Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease

Abstract

Background: The normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease.

Results: In this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis.

Conclusions: By integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Keywords: Molecular mechanism; Proteins; R. glutinosa; Replant disease; Transcriptome; iTRAQ.

Fig. 1

a The construction of the isolation plots for R. glutinosa plants. The soil was first removed from the walkways between any two plots (left and right sides), to a depth greater than 1 m, as was soil from the other two sides of each plot. The soil that was dug from these locations was moved away from the plots. The separation walls were then built on the four sides of each plot using brick (Step 2), and the separation walls were then evenly covered with impermeable membranes (Step 3) that were placed tightly against the walls (Step 4). After the separation walls of each plot were built, the soil was backfilled into the plots and walkways (Step 5). Finally, the surfaces of the plots were levelled and made consistent with each other (Step 6). b Construction of R. glutinosa transcriptome libraries and c assembly of unigene sequences generated from different libraries

Fig. 2

a Effects of replant disease on the biomass and related physiological indexes of R. glutinosa. b Comparison of the appearance of replanted and normal-growth R. glutinosa at the most critical harmful stage (90 DAP) caused by replanting and (c) an overview of the iTRAQ experimental protocol. Error bars represent standard deviations (SD). * and ** represent significant differences of tested indexes between replanted and normal-growth R. glutinosa at p < 0.05 and p < 0.01, respectively, based on LSD test

Fig. 3

Functional categories of DEPs in replanted R. glutinosa compared with normal-growth plants. a Total GO biological process categories and b GO biological process categories with the top 20 enriched terms. c Total KEGG and d the top 20 enriched KEGG pathways. * Significantly enriched GO and KEGG categories

Fig. 4

Cellular component and molecular function categories in GO analysis of differentially expressed proteins (DEPs). a Total cellular component categories, b total GO molecular function categories and (c) significantly enriched molecular function categories. * Significantly enriched GO categories

Fig. 5

a DEPs in replanted R. glutinosa compared with normal-growth plants, and key molecular events caused by replant disease. Proteins listed in black were significantly upregulated in replanted R. glutinosa; those in blue were significantly upregulated in normal-growth R. glutinosa. b-f Comparison between the present study and previous studies conducted at different molecular levels. Some important molecular processes responding to replanting identified in this study were also found in previous studies. For example, proteins related to immunity systems were significantly upregulated in replanted R. glutinosa in the present study (a), reflecting a proliferation of microbes that might been mediated by phenolic allelochemicals (b) [11]. Some metabolic pathways (PPP and TCA) enhanced in replanted R. glutinosa (a) were observed in the rhizosphere of replanted R. glutinosa that was analysed by soil metaproteomics (c) [53]. The vital enzymes in phenylalanine metabolism (a), which generated various phenolic acids that have been identified as important allelochemicals (d) [12] were also highly expressed in replanted R. glutinosa. The ethylene and calcium signalling involved in sensing replant disease in the present study (a) were identified at the transcript level (e, f) [26, 27]

Fig. 6

Expression profile of critical DEPs at the transcript level in replanted R. glutinosa compared with normal-growth plants during formation of replant disease. Error bars represent standard deviations (SD)

Fig. 7

Differences in H₂O₂, Ca²⁺ density, and CAT and POD activities between replanted and normal-growth R. glutinosa at the most critical harmful stage caused by replanting (90 DAP). Error bars represent standard deviations (SD). * and ** represent significant differences of tested indexes between replanted and normal-growth R. glutinosa at p < 0.05 and p < 0.01, respectively, based on LSD test

Fig. 8

Hypothetical model for the mechanisms causing damage in replant disease of R. glutinosa