Comparative Transcriptome Combined with Proteome Analyses Revealed Key Factors Involved in Alfalfa (Medicago sativa) Response to Waterlogging Stress

Abstract

Alfalfa (Medicago sativa) is the most widely grown and most important forage crop in the world. However, alfalfa is susceptible to waterlogging stress, which is the major constraint for its cultivation area and crop production. So far, the molecular mechanism of alfalfa response to the waterlogging is largely unknown. Here, comparative transcriptome combined with proteomic analyses of two cultivars (M12, tolerant; M25, sensitive) of alfalfa showing contrasting tolerance to waterlogging were performed to understand the mechanism of alfalfa in response to waterlogging stress. Totally, 748 (581 up- and 167 down-regulated) genes were differentially expressed in leaves of waterlogging-stressed alfalfa compared with the control (M12_W vs. M12_CK), whereas 1193 (740 up- and 453 down-regulated) differentially abundant transcripts (DATs) were detected in the leaves of waterlogging-stressed plants in comparison with the control plants (M25_W vs. M25_CK). Furthermore, a total of 187 (122 up- and 65 down-regulated) and 190 (105 up- and 85 down-regulated) differentially abundant proteins (DAPs) were identified via isobaric tags for relative and absolute quantification (iTRAQ) method in M12_W vs. M12_CK and M25_W vs. M25_CK comparison, respectively. Compared dataset analysis of proteomics and transcriptomics revealed that 27 and eight genes displayed jointly up-regulated or down-regulated expression profiles at both mRNA and protein levels in M12_W vs. M12_CK comparison, whereas 30 and 27 genes were found to be co-up-regulated or co-down-regulated in M25_W vs. M25_CK comparison, respectively. The strongly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for co-up-regulated genes at mRNA and protein levels in M12_W vs. M12_CK comparison were 'Amino sugar and nucleotide sugar metabolism', 'Arginine and proline metabolism' and 'Starch and sucrose metabolism', whereas co-up-regulated protein-related pathways including 'Arginine and proline metabolism' and 'Valine, leucine and isoleucine degradation' were largely enriched in M25_W vs. M25_CK comparison. Importantly, the identified genes related to beta-amylase, Ethylene response Factor (ERF), Calcineurin B-like (CBL) interacting protein kinases (CIPKs), Glutathione peroxidase (GPX), and Glutathione-S-transferase (GST) may play key roles in conferring alfalfa tolerance to waterlogging stress. The present study may contribute to our understanding the molecular mechanism underlying the responses of alfalfa to waterlogging stress, and also provide important clues for further study and in-depth characterization of waterlogging-resistance breeding candidate genes in alfalfa.

Keywords: alfalfa; molecular mechanism; proteome; transcriptome; waterlogging.

Figure 1

Effect of waterlogging on the phenotypic trait (A), leaf chlorophyll content (B), maximum quantum yield of photosystem II efficiency (Fv/Fm, (C) and net photosynthetic rate (P_n) (D) in two alfalfa cultivars with contrasting waterlogging tolerance (M12: tolerant; M25, sensitive). Vertical bars on the top indicate standard deviation, and bars with the same letter indicate no significant difference at p < 0.05 for the comparison of different treatments (Duncan’s multiple range test).

Figure 2

Histogram of gene ontology (GO) classification. The results are summarized in three main categories: biological process, cellular component, and molecular function.

Figure 3

The euKaryotic Ortholog Groups (KOG) annotation of putative proteins. All 13,371 putative proteins assigned to KOG classification and classified into 25 molecular families.

Figure 4

Volcano plots of differentially abundant transcripts in waterlogging-tolerant (A, M12) and waterlogging-sensitive (B, M25) plants after RNA-seq analysis. The x-axis represents the natural logarithm of fold change (Fc) and the y-axis represents log10 of the p-value of each transcript; (C) Differentially abundant transcripts showed in Venn diagram form; (D) hierarchical clustering analysis of waterlogging-induced changes in transcripts in leaves of alfalfa (M12CK indicates M12 under control condition; M12W indicates M12 under waterlogged condition; M25CK indicates M25 under control condition; M25W indicates M25 under waterlogged condition).

Figure 5

The number of differentially abundant proteins (fold change ≥ 1.3 or fold change ≤ 0.77 and p-value < 0.05) in two alfalfa cultivars (M12 and M25) under waterlogged and control conditions. M12CK indicate M12 under control condition; M12W indicate M12 under waterlogged condition; M25CK indicate M25 under control condition; M25W indicate M25 under waterlogged condition.

Figure 6

KEGG pathway analysis of differentially abundant proteins in two alfalfa cultivars (M12 and M25) under waterlogged and control conditions. M12CK indicates M12 under control condition; M12W indicates M12 under waterlogged condition; M25CK indicates M25 under control condition; M25W indicates M25 under waterlogged condition.

Figure 7

KEGG pathways for co-regulated genes at mRNA and protein levels in two alfalfa cultivars (A, M12W vs. M12CK; B, M25W vs. M25CK) under waterlogged and control conditions. M12CK indicates M12 under control condition; M12W indicates M12 under waterlogged condition; M25CK indicates M25 under control condition; M25W indicates M25 under waterlogged condition.

Figure 8

A schematic model for waterlogging stress responses in alfalfa plants.