitute for each other in their respective SNARE complexes, at both the molecular and functional levels. VTI11 has been shown to have a role in the gravitropic response and also contributes to the establishment and maintenance of tissue identity. VTI12 participates in autophagosome formation and/or autophagosome docking and fusion with the central vacuole. The compositions of the individual SNARE complexes that are associated with these different pathways are not known. a) Gravitropism pathway. b) Cell-type-specific pathway. c) Cytoplasm-to-vacuole transport (CVT)/autophagy pathways. A, autophagosome; CV, central vacuole; ER, endoplasmic reticulum; GA, Golgi apparatus; LV, lytic vacuole; N, nucleus. Surpin M and Raikhel NV (2004) Traffic Jams Affect Plant Development and Signal Transduction. Nature Reviews/Molecular Cell Biology 5:100-109.

Figure 8.   Plant growth and development depends upon 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 non-cell autonomously 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 through the secretory pathway 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 (Rojo et al., 2002) [Full Text PDF]. CLV3 protein that is fused to a CTPP vacuolar targeting signal is localized to the vacuole and does not complement the clv3 mutant (Figure 8).

Figure 9.         We have initiated a genetic screen using clv3 for mutants defective in vacuolar targeting of CTPP-bearing protein reporters and are now working to characterize several mutants derived from that screen (Figure 9).

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A Chemical Genomics Screen for Compounds that Affect Gravitropism and Vacuolar Biogenesis

A link between vacuolar function and gravitropism has recently been established with the characterization of agravitropic mutants, such as sgr2 and zig1/vti11. These mutants have lesions in components of the endomembrane system and have abnormal vacuolar morphology (Kato et al., 2002; Morita et al., 2002; Surpin et al., 2003 [Full Text PDF]). Based on these results, it has been proposed that the vacuole is involved in perception or signaling of gravitropism in plants. The mechanism of gravity sensing and the events leading to gravitropic bending are poorly understood in spite of many years of study. This recently discovered link presents exciting opportunities to understand both the endomembrane system and gravitropism from an entirely new perspective.
The limitations of classical genetic approaches to study vacuoles was demonstrated by the isolation of a single vcl1 mutant in Arabidopsis from a screen of over 5,000 T-DNA insertion lines (Rojo et al., 2001) [Full Text PDF]. As has been mentioned above, in addition to the vcl1 mutant, several T-DNA insertions in other components of the endomembrane system result in gametophytic or embryo lethality. To overcome these limitations, a chemical genomics approach has been initiated to facilitate our understanding of vacuolar biogenesis. A screen for compounds that affect shoot gravitropism in Arabidopsis is currently under way using a diverse chemical library of small compounds. The effect of 10,000 chemicals on the gravitropic response has been tested in a semi-high-throughput system (Figure 10). A secondary screen has been initiated using a tonoplast reporter line that carries the δ-TIP::GFP fusion to identify compounds that affect vacuolar morphology by confocal microscopy (Figure 11). The effect of these compounds on gravitropism and endomembrane system morphology is currently being characterized, and screens for resistant (Figure 12) and hypersensitive mutants are under way in my laboratory.     Figure 10.
	Figure 11.       The large-scale use of chemical-genomics screens will require the concomitant development of high-throughput methods to identify chemicals that modify trafficking components. Ultimately, the knowledge accumulated from traditional and chemical genomics screens, along with the development of additional high-throughput methods and computer-modelling techniques can be used to better understand the interactions between trafficking pathways. Such systems biology methods could be used to study cargo loading and fluxes in endomembrane trafficking pathways.

It is clear that the endomembrane trafficking system does not just deliver cargo. It is intimately involved in signal transduction and development. The new approaches undertaken by our and other laboratories increase our opportunities to discover new connections between trafficking, plant development and signal transduction, and mark the beginning of our understanding of these networks of pathways. (Figure 13).

Figure 13. Holistic view of a simplified plant cell. Girke T, Ozkan M, Carter D, Raikhel NV (2003) Towards a Modeling Infrastructure for Studying Plant Cells. Plant Physiol 132: 410-414

Selected Publications: Vacuolar Trafficking (Bibliography Page)

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How Are the Cell Wall Components Organized?

Plant cell walls play a crucial role in plant development, signal transduction, and disease resistance. Although much is known about the structure of various cell wall components, their biosynthesis is largely unknown. Hemicellulose is a heterogeneous group of branched matrix polysaccharides that bind noncovalently to the surface of the cellulose microfibrils and, therefore, shape the cell wall. Xyloglucan is the principal hemicellulose of higher plants that bind tightly but noncovalently to cellulose microfibrils, cross-linking them into a complex network. We are interested in identifying the genes whose products are involved in xyloglucan biosynthesis and for developing testable hypothesis regarding their biochemical function.

Figure 3. Schematic representation of xyloglucan structure.

Our efforts on cell wall biosynthesis are being performed jointly with the research group of Kenneth Keegstra at MSU-DOE Plant Research Laboratory at Michigan State University. Our long-term goal is to understand both the biochemical pathways that result in cell wall biosynthesis and the regulatory events that control them. Our immediate objectives are to investigate the biosynthesis of xyloglucan (Figure 3), the major hemicellulosic polysaccharide in dicots. Our first efforts have focused on fucosyltransferase, an enzyme that adds fucose, a terminal sugar, in xyloglucan. The enzyme has been purified and cDNA clones (At FUT1 and PsFUT1 have been isolated (Perrin et al., 1999; Faik et al., 2000, respectively). A combination of molecular and bioinformatic methods were used to analyze a family of genes homologous to AtFUT1. Nine genes (AtFUT2-10) were identified that share between 47 and 62% amino acid similarity with the xyloglucan-specific fucosyltransferase AtFUT1. RT-PCR analysis indicates that all these genes are expressed. Bioinformatic analysis predicts that these family members are fucosyltransferases, and we first hypothesized that some may also be involved in xyloglucan biosynthesis. We screened for T-DNA insertions in members of this family and identified a plant with an insertion in AtFUT5. Plants homozygous for the insertion appeared phenotypically normal and had no discernible changes in cell wall carbohydrate composition. AtFUT3, AtFUT4, and AtFUT5 were expressed in tobacco suspension culture cells, and the resulting proteins did not transfer fucose from GDP-Fuc to tamarind xyloglucan. AtFUT3, AtFUT4, and AtFUT5 were overexpressed in Arabidopsis plants. Although there was no effect on leaf cell wall carbohydrate composition, stems of plants overexpressing AtFUT4 or AtFUT5 contained more xylose, less arabinose and less galactose than wild-type plants. We suggest that the AtFUT family is likely to include fucosyltransferases important for the synthesis of wall carbohydrates. A targeted analysis of isolated cell wall matrix components from plants altered in expression of these proteins will help determine their specificity and biological function (Sarria et al., 2001).

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of a-xylosyltransferase and b-glucan synthase activities. We were able to show that etiolated pea microsomes contain an a-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto b(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 a-xylosyltransferases involved in xyloglucan biosynthesis (see more details in Plant Genome Project below). To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured using the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained using cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the 14C-labeled xylose mainly to the second glucose residue from the non-reducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an a(1,6)- linkage to the glucan chain. We conclude that this Arabidopsis gene encode an a-xylosyltransferase activity involved in xyloglucan biosynthesis (Faik et al., 2002). We are now using a combination of genomic, bioinformatic, and biochemical approaches to identify and characterize additional genes required for the biosynthesis of xyloglucan.

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Functional Genomics of Hemicellulose Biosynthesis (Plant Genome Project DBI-9975815)

Despite the importance of cell walls to the biology of plants, little is known about the biosynthesis of their major macromolecular components. From the known complexity of cell-wall structure we can predict that wall synthesis requires hundreds of enzymes, but biochemical approaches have been unsuccessful in identifying and characterizing more than a few of them. Comparative molecular genetic studies have not been useful because the walls of other organisms, such as bacteria and yeast, are fundamentally different in composition, structure, and function from those of plants. We posit that a genomics-based approach is particularly appropriate for attacking intractable problems in plant biology such as cell-wall architecture and biosynthesis. Recent advances in genomics make it possible to identify large numbers of genes as being candidates for involvement in particular processes. With the identification of candidate genes for biosynthetic enzymes and regulatory proteins comes the challenge of analyzing the functions of these genes and of the proteins they encode. This task is particularly critical for understanding the numerous genes whose functions are unique to plants [Keegstra and Raikhel, 2001].

Our NSF Genomic grant was initiated on December 1, 1999, and is now in its third year. Three investigators are collaborating on this project: Natasha Raikhel (University of California, Riverside) and Kenneth Keegstra and Jonathan Walton (Michigan State University). Our long-term goal is to understand how hemicelluloses are synthesized, delivered to the cell surface, and incorporated into the wall matrix. Our first step toward this goal is to identify and characterize the polypeptides that mediate polysaccharide biosynthesis. We are working with several plant species, with emphasis on Arabidopsis as a dicot model to investigate xyloglucan biosynthesis (Raikhel and Keegstra) and maize and rice as monocot models to study the hemicelluloses of grasses (Walton). Here we will discuss only our main accomplishments with Arabidopsis.

Prior to the initiation of our genomic grant, we identified the fucosyltransferase involved in xyloglucan biosynthesis, AtFUT1 (Perrin et al., 1999). Identification of AtFUT1 made it possible to use bioinformatic approaches to identify nine additional Arabidopsis genes related to AtFUT1 (Sarria et al., 2001). All these genes, named AtFUT1-10, are located on chromosome 1 or 2 and are clustered on four BAC clones (Table 1). RT-PCR analysis revealed that all members of the AtFUT gene family are expressed, with overlapping expression patterns in roots, stems, and leaves. AtFUT1-10 have been assigned to glycosyltransferase family 37, a group that is distinct from most fucosyltransferases from fungi, bacteria, and animals. The level of amino acid identity between AtFUT family members and fucosyltransferases from non-plant species is lower than 12%, supporting the assignment of the AtFUT family to a new group of fucosyltransferases. Phylogenetic analysis based on amino acid sequences indicate that the AtFUT family can be subdivided further: AtFUT1, AtFUT2, and AtFUT3 each belong in their own group, and AtFUT9 belongs either in its own group or in a larger group containing AtFUT4, -5, -6, -7, -8, and -10. Both biochemical studies to date (Sarria et al, 2001) and genetic studies (Vanzin et al., 2002) support the conclusion that AtFUT1 is the only enzyme required for fucosylation of xyloglucan and that the other enzymes fucosylate other polysaccharides. Because bioinformatics cannot predict the acceptor substrates for these enzymes, further reverse genetic and biochemical studies will be needed to clarify their function.

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TABLE 1
Family of Arabidopsis genes related to Xyloglucan Fucosyltransferase1

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Genomic strategies for determining gene function require two independent steps: the identification of candidate genes and evaluation of the function of the candidates. One effective strategy for evaluating the function of candidate genes is to measure the enzymatic activity of the gene products. Such a strategy requires a reliable enzymatic assay and until recently, an acceptor-dependent assay was not available for the XyG xylosyltransferases. Thus, we invested considerable effort in establishing a biochemical assay for the XyG alpha-(1,6)-xylosyltransferase. Using pea microsomes that are capable of XyG biosynthesis (White et al., 1993) we solubilized an alpha-(1,6)-xylosyltransferase that catalyzes the transfer of xylose]xylose onto alpha-(1,4)-linked glucan oligosaccharides. When cellopentaose was used as acceptor, product analysis revealed that the xylose was present in an alpha-(1,6)-linkage to a glucosyl residue, as expected for an enzyme involved in XyG biosynthesis.

Bioinformatic analyses revealed several candidates for the xylosyltransferases, but the biochemical characterization of the xylosyltransferase activity from peas led us to focus on seven Arabidopsis genes with sequence similarity to a fenugreek alpha-(1,6)-galactosyltransferase that is involved in galactomannan biosynthesis (Edwards et al., 1999). Although the Arabidopsis genes were annotated as á-galactosyltransferases (family 34), we postulated that they might be alpha-xylosyltransferases because of predicted enzymatic similarities between the pea alpha-xylosyltransferase and the fenugreek alpha-galactosyltransferase. Full-length cDNA clones of six of the putative xylosyltransferase genes were expressed in the yeast Pichia pastoris, and the resulting proteins were tested using the biochemical assay described above. Products of one of these candidate genes (AtXT1) showed cello-oligosaccharide-dependent xylosyltransferase activity and produced products similar or identical to those generated by the pea enzyme. Thus, we conclude that AtXT1 encodes xylosyltransferases involved in XyG biosynthesis (Faik et al., 2002) (Table 2).

Although xylosyltransferase activity was not observed with five other putative AtPXTs when they were tested in this assay, it is possible that they require acceptors that already contain a xylosyl residue and are involved in adding other xylosyl residues to the XyG backbone. We are continuing genetic and biochemical analysis of these putative glycosyltransferase genes and their products in search of their biological function.

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TABLE 2
Family of Arabidopsis genes related to Xyloglucan Xylosyltransferase 1

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Raikhel lab, April 2004 (left to right, standing): Glenn Hicks, Valya Kovaleva, Marci Surpin, Olga Zabotina, Mien van de Ven, David Carter (CEPCEB Academic Coordinator, Microscopy/Imaging), Lorena Norambuena, Thomas Girke (CEPCEB Academic Coordinator, Bioinformatics), Jocelyn Brimo, Natasha Raikhel, Clay Carter, Jan Zouhar, Songqin Pan (CEPCEB Academic Coordinator, Proteomics), Narasimha C. Samboju, Jacob Vasquez (left to right, sitting): Emily Avila, Eun-Ju (Julie) Sohn, Georgia Drakakaki, April Agee, Marcela Rojas-Pierce (and Tomás)

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Raikhel Lab at Joshua Tree (2002) (left to right): Nick Price, Jan Zouhar, Valya Kovaleva, Natasha Raikhel, Olga Zabotina, Enrique Rojo, Emily Avila, David Carter (CEPCEB Academic Coordinator, Microscopy/Imaging), Seho Hong, Georgia Drakakaki

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