Proteomic analysis of peach fruit mesocarp softening and chilling injury using difference gel electrophoresis (DIGE).

BACKGROUND: Peach fruit undergoes a rapid softening process that involves a number of metabolic changes. Storing fruit at low temperatures has been widely used to extend its postharvest life. However, this leads to undesired changes, such as mealiness and browning, which affect the quality of the fruit. In this study, a 2-D DIGE approach was designed to screen for differentially accumulated proteins in peach fruit during normal softening as well as under conditions that led to fruit chilling injury. RESULTS: The analysis allowed us to identify 43 spots -representing about 18% of the total number analyzed- that show statistically significant changes. Thirty-nine of the proteins could be identified by mass spectrometry. Some of the proteins that changed during postharvest had been related to peach fruit ripening and cold stress in the past. However, we identified other proteins that had not been linked to these processes. A graphical display of the relationship between the differentially accumulated proteins was obtained using pairwise average-linkage cluster analysis and principal component analysis. Proteins such as endopolygalacturonase, catalase, NADP-dependent isocitrate dehydrogenase, pectin methylesterase and dehydrins were found to be very important for distinguishing between healthy and chill injured fruit. A categorization of the differentially accumulated proteins was performed using Gene Ontology annotation. The results showed that the 'response to stress', 'cellular homeostasis', 'metabolism of carbohydrates' and 'amino acid metabolism' biological processes were affected the most during the postharvest. CONCLUSIONS: Using a comparative proteomic approach with 2-D DIGE allowed us to identify proteins that showed stage-specific changes in their accumulation pattern. Several proteins that are related to response to stress, cellular homeostasis, cellular component organization and carbohydrate metabolism were detected as being differentially accumulated. Finally, a significant proportion of the proteins identified had not been associated with softening, cold storage or chilling injury-altered fruit before; thus, comparative proteomics has proven to be a valuable tool for understanding fruit softening and postharvest.

AtUTr1, a UDP-glucose/UDP-galactose transporter from Arabidopsis thaliana, is located in the endoplasmic reticulum and up-regulated by the unfolded protein response.

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.

AtUTr2 is an Arabidopsis thaliana nucleotide sugar transporter located in the Golgi apparatus capable of transporting UDP-galactose.

The synthesis of noncellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as a substrate. We have cloned and characterized a nucleotide sugar transporter from Arabidopsis thaliana (L.) Heynh. named AtUTr2. Expression in tobacco and Saccharomyces cerevisiae and subsequent biochemical characterization indicate that AtUTr2 transports UDP-galactose, but not UDP-glucose, UDP-N-acetyl glucosamine, UDP-xylose, UDP-glucuronic acid, GDP-fucose or GDP-mannose. Experiments expressing an AtUTr2-GFP fusion protein in onion epidermal cells suggest that AtUTr2 is located in the Golgi apparatus. Finally, northern analysis indicates that the AtUTr2 transcript was more abundant in roots and calli although it was also present in other Arabidopsis organs but at lower levels. Therefore, AtUTr2 is a nucleotide sugar transporter capable of transporting UDP-galactose that may play an important role in the synthesis of galactose-containing glycoconjugates in Arabidopsis.