Proteomic Analysis of Coprinopsis cinerea under Conditions of Horizontal and Perpendicular Gravity

Abstract

Coprinopsis cinerea was employed to investigate the fungal response to gravity. Mycelium growth revealed a consistent growth pattern, irrespective of the direction of gravity (i.e., horizontal vs. perpendicular). However, the fruiting body grew in the direction opposite to that of gravity once the primordia had formed. For the proteomic analysis, only curved-stem samples were used. Fifty-one proteins were identified and classified into 13 groups according to function. The major functional groups were hydrolases and transferases (16%), signal transduction (15%), oxidoreductases and isomerases (11%), carbohydrate metabolism (9%), and transport (5%). To the best of our knowledge, this is the first report on a proteomic approach to evaluate the molecular response of C. cinerea to gravity.

Coprinopsis cinerea is a model basidiomycete fungus used for many basic research studies, including research of fungal developmental stages. As with other model organisms, C. cinerea is easy to maintain, has a short life cycle, and can be induced to develop fruit bodies within the laboratory. C. cinerea requires only 2 wk to develop mature fruit bodies [1,2,3]. Once mycelia have reached full growth stage, C. cinerea first develops hyphal knots and thereafter produces primordia followed by the development of fruit bodies. Stalk structures of the fruit body are empty and shaped as thin cylinders. Young fruit bodies contain many glycogen and insoluble proteins as nutrient sources used in the growth and development of the mature fruit bodies [4,5,6]. Traditionally, the Coprinopsis genus has been referred to as an ink cap mushroom, since disintegration of the mushroom results in the development of a black ink when the fruit body has reached full maturation [7,8,9]. This unique characteristic of C. cinerea differs in comparison to other mushrooms, which normally scatter spores by air or raindrops.

All creatures on Earth are affected by and respond to environmental factors, especially gravity. Most biological research involving gravity has to date made use of plant model systems. Parts of the plants that grow above ground show negative gravitropism when compared to the root, which shows positive gravitropism. Gravitropism can be split into three processes, namely gravity perception, signal transduction, and asymmetric growth response [10]. Plants have been shown to recognize gravity through starch-filled cells in the root tissue. The starch-filled hypothesis suggests that plants recognize gravity by the starch particles moving in the direction of the gravity. Starch granule movement is considered to initiate the signal transduction processes in response to gravity [10,11]. Plants have been reported to recognize gravity through the endodermis layer inside the plant cortex [11,12]. Research regarding the gravity response and its mechanism of action in fungi has rarely been conducted over past few decades. Therefore, in the present study, we employed C. cinerea as the model fungus and adopted a proteomics approach to observe the physical and molecular responses of the mushroom to gravity.

C. cinerea was maintained on yeast malt glucose media (yeast extract 3 g, malt extract 3 g, glucose 10 g, agar 20 g per 1 L, pH 6.2) and grown at 30℃. Mycelia were grown on plates for 5 days. For fruit body formation, cultures were incubated at room temperature for 3–4 days under a 12 hr light/dark cycle in either a horizontal or perpendicular orientation. Regardless of the growth conditions, fruit bodies grew in the direction opposite to that of gravity, unlike growth of the hyphae (Fig. 1A). Fruit bodies grown in a perpendicular orientation, grew in a skyward direction and developed bent stalks (Fig. 1B). This demonstrates that the mycelium of C. cinerea did not recognize gravity, whereas the fruiting body actively responded to gravity.

Fig. 1. Formation of fruiting bodies under different gravity growth conditions. A, Horizontal growth conditions; B, Perpendicular growth conditions.

Proteomic analyses were undertaken to identify protein expression under each growth condition. To extract protein, only banded-stalks (less than 1 cm) were harvested from mushrooms grown under horizontal and perpendicular conditions. A total of three biological replicates were employed for the two-dimensional gel electrophoresis (2-DE) experiments and for each experiment, 10–12 stalks were harvested for protein extraction. Total protein was extracted using the trichloroacetic acid/acetone/phenol protein extraction protocol, as previously described [13]. Extracted protein concentrations were measured using a 2-DE Quant kit (Amersham Bioscience, San Francisco, CA, USA). For the 2-DE experiments, 200 µg of protein was loaded onto immobilized strips (17 cm, pH 5–8) and subjected to isoelectric focusing for 15 min at 250 V, 3 hr at 8,000 V, and 5 hr at 50,000 V. The equilibrated strips were subsequently separated on 12% polyacrylamide gels at 25 mA at room temperature and the gels stained with Coomassie brilliant blue. The C. cinerea fruiting bodies that were grown under either the horizontal or perpendicular conditions were evaluated as three independent, biological replicates. Each gel image was captured using a GS-800 Imaging Densitometer Scanner (Bio-Rad, Hercules, CA, USA) (Fig. 2). The 2-DE image of C. cinerea stalks grown in a horizontal orientation served as the control for quantitative analysis of stalks grown in a perpendicular orientation. With the differential expression threshold cut-off set as a change of 1.5-fold, a total of 69 differentially expressed protein spots were identified (Table 1). Irrespective of fold change, 10 proteins were up-regulated and 59 proteins down-regulated when comparing expression levels under perpendicular growth conditions to those under horizontal conditions. These 69 proteins were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for further identification (Table 1). With the cut-off for differential expression set as a change of 1.5-fold, 51 proteins were identified as being differentially expressed in the respective gravity states. Under perpendicular growth conditions, six proteins were up-regulated and 45 proteins were down-regulated compared with their expression levels under horizontal growth conditions.

Fig. 2. Representative two-dimensional gel electrophoresis (2-DE) image of Coprinopsis cinerea. A, Horizontal growth conditions; B, Perpendicular growth conditions; C, Overlay of the horizontal and perpendicular 2-DE images to identify spots indicating differential protein expression. The numbers on the gel image indicate differentially expressed proteins.

Table 1. Differentially expressed proteins identified under each gravity growth condition by 2-DE and MALDI-TOF MS.

^a2-DE, two-dimensional gel electrophoresis; MALDI-TOF/TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MW, molecular weight; MP, ratio of matched peptides; SC, sequence coverage.

^b‘+’ Up-regulation, ‘−’ down regulation; 1.5-fold change was set as the cut-off for differential protein expression.

Among the 69 proteins showing significantly different expression levels as a result of the gravity growth conditions, 51 were identified in a protein database. The identified proteins were subsequently classified into 13 groups according to their molecular function. The major functional groups were as follows: hydrolases and transferases (16%), signal transduction (15%), oxidoreductases and isomerases (11%), and carbohydrate metabolism (9%) (Fig. 3). We confirmed that the level of protein expression declined under perpendicular growth conditions, with those proteins showing differential expression under perpendicular growth conditions being primarily related to signal transduction processes. This finding suggests that gravity triggers a molecular response that either increases or decreases the levels of protein expression. Tyrosinases and serine-proteases are also known to be specifically expressed in different stalk and cap tissues during senescence [14]. During the sexual development of C. cinerea, xenobiotic detoxification-related proteins (cytochrome p450, glutathione S-transferase, transporter) and several types of proteases are known to be differentially expressed in specific tissues [15]. In the present study, glutathione S-transferase, several transporters, and serinetype peptidases were detected as differentially expressed under the different gravity growth conditions. This suggests that the response of C. cinerea to gravity may be related to the fruiting body development mechanism of the fungus.

Fig. 3. Functional classification of proteins that were differentially expressed under each growth condition. A, up-regulated proteins; B, down-regulated proteins.

In conclusion, we identified 51 proteins that were differentially expressed in C. cinerea under horizontal and perpendicular growth conditions. The functions of the identified proteins were related to hydrolases and transferases, signal transduction, oxidoreductases and isomerases, and carbohydrate metabolism. This study, which examined fungi gravitropism through a proteomic analysis of C. cinerea, will help contribute towards our understanding of fungal responses to gravity.