Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars

Abstract

Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants.In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at threetrifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mM NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier

Quantitative Phosphoproteomic and Metabolomic Analyses Reveal GmMYB173 Optimizes Flavonoid Metabolism in Soybean under Salt Stress

“…Martens Yan et al found that the RNA interference of GmFNSII (coding for CYP93B, an enzyme in cytochrome P450 monooxygenase subfamily) significantly reduced the tolerance of soybean roots to salinity because of the decreased flavone level and higher H 2 O 2 accumulation (9). Our recent study documented that salt tolerance of Arabidopsis and soybean were positively regulated by CHS and negatively regulated by CHI and CPM (28). It is noteworthy that CHS is a single copy gene in the Arabidopsis genome (29), and our results confirmed that the AtCHS knockout mutant is extremely sensitive to salt stress, demonstrating that chalcone and its downstream derivatives play a key role in protecting plants from the damage caused by saline stresses (28).…”

“…Using phosphoproteomic and proteomic approaches, our previous studies revealed that several key proteins (such as MYB TFs, CHS, CHI and CPM) are involved in soybean tolerance to salinity (28). We also proposed a salt tolerance pathway involving flavonoid metabolism, mostly mediated by phosphorylated MYB TFs.…”

“…Our recent study documented that salt tolerance of Arabidopsis and soybean were positively regulated by CHS and negatively regulated by CHI and CPM (28). It is noteworthy that CHS is a single copy gene in the Arabidopsis genome (29), and our results confirmed that the AtCHS knockout mutant is extremely sensitive to salt stress, demonstrating that chalcone and its downstream derivatives play a key role in protecting plants from the damage caused by saline stresses (28). CHS genes in leguminous plants is comprised of a multiple gene family, for instance there are at least nine CHS genes in soybean (29).…”

“…The physiological responses to salinity rely on diverse interplays, such as those between kinases and protein phosphorylation (28), between MYB-type TFs and salt-responsive enzymes (e.g. CHS, CHI, and CPM) (32,40), and those between enzymes and metabolites (24).…”

The stability and ABA import activity of NRT1.2 in Arabidopsis are regulated by CEPR2 via phosphorylation modification

“…Phosphoproteome analysis was performed by capitalBio technology (Beijing, China). For total protein extraction, the TCA/Acetone extraction method was used to isolate total proteins from CEPR2-OE-9 and WT seedlings grown in 1/2 MS for seven days as described by Pi et al (Pi et al, 2016). See Supplementary Figure 1a for a detailed description.…”