Proteomic Responses of Dark-Adapted Euglena gracilis and Bleached Mutant Against Light Stimuli

Abstract

Euglena gracilis (E. gracilis) has secondary endosymbiotic chloroplasts derived from ancient green algae. Its chloroplasts are easily lost under numerous conditions to become permanently bleached mutants. Green cells adapted in the dark contain undeveloped proplastids and they will develop into mature chloroplasts after 3 days of light exposure. Thus, E. gracilis is an ideal model species for a chloroplast development study. Previous studies about chloroplast development in E. gracilis focused on morphology and physiology, whereas few studies have addressed the regulatory processes induced by light in the proteome. In this study, the whole-genome proteome of dark-adapted E. gracilis (WT) and permanently ofloxacin-bleached mutant (B2) was compared under the light exposure after 0, 12, and 72 h. The results showed that the photosynthesis-related proteins were up-regulated over time in both WT and B2. The B2 strain, with losing functional chloroplasts, seemed to possess a complete photosynthetic function system. Both WT and B2 exhibited significant light responses with similar alternation patterns, suggesting the sensitive responses to light in proteomic levels. The main metabolic activities for the utilization of carbon and energy in WT were up-regulated, while the proteins with calcium ion binding, cell cycle, and non-photosynthetic carbon fixation were down-regulated in B2. This study confirmed light-induced chloroplast development in WT from dark, and also for the first time investigates the light responses of a bleached mutant B2, providing more information about the unknown functions of residual plastids in Euglena bleached mutants.

Keywords: Euglena gracilis; bleached strain; chloroplast development; light exposure restoration; proteome.

FIGURE 1

Photos of chloroplast development in wild type (WT) and bleached mutant (B2) of E. gracilis after light exposure restoration. Two groups (WT and B2) that included cell pellets images and microscopy images from visible light and fluorescent light were shown at the time point of 0, 12, and 72 h, respectively.

FIGURE 2

Principle component analysis (PCA) plot of proteomic samples in WT and B2. In the score plot, samples were distinguished by different shapes and colors of symbol.

FIGURE 3

Heatmap of protein profile in WT and B2. All the proteins can be clustered as 10 groups based on their distribution similarity.

FIGURE 4

GO annotation and KEGG pathways profile of the differential protein clusters in WT and B2. (A) The GO annotation of the differential proteins in cluster. (B) KEGG pathways profile of the differential proteins in cluster. The axis of abscissa was the groups’ name and the ordinate represented the GO annotation or KEGG pathways. The size of bubbles was calculated by the number of matched proteins. p-value was shown on the left color bar.

FIGURE 5

GO items in biological process (BP), cellular component (CC), and molecular function (MF) categories for the differential genes. The axis of abscissa was the groups’ name and the ordinate represented the GO annotation. The size of bubbles was calculated by the number of matched proteins. p-value was shown on the left color bar.

FIGURE 6

Up/down-regulated GO categories for WT and B2 during the chloroplast development. The number of matched proteins was in parentheses. The size of bubbles represented the GeneRatio of mapping GO. p-value was shown on the left color bar.

FIGURE 7

Up/down-regulated KEGG pathways for WT and B2 during chloroplast development. The number of matched proteins was in parentheses. The size of bubbles represented the GeneRatio of mapping KEGG pathways. p-value was shown on the left color bar.

FIGURE 8

Comparison of WT and B2 in the photosynthesis KEGG pathway map after light exposure restoration. The map of the KEGG photosynthesis pathway is capable of the website on http://www.kegg.jp/pathway/map00195. The green boxes indicate the genes of encoded proteins. The red boxes indicate the genes of encoded proteins that are up-regulated, while the blue boxes are down-regulated.