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 ␤-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 ␤-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.nasturtium ͉ cell wall ͉ xyloglucan ͉ polysaccharide

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes.

The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internallytagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.