Tomato (Solanum lycopersicum) is an established model for studying fruit biology; however, most studies of tomato fruit growth and ripening are based on homogenized pericarp, and do not consider the internal tissues, or the expression signatures of individual cell and tissue types. We present a spatiotemporally resolved transcriptome analysis of tomato fruit ontogeny, using laser microdissection (LM) or hand dissection coupled with RNA-Seq analysis. Regulatory and structural gene networks, including families of transcription factors and hormone synthesis and signaling pathways, are defined across tissue and developmental spectra. The ripening program is revealed as comprising gradients of gene expression, initiating in internal tissues then radiating outward, and basipetally along a latitudinal axis. We also identify spatial variations in the patterns of epigenetic control superimposed on ripening gradients. Functional studies elucidate previously masked regulatory phenomena and relationships, including those associated with fruit quality traits, such as texture, color, aroma, and metabolite profiles.

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the ؊1 and ؉1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example. Molecular & Cellular

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

N-Glycosylation is a common form of eukaryotic protein post-translational modification, and one that is particularly prevalent in plant cell wall proteins. Large scale and detailed characterization of N-glycoproteins therefore has considerable potential in better understanding the composition and functions of the cell wall proteome, as well as those proteins that reside in other compartments of the secretory pathway. While there have been numerous studies of mammalian and yeast N-glycoproteins, less is known about the population complexity, biosynthesis, structural variation, and trafficking of their plant counterparts. However, technical developments in the analysis of glycoproteins and the structures the glycans that they bear, as well as valuable comparative analyses with non-plant systems, are providing new insights into features that are common among eukaryotes and those that are specific to plants, some of which may reflect the unique nature of the plant cell wall. In this review we present an overview of the current knowledge of plant N-glycoprotein synthesis and trafficking, with particular reference to those that are cell wall localized.

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